Danny Reinberg, Ph.D.

Chromatin higher-order structures and gene regulation
Li, Guohong; Reinberg, Danny
2011 Apr;21(2):175-186, Current opinion in genetics & development
Genomic DNA in the eukaryotic nucleus is hierarchically packaged by histones into chromatin to fit inside the nucleus. The dynamics of higher-order chromatin compaction play a crucial role in transcription and other biological processes inherent to DNA. Many factors, including histone variants, histone modifications, DNA methylation, and the binding of non-histone architectural proteins regulate the structure of chromatin. Although the structure of nucleosomes, the fundamental repeating unit of chromatin, is clear, there is still much discussion on the higher-order levels of chromatin structure. In this review, we focus on the recent progress in elucidating the structure of the 30-nm chromatin fiber. We also discuss the structural plasticity/dynamics and epigenetic inheritance of higher-order chromatin and the roles of chromatin higher-order organization in eukaryotic gene regulation
— id: 133353, year: 2011, vol: 21, page: 175, stat: Journal Article,

The Polycomb complex PRC2 and its mark in life
Margueron, Raphael; Reinberg, Danny
2011 Jan 20;469(7330):343-349, Nature
Polycomb group proteins maintain the gene-expression pattern of different cells that is set during early development by regulating chromatin structure. In mammals, two main Polycomb group complexes exist - Polycomb repressive complex 1 (PRC1) and 2 (PRC2). PRC1 compacts chromatin and catalyses the monoubiquitylation of histone H2A. PRC2 also contributes to chromatin compaction, and catalyses the methylation of histone H3 at lysine 27. PRC2 is involved in various biological processes, including differentiation, maintaining cell identity and proliferation, and stem-cell plasticity. Recent studies of PRC2 have expanded our perspectives on its function and regulation, and uncovered a role for non-coding RNA in the recruitment of PRC2 to target genes
— id: 121294, year: 2011, vol: 469, page: 343, stat: Journal Article,

The structure of NSD1 reveals an autoregulatory mechanism underlying histone H3K36 methylation
Qiao, Qi; Li, Yan; Chen, Zhi; Wang, Mingzhu; Reinberg, Danny; Xu, Rui-Ming
2011 Mar 11;286(10):8361-8368, Journal of biological chemistry
The Sotos syndrome gene product, NSD1, is a SET domain histone methyltransferase that primarily dimethylates nucleosomal histone H3 lysine 36 (H3K36). To date, the intrinsic properties of NSD1 that determine its nucleosomal substrate selectivity and dimethyl H3K36 product specificity remain unknown. The 1.7 A structure of the catalytic domain of NSD1 presented here shows that a regulatory loop adopts a conformation that prevents free access of H3K36 to the bound S-adenosyl-L-methionine. Molecular dynamics simulation and computational docking revealed that this normally inhibitory loop can adopt an active conformation, allowing H3K36 access to the active site, and that the nucleosome may stabilize the active conformation of the regulatory loop. Hence, our study reveals an autoregulatory mechanism of NSD1 and provides insight into the molecular mechanism of the nucleosomal substrate selectivity of this disease-related H3K36 methyltransferase
— id: 138314, year: 2011, vol: 286, page: 8361, stat: Journal Article,

A dual flip-out mechanism for 5mC recognition by the Arabidopsis SUVH5 SRA domain and its impact on DNA methylation and H3K9 dimethylation in vivo
Rajakumara, Eerappa; Law, Julie A; Simanshu, Dhirendra K; Voigt, Philipp; Johnson, Lianna M; Reinberg, Danny; Patel, Dinshaw J; Jacobsen, Steven E
2011 Jan 15;25(2):137-152, Genes & development
Cytosine DNA methylation is evolutionarily ancient, and in eukaryotes this epigenetic modification is associated with gene silencing. Proteins with SRA (SET- or RING-associated) methyl-binding domains are required for the establishment and/or maintenance of DNA methylation in both plants and mammals. The 5-methyl-cytosine (5mC)-binding specificity of several SRA domains have been characterized, and each one has a preference for DNA methylation in different sequence contexts. Here we demonstrate through mobility shift assays and calorimetric measurements that the SU(VAR)3-9 HOMOLOG 5 (SUVH5) SRA domain differs from other SRA domains in that it can bind methylated DNA in all contexts to similar extents. Crystal structures of the SUVH5 SRA domain bound to 5mC-containing DNA in either the fully or hemimethylated CG context or the methylated CHH context revealed a dual flip-out mechanism where both the 5mC and a base (5mC, C, or G, respectively) from the partner strand are simultaneously extruded from the DNA duplex and positioned within binding pockets of individual SRA domains. Our structure-based in vivo studies suggest that a functional SUVH5 SRA domain is required for both DNA methylation and accumulation of the H3K9 dimethyl modification in vivo, suggesting a role for the SRA domain in recruitment of SUVH5 to genomic loci
— id: 121295, year: 2011, vol: 25, page: 137, stat: Journal Article,

The C-terminal domain of RNA polymerase II is modified by site-specific methylation
Sims, Robert J 3rd; Rojas, Luis Alejandro; Beck, David; Bonasio, Roberto; Schuller, Roland; Drury, William J 3rd; Eick, Dirk; Reinberg, Danny
2011 Apr 1;332(6025):99-103, Science
The carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) in mammals undergoes extensive posttranslational modification, which is essential for transcriptional initiation and elongation. Here, we show that the CTD of RNAPII is methylated at a single arginine (R1810) by the coactivator-associated arginine methyltransferase 1 (CARM1). Although methylation at R1810 is present on the hyperphosphorylated form of RNAPII in vivo, Ser2 or Ser5 phosphorylation inhibits CARM1 activity toward this site in vitro, suggesting that methylation occurs before transcription initiation. Mutation of R1810 results in the misexpression of a variety of small nuclear RNAs and small nucleolar RNAs, an effect that is also observed in Carm1(-/-) mouse embryo fibroblasts. These results demonstrate that CTD methylation facilitates the expression of select RNAs, perhaps serving to discriminate the RNAPII-associated machinery recruited to distinct gene types
— id: 130305, year: 2011, vol: 332, page: 99, stat: Journal Article,

L3MBTL2 Protein Acts in Concert with PcG Protein-Mediated Monoubiquitination of H2A to Establish a Repressive Chromatin Structure
Trojer, Patrick; Cao, Alina R; Gao, Zhonghua; Li, Yan; Zhang, Jin; Xu, Xiaoqin; Li, Guohong; Losson, Regine; Erdjument-Bromage, Hediye; Tempst, Paul; Farnham, Peggy J; Reinberg, Danny
2011 May 20;42(4):438-450, Molecular cell
We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb repressive complex 1 (PRC1)-like 4 (PRC1L4), given the copresence of PcG proteins RING1, RING2, and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/HP1gamma, known to function in transcriptional repression. PRC1L4-mediated repression necessitated L3MBTL2 that compacted chromatin in a histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT domain-containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genes that exhibited a significant reduction in H2A lysine 119 monoubiquitination. Our findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks
— id: 132884, year: 2011, vol: 42, page: 438, stat: Journal Article,

BRD4 jump-starts transcription after mitotic silencing
Voigt P; Reinberg D
2011 Nov 29;12(11):133-133, Genome biology
ABSTRACT: The chromatin adapter BRD4 may be crucial for transmitting epigenetic information by acting as a histone acetylation-dependent gene bookmark and accelerating post-mitotic transcriptional reactivation
— id: 145774, year: 2011, vol: 12, page: 133, stat: Journal Article,

Histone Tails: Ideal Motifs for Probing Epigenetics through Chemical Biology Approaches
Voigt, Philipp; Reinberg, Danny
2011 Jan 24;12(2):236-252, Chembiochem : a European journal of chemical biology
Post-translational modifications (PTMs) on histone proteins have emerged as a central theme in the regulation of gene expression and other chromatin-associated processes. The discovery that certain protein domains can recognize acetylated and methylated lysine residues of histones has spurred efforts to uncover and characterize histone PTM-binding proteins. In this task, chromatin biology has strongly benefited from synthetic approaches stemming from chemical biology. Peptide-based techniques have been instrumental in identifying histone mark-binding proteins and analyzing their binding specificities. To explore how histone PTMs carry out their function in the context of chromatin, reconstituted systems based on recombinant histones carrying defined modifications are increasingly being used. They constitute promising tools to analyze mechanistic aspects of histone PTMs, including their role in transcription and their transmission in replication. In this review, we present strategies that have been used successfully to investigate the role of histone modifications, concepts that have emerged from their application, and their potential to contribute to current developments in the field
— id: 120654, year: 2011, vol: 12, page: 236, stat: Journal Article,

Epigenetic inheritance: uncontested?
Zhu, Bing; Reinberg, Danny
2011 Mar;21(3):435-441, Cell research
'Epigenetics' is currently defined as 'the inheritance of variation (-genetics) above and beyond (epi-) changes in the DNA sequence'. Despite the fact that histones are believed to carry important epigenetic information, little is known about the molecular mechanisms of the inheritance of histone-based epigenetic information, including histone modifications and histone variants. Here we review recent progress and discuss potential models for the mitotic inheritance of histone modifications-based epigenetic information
— id: 134120, year: 2011, vol: 21, page: 435, stat: Journal Article,

MBT domain proteins in development and disease
Bonasio, Roberto; Lecona, Emilio; Reinberg, Danny
2010 Apr;21(2):221-230, Seminars in cell & developmental biology
The Malignant Brain Tumor (MBT) domain is a 'chromatin reader', a protein module that binds to post-translational modifications on histone tails that are thought to affect a variety of chromatin processes, including transcription. More specifically, MBT domains recognize mono- and di-methylated lysines at a number of different positions on histone H3 and H4 tails. Three Drosophila proteins, SCM, L(3)MBT and SFMBT contain multiple adjacent MBT repeats and have critical roles in development, maintenance of cell identity, and tumor suppression. Although they function in different pathways, these proteins all localize to chromatin in vivo and repress transcription by a currently unknown molecular mechanism that requires the MBT domains. The human genome contains several homologues of these MBT proteins, some of which have been linked to important gene regulatory pathways, such as E2F/Rb- and Polycomb-mediated repression, and to the insurgence of certain neurological tumors. Here, we review the genetics, biochemistry, and cell biology of MBT proteins and their role in development and disease
— id: 121299, year: 2010, vol: 21, page: 221, stat: Journal Article,

Molecular signals of epigenetic states
Bonasio, Roberto; Tu, Shengjiang; Reinberg, Danny
2010 Oct 29;330(6004):612-616, Science
Epigenetic signals are responsible for the establishment, maintenance, and reversal of metastable transcriptional states that are fundamental for the cell's ability to 'remember' past events, such as changes in the external environment or developmental cues. Complex epigenetic states are orchestrated by several converging and reinforcing signals, including transcription factors, noncoding RNAs, DNA methylation, and histone modifications. Although all of these pathways modulate transcription from chromatin in vivo, the mechanisms by which epigenetic information is transmitted through cell division remain unclear. Because epigenetic states are metastable and change in response to the appropriate signals, a deeper understanding of their molecular framework will allow us to tackle the dysregulation of epigenetics in disease
— id: 121296, year: 2010, vol: 330, page: 612, stat: Journal Article,

Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator
Bonasio, Roberto; Zhang, Guojie; Ye, Chaoyang; Mutti, Navdeep S; Fang, Xiaodong; Qin, Nan; Donahue, Greg; Yang, Pengcheng; Li, Qiye; Li, Cai; Zhang, Pei; Huang, Zhiyong; Berger, Shelley L; Reinberg, Danny; Wang, Jun; Liebig, Jurgen
2010 Aug 27;329(5995):1068-1071, Science
The organized societies of ants include short-lived worker castes displaying specialized behavior and morphology and long-lived queens dedicated to reproduction. We sequenced and compared the genomes of two socially divergent ant species: Camponotus floridanus and Harpegnathos saltator. Both genomes contained high amounts of CpG, despite the presence of DNA methylation, which in non-Hymenoptera correlates with CpG depletion. Comparison of gene expression in different castes identified up-regulation of telomerase and sirtuin deacetylases in longer-lived H. saltator reproductives, caste-specific expression of microRNAs and SMYD histone methyltransferases, and differential regulation of genes implicated in neuronal function and chemical communication. Our findings provide clues on the molecular differences between castes in these two ants and establish a new experimental model to study epigenetics in aging and behavior
— id: 112045, year: 2010, vol: 329, page: 1068, stat: Journal Article,

The program for processing newly synthesized histones H3.1 and H4
Campos, Eric I; Fillingham, Jeffrey; Li, Guohong; Zheng, Haiyan; Voigt, Philipp; Kuo, Wei-Hung W; Seepany, Harshika; Gao, Zhonghua; Day, Loren A; Greenblatt, Jack F; Reinberg, Danny
2010 Nov;17(11):1343-1351, Nature structural & molecular biology
The mechanism by which newly synthesized histones are imported into the nucleus and deposited onto replicating chromatin alongside segregating nucleosomal counterparts is poorly understood, yet this program is expected to bear on the putative epigenetic nature of histone post-translational modifications. To define the events by which naive pre-deposition histones are imported into the nucleus, we biochemically purified and characterized the full gamut of histone H3.1-containing complexes from human cytoplasmic fractions and identified their associated histone post-translational modifications. Through reconstitution assays, biophysical analyses and live cell manipulations, we describe in detail this series of events, namely the assembly of H3-H4 dimers, the acetylation of histones by the HAT1 holoenzyme and the transfer of histones between chaperones that culminates with their karyopherin-mediated nuclear import. We further demonstrate the high degree of conservation for this pathway between higher and lower eukaryotes
— id: 114589, year: 2010, vol: 17, page: 1343, stat: Journal Article,

New chaps in the histone chaperone arena
Campos, Eric I; Reinberg, Danny
2010 Jul 1;24(13):1334-1338, Genes & development
Understanding exactly how chromatin is assembled is paramount to addressing how select histone modifications may be transmitted, a putative epigenetic process. In the June 15, 2010, issue of Genes & Development, Drane and colleagues (pp. 1253-1265) identified DAXX as a novel H3.3-specific chaperone. This finding, in the context of others published by Goldberg and colleagues in Cell and Sawatsubashi and colleagues (pp. 159-170) in the January 15, 2010, issue of Genes & Development, provides the impetus for uncovering the mechanistic and functional properties of alternative histone deposition pathways
— id: 110689, year: 2010, vol: 24, page: 1334, stat: Journal Article,

G9a and Glp methylate lysine 373 in the tumor suppressor p53
Huang, Jing; Dorsey, Jean; Chuikov, Sergei; Perez-Burgos, Laura; Zhang, Xinyue; Jenuwein, Thomas; Reinberg, Danny; Berger, Shelley L
2010 Mar 26;285(13):9636-9641, Journal of biological chemistry
The tumor suppressor p53 is regulated by numerous post-translational modifications. Lysine methylation has recently emerged as a key post-translational modification that alters the activity of p53. Here, we describe a novel lysine methylation site in p53 that is carried out by two homologous histone methyltransferases, G9a and Glp. G9a and Glp specifically methylate p53 at Lys(373), resulting mainly in dimethylation. During DNA damage, the overall level of p53 modified at Lys(373)me2 does not increase, despite the dramatic increase in total p53, indicating that Lys(373)me2 correlates with inactive p53. Further, reduction of G9a and/or Glp levels leads to a larger population of apoptotic cells. Examination of the Oncomine data base shows that G9a and Glp are overexpressed in various cancers compared with corresponding normal tissues, suggesting that they are putative oncogenes. These data reveal a new methylation site within p53 mediated by the methylases G9a and Glp and indicate that G9a is a potential inhibitory target for cancer treatment
— id: 121298, year: 2010, vol: 285, page: 9636, stat: Journal Article,

Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA
Kaneko, Syuzo; Li, Gang; Son, Jinsook; Xu, Chong-Feng; Margueron, Raphael; Neubert, Thomas A; Reinberg, Danny
2010 Dec 1;24(23):2615-2620, Genes & development
Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5' end of Xist
— id: 114862, year: 2010, vol: 24, page: 2615, stat: Journal Article,

Jarid2 and PRC2, partners in regulating gene expression
Li, Gang; Margueron, Raphael; Ku, Manching; Chambon, Pierre; Bernstein, Bradley E; Reinberg, Danny
2010 Feb 15;24(4):368-380, Genes & development
The Polycomb group proteins foster gene repression profiles required for proper development and unimpaired adulthood, and comprise the components of the Polycomb-Repressive Complex 2 (PRC2) including the histone H3 Lys 27 (H3K27) methyltransferase Ezh2. How mammalian PRC2 accesses chromatin is unclear. We found that Jarid2 associates with PRC2 and stimulates its enzymatic activity in vitro. Jarid2 contains a Jumonji C domain, but is devoid of detectable histone demethylase activity. Instead, its artificial recruitment to a promoter in vivo resulted in corecruitment of PRC2 with resultant increased levels of di- and trimethylation of H3K27 (H3K27me2/3). Jarid2 colocalizes with Ezh2 and MTF2, a homolog of Drosophila Pcl, at endogenous genes in embryonic stem (ES) cells. Jarid2 can bind DNA and its recruitment in ES cells is interdependent with that of PRC2, as Jarid2 knockdown reduced PRC2 at its target promoters, and ES cells devoid of the PRC2 component EED are deficient in Jarid2 promoter access. In addition to the well-documented defects in embryonic viability upon down-regulation of Jarid2, ES cell differentiation is impaired, as is Oct4 silencing
— id: 107274, year: 2010, vol: 24, page: 368, stat: Journal Article,

Highly compacted chromatin formed in vitro reflects the dynamics of transcription activation in vivo
Li, Guohong; Margueron, Raphael; Hu, Guobin; Stokes, David; Wang, Yuh-Hwa; Reinberg, Danny
2010 Apr 9;38(1):41-53, Molecular cell
High-order chromatin was reconstituted in vitro. This species reflects the criteria associated with transcriptional regulation in vivo. Histone H1 was determinant to formation of condensed structures, with deacetylated histones giving rise to highly compacted chromatin that approximated 30 nm fibers as evidenced by electron microscopy. Using the PEPCK promoter, we validated the integrity of these templates that were refractory to transcription by attaining transcription through the progressive action of the pertinent factors. The retinoic acid receptor binds to highly compacted chromatin, but the NF1 transcription factor binds only after histone acetylation by p300 and SWI/SNF-mediated nucleosome mobilization, reflecting the in vivo case. Mapping studies revealed the same pattern of nucleosomal repositioning on the PEPCK promoter in vitro and in vivo, correlating with NF1 binding and transcription. The reconstitution of such highly compacted '30 nm' chromatin that mimics in vivo characteristics should advance studies of its conversion to a transcriptionally active form
— id: 121297, year: 2010, vol: 38, page: 41, stat: Journal Article,

Chromatin structure and the inheritance of epigenetic information
Margueron, Raphael; Reinberg, Danny
2010 Apr;11(4):285-296, Nature reviews. Genetics
Although it is widely accepted that the regulation of the chromatin landscape is pivotal to conveying the epigenetic program, it is still unclear how a defined chromatin domain is reproduced following DNA replication and transmitted from one cell generation to the next. Here, we review the multiple mechanisms that potentially affect the inheritance of epigenetic information in somatic cells. We consider models of how histones might be recycled following replication, and discuss the importance of positive-feedback loops, long-range gene interactions and the complex network of trans-acting factors in the transmission of chromatin states
— id: 108797, year: 2010, vol: 11, page: 285, stat: Journal Article,

Regulation of the Histone H4 Monomethylase PR-Set7 by CRL4(Cdt2)-Mediated PCNA-Dependent Degradation during DNA Damage
Oda, Hisanobu; Hubner, Michael R; Beck, David B; Vermeulen, Michiel; Hurwitz, Jerard; Spector, David L; Reinberg, Danny
2010 Nov 12;40(3):364-376, Molecular cell
The histone methyltransferase PR-Set7/Set8 is the sole enzyme that catalyzes monomethylation of histone H4 at K20 (H4K20me1). Previous reports document disparate evidence regarding PR-Set7 expression during the cell cycle, the biological relevance of PR-Set7 interaction with PCNA, and its role in the cell. We find that PR-Set7 is indeed undetectable during S phase and instead is detected during late G2, mitosis, and early G1. PR-Set7 is transiently recruited to laser-induced DNA damage sites through its interaction with PCNA, after which 53BP1 is recruited dependent on PR-Set7 catalytic activity. During the DNA damage response, PR-Set7 interaction with PCNA through a specialized 'PIP degron' domain targets it for PCNA-coupled CRL4(Cdt2)-dependent proteolysis. PR-Set7 mutant in its 'PIP degron' is now detectable during S phase, during which the mutant protein accumulates. Outside the chromatin context, Skp2 promotes PR-Set7 degradation as well. These findings demonstrate a stringent spatiotemporal control of PR-Set7 that is essential for preserving the genomic integrity of mammalian cells
— id: 114509, year: 2010, vol: 40, page: 364, stat: Journal Article,

Histone Tails: Ideal Motifs for Probing Epigenetics through Chemical Biology Approaches
Voigt P; Reinberg D
2010 Nov 24;:?-? #, Chembiochem : a European journal of chemical biology
Post-translational modifications (PTMs) on histone proteins have emerged as a central theme in the regulation of gene expression and other chromatin-associated processes. The discovery that certain protein domains can recognize acetylated and methylated lysine residues of histones has spurred efforts to uncover and characterize histone PTM-binding proteins. In this task, chromatin biology has strongly benefited from synthetic approaches stemming from chemical biology. Peptide-based techniques have been instrumental in identifying histone mark-binding proteins and analyzing their binding specificities. To explore how histone PTMs carry out their function in the context of chromatin, reconstituted systems based on recombinant histones carrying defined modifications are increasingly being used. They constitute promising tools to analyze mechanistic aspects of histone PTMs, including their role in transcription and their transmission in replication. In this review, we present strategies that have been used successfully to investigate the role of histone modifications, concepts that have emerged from their application, and their potential to contribute to current developments in the field
— id: 138315, year: 2010, vol: , page: ?, stat: Journal Article,

Histones: annotating chromatin
Campos, Eric I; Reinberg, Danny
2009 ;43:559-599, Annual review of genetics
Chromatin is a highly regulated nucleoprotein complex through which genetic material is structured and maneuvered to elicit cellular processes, including transcription, cell division, differentiation, and DNA repair. In eukaryotes, the core of this structure is composed of nucleosomes, or repetitive histone octamer units typically enfolded by 147 base pairs of DNA. DNA is arranged and indexed through these nucleosomal structures to adjust local chromatin compaction and accessibility. Histones are subject to multiple covalent posttranslational modifications, some of which alter intrinsic chromatin properties, others of which present or hinder binding modules for non-histone, chromatin-modifying complexes. Although certain histone marks correlate with different biological outputs, we have yet to fully appreciate their effects on transcription and other cellular processes. Tremendous advancements over the past years have uncovered intriguing histone-related matters and raised important related questions. This review revisits past breakthroughs and discusses novel developments that pertain to histone posttranslational modifications and the affects they have on transcription and DNA packaging
— id: 105363, year: 2009, vol: 43, page: 559, stat: Journal Article,

The target of the NSD family of histone lysine methyltransferases depends on the nature of the substrate
Li, Yan; Trojer, Patrick; Xu, Chong-Feng; Cheung, Peggie; Kuo, Alex; Drury, William J 3rd; Qiao, Qi; Neubert, Thomas A; Xu, Rui-Ming; Gozani, Or; Reinberg, Danny
2009 Dec 4;284(49):34283-34295, Journal of biological chemistry
The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target
— id: 105498, year: 2009, vol: 284, page: 34283, stat: Journal Article,

Role of the polycomb protein EED in the propagation of repressive histone marks
Margueron, Raphael; Justin, Neil; Ohno, Katsuhito; Sharpe, Miriam L; Son, Jinsook; Drury, William J 3rd; Voigt, Philipp; Martin, Stephen R; Taylor, William R; De Marco, Valeria; Pirrotta, Vincenzo; Reinberg, Danny; Gamblin, Steven J
2009 Oct 8;461(7265):762-767, Nature
Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells
— id: 103154, year: 2009, vol: 461, page: 762, stat: Journal Article,

Monomethylation of Histone H4-Lysine 20 is involved in Chromosome Structure and Stability and is essential for Mouse Development
Oda, Hisanobu; Okamoto, Ikuhiro; Murphy, Niall; Chu, Jianhua; Price, Sandy M; Shen, Michael M; Torres-Padilla, Maria Elena; Heard, Edith; Reinberg, Danny
2009 Apr;29(8):2278-2295, Molecular & cellular biology
PR-Set7/Set8/KMT5A is the sole enzyme known to catalyze mono-methylation of histone H4 lysine-20 (H4K20) and is present only in multicellular organisms that compact a large fraction of their DNA. We found that mouse embryos that are homozygous null mutant for PR-Set7 gene display in early embryonic lethality, prior to the 8-cell stage. Death was due to the absence of PR-Set7 catalytic activity, since microinjection of wild-type, but not a catalytically inactive version into 2-cell embryos rescued the phenotype. A lack of PR-Set7 activity resulted not only in depletion of H4K20me1 but also in reduced levels of the H4K20me2/3 marks catalyzed by the Suv4-20h1/h2 enzymes implying that H4K20me1 may be essential for the function of these enzymes to ensure the di- and tri-methylated states. ES cells that were inducibly deleted for PR-Set7 passed through an initial G2/M phase but the progeny were defective at the subsequent S and G2/M phases exhibiting a delay in their cell cycle, accumulation at G2/M, massive DNA damage and improper mitotic chromosome condensation. Cell cycle analysis after synchronization indicated that the defects were a consequence of decreased H4K20me1 due to the absence of PR-Set7. Most importantly, the lack of H4K20me1 also resulted in defects in chromosome condensation in interphase nuclei. These results demonstrate the critical role of H4K20 mono-methylation in mammals in a developmental context
— id: 94406, year: 2009, vol: 29, page: 2278, stat: Journal Article,

Processing the H3K36me3 signature
Sims, Robert J 3rd; Reinberg, Danny
2009 Mar;41(3):270-271, Nature genetics
— id: 94405, year: 2009, vol: 41, page: 270, stat: Journal Article,

Stem cells: Escaping fates with open states
Sims, Robert J 3rd; Reinberg, Danny
2009 Aug 13;460(7257):802-803, Nature
— id: 121300, year: 2009, vol: 460, page: 802, stat: Journal Article,

Dynamic histone H1 isotype 4 methylation and demethylation by histone lysine methyltransferase G9A/KMT1C and the jumonji domain containing JMJD2/KDM4 proteins
Trojer, Patrick; Zhang, Jin; Yonezawa, Masato; Schmidt, Andreas; Zheng, Haiyan; Jenuwein, Thomas; Reinberg, Danny
2009 Mar 27;284(13):8395-8405, Journal of biological chemistry
The linker histone H1 generally participates in the establishment of chromatin structure. However, of the seven somatic H1 isotypes in humans some are also implicated in the regulation of local gene expression. Histone H1 isotype 4 (H1.4) represses transcription and its lysine residue 26 (K26) was found to be important in this aspect. H1.4K26 is known to be methylated and acetylated in vivo but the enzymes responsible for these post-translational modifications and the regulatory cues that promote H1.4 residence on chromatin are poorly characterized. Here we report that the euchromatic histone lysine methyltransferase G9a/KMT1C mediates H1.4K26 mono- and di-methylation in vitro and in vivo and thereby provides a recognition surface for the chromatin binding proteins HP1 and L3MBTL1. Moreover, we show evidence that G9a promotes H1 deposition and is required for retention of H1 on chromatin. We also identify members of the JMJD2/KDM4 subfamily of jumonji-C type histone demethylases as being responsible for the removal of H1.4K26 methylation
— id: 94407, year: 2009, vol: 284, page: 8395, stat: Journal Article,

Calorie restriction and the exercise of chromatin
Vaquero, Alejandro; Reinberg, Danny
2009 Aug 15;23(16):1849-1869, Genes & development
Since the earliest stages of evolution, organisms have faced the challenge of sensing and adapting to environmental changes for their survival under compromising conditions such as food depletion or stress. Implicit in these responses are mechanisms developed during evolution that include the targeting of chromatin to allow or prevent expression of fundamental genes and to protect genome integrity. Among the different approaches to study these mechanisms, the analysis of the response to a moderate reduction of energy intake, also known as calorie restriction (CR), has become one of the best sources of information regarding the factors and pathways involved in metabolic adaptation from lower to higher eukaryotes. Furthermore, responses to CR are involved in life span regulation-conserved from yeast to mammals-and therefore have garnered major research interest. Herein we review current knowledge of responses to CR at the molecular level and their functional link to chromatin
— id: 121301, year: 2009, vol: 23, page: 1849, stat: Journal Article,

Heterogeneous nuclear ribonucleoprotein L Is a subunit of human KMT3a/Set2 complex required for H3 Lys-36 trimethylation activity in vivo
Yuan, Wen; Xie, Jingwei; Long, Chengzu; Erdjument-Bromage, Hediye; Ding, Xiaojun; Zheng, Yong; Tempst, Paul; Chen, She; Zhu, Bing; Reinberg, Danny
2009 Jun 5;284(23):15701-15707, Journal of biological chemistry
The presence of histone H3 lysine 36 methylation (H3K36me) correlates with actively transcribed genes. In yeast, histone H3K36me mediated by KMT3 (also known as Set2) recruits a histone deacetylase complex, Rpd3s, to ensure the fidelity of transcription initiation. We report the purification of human KMT3a (also known as HYPB or hSet2) complex and the identification of a novel, higher eukaryotic specific subunit, heterogeneous nuclear ribonucleoprotein L (HnRNP-L). Interestingly, although KMT3a has intrinsic activity in vitro, HnRNP-L is essential in vivo. Moreover, KMT3a generates mono-, di-, and trimethylated products in vitro, but RNA interference against KMT3a or HnRNP-L down-regulates exclusively the H3K36me3 mark in vivo
— id: 121302, year: 2009, vol: 284, page: 15701, stat: Journal Article,

Nonradioactive, ultrasensitive site-specific protein-protein photocrosslinking: interactions of alpha-helix 2 of TATA-binding protein with general transcription factor TFIIA and transcriptional repressor NC2
Kim, Younggyu; Ebright, Yon W; Goodman, Adam R; Reinberg, Danny; Ebright, Richard H
2008 Nov;36(19):6143-6154, Nucleic acids research
We have developed an approach that enables nonradioactive, ultrasensitive (attamole sensitivity) site-specific protein-protein photocrosslinking, and we have applied the approach to the analysis of interactions of alpha-helix 2 (H2) of human TATA-element binding protein (TBP) with general transcription factor TFIIA and transcriptional repressor NC2. We have found that TBP H2 can be crosslinked to TFIIA in the TFIIA-TBP-DNA complex and in higher order transcription-initiation complexes, and we have mapped the crosslink to the 'connector' region of the TFIIA alpha/beta subunit (TFIIAalpha/beta). We further have found that TBP H2 can be crosslinked to NC2 in the NC2-TBP-DNA complex, and we have mapped the crosslink to the C-terminal 'tail' of the NC2 alpha-subunit (NC2alpha). Interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail were not observed in crystal structures of TFIIA-TBP-DNA and NC2-TBP-DNA complexes, since relevant segments of TFIIA and NC2 were not present in truncated TFIIA and NC2 derivatives used for crystallization. We propose that interactions of TBP H2 with the TFIIAalpha/beta connector and the NC2alpha C-terminal tail provide an explanation for genetic results suggesting importance of TBP H2 in TBP-TFIIA interactions and TBP-NC2 interactions, and provide an explanation-steric exclusion-for competition between TFIIA and NC2
— id: 94408, year: 2008, vol: 36, page: 6143, stat: Journal Article,

Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms
Margueron, Raphael; Li, Guohong; Sarma, Kavitha; Blais, Alexandre; Zavadil, Jiri; Woodcock, Christopher L; Dynlacht, Brian D; Reinberg, Danny
2008 Nov 21;32(4):503-518, Molecular cell
Polycomb group proteins are critical to maintaining gene repression established during Drosophila development. Part of this group forms the PRC2 complex containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27 (H3K37me2/3), marks repressive to transcription. We report that the mammalian homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead, PRC2-Ezh1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2 genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2 expression is tightly associated with proliferation, as evidenced when analyzing aging mouse kidney. These results might reflect subfunctionalization of a PcG protein during evolution
— id: 91489, year: 2008, vol: 32, page: 503, stat: Journal Article,

Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 trimethylation in vivo
Sarma, Kavitha; Margueron, Raphael; Ivanov, Alexey; Pirrotta, Vincenzo; Reinberg, Danny
2008 Apr;28(8):2718-2731, Molecular & cellular biology
The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression
— id: 78359, year: 2008, vol: 28, page: 2718, stat: Journal Article,

Is there a code embedded in proteins that is based on post-translational modifications?
Sims, Robert J 3rd; Reinberg, Danny
2008 Oct;9(10):815-820, Nature reviews. Molecular cell biology
Covalent post-translational modifications (PTMs) provide vast indexing potential and expanded protein use. The 'histone code' hypothesis has inspired rapid advances throughout chromatin biology, and has recently been tapped for its relevance to non-histone proteins. Comprehensive analyses suggest that rather than constituting a general code, the covalent modifications of proteins (including histones) provide surfaces that are recognized by effectors that can give rise to intricate interactions and downstream events. These are reminiscent of other regulatory cascades in transcription and cell signalling
— id: 94409, year: 2008, vol: 9, page: 815, stat: Journal Article,

A gateway to study protein lysine methylation
Trojer, Patrick; Reinberg, Danny
2008 Jun;4(6):332-334, Nature Chemical Biology
— id: 94410, year: 2008, vol: 4, page: 332, stat: Journal Article,

Beyond histone methyl-lysine binding: how malignant brain tumor (MBT) protein L3MBTL1 impacts chromatin structure
Trojer, Patrick; Reinberg, Danny
2008 Mar;7(5):578-585, Cell cycle
Alterations in gene expression are commonly accompanied by changes in chromatin structure. Histone lysine residues of the so called 'histone tails' are subject to various post-translational modifications among which methylation has been extensively studied over the past years. The presence and the extent of methylation on histone lysine residues somehow mediate chromatin structural changes that contribute to activation or repression of gene expression. Chromatin states are functionally linked with cellular processes including the regulation of gene expression during the cell cycle. For nearly a decade, however, it proved difficult to explain mechanistically how methyl moieties on histone lysine residues impact chromatin structure. We recently found that a member of the malignant brain tumor (MBT) protein family, L3MBTL1, directly compacts chromatin in a strictly histone lysine methylation dependent fashion. Below, we briefly discuss our observations and those of others to provide an overview of how L3MBTL1, partially by chromatin condensation, regulates transcription and functions in cell cycle control
— id: 78357, year: 2008, vol: 7, page: 578, stat: Journal Article,

New nomenclature for chromatin-modifying enzymes
Allis, C David; Berger, Shelley L; Cote, Jacques; Dent, Sharon; Jenuwien, Thomas; Kouzarides, Tony; Pillus, Lorraine; Reinberg, Danny; Shi, Yang; Shiekhattar, Ramin; Shilatifard, Ali; Workman, Jerry; Zhang, Yi
2007 Nov 16;131(4):633-636, Cell
— id: 94411, year: 2007, vol: 131, page: 633, stat: Journal Article,

Methylation-acetylation interplay activates p53 in response to DNA damage
Ivanov, Gleb S; Ivanova, Tatyana; Kurash, Julia; Ivanov, Alexey; Chuikov, Sergey; Gizatullin, Farid; Herrera-Medina, Enrique M; Rauscher, Frank 3rd; Reinberg, Danny; Barlev, Nickolai A
2007 Oct;27(19):6756-6769, Molecular & cellular biology
p53, an important tumor suppressor protein, exerts its function mostly as a sequence-specific transcription factor and is subjected to multiple posttranslational modifications in response to genotoxic stress. Recently, we discovered that lysine methylation of p53 at K372 by Set7/9 (also known as SET7 and Set9) is important for transcriptional activation and stabilization of p53. In this report we provide a molecular mechanism for the effect of p53 methylation on transcription. We demonstrate that Set7/9 activity toward p53, but not the nucleosomal histones, is modulated by DNA damage. Significantly, we show that lysine methylation of p53 is important for its subsequent acetylation, resulting in stabilization of the p53 protein. These p53 modification events can be observed on the promoter of p21 gene, a known transcriptional target of p53. Finally, we show that methylation-acetylation interplay in p53 augments acetylation of histone H4 in the promoter of p21 gene, resulting in its subsequent transcriptional activation and, hence, cell cycle arrest. Collectively, these results suggest that the cross talk between lysine methylation and acetylation is critical for p53 activation in response to DNA damage and that Set7/9 may play an important role in tumor suppression
— id: 94414, year: 2007, vol: 27, page: 6756, stat: Journal Article,

Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination
Lee, Min Gyu; Villa, Raffaella; Trojer, Patrick; Norman, Jessica; Yan, Kai-Ping; Reinberg, Danny; Di Croce, Luciano; Shiekhattar, Ramin
2007 Oct 19;318(5849):447-450, Science
Methylation of histone H3 lysine 27 (H3K27) is a posttranslational modification that is highly correlated with genomic silencing. Here we show that human UTX, a member of the Jumonji C family of proteins, is a di- and trimethyl H3K27 demethylase. UTX occupies the promoters of HOX gene clusters and regulates their transcriptional output by modulating the recruitment of polycomb repressive complex 1 and the monoubiquitination of histone H2A. Moreover, UTX associates with mixed-lineage leukemia (MLL) 2/3 complexes, and during retinoic acid signaling events, the recruitment of the UTX complex to HOX genes results in H3K27 demethylation and a concomitant methylation of H3K4. Our results suggest a concerted mechanism for transcriptional activation in which cycles of H3K4 methylation by MLL2/3 are linked with the demethylation of H3K27 through UTX
— id: 94413, year: 2007, vol: 318, page: 447, stat: Journal Article,

SirT3 is a nuclear NAD+-dependent histone deacetylase that translocates to the mitochondria upon cellular stress
Scher, Michael B; Vaquero, Alejandro; Reinberg, Danny
2007 Apr 15;21(8):920-928, Genes & development
In humans, there are at least seven Sir2-like proteins (SirT1-7) with diverse functions, including the regulation of chromatin structure, and metabolism. SirT3 levels have been shown to correlate with extended life span, to localize to the mitochondria, and to be highly expressed in brown adipose tissue. In humans, SirT3 exists in two forms, a full-length protein of approximately 44 kDa and a processed polypeptide lacking 142 amino acids at its N terminus. We found that SirT3 not only localizes to the mitochondria, but also to the nucleus under normal cell growth conditions. Both the full-length and processed forms of SirT3 target H4-K16 for deacetylation in vitro and can deacetylate H4-K16 in vivo when recruited to a gene. Using a highly specific antibody against the N terminus of SirT3, we found that SirT3 is transported from the nucleus to the mitochondria upon cellular stress. This includes DNA damage induced by Etoposide and UV-irradiation, as well as overexpression of SirT3 itself
— id: 72041, year: 2007, vol: 21, page: 920, stat: Journal Article,

Recognition of trimethylated histone H3 lysine 4 facilitates the recruitment of transcription postinitiation factors and pre-mRNA splicing
Sims, Robert J 3rd; Millhouse, Scott; Chen, Chi-Fu; Lewis, Brian A; Erdjument-Bromage, Hediye; Tempst, Paul; Manley, James L; Reinberg, Danny
2007 Nov 30;28(4):665-676, Molecular cell
Trimethylation of histone H3 on lysine 4 (H3K4me3) localizes near the 5' region of genes and is tightly associated with active loci. Several proteins, such as CHD1, BPTF, JMJD2A, and the ING tumor suppressor family, directly recognize this lysine methyl mark. However, how H3K4me3 recognition participates in active transcription remains poorly characterized. Here we identify specific CHD1-interacting proteins via H3K4me3 affinity purification, including numerous factors mediating postinitiation events. Conventional biochemical purification revealed a stable complex between CHD1 and components of the spliceosome. Depletion of CHD1 in extracts dramatically reduced splicing efficiency in vitro, indicating a functional link between CHD1 and the spliceosome. Knockdown of CHD1 and H3K4me3 levels by siRNA reduced association of U2 snRNP components with chromatin and, more importantly, altered the efficiency of pre-mRNA splicing on active genes in vivo. These findings suggest that methylated H3K4 serves to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components to H3K4me3 via CHD1
— id: 75673, year: 2007, vol: 28, page: 665, stat: Journal Article,

L3MBTL1, a histone-methylation-dependent chromatin lock
Trojer, Patrick; Li, Guohong; Sims, Robert J 3rd; Vaquero, Alejandro; Kalakonda, Nagesh; Boccuni, Piernicola; Lee, Donghoon; Erdjument-Bromage, Hediye; Tempst, Paul; Nimer, Stephen D; Wang, Yuh-Hwa; Reinberg, Danny
2007 Jun 1;129(5):915-928, Cell
Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb
— id: 94415, year: 2007, vol: 129, page: 915, stat: Journal Article,

Facultative heterochromatin: is there a distinctive molecular signature?
Trojer, Patrick; Reinberg, Danny
2007 Oct 12;28(1):1-13, Molecular cell
The Latin word 'facultas' literally means 'opportunity.' Facultative heterochromatin (fHC) then designates genomic regions in the nucleus of a eukaryotic cell that have the opportunity to adopt open or compact conformations within temporal and spatial contexts. This review focuses on the molecular and functional aspects of fHC that distinguish it from constitutive heterochromatin (cHC) and euchromatin (EC) and discusses various concepts regarding the regulation of fHC structure. We begin by revisiting the historical developments that gave rise to our current appreciation of fHC
— id: 75844, year: 2007, vol: 28, page: 1, stat: Journal Article,

NAD+-dependent deacetylation of H4 lysine 16 by class III HDACs
Vaquero, A; Sternglanz, R; Reinberg, D
2007 Aug 13;26(37):5505-5520, Oncogene
Histone deacetylases (HDACs) catalyse the removal of acetyl groups from the N-terminal tails of histones. All known HDACs can be categorized into one of four classes (I-IV). The class III HDAC or silencing information regulator 2 (Sir2) family exhibits characteristics consistent with a distinctive role in regulation of chromatin structure. Accumulating data suggest that these deacetylases acquired new roles as genomic complexity increased, including deacetylation of non-histone proteins and functional diversification in mammals. However, the intrinsic regulation of chromatin structure in species as diverse as yeast and humans, underscores the pressure to conserve core functions of class III HDACs, which are also known as Sirtuins. One of the key factors that might have contributed to this preservation is the intimate relationship between some members of this group of proteins (SirT1, SirT2 and SirT3) and deacetylation of a specific residue in histone H4, lysine 16 (H4K16). Evidence accumulated over the years has uncovered a unique role for H4K16 in chromatin structure throughout eukaryotes. Here, we review the recent findings about the functional relationship between H4K16 and the Sir2 class of deacetylases and how that relationship might impact aging and diseases including cancer and diabetes
— id: 73924, year: 2007, vol: 26, page: 5505, stat: Journal Article,

SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation
Vaquero, Alejandro; Scher, Michael; Erdjument-Bromage, Hediye; Tempst, Paul; Serrano, Lourdes; Reinberg, Danny
2007 Nov 15;450(7168):440-444, Nature
In contrast to stably repressive, constitutive heterochromatin and stably active, euchromatin, facultative heterochromatin has the capacity to alternate between repressive and activated states of transcription. As such, it is an instructive source to understand the molecular basis for changes in chromatin structure that correlate with transcriptional status. Sirtuin 1 (SIRT1) and suppressor of variegation 3-9 homologue 1 (SUV39H1) are amongst the enzymes responsible for chromatin modulations associated with facultative heterochromatin formation. SUV39H1 is the principal enzyme responsible for the accumulation of histone H3 containing a tri-methyl group at its lysine 9 position (H3K9me3) in regions of heterochromatin. SIRT1 is an NAD+-dependent deacetylase that targets histone H4 at lysine 16 (refs 3 and 4), and through an unknown mechanism facilitates increased levels of H3K9me3 (ref. 3). Here we show that the mammalian histone methyltransferase SUV39H1 is itself targeted by the histone deacetylase SIRT1 and that SUV39H1 activity is regulated by acetylation at lysine residue 266 in its catalytic SET domain. SIRT1 interacts directly with, recruits and deacetylates SUV39H1, and these activities independently contribute to elevated levels of SUV39H1 activity resulting in increased levels of the H3K9me3 modification. Loss of SIRT1 greatly affects SUV39H1-dependent H3K9me3 and impairs localization of heterochromatin protein 1. These findings demonstrate a functional link between the heterochromatin-related histone methyltransferase SUV39H1 and the histone deacetylase SIRT1
— id: 94412, year: 2007, vol: 450, page: 440, stat: Journal Article,

Drosophila Paf1 modulates chromatin structure at actively transcribed genes
Adelman, Karen; Wei, Wenxiang; Ardehali, M Behfar; Werner, Janis; Zhu, Bing; Reinberg, Danny; Lis, John T
2006 Jan;26(1):250-260, Molecular & cellular biology
The Paf1 complex in yeast has been reported to influence a multitude of steps in gene expression through interactions with RNA polymerase II (Pol II) and chromatin-modifying complexes; however, it is unclear which of these many activities are primary functions of Paf1 and are conserved in metazoans. We have identified and characterized the Drosophila homologs of three subunits of the yeast Paf1 complex and found striking differences between the yeast and Drosophila Paf1 complexes. We demonstrate that although Drosophila Paf1, Rtf1, and Cdc73 colocalize broadly with actively transcribing, phosphorylated Pol II, and all are recruited to activated heat shock genes with similar kinetics; Rtf1 does not appear to be a stable part of the Drosophila Paf1 complex. RNA interference (RNAi)-mediated depletion of Paf1 or Rtf1 leads to defects in induction of Hsp70 RNA, but tandem RNAi-chromatin immunoprecipitation assays show that loss of neither Paf1 nor Rtf1 alters the density or distribution of phosphorylated Pol II on the active Hsp70 gene. However, depletion of Paf1 reduces trimethylation of histone H3 at lysine 4 in the Hsp70 promoter region and significantly decreases the recruitment of chromatin-associated factors Spt6 and FACT, suggesting that Paf1 may manifest its effects on transcription through modulating chromatin structure
— id: 69848, year: 2006, vol: 26, page: 250, stat: Journal Article,

Promoter activation when the ChIPs are down
Lewis, Brian A; Reinberg, Danny
2006 Feb;13(2):96-97, Nature structural & molecular biology
— id: 68410, year: 2006, vol: 13, page: 96, stat: Journal Article,

Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II
Pavri, Rushad; Zhu, Bing; Li, Guohong; Trojer, Patrick; Mandal, Subhrangsu; Shilatifard, Ali; Reinberg, Danny
2006 May 19;125(4):703-717, Cell
Over the past years, a large number of histone posttranslational modifications have been described, some of which function to attain a repressed chromatin structure, while others facilitate activation by allowing access of regulators to DNA. Histone H2B monoubiquitination is a mark associated with transcriptional activity. Using a highly reconstituted chromatin-transcription system incorporating the inducible RARbeta2 promoter, we find that the establishment of H2B monoubiquitination by RNF20/40 and UbcH6 is dependent on the transcription elongation regulator complex PAF, the histone chaperone FACT, and transcription. H2B monoubiquitination facilitates FACT function, thereby stimulating transcript elongation and the generation of longer transcripts. These in vitro analyses and corroborating in vivo experiments demonstrate that elongation by RNA polymerase II through the nucleosomal barrier is minimally dependent upon (1) FACT and (2) the recruitment of PAF and the H2B monoubiquitination machinery
— id: 69845, year: 2006, vol: 125, page: 703, stat: Journal Article,

de FACTo nucleosome dynamics
Reinberg, Danny; Sims, Robert J 3rd
2006 Aug 18;281(33):23297-23301, Journal of biological chemistry
The factors required for the delivery of RNA polymerase II to class II promoters using naked DNA were all identified by 1998, yet their exact mechanisms of action were not fully understood in all cases, and in some instances, their precise function still remains unknown. Nonetheless, a complete understanding of the complexity of the RNA polymerase II transcription cycle necessitated the development of assays that include chromatinized DNA templates. At this time, the field was actively searching for factors that allow transcription initiation on chromatinized templates. We began studies using chromatin templates in an attempt to identify factor(s) that permit RNA polymerase II to traverse nucleosomes, i.e. that allow elongation on chromatinized DNA templates. The challenge herein was to develop an assay that directly measured the ability of transcriptionally engaged RNA polymerase II to traverse nucleosomes. This approach resulted in the isolation of FACT, a heterodimer in humans comprised of Spt16 and SSRP1. Defined functional biochemical assays corroborated genetic studies in yeast that allowed the elucidation of FACT function in vivo. Collectively, these approaches demonstrate that FACT is a factor that allows RNA polymerase II to traverse nucleosomes in vitro and in vivo by removing one H2A/H2B dimer. More recent studies using a fully defined chromatin reconstitution/transcription assay revealed that FACT activity is greatly stimulated by post-translational modification of the histone polypeptides, specifically by monoubiquitination of lysine 120 of human histone H2B
— id: 69843, year: 2006, vol: 281, page: 23297, stat: Journal Article,

Histone H3 Lys 4 methylation: caught in a bind?
Sims, Robert J 3rd; Reinberg, Danny
2006 Oct 15;20(20):2779-2786, Genes & development
— id: 69842, year: 2006, vol: 20, page: 2779, stat: Journal Article,

Methods to identify and functionally analyze factors that specifically recognize histone lysine methylation
Sims, Robert J 3rd; Trojer, Patrick; Li, Guohong; Reinberg, Danny
2006 Dec;40(4):331-338, Methods
Histone lysine methylation plays an important role in the regulation of gene expression and impacts many fundamental biological processes, such as cellular identity. Despite great efforts, the mechanisms behind the downstream consequences of histone methyl-recognition remain poorly understood. Here, we describe various methods to investigate specific histone lysine-methyl recognition, including the use of short peptides, histone octamers, and the more physiological nucleosomal arrays. We also discuss techniques that are well suited to assess functional aspects of binding as it relates to transcriptional regulation
— id: 69841, year: 2006, vol: 40, page: 331, stat: Journal Article,

Suz12 binds to silenced regions of the genome in a cell-type-specific manner
Squazzo, Sharon L; O'Geen, Henriette; Komashko, Vitalina M; Krig, Sheryl R; Jin, Victor X; Jang, Sung-wook; Margueron, Raphael; Reinberg, Danny; Green, Roland; Farnham, Peggy J
2006 Jul;16(7):890-900, Genome research
Suz12 is a component of the Polycomb group complexes 2, 3, and 4 (PRC 2/3/4). These complexes are critical for proper embryonic development, but very few target genes have been identified in either mouse or human cells. Using a variety of ChIP-chip approaches, we have identified a large set of Suz12 target genes in five different human and mouse cell lines. Interestingly, we found that Suz12 target promoters are cell type specific, with transcription factors and homeobox proteins predominating in embryonal cells and glycoproteins and immunoglobulin-related proteins predominating in adult tumors. We have also characterized the localization of other components of the PRC complex with Suz12 and investigated the overall relationship between Suz12 binding and markers of active versus inactive chromatin, using both promoter arrays and custom tiling arrays. Surprisingly, we find that the PRC complexes can be localized to discrete binding sites or spread through large regions of the mouse and human genomes. Finally, we have shown that some Suz12 target genes are bound by OCT4 in embryonal cells and suggest that OCT4 maintains stem cell self-renewal, in part, by recruiting PRC complexes to certain genes that promote differentiation
— id: 69844, year: 2006, vol: 16, page: 890, stat: Journal Article,

Histone lysine demethylases and their impact on epigenetics
Trojer, Patrick; Reinberg, Danny
2006 Apr 21;125(2):213-217, Cell
Methylation marks on the lysine residues of histone proteins are thought to contribute to epigenetic phenomena in part because of their apparent irreversibility. Will this view change with the recent discovery of histone lysine demethylases that reversibly remove methyl marks?
— id: 69847, year: 2006, vol: 125, page: 213, stat: Journal Article,

SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis
Vaquero, Alejandro; Scher, Michael B; Lee, Dong Hoon; Sutton, Ann; Cheng, Hwei-Ling; Alt, Frederick W; Serrano, Lourdes; Sternglanz, Rolf; Reinberg, Danny
2006 May 15;20(10):1256-1261, Genes & development
The mammalian cytoplasmic protein SirT2 is a member of the Sir2 family of NAD+-dependent protein deacetylases involved in caloric restriction-dependent life span extension. We found that SirT2 and its yeast counterpart Hst2 have a strong preference for histone H4K16Ac in their deacetylation activity in vitro and in vivo. We have pinpointed the decrease in global levels of H4K16Ac during the mammalian cell cycle to the G2/M transition that coincides with SirT2 localization on chromatin. Mouse embryonic fibroblasts (MEFs) deficient for SirT2 show higher levels of H4K16Ac in mitosis, in contrast to the normal levels exhibited by SirT1-deficient MEFs. The enzymatic conversion of H4K16Ac to its deacetylated form may be pivotal to the formation of condensed chromatin. Thus, SirT2 is a major contributor to this enzymatic conversion at the time in the cell's life cycle when condensed chromatin must be generated anew
— id: 69846, year: 2006, vol: 20, page: 1256, stat: Journal Article,

PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis
Karachentsev, Dmitry; Sarma, Kavitha; Reinberg, Danny; Steward, Ruth
2005 Feb 15;19(4):431-435, Genes & development
The histone methyl transferase PR-Set7 mediates histone H4 Lys 20 methylation, a mark of constitutive and facultative heterochromatin. We isolated a null mutation in Drosophila PR-Set7 that suppresses position effect variegation, indicating that PR-Set7 indeed functions in silencing general gene expression. In PR-Set7 larval leg and eye discs, the number of cells is lower than normal, and the DNA content in these cells is significantly increased. These data show that PR-Set7-dependent methylation is essential for the process of mitosis. The methylation mark is highly stable and is maintained even in the absence of PR-Set7 protein
— id: 69857, year: 2005, vol: 19, page: 431, stat: Journal Article,

Composition and histone substrates of polycomb repressive group complexes change during cellular differentiation
Kuzmichev, Andrei; Margueron, Raphael; Vaquero, Alejandro; Preissner, Tanja S; Scher, Michael; Kirmizis, Antonis; Ouyang, Xuesong; Brockdorff, Neil; Abate-Shen, Cory; Farnham, Peggy; Reinberg, Danny
2005 Feb 8;102(6):1859-1864, Proceedings of the National Academy of Sciences of the United States of America
Changes in the substrate specificities of factors that irreversibly modify the histone components of chromatin are expected to have a profound effect on gene expression through epigenetics. Ezh2 is a histone-lysine methyltransferase with activity dependent on its association with other components of the Polycomb Repressive Complexes 2 and 3 (PRC2/3). Ezh2 levels are increasingly elevated during prostate cancer progression. Other PRC2/3 components also are elevated in cancer cells. Overexpression of Ezh2 in tissue culture promotes formation of a previously undescribed PRC complex, PRC4, that contains the NAD+-dependent histone deacetylase SirT1 and isoform 2 of the PRC component Eed. Eed2 is expressed in cancer and undifferentiated embryonic stem (ES) cells but is undetectable in normal and differentiated ES cells. The distinct PRCs exhibit differential histone substrate specificities. These findings suggest that formation of a transformation-specific PRC complex may have a major role in resetting patterns of gene expression by regulating chromatin structure
— id: 69856, year: 2005, vol: 102, page: 1859, stat: Journal Article,

Functional characterization of core promoter elements: the downstream core element is recognized by TAF1
Lee, Dong-Hoon; Gershenzon, Naum; Gupta, Malavika; Ioshikhes, Ilya P; Reinberg, Danny; Lewis, Brian A
2005 Nov;25(21):9674-9686, Molecular & cellular biology
Downstream elements are a newly appreciated class of core promoter elements of RNA polymerase II-transcribed genes. The downstream core element (DCE) was discovered in the human beta-globin promoter, and its sequence composition is distinct from that of the downstream promoter element (DPE). We show here that the DCE is a bona fide core promoter element present in a large number of promoters and with high incidence in promoters containing a TATA motif. Database analysis indicates that the DCE is found in diverse promoters, supporting its functional relevance in a variety of promoter contexts. The DCE consists of three subelements, and DCE function is recapitulated in a TFIID-dependent manner. Subelement 3 can function independently of the other two and shows a TFIID requirement as well. UV photo-cross-linking results demonstrate that TAF1/TAF(II)250 interacts with the DCE subelement DNA in a sequence-dependent manner. These data show that downstream elements consist of at least two types, those of the DPE class and those of the DCE class; they function via different DNA sequences and interact with different transcription activation factors. Finally, these data argue that TFIID is, in fact, a core promoter recognition complex
— id: 68411, year: 2005, vol: 25, page: 9674, stat: Journal Article,

Functional characterization of core promoter elements: DPE-specific transcription requires the protein kinase CK2 and the PC4 coactivator
Lewis, Brian A; Sims, Robert J 3rd; Lane, William S; Reinberg, Danny
2005 May 13;18(4):471-481, Molecular cell
Downstream core promoter elements are an expanding class of regulatory sequences that add considerable diversity to the promoter architecture of RNA polymerase II-transcribed genes. We set out to determine the factors necessary for downstream promoter element (DPE)-dependent transcription and find that, against expectations, TFIID and the GTFs are not sufficient. Instead, the protein kinase CK2 and the coactivator PC4 establish DPE-specific transcription in an in vitro transcription system containing TFIID, Mediator, and the GTFs. Chromatin immunoprecipitation analyses using the DPE-dependent IRF-1 and TAF7 promoters demonstrated that CK2, and PC4 are present on these promoters in vivo. In contrast, neither PC4 nor CK2 were detected on the TAF1-dependent cyclin D promoter, which contains a DCE type of downstream element. Our findings also demonstrate that CK2 activity alters TFIID-dependent recognition of DCE sequences. These data establish that CK2 acts as a switch, converting the transcriptional machinery from functioning on one type of downstream element to another
— id: 68412, year: 2005, vol: 18, page: 471, stat: Journal Article,

The key to development: interpreting the histone code?
Margueron, Raphael; Trojer, Patrick; Reinberg, Danny
2005 Apr;15(2):163-176, Current opinion in genetics & development
Developmental stages in multicellular organisms proceed according to a temporally and spatially precise pattern of gene expression. It has become evident that changes within the chromatin structure brought about by covalent modifications of histones are of crucial importance in determining many biological processes, including development. Numerous studies have provided evidence that the enzymes responsible for the modifications of histones function in a coordinated pattern to control gene expression in the short term and, through the transferral of these modifications by inheritance to their progeny, in the long term
— id: 69854, year: 2005, vol: 15, page: 163, stat: Journal Article,

PARP-1 determines specificity in a retinoid signaling pathway via direct modulation of mediator
Pavri, Rushad; Lewis, Brian; Kim, Tae-Kyung; Dilworth, F Jeffrey; Erdjument-Bromage, Hediye; Tempst, Paul; de Murcia, Gilbert; Evans, Ronald; Chambon, Pierre; Reinberg, Danny
2005 Apr 1;18(1):83-96, Molecular cell
We show that PARP-1 is indispensable to retinoic acid receptor (RAR)-mediated transcription from the RARbeta2 promoter in a highly purified, reconstituted transcription system and that RA-inducible expression of all RARbeta isoforms is abrogated in PARP-1(-/-) cells in vivo. Importantly, PARP-1 activity was independent of its catalytic domain. PARP-1 directly interacts with RAR and Mediator. Chromatin immunoprecipitation experiments confirmed the presence of PARP-1 and Mediator on RAR-responsive promoters in vivo. Importantly, Mediator was inactive (Cdk8+) under basal conditions but was activated (Cdk8-) upon induction. However, in PARP-1(-/-) cells, Mediator was retained in its inactive state (Cdk8+) upon induction consistent with the absence of gene expression. PARP-1 became dispensable for ligand-dependent transcription in a chromatin reconstituted transcription assay when Mediator was devoid of the Cdk8 module (CRSP). PARP-1 appears to function as a specificity factor regulating the RA-induced switch of Mediator from the inactive (Cdk8+) to the active (Cdk8-) state in RAR-dependent transcription
— id: 68413, year: 2005, vol: 18, page: 83, stat: Journal Article,

Histone variants meet their match
Sarma, Kavitha; Reinberg, Danny
2005 Feb;6(2):139-149, Nature reviews. Molecular cell biology
A fascinating aspect of how chromatin structure impacts on gene expression and cellular identity is the transmission of information from mother to daughter cells, independently of the primary DNA sequence. This epigenetic information seems to be contained within the covalent modifications of histone polypeptides and the distinctive characteristics of variant histone subspecies. There are specific deposition pathways for some histone variants, which provide invaluable mechanistic insights into processes whereby the major histones are exchanged for their more specialized counterparts
— id: 69855, year: 2005, vol: 6, page: 139, stat: Journal Article,

Human but not yeast CHD1 binds directly and selectively to histone H3 methylated at lysine 4 via its tandem chromodomains
Sims, Robert J 3rd; Chen, Chi-Fu; Santos-Rosa, Helena; Kouzarides, Tony; Patel, Smita S; Reinberg, Danny
2005 Dec 23;280(51):41789-41792, Journal of biological chemistry
Defining the protein factors that directly recognize post-translational, covalent histone modifications is essential toward understanding the impact of these chromatin 'marks' on gene regulation. In the current study, we identify human CHD1, an ATP-dependent chromatin remodeling protein, as a factor that directly and selectively recognizes histone H3 methylated on lysine 4. In vitro binding studies identified that CHD1 recognizes di- and trimethyl H3K4 with a dissociation constant (Kd) of approximately 5 microm, whereas monomethyl H3K4 binds CHD1 with a 3-fold lower affinity. Surprisingly, human CHD1 binds to methylated H3K4 in a manner that requires both of its tandem chromodomains. In vitro analyses demonstrate that unlike human CHD1, yeast Chd1 does not bind methylated H3K4. Our findings indicate that yeast and human CHD1 have diverged in their ability to discriminate covalently modified histones and link histone modification-recognition and non-covalent chromatin remodeling activities within a single human protein
— id: 69850, year: 2005, vol: 280, page: 41789, stat: Journal Article,

Polycomb group protein ezh2 controls actin polymerization and cell signaling
Su, I-hsin; Dobenecker, Marc-Werner; Dickinson, Ephraim; Oser, Matthew; Basavaraj, Ashwin; Marqueron, Raphael; Viale, Agnes; Reinberg, Danny; Wulfing, Christoph; Tarakhovsky, Alexander
2005 May 6;121(3):425-436, Cell
Polycomb group protein Ezh2, one of the key regulators of development in organisms from flies to mice, exerts its epigenetic function through regulation of histone methylation. Here, we report the existence of the cytosolic Ezh2-containing methyltransferase complex and tie the function of this complex to regulation of actin polymerization in various cell types. Genetic evidence supports the essential role of cytosolic Ezh2 in actin polymerization-dependent processes such as antigen receptor signaling in T cells and PDGF-induced dorsal circular ruffle formation in fibroblasts. Revealed function of Ezh2 points to a broader usage of lysine methylation in regulation of both nuclear and extra-nuclear signaling processes
— id: 69853, year: 2005, vol: 121, page: 425, stat: Journal Article,

Specificity and mechanism of the histone methyltransferase Pr-Set7
Xiao, Bing; Jing, Chun; Kelly, Geoff; Walker, Philip A; Muskett, Frederick W; Frenkiel, Thomas A; Martin, Stephen R; Sarma, Kavitha; Reinberg, Danny; Gamblin, Steven J; Wilson, Jonathan R
2005 Jun 15;19(12):1444-1454, Genes & development
Methylation of lysine residues of histones is an important epigenetic mark that correlates with functionally distinct regions of chromatin. We present here the crystal structure of a ternary complex of the enzyme Pr-Set7 (also known as Set8) that methylates Lys 20 of histone H4 (H4-K20). We show that the enzyme is exclusively a mono-methylase and is therefore responsible for a signaling role quite distinct from that established by other enzymes that target this histone residue. We provide evidence from NMR for the C-flanking domains of SET proteins becoming ordered upon addition of AdoMet cofactor and develop a model for the catalytic cycle of these enzymes. The crystal structure reveals the basis of the specificity of the enzyme for H4-K20 because a histidine residue within the substrate, close to the target lysine, is required for completion of the active site. We also show how a highly variable component of the SET domain is responsible for many of the enzymes' interactions with its target histone peptide and probably also how this part of the structure ensures that Pr-Set7 is nucleosome specific
— id: 69852, year: 2005, vol: 19, page: 1444, stat: Journal Article,

The human PAF complex coordinates transcription with events downstream of RNA synthesis
Zhu, Bing; Mandal, Subhrangsu S; Pham, Anh-Dung; Zheng, Yong; Erdjument-Bromage, Hediye; Batra, Surinder K; Tempst, Paul; Reinberg, Danny
2005 Jul 15;19(14):1668-1673, Genes & development
The yeast PAF (yPAF) complex interacts with RNA polymerase II and coordinates the setting of histone marks associated with active transcription. We report the isolation and functional characterization of the human PAF (hPAF) complex. hPAF shares four subunits with yPAF (hCtr9, hPaf1, hLeo1, and hCdc73), but contains a novel higher eukaryotic-specific subunit, hSki8. RNAi against hSki8 or hCtr9 reduces the cellular levels of other hPAF subunits and of mono- and trimethylated H3-Lys 4 and dimethylated H3-Lys 79. The hSki8 subunit is also a component of the human SKI (hSKI) complex. Yeast SKI complex is cytoplasmic and together with Exosome mediates 3'-5' mRNA degradation. However, hSKI complex localizes to both nucleus and cytoplasm. Immunoprecipitation experiments revealed that hPAF and hSKI complexes interact, and ChIP experiments demonstrated that hSKI associates with transcriptionally active genes dependent on the presence of hPAF. Thus, in addition to coordinating events during transcription (initiation, promoter clearance, and elongation), hPAF also coordinates events in RNA quality control
— id: 69851, year: 2005, vol: 19, page: 1668, stat: Journal Article,

Monoubiquitination of human histone H2B: the factors involved and their roles in HOX gene regulation
Zhu, Bing; Zheng, Yong; Pham, Anh-Dung; Mandal, Subhrangsu S; Erdjument-Bromage, Hediye; Tempst, Paul; Reinberg, Danny
2005 Nov 23;20(4):601-611, Molecular cell
In yeast, histone H2B monoubiquitination is a cotranscriptional event regulating histone H3 methylation at lysines 4 and 79. However, mammalian H2B monoubiquitination remains poorly understood. We report that in humans, the 600 kDa RNF20/40 complex is the E3 ligase and UbcH6 is the ubiquitin E2-conjugating enzyme for H2B-Lys120 monoubiquitination. RNF20 and RNF40 are both homologs of Bre1, the E3 ligase in the yeast case. UbcH6 physically interacts with RNF20/40 and with the hPAF complex. Formation of a trimeric complex with hPAF stimulates H2B monoubiquitination activity in vitro. Accordingly, UbcH6, RNF20/40, and the hPAF complex are recruited to transcriptionally active genes in vivo. RNF20 overexpression leads to elevated H2B monoubiquitination, subsequently higher levels of methylation at H3 lysines 4 and 79, and stimulation of HOX gene expression. In contrast, RNAi against the RNF20/40 complex or hPAF complex reduces H2B monoubiquitination, lowers methylation levels at H3 lysines 4 and 79, and represses HOX gene expression
— id: 69849, year: 2005, vol: 20, page: 601, stat: Journal Article,

Facts about FACT and transcript elongation through chromatin
Belotserkovskaya, Rimma; Reinberg, Danny
2004 Apr;14(2):139-146, Current opinion in genetics & development
The regulation of transcription elongation within the context of chromatin is a topic of great interest. Even though chromatin presents a barrier to transcription by the PolII machinery in vitro, this process is rather efficient in vivo. Importantly, the chromatin structure of the actively transcribed genes is altered as part of this process. A large number of factors implicated in the control of transcript elongation have been identified through genetics, biochemistry and targeted proteomics approaches. However the precise roles and mechanisms of action of these factors remain obscure. A significant advance came about this past year with the elucidation of the roles of FACT and Spt6 in transcription elongation. These factors facilitate PolII passage through chromatin by destabilizing the nucleosome structure as well as reassemble nucleosomes traversed by PolII
— id: 69863, year: 2004, vol: 14, page: 139, stat: Journal Article,

Transcription through chromatin: understanding a complex FACT
Belotserkovskaya, Rimma; Saunders, Abbie; Lis, John T; Reinberg, Danny
2004 Mar 15;1677(1-3):87-99, Biochimica & biophysica acta
In eukaryotic cells, genomic DNA is assembled with chromosomal proteins, mainly histones, in a highly compact structure termed chromatin. In this form, DNA is not readily accessible to the cellular machineries, which require DNA as a template. Dynamic changes in chromatin organization play a critical role in regulation of DNA-dependent processes such as transcription, DNA replication, recombination and repair. Chromatin structure is altered in transcriptionally active loci: the basic chromatin unit, the nucleosome, appears to be depleted for one histone H2A/H2B dimer. Previously, reconstitution of RNA polymerase II (PolII)-driven transcription on chromatin templates in a highly purified in vitro system led to identification of FACT (for facilitates chromatin transcription), which was required for productive transcript elongation through nucleosomes. FACT was proposed to promote PolII transcription through nucleosomes by removing either one or both H2A/H2B dimers. Here we present an overview of the earlier studies, which resulted in the initial identification and characterization of FACT, as well as the recent findings that refine the model for the mechanism of FACT function in transcription
— id: 69871, year: 2004, vol: 1677, page: 87, stat: Journal Article,

A unified nomenclature for protein subunits of mediator complexes linking transcriptional regulators to RNA polymerase II
Bourbon, Henri-Marc; Aguilera, Andres; Ansari, Aseem Z; Asturias, Francisco J; Berk, Arnold J; Bjorklund, Stefan; Blackwell, T Keith; Borggrefe, Tilman; Carey, Michael; Carlson, Marian; Conaway, Joan W; Conaway, Ronald C; Emmons, Scott W; Fondell, Joseph D; Freedman, Leonard P; Fukasawa, Toshio; Gustafsson, Claes M; Han, Min; He, Xi; Herman, Paul K; Hinnebusch, Alan G; Holmberg, Steen; Holstege, Frank C; Jaehning, Judith A; Kim, Young-Joon; Kuras, Laurent; Leutz, Achim; Lis, John T; Meisterernest, Michael; Naar, Anders M; Nasmyth, Kim; Parvin, Jeffrey D; Ptashne, Mark; Reinberg, Danny; Ronne, Hans; Sadowski, Ivan; Sakurai, Hiroshi; Sipiczki, Matthias; Sternberg, Paul W; Stillman, David J; Strich, Randy; Struhl, Kevin; Svejstrup, Jasper Q; Tuck, Simon; Winston, Fred; Roeder, Robert G; Kornberg, Roger D
2004 Jun 4;14(5):553-557, Molecular cell
— id: 69864, year: 2004, vol: 14, page: 553, stat: Journal Article,

Regulation of p53 activity through lysine methylation
Chuikov, Sergei; Kurash, Julia K; Wilson, Jonathan R; Xiao, Bing; Justin, Neil; Ivanov, Gleb S; McKinney, Kristine; Tempst, Paul; Prives, Carol; Gamblin, Steven J; Barlev, Nickolai A; Reinberg, Danny
2004 Nov 18;432(7015):353-360, Nature
p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase
— id: 69858, year: 2004, vol: 432, page: 353, stat: Journal Article,

Human Spt6 stimulates transcription elongation by RNA polymerase II in vitro
Endoh, Masaki; Zhu, Wenyan; Hasegawa, Jun; Watanabe, Hajime; Kim, Dong-Ki; Aida, Masatoshi; Inukai, Naoto; Narita, Takashi; Yamada, Tomoko; Furuya, Akiko; Sato, Hiroe; Yamaguchi, Yuki; Mandal, Subhrangsu S; Reinberg, Danny; Wada, Tadashi; Handa, Hiroshi
2004 Apr;24(8):3324-3336, Molecular & cellular biology
Recent studies have suggested that Spt6 participates in the regulation of transcription by RNA polymerase II (RNAPII). However, its underlying mechanism remains largely unknown. One possibility, which is supported by genetic and biochemical studies of Saccharomyces cerevisiae, is that Spt6 affects chromatin structure. Alternatively, Spt6 directly controls transcription by binding to the transcription machinery. In this study, we establish that human Spt6 (hSpt6) is a classic transcription elongation factor that enhances the rate of RNAPII elongation. hSpt6 is capable of stimulating transcription elongation both individually and in concert with DRB sensitivity-inducing factor (DSIF), comprising human Spt5 and human Spt4. We also provide evidence showing that hSpt6 interacts with RNAPII and DSIF in human cells. Thus, in vivo, hSpt6 may regulate multiple steps of mRNA synthesis through its interaction with histones, elongating RNAPII, and possibly other components of the transcription machinery
— id: 69870, year: 2004, vol: 24, page: 3324, stat: Journal Article,

Silencing of human polycomb target genes is associated with methylation of histone H3 Lys 27
Kirmizis, Antonis; Bartley, Stephanie M; Kuzmichev, Andrei; Margueron, Raphael; Reinberg, Danny; Green, Roland; Farnham, Peggy J
2004 Jul 1;18(13):1592-1605, Genes & development
Polycomb group (PcG) complexes 2 and 3 are involved in transcriptional silencing. These complexes contain a histone lysine methyltransferase (HKMT) activity that targets different lysine residues on histones H1 or H3 in vitro. However, it is not known if these histones are methylation targets in vivo because the human PRC2/3 complexes have not been studied in the context of a natural promoter because of the lack of known target genes. Here we report the use of RNA expression arrays and CpG-island DNA arrays to identify and characterize human PRC2/3 target genes. Using oligonucleotide arrays, we first identified a cohort of genes whose expression changes upon siRNA-mediated removal of Suz12, a core component of PRC2/3, from colon cancer cells. To determine which of the putative target genes are directly bound by Suz12 and to precisely map the binding of Suz12 to those promoters, we combined a high-resolution chromatin immunoprecipitation (ChIP) analysis with custom oligonucleotide promoter arrays. We next identified additional putative Suz12 target genes by using ChIP coupled to CpG-island microarrays. We showed that HKMT-Ezh2 and Eed, two other components of the PRC2/3 complexes, colocalize to the target promoters with Suz12. Importantly, recruitment of Suz12, Ezh2 and Eed to target promoters coincides with methylation of histone H3 on Lys 27
— id: 69862, year: 2004, vol: 18, page: 1592, stat: Journal Article,

Different EZH2-containing complexes target methylation of histone H1 or nucleosomal histone H3
Kuzmichev, Andrei; Jenuwein, Thomas; Tempst, Paul; Reinberg, Danny
2004 Apr 23;14(2):183-193, Molecular cell
Human Enhancer of Zeste homolog (Ezh2) is a histone lysine methyltransferase (HKMT) associated with transcriptional repression. Ezh2 is present in several distinct complexes, one of which, PRC2, we characterized previously. Here we report an additional Ezh2 complex, PRC3. We show that the Ezh2 complexes exhibit differential targeting of specific histones for lysine methylation dependent upon the context of the histone substrates. This differential targeting is a function of the associated Eed protein within each complex. We found that Eed protein is present in four isoforms, which represent alternate translation start site usage from the same mRNA. These Eed isoforms selectively associate with distinct Ezh2-containing complexes with resultant differential targeting of their associated HKMT activity toward histone H3-K27 or histone H1-K26. Our data provide evidence for a novel mechanism regulating the substrate specificity of a chromatin-modifying enzyme through disparate translational products of a regulatory subunit
— id: 69869, year: 2004, vol: 14, page: 183, stat: Journal Article,

Techniques used to study transcription on chromatin templates
Loyola, Alejandra; He, Shu; Oh, Santaek; McCafferty, Dewey G; Reinberg, Danny
2004 ;377:474-499, Methods in enzymology
— id: 69872, year: 2004, vol: 377, page: 474, stat: Journal Article,

Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II
Mandal, Subhrangsu S; Chu, Chun; Wada, Tadashi; Handa, Hiroshi; Shatkin, Aaron J; Reinberg, Danny
2004 May 18;101(20):7572-7577, Proceedings of the National Academy of Sciences of the United States of America
Capping of the 5' ends of nascent RNA polymerase II transcripts is the first pre-mRNA processing event in all eukaryotic cells. Capping enzyme (CE) is recruited to transcription complexes soon after initiation by the phosphorylation of Ser-5 of the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here, we analyze the role of CE in promoter clearance and its functional interactions with different factors that are involved in promoter clearance. FCP1-mediated dephosphorylation of the carboxyl-terminal domain results in a drastic decrease in cotranscriptional capping efficiency but is reversed by the presence of DRB sensitivity-inducing factor (DSIF). These results suggest involvement of DSIF in CE recruitment. Importantly, CE relieves transcriptional repression by the negative elongation factor, indicating a critical role of CE in the elongation checkpoint control mechanism during promoter clearance. This functional interaction between CE and the negative elongation factor documents a dynamic role of CE in promoter clearance beyond its catalytic activities
— id: 69868, year: 2004, vol: 101, page: 7572, stat: Journal Article,

Epigenetic dynamics of imprinted X inactivation during early mouse development
Okamoto, Ikuhiro; Otte, Arie P; Allis, C David; Reinberg, Danny; Heard, Edith
2004 Jan 30;303(5658):644-649, Science
The initiation of X-chromosome inactivation is thought to be tightly correlated with early differentiation events during mouse development. Here, we show that although initially active, the paternal X chromosome undergoes imprinted inactivation from the cleavage stages, well before cellular differentiation. A reversal of the inactive state, with a loss of epigenetic marks such as histone modifications and polycomb proteins, subsequently occurs in cells of the inner cell mass (ICM), which give rise to the embryo-proper in which random X inactivation is known to occur. This reveals the remarkable plasticity of the X-inactivation process during preimplantation development and underlines the importance of the ICM in global reprogramming of epigenetic marks in the early embryo
— id: 69874, year: 2004, vol: 303, page: 644, stat: Journal Article,

Steps toward understanding the inheritance of repressive methyl-lysine marks in histones
Reinberg, D; Chuikov, S; Farnham, P; Karachentsev, D; Kirmizis, A; Kuzmichev, A; Margueron, R; Nishioka, K; Preissner, T S; Sarma, K; Abate-Shen, C; Steward, R; Vaquero, A
2004 ;69:171-182, Cold Spring Harbor symposia on quantitative biology
— id: 69895, year: 2004, vol: 69, page: 171, stat: Journal Article,

Differential histone H3 Lys-9 and Lys-27 methylation profiles on the X chromosome
Rougeulle, Claire; Chaumeil, Julie; Sarma, Kavitha; Allis, C David; Reinberg, Danny; Avner, Philip; Heard, Edith
2004 Jun;24(12):5475-5484, Molecular & cellular biology
Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5' to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases
— id: 69865, year: 2004, vol: 24, page: 5475, stat: Journal Article,

Tips in analyzing antibodies directed against specific histone tail modifications
Sarma, Kavitha; Nishioka, Kenichi; Reinberg, Danny
2004 ;376:255-269, Methods in enzymology
— id: 69873, year: 2004, vol: 376, page: 255, stat: Journal Article,

A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin
Schotta, Gunnar; Lachner, Monika; Sarma, Kavitha; Ebert, Anja; Sengupta, Roopsha; Reuter, Gunter; Reinberg, Danny; Jenuwein, Thomas
2004 Jun 1;18(11):1251-1262, Genes & development
Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin
— id: 69866, year: 2004, vol: 18, page: 1251, stat: Journal Article,

Elongation by RNA polymerase II: the short and long of it
Sims, Robert J 3rd; Belotserkovskaya, Rimma; Reinberg, Danny
2004 Oct 15;18(20):2437-2468, Genes & development
Appreciable advances into the process of transcript elongation by RNA polymerase II (RNAP II) have identified this stage as a dynamic and highly regulated step of the transcription cycle. Here, we discuss the many factors that regulate the elongation stage of transcription. Our discussion includes the classical elongation factors that modulate the activity of RNAP II, and the more recently identified factors that facilitate elongation on chromatin templates. Additionally, we discuss the factors that associate with RNAP II, but do not modulate its catalytic activity. Elongation is highlighted as a central process that coordinates multiple stages in mRNA biogenesis and maturation
— id: 69859, year: 2004, vol: 18, page: 2437, stat: Journal Article,

Recent highlights of RNA-polymerase-II-mediated transcription
Sims, Robert J 3rd; Mandal, Subhrangsu S; Reinberg, Danny
2004 Jun;16(3):263-271, Current opinion in cell biology
Considerable advances into the basis of RNA-polymerase-II-mediated transcriptional regulation have recently emerged. Biochemical, genetic and structural studies have contributed to novel insights into transcription, as well as the functional significance of covalent histone modifications. New details regarding transcription elongation through chromatin have further defined the mechanism behind this action, and identified how chromatin structure may be maintained after RNAP II traverses a nucleosome. ATP-dependent chromatin remodeling complexes, along with histone chaperone complexes, were recently discovered to facilitate histone exchange. In addition, it has become increasingly clear that transcription by RNA polymerase II extends beyond RNA synthesis, towards a more active role in mRNA maturation, surveillance and export to the cytoplasm
— id: 69867, year: 2004, vol: 16, page: 263, stat: Journal Article,

From chromatin to cancer: a new histone lysine methyltransferase enters the mix
Sims, Robert J 3rd; Reinberg, Danny
2004 Aug;6(8):685-687, Nature cell biology
— id: 69861, year: 2004, vol: 6, page: 685, stat: Journal Article,

Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin
Vaquero, Alejandro; Scher, Michael; Lee, Donghoon; Erdjument-Bromage, Hediye; Tempst, Paul; Reinberg, Danny
2004 Oct 8;16(1):93-105, Molecular cell
We characterized human SirT1, one of the human homologs of the budding yeast Sir2p, an NAD+-dependent histone deacetylase involved in establishing repressive chromatin and increased life span. SirT1 deacetylates histone polypeptides with a preference for histone H4 lysine 16 (H4-K16Ac) and H3 lysine 9 (H3-K9Ac) in vitro. RNAi-mediated decreased expression of SirT1 in human cells causes hyperacetylation of H4-K16 and H3-K9 in vivo. SirT1 interacts with and deacetylates histone H1 at lysine 26. Using an inducible system directing expression of SirT1 fused to the Gal4-DNA binding domain and a Gal4-reporter integrated in euchromatin, Gal4-SirT1 expression resulted in the deacetylation of H4-K16 and H3-K9, recruitment of H1 within the promoter vicinity, drastically reduced reporter expression, and loss of H3-K79 methylation, a mark restricting silenced chromatin. We propose a model for SirT1-mediated heterochromatin formation that includes deacetylation of histone tails, recruitment and deacetylation of histone H1, and spreading of hypomethylated H3-K79 with resultant silencing
— id: 69860, year: 2004, vol: 16, page: 93, stat: Journal Article,

FACT facilitates transcription-dependent nucleosome alteration
Belotserkovskaya, Rimma; Oh, Sangtaek; Bondarenko, Vladimir A; Orphanides, George; Studitsky, Vasily M; Reinberg, Danny
2003 Aug 22;301(5636):1090-1093, Science
The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT possesses intrinsic histone chaperone activity and can deposit core histones onto DNA. Importantly, FACT activity requires both of its constituent subunits and is dependent on the highly acidic C terminus of its larger subunit, Spt16. These findings define the mechanism by which Pol II can transcribe through chromatin without disrupting its epigenetic status
— id: 69879, year: 2003, vol: 301, page: 1090, stat: Journal Article,

The C-terminal domain phosphatase and transcription elongation activities of FCP1 are regulated by phosphorylation
Friedl, Erika M; Lane, William S; Erdjument-Bromage, Hediye; Tempst, Paul; Reinberg, Danny
2003 Mar 4;100(5):2328-2333, Proceedings of the National Academy of Sciences of the United States of America
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is heavily phosphorylated during the transition from transcription initiation to the establishment of an elongation-competent transcription complex. FCP1 is the only phosphatase known to be specific for the CTD of the largest subunit of RNAPII, and its activity is believed to be required to reactivate RNAPII, so that RNAPII can enter another round of transcription. We demonstrate that FCP1 is a phosphoprotein, and that phosphorylation regulates FCP1 activities. FCP1 is phosphorylated at multiple sites in vivo. The CTD phosphatase activity of phosphorylated FCP1 is stimulated by TFIIF, whereas dephosphorylated FCP1 is not. In addition to its role in the recycling of RNAPII, FCP1 also affects transcription elongation. Phosphorylated FCP1 is more active in stimulating transcription elongation than the dephosphorylated form of FCP1. We found that only phosphorylated FCP1 can physically interact with TFIIF. We set out to purify an FCP1 kinase from HeLa cells and identified casein kinase 2, which, surprisingly, displayed a negative effect on FCP1-associated activities
— id: 69886, year: 2003, vol: 100, page: 2328, stat: Journal Article,

Tails of intrigue: phosphorylation of RNA polymerase II mediates histone methylation
Hampsey, Michael; Reinberg, Danny
2003 May 16;113(4):429-432, Cell
Histone lysine methylation plays a key role in the organization of chromatin structure and the regulation of gene expression. Recent studies demonstrated that the yeast Set1 and Set2 histone methyltransferases are recruited to mRNA coding regions by the PAF transcription elongation complex in a manner dependent upon the phosphorylation state of the carboxy-terminal domain of RNA polymerase II. These studies define an unexpected link between transcription elongation and histone methylation
— id: 69884, year: 2003, vol: 113, page: 429, stat: Journal Article,

Facile synthesis of site-specifically acetylated and methylated histone proteins: reagents for evaluation of the histone code hypothesis
He, Shu; Bauman, David; Davis, Jamaine S; Loyola, Alejandra; Nishioka, Kenichi; Gronlund, Jennifer L; Reinberg, Danny; Meng, Fanyu; Kelleher, Neil; McCafferty, Dewey G
2003 Oct 14;100(21):12033-12038, Proceedings of the National Academy of Sciences of the United States of America
The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a 'histone code' for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc/HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3/H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing Factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms
— id: 69876, year: 2003, vol: 100, page: 12033, stat: Journal Article,

The mediator coactivator complex: functional and physical roles in transcriptional regulation
Lewis, Brian A; Reinberg, Danny
2003 Sep 15;116(Pt 18):3667-3675, Journal of cell science
In vivo, the DNA is packed into chromatin and transcription is dependent upon activators that recruit other factors to reverse the repressive effects of chromatin. The response to activators requires additional factors referred to as coactivators. One such coactivator, mediator, is a multi-subunit complex capable of responding to different activators. It plays an key role in activation, bridging DNA-bound activators, the general transcriptional machinery, especially RNA polymerase II, and the core promoter. Its subunits are necessary for a variety of positive and negative regulatory processes and serve as the direct targets of activators themselves. In vivo and in vitro studies support various roles for mediator in transcription initiation, while structural studies demonstrate that it engages in multiple interactions with RNA polymerase II, and adopts conformations that are activator specific
— id: 69880, year: 2003, vol: 116, page: 3667, stat: Journal Article,

Functional analysis of the subunits of the chromatin assembly factor RSF
Loyola, Alejandra; Huang, Jing-Yi; LeRoy, Gary; Hu, Sherrie; Wang, Yuh-Hwa; Donnelly, Robert J; Lane, William S; Lee, Sheng-Chung; Reinberg, Danny
2003 Oct;23(19):6759-6768, Molecular & cellular biology
The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different
— id: 69877, year: 2003, vol: 23, page: 6759, stat: Journal Article,

Histone deposition and chromatin assembly by RSF
Loyola, Alejandra; Reinberg, Danny
2003 Sep;31(1):96-103, Methods
It is becoming clear that the structure of cellular chromatin is dynamic and capable of undergoing rapid changes to respond to the metabolic requirements of the cell. These changes have a direct impact on gene expression and, therefore, the chromatin context must be considered when biochemical reactions that involve DNA are studied. Over the past several decades, a number of methods for assembling chromatin in vitro have been described. Some of them use chemical compounds to deposit histone octamers onto the DNA. Others take advantage of cellular protein complexes that have the ability to assemble chromatin. Some of these complexes have been identified and purified. This article focuses on one of these factors, RSF (remodeling and spacing factor), which was identified in our laboratory. We describe how the chromatin assembly reaction is performed and how it can be monitored to evaluate its efficiency
— id: 69881, year: 2003, vol: 31, page: 96, stat: Journal Article,

Methods and tips for the purification of human histone methyltransferases
Nishioka, Kenichi; Reinberg, Danny
2003 Sep;31(1):49-58, Methods
Recently developed biochemical techniques have enabled researchers to study histone modifications more easily and accurately. One of these modifications, histone lysine methylation, has been shown to be highly stable and to represent an epigenetic alteration. Extensive biochemical analyses have led to discoveries about the nature and functions of this modification, thus accelerating our understanding of this crucial epigenetic event. Here we describe basic methods for purification and biochemical analysis of lysine-directed, histone methyltransferases from HeLa cell-derived extracts. In the section on substrate preparation, we describe a simple method for the preparation of recombinant substrates, although we recommend using native substrates for initial detection of the activities. The purification protocols for several histone methyltransferases have been streamlined so that those researchers with a basic understanding of biochemistry can perform them. We also describe many tips and provide suggestions to avoid common pitfalls in the biochemical analysis of histone methyltransferases
— id: 69882, year: 2003, vol: 31, page: 49, stat: Journal Article,

Tracking FACT and the RNA polymerase II elongation complex through chromatin in vivo
Saunders, Abbie; Werner, Janis; Andrulis, Erik D; Nakayama, Takahiro; Hirose, Susumu; Reinberg, Danny; Lis, John T
2003 Aug 22;301(5636):1094-1096, Science
RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). Here we show that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III-transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. Our observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms
— id: 69878, year: 2003, vol: 301, page: 1094, stat: Journal Article,

ASAP, a novel protein complex involved in RNA processing and apoptosis
Schwerk, Christian; Prasad, Jayendra; Degenhardt, Kurt; Erdjument-Bromage, Hediye; White, Eileen; Tempst, Paul; Kidd, Vincent J; Manley, James L; Lahti, Jill M; Reinberg, Danny
2003 Apr;23(8):2981-2990, Molecular & cellular biology
Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis
— id: 69885, year: 2003, vol: 23, page: 2981, stat: Journal Article,

Histone lysine methylation: a signature for chromatin function
Sims, Robert J 3rd; Nishioka, Kenichi; Reinberg, Danny
2003 Nov;19(11):629-639, Trends in genetics
— id: 69875, year: 2003, vol: 19, page: 629, stat: Journal Article,

The constantly changing face of chromatin
Vaquero, Alejandro; Loyola, Alejandra; Reinberg, Danny
2003 Apr 9;2003(14):RE4-RE4, Science of aging knowledge environment
Many recent findings have altered our vision of chromatin and its role in the regulation of cellular functions. Our perspective concerning chromatin has changed to a much more complex, but also more dynamic, view of chromatin as an entity that is intimately involved in the regulation of a variety of cellular functions. In this review, we describe the various types of proteins that alter the structure and, therefore, the function of chromatin and discuss the possible role of chromatin in cell aging. The elucidation of the mechanisms that link chromatin to aging will be one of the most exciting and striking advancements in the coming years
— id: 69883, year: 2003, vol: 2003, page: RE4, stat: Journal Article,

Inhibition of excess nodal signaling during mouse gastrulation by the transcriptional corepressor DRAP1
Iratni, Rabah; Yan, Yu-Ting; Chen, Canhe; Ding, Jixiang; Zhang, Yi; Price, Sandy M; Reinberg, Danny; Shen, Michael M
2002 Dec 6;298(5600):1996-1999, Science
The formation and patterning of mesoderm during mammalian gastrulation require the activity of Nodal, a secreted mesoderm-inducing factor of the transforming growth factor-beta (TGF-beta) family. Here we show that the transcriptional corepressor DRAP1 has a very specific role in regulation of Nodal activity during mouse embryogenesis. We find that loss of Drap1 leads to severe gastrulation defects that are consistent with increased expression of Nodal and can be partially suppressed by Nodal heterozygosity. Biochemical studies indicate that DRAP1 interacts with and inhibits DNA binding by the winged-helix transcription factor FoxH1 (FAST), a critical component of a positive feedback loop for Nodal activity. We propose that DRAP1 limits the spread of a morphogenetic signal by down-modulating the response to the Nodal autoregulatory loop
— id: 69887, year: 2002, vol: 298, page: 1996, stat: Journal Article,

Human Elongator facilitates RNA polymerase II transcription through chromatin
Kim, Jae-Hyun; Lane, William S; Reinberg, Danny
2002 Feb 5;99(3):1241-1246, Proceedings of the National Academy of Sciences of the United States of America
A human Elongator complex was purified from HeLa cells and found to be composed of three polypeptides. Human Elongator contains histone acetyltransferase activity with specificity to histone H3 and, to a much lesser extent, to histone H4. Although many reports have suggested a role for the yeast Elongator in transcription elongation through chromatin templates, no direct evidence supporting this function exists. In the present study, we demonstrate that the human Elongator facilitates transcription by RNA polymerase II in a chromatin- and acetyl-CoA-dependent manner. The complex was found to directly interact with RNA polymerase II but failed to interact with other factors that facilitated RNA polymerase II to traverse through nucleosomes. From our results, we postulate that different mechanisms operate to ensure efficient transcription by RNA polymerase II on chromatin templates
— id: 69894, year: 2002, vol: 99, page: 1241, stat: Journal Article,

Role of the Sin3-histone deacetylase complex in growth regulation by the candidate tumor suppressor p33(ING1)
Kuzmichev, A; Zhang, Y; Erdjument-Bromage, H; Tempst, P; Reinberg, D
2002 Feb;22(3):835-848, Molecular & cellular biology
Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain
— id: 69896, year: 2002, vol: 22, page: 835, stat: Journal Article,

Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein
Kuzmichev, Andrei; Nishioka, Kenichi; Erdjument-Bromage, Hediye; Tempst, Paul; Reinberg, Danny
2002 Nov 15;16(22):2893-2905, Genes & development
Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. Here we report the isolation of a multiprotein E(z) complex that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48. This complex, which we termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. We hypothesize that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing
— id: 69888, year: 2002, vol: 16, page: 2893, stat: Journal Article,

FCP1, a phosphatase specific for the heptapeptide repeat of the largest subunit of RNA polymerase II, stimulates transcription elongation
Mandal, Subhrangsu S; Cho, Helen; Kim, Sungjoon; Cabane, Kettly; Reinberg, Danny
2002 Nov;22(21):7543-7552, Molecular & cellular biology
FCP1, a phosphatase specific for the carboxy-terminal domain of RNA polymerase II (RNAP II), was found to stimulate transcript elongation by RNAP II in vitro and in vivo. This activity is independent of and distinct from the elongation-stimulatory activity associated with transcription factor IIF (TFIIF), and the elongation effects of TFIIF and FCP1 were found to be additive. Genetic experiments resulted in the isolation of several distinct fcp1 alleles. One of these alleles was found to suppress the slow-growth phenotype associated with either the reduction of intracellular nucleotide concentrations or the inhibition of other transcription elongation factors. Importantly, this allele of fcp1 was found to be lethal when combined individually with two mutations in the second-largest subunit of RNAP II, which had been shown previously to affect transcription elongation
— id: 69889, year: 2002, vol: 22, page: 7543, stat: Journal Article,

Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation
Nishioka, Kenichi; Chuikov, Sergei; Sarma, Kavitha; Erdjument-Bromage, Hediye; Allis, C David; Tempst, Paul; Reinberg, Danny
2002 Feb 15;16(4):479-489, Genes & development
A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, as the methylation of H3-K9 by the histone methyltransferase G9a was not affected by Set9 methylation of H3-K4. Our studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. Our results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification
— id: 69893, year: 2002, vol: 16, page: 479, stat: Journal Article,

PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin
Nishioka, Kenichi; Rice, Judd C; Sarma, Kavitha; Erdjument-Bromage, Hediye; Werner, Janis; Wang, Yanming; Chuikov, Sergei; Valenzuela, Pablo; Tempst, Paul; Steward, Ruth; Lis, John T; Allis, C David; Reinberg, Danny
2002 Jun;9(6):1201-1213, Molecular cell
We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail
— id: 69891, year: 2002, vol: 9, page: 1201, stat: Journal Article,

A unified theory of gene expression
Orphanides, George; Reinberg, Danny
2002 Feb 22;108(4):439-451, Cell
The human genome has been called 'the blueprint for life.' This master plan is realized through the process of gene expression. Recent progress has revealed that many of the steps in the pathway from gene sequence to active protein are connected, suggesting a unified theory of gene expression
— id: 69892, year: 2002, vol: 108, page: 439, stat: Journal Article,

Mitotic-specific methylation of histone H4 Lys 20 follows increased PR-Set7 expression and its localization to mitotic chromosomes
Rice, Judd C; Nishioka, Kenichi; Sarma, Kavitha; Steward, Ruth; Reinberg, Danny; Allis, C David
2002 Sep 1;16(17):2225-2230, Genes & development
We describe distinct patterns of histone methylation during human cell cycle progression. Histone H4 methyltransferase activity was found to be cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely followed the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted
— id: 69890, year: 2002, vol: 16, page: 2225, stat: Journal Article,

Role of histone deacetylase complexes in the regulation of chromatin metabolism
Kuzmichev, A; Reinberg, D
2001 ;254:35-58, Current topics in microbiology & immunology
— id: 69903, year: 2001, vol: 254, page: 35, stat: Journal Article,

RBP1 recruits the mSIN3-histone deacetylase complex to the pocket of retinoblastoma tumor suppressor family proteins found in limited discrete regions of the nucleus at growth arrest
Lai, A; Kennedy, B K; Barbie, D A; Bertos, N R; Yang, X J; Theberge, M C; Tsai, S C; Seto, E; Zhang, Y; Kuzmichev, A; Lane, W S; Reinberg, D; Harlow, E; Branton, P E
2001 Apr;21(8):2918-2932, Molecular & cellular biology
Retinoblastoma (RB) tumor suppressor family pocket proteins induce cell cycle arrest by repressing transcription of E2F-regulated genes through both histone deacetylase (HDAC)-dependent and -independent mechanisms. In this study we have identified a stable complex that accounts for the recruitment of both repression activities to the pocket. One component of this complex is RBP1, a known pocket-binding protein that exhibits both HDAC-dependent and -independent repression functions. RB family proteins were shown to associate via the pocket with previously identified mSIN3-SAP30-HDAC complexes containing exclusively class I HDACs. Such enzymes do not interact directly with RB family proteins but rather utilize RBP1 to target the pocket. This mechanism was shown to account for the majority of RB-associated HDAC activity. We also show that in quiescent normal human cells this entire RBP1-mSIN3-SAP30-HDAC complex colocalizes with both RB family members and E2F4 in a limited number of discrete regions of the nucleus that in other studies have been shown to represent the initial origins of DNA replication following growth stimulation. These results suggest that RB family members, at least in part, drive exit from the cell cycle by recruitment of this HDAC complex via RBP1 to repress transcription from E2F-dependent promoters and possibly to alter chromatin structure at DNA origins
— id: 69901, year: 2001, vol: 21, page: 2918, stat: Journal Article,

Defective interplay of activators and repressors with TFIH in xeroderma pigmentosum
Liu, J; Akoulitchev, S; Weber, A; Ge, H; Chuikov, S; Libutti, D; Wang, X W; Conaway, J W; Harris, C C; Conaway, R C; Reinberg, D; Levens, D
2001 Feb 9;104(3):353-363, Cell
Inherited mutations of the TFIIH helicase subunits xeroderma pigmentosum (XP) B or XPD yield overlapping DNA repair and transcription syndromes. The high risk of cancer in these patients is not fully explained by the repair defect. The transcription defect is subtle and has proven more difficult to evaluate. Here, XPB and XPD mutations are shown to block transcription activation by the FUSE Binding Protein (FBP), a regulator of c-myc expression, and repression by the FBP Interacting Repressor (FIR). Through TFIIH, FBP facilitates transcription until promoter escape, whereas after initiation, FIR uses TFIIH to delay promoter escape. Mutations in TFIIH that impair regulation by FBP and FIR affect proper regulation of c-myc expression and have implications in the development of malignancy
— id: 69902, year: 2001, vol: 104, page: 353, stat: Journal Article,

Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription
Loyola, A; LeRoy, G; Wang, Y H; Reinberg, D
2001 Nov 1;15(21):2837-2851, Genes & development
The human ISWI-containing factor RSF (remodeling and spacing factor) was found to mediate nucleosome deposition and, in the presence of ATP, generate regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation was impaired in directing transcription. Histone-tail modifications were found to regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 was found to increase transcription. Methylation of the histone H3 tail by Suv39H1 was found to repress transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system was refractory to the effect imposed by acetylation and methylation
— id: 69898, year: 2001, vol: 15, page: 2837, stat: Journal Article,

Transcription. Switching partners in a regulatory tango
Nishioka, K; Reinberg, D
2001 Dec 21;294(5551):2497-2498, Science
— id: 69897, year: 2001, vol: 294, page: 2497, stat: Journal Article,

Interaction of FACT, SSRP1, and the high mobility group (HMG) domain of SSRP1 with DNA damaged by the anticancer drug cisplatin
Yarnell, A T; Oh, S; Reinberg, D; Lippard, S J
2001 Jul 13;276(28):25736-25741, Journal of biological chemistry
The structure-specific recognition protein SSRP1, initially isolated from expression screening of a human B-cell cDNA library for proteins that bind to cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a single DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal block to transcription elongation by RNAPII in vitro. The affinity and specificity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were investigated by gel mobility shift assays. FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared with unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer. FACT binds the major 1,2-d(GpG) intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to form discrete, high affinity complexes with cisplatin-modified DNA under similar conditions. These results suggest that Spt16 primes SSRP1 for cisplatin-damaged DNA recognition by unveiling its HMG domain. As expected, the isolated HMG domain of SSRP1 is sufficient for specific binding to cisplatin-damaged DNA and binds the major cisplatin 1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT for cisplatin-modified DNA, as well as its importance for transcription of chromatin, suggests that the interaction of FACT and cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin
— id: 69900, year: 2001, vol: 276, page: 25736, stat: Journal Article,

Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails
Zhang, Y; Reinberg, D
2001 Sep 15;15(18):2343-2360, Genes & development
— id: 69899, year: 2001, vol: 15, page: 2343, stat: Journal Article,

TFIIH is negatively regulated by cdk8-containing mediator complexes
Akoulitchev, S; Chuikov, S; Reinberg, D
2000 Sep 7;407(6800):102-106, Nature
The mammalian cyclin-dependent kinase 8 (cdk8) gene has been linked with a subset of acute lymphoblastic leukaemias, and its corresponding protein has been functionally implicated in regulation of transcription. Mammalian cdk8 and cyclin C, and their respective yeast homologues, Srb10 and Srb11, are components of the RNA polymerase II holoenzyme complex where they function as a protein kinase that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref. 7). The yeast SRB10 and SRB11 genes have been implicated in the negative regulation of transcription. The cdk8/cyclin C protein complex is also found in a number of mammalian Mediator-like protein complexes, which repress activated transcription independently of the CTD in vitro. Here we show that cdk8/cyclin C can regulate transcription by targeting the cdk7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH). cdk8 phosphorylates mammalian cyclin H in the vicinity of its functionally unique amino-terminal and carboxy-terminal alpha-helical domains. This phosphorylation represses both the ability of TFIIH to activate transcription and its CTD kinase activity. In addition, mimicking cdk8 phosphorylation of cyclin H in vivo has a dominant-negative effect on cell growth. Our results link the Mediator complex and the basal transcription machinery by a regulatory pathway involving two cyclin-dependent kinases. This pathway appears to be unique to higher organisms
— id: 69905, year: 2000, vol: 407, page: 102, stat: Journal Article,

Genetic analysis of the YDR1-BUR6 repressor complex reveals an intricate balance among transcriptional regulatory proteins in yeast
Kim, S; Cabane, K; Hampsey, M; Reinberg, D
2000 Apr;20(7):2455-2465, Molecular & cellular biology
A transcriptional repressor complex encoded by two essential genes, YDR1 and BUR6, was isolated from Saccharomyces cerevisiae and shown to be the functional counterpart of the human repressor complex Dr1-DRAP1. To elucidate the mechanism of repression by this complex, altered forms of Ydr1 and Bur6 were studied in vitro and in vivo. Deletion of the C-terminal 41 amino acids of Ydr1 resulted in loss of repressor activity and a growth defect, suggesting that the C-terminal domain of Ydr1 functions as a potent transcriptional repressor. A screen for extragenic suppressors of a cold-sensitive ydr1 (ydr1(cs)) mutant led to the identification of recessive mutations in the SIN4 gene, which encodes a component of the SRB-MED complex. The sin4 alleles suppressed not only ydr1(cs) mutations but also bur6(cs) mutations. In contrast, deletion of the gal11 gene, whose product is also a member of the SRB-MED complex, failed to suppress ydr1(cs) and bur6(cs) mutations, indicating that suppression is not due to general defects in the SRB-MED complex. Moreover, one of the sin4 alleles, but not the sin4 deletion, was found to specifically suppress the inviability of a ydr1 deletion, demonstrating that the essential function of Ydr1 becomes dispensable in a sin4 mutant background. Biochemical analysis of the SRB-MED complex from the sin4 suppressor strain revealed a structurally distinct form of the SRB-MED complex that lacks a subset of mediator subunits. These results define a delicate balance between positive and negative regulators of transcription operating through the Ydr1-Bur6 repressor complex
— id: 69909, year: 2000, vol: 20, page: 2455, stat: Journal Article,

Mechanism of ATP-dependent promoter melting by transcription factor IIH
Kim, T K; Ebright, R H; Reinberg, D
2000 May 26;288(5470):1418-1422, Science
We show that transcription factor IIH ERCC3 subunit, the DNA helicase responsible for adenosine triphosphate (ATP)-dependent promoter melting during transcription initiation, does not interact with the promoter region that undergoes melting but instead interacts with DNA downstream of this region. We show further that promoter melting does not change protein-DNA interactions upstream of the region that undergoes melting but does change interactions within and downstream of this region. Our results rule out the proposal that IIH functions in promoter melting through a conventional DNA-helicase mechanism. We propose that IIH functions as a molecular wrench: rotating downstream DNA relative to fixed upstream protein-DNA interactions, thereby generating torque on, and melting, the intervening DNA
— id: 69907, year: 2000, vol: 288, page: 1418, stat: Journal Article,

Purification and characterization of a human factor that assembles and remodels chromatin
LeRoy, G; Loyola, A; Lane, W S; Reinberg, D
2000 May 19;275(20):14787-14790, Journal of biological chemistry
We have previously reported the isolation and characterization of a nucleosome remodeling and spacing factor, RSF. One of the RSF subunits is hSNF2h, a SNF2 homologue. Here we set out to isolate and characterize other hSNF2h-containing complexes. We have identified a novel hSNF2h complex that facilitates ATP-dependent chromatin assembly with the histone chaperone NAP-1. The complex possesses ATPase activity that is DNA-dependent and nucleosome-stimulated. This complex is capable of facilitating ATP-dependent nucleosome remodeling and transcription initiation from chromatin templates. In addition to hSNF2h, this complex also contains a 190-kDa protein encoded by the BAZ1A gene. Since both subunits are homologues of the Drosophila ACF complex (ATP-utilizing chromatin assembly and remodeling factor), we have named this factor human ACF or hACF
— id: 69908, year: 2000, vol: 275, page: 14787, stat: Journal Article,

RNA polymerase II elongation through chromatin
Orphanides, G; Reinberg, D
2000 Sep 28;407(6803):471-475, Nature
The machinery that transcribes protein-coding genes in eukaryotic cells must contend with repressive chromatin structures in order to find its target DNA sequences. Diverse arrays of proteins modify the structure of chromatin at gene promoters to help transcriptional regulatory proteins access their DNA recognition sites. The way in which disruption of chromatin structure at a promoter is transmitted through a whole gene has not been defined. Recent breakthroughs suggest that the passage of an RNA polymerase through a gene is coupled to mechanisms that propagate the breakdown of chromatin
— id: 69904, year: 2000, vol: 407, page: 471, stat: Journal Article,

FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH
Wada, T; Orphanides, G; Hasegawa, J; Kim, D K; Shima, D; Yamaguchi, Y; Fukuda, A; Hisatake, K; Oh, S; Reinberg, D; Handa, H
2000 Jun;5(6):1067-1072, Molecular cell
We report that the chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). We find that the kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, we propose a novel transcriptional regulatory network in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT. This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription
— id: 69906, year: 2000, vol: 5, page: 1067, stat: Journal Article,

A protein phosphatase functions to recycle RNA polymerase II
Cho, H; Kim, T K; Mancebo, H; Lane, W S; Flores, O; Reinberg, D
1999 Jun 15;13(12):1540-1552, Genes & development
Transcription is regulated by the state of phosphorylation of a heptapeptide repeat known as the carboxy-terminal domain (CTD) present in the largest subunit of RNA polymerase II (RNAPII). RNAPII that associates with transcription initiation complexes contains an unphosphorylated CTD, whereas the elongating polymerase has a phosphorylated CTD. Transcription factor IIH has a kinase activity specific for the CTD that is stimulated by the formation of a transcription initiation complex. Here, we report the isolation of a cDNA clone encoding a 150-kD polypeptide, which, together with RNAPII, reconstitutes a highly specific CTD phosphatase activity. Functional analysis demonstrates that the CTD phosphatase allows recycling of RNAPII. The phosphatase dephosphorylates the CTD allowing efficient incorporation of RNAPII into transcription initiation complexes, which results in increased transcription. The CTD phosphatase was found to be active in ternary elongation complexes. Moreover, the phosphatase stimulates elongation by RNAPII; however, this function is independent of its catalytic activity
— id: 69914, year: 1999, vol: 13, page: 1540, stat: Journal Article,

RNA polymerase II as a control panel for multiple coactivator complexes
Hampsey, M; Reinberg, D
1999 Apr;9(2):132-139, Current opinion in genetics & development
1999 marks the 30th anniversary of the reported discovery of sigma factor and the bacterial RNA polymerase holoenzyme. In 1994, an RNA polymerase II complex was discovered in yeast - mammalian complexes were subsequently identified. Recent developments regarding the composition and function of RNA polymerase II complexes suggest, however, that the concept of the holoenzyme, as defined in bacteria, might not be relevant to eukaryotes
— id: 69916, year: 1999, vol: 9, page: 132, stat: Journal Article,

The general transcription factors IIA, IIB, IIF, and IIE are required for RNA polymerase II transcription from the human U1 small nuclear RNA promoter
Kuhlman, T C; Cho, H; Reinberg, D; Hernandez, N
1999 Mar;19(3):2130-2141, Molecular & cellular biology
RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs
— id: 69917, year: 1999, vol: 19, page: 2130, stat: Journal Article,

Repression: targeting the heart of the matter
Maldonado, E; Hampsey, M; Reinberg, D
1999 Nov 24;99(5):455-458, Cell
— id: 69910, year: 1999, vol: 99, page: 455, stat: Journal Article,

MBD2 is a transcriptional repressor belonging to the MeCP1 histone deacetylase complex
Ng, H H; Zhang, Y; Hendrich, B; Johnson, C A; Turner, B M; Erdjument-Bromage, H; Tempst, P; Reinberg, D; Bird, A
1999 Sep;23(1):58-61, Nature genetics
Mammalian DNA is methylated at many CpG dinucleotides. The biological consequences of methylation are mediated by a family of methyl-CpG binding proteins. The best characterized family member is MeCP2, a transcriptional repressor that recruits histone deacetylases. Our report concerns MBD2, which can bind methylated DNA in vivo and in vitro and has been reported to actively demethylate DNA (ref. 8). As DNA methylation causes gene silencing, the MBD2 demethylase is a candidate transcriptional activator. Using specific antibodies, however, we find here that MBD2 in HeLa cells is associated with histone deacetylase (HDAC) in the MeCP1 repressor complex. An affinity-purified HDAC1 corepressor complex also contains MBD2, suggesting that MeCP1 corresponds to a fraction of this complex. Exogenous MBD2 represses transcription in a transient assay, and repression can be relieved by the deacetylase inhibitor trichostatin A (TSA; ref. 12). In our hands, MBD2 does not demethylate DNA. Our data suggest that HeLa cells, which lack the known methylation-dependent repressor MeCP2, use an alternative pathway involving MBD2 to silence methylated genes
— id: 69911, year: 1999, vol: 23, page: 58, stat: Journal Article,

The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins
Orphanides, G; Wu, W H; Lane, W S; Hampsey, M; Reinberg, D
1999 Jul 15;400(6741):284-288, Nature
The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones
— id: 69913, year: 1999, vol: 400, page: 284, stat: Journal Article,

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation
Zhang, Y; Ng, H H; Erdjument-Bromage, H; Tempst, P; Bird, A; Reinberg, D
1999 Aug 1;13(15):1924-1935, Genes & development
ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation
— id: 69912, year: 1999, vol: 13, page: 1924, stat: Journal Article,

The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7
Akoulitchev, S; Reinberg, D
1998 Nov 15;12(22):3541-3550, Genes & development
TFIIH is a multisubunit complex, containing ATPase, helicases, and kinase activities. Functionally, TFIIH has been implicated in transcription by RNA polymerase II (RNAPII) and in nucleotide excision repair. A member of the cyclin-dependent kinase family, CDK7, is the kinase subunit of TFIIH. Genetically, CDK7 homologues have been implicated in transcription in Saccharomyces cerevisiae, and in mitotic regulation in Schizosaccharomyces pombe. Here we show that in mitosis the CDK7 subunit of TFIIH and the largest subunit of RNAPII become hyperphosphorylated. MPF-induced phosphorylation of CDK7 results in inhibition of the TFIIH-associated kinase and transcription activities. Negative and positive regulation of TFIIH requires phosphorylation within the T-loop of CDK7. Our data establishes TFIIH and its subunit CDK7 as a direct link between the regulation of transcription and the cell cycle
— id: 69919, year: 1998, vol: 12, page: 3541, stat: Journal Article,

A human RNA polymerase II complex containing factors that modify chromatin structure
Cho, H; Orphanides, G; Sun, X; Yang, X J; Ogryzko, V; Lees, E; Nakatani, Y; Reinberg, D
1998 Sep;18(9):5355-5363, Molecular & cellular biology
We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II
— id: 69922, year: 1998, vol: 18, page: 5355, stat: Journal Article,

Human general transcription factor TFIIB: conformational variability and interaction with VP16 activation domain
Hayashi, F; Ishima, R; Liu, D; Tong, K I; Kim, S; Reinberg, D; Bagby, S; Ikura, M
1998 Jun 2;37(22):7941-7951, Biochemistry
Human TFIIB, an essential factor in transcription of protein-coding genes by RNA polymerase II, consists of an amino-terminal zinc binding domain (TFIIBn) connected by a linker of about 60 residues to a carboxy-terminal core domain (TFIIBc). The TFIIB core domain has two internally repeated motifs, each comprising five alpha-helices arranged as in the cyclin box. Compared to the crystal structure of TFIIBc in complex with TBP and a TATA-containing oligonucleotide, the NMR-derived solution structure of free TFIIBc is more compact, with a different repeat-repeat orientation and a significantly shorter first helix in the second repeat. Analysis of backbone 15N relaxation parameters indicates the presence of relatively large amplitude, nanosecond time-scale motions in the TFIIBc interrepeat linker and structural fluctuations throughout the backbone. Interaction of TFIIBc with the acidic activation domain of VP16 or with TFIIBn induces 1H-15N chemical shift and line width changes concentrated in the first repeat, interrepeat linker and the first helix of the second repeat. These results suggest that TFIIB is somewhat pliable and that the conformation of the C-terminal core domain can be modulated by interaction with the N-terminal zinc binding domain. Furthermore, binding of the VP16 activation domain may promote TFIIBc conformations primed for binding to a TBP-DNA complex
— id: 69925, year: 1998, vol: 37, page: 7941, stat: Journal Article,

Promoter-proximal stalling results from the inability to recruit transcription factor IIH to the transcription complex and is a regulated event
Kumar, K P; Akoulitchev, S; Reinberg, D
1998 Aug 18;95(17):9767-9772, Proceedings of the National Academy of Sciences of the United States of America
Promoter-proximal stalling, a general phenomenon observed during the expression of many RNA polymerase II transcribed genes, is dependent on transcription factor IIH (TFIIH). Reactions lacking TFIIH initiated transcription, but the transcription complex encountered a block to elongation proximal to the promoter. The accumulation of promoter-proximal stalled complexes was reduced in the presence of TFIIH and efficient escape from this site also required an activator. Promoter-proximal stalled complexes could not be induced to resume elongation. Our results indicate that effective recruitment of TFIIH into transcription complexes is achieved during formation of the preinitiation complex at the promoter. The studies establish that promoter clearance is a regulated event that requires TFIIH
— id: 69923, year: 1998, vol: 95, page: 9767, stat: Journal Article,

New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB
Lagrange, T; Kapanidis, A N; Tang, H; Reinberg, D; Ebright, R H
1998 Jan 1;12(1):34-44, Genes & development
A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5'-G/C-G/C-G/A-C-G-C-C-3', affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves alpha-helices 4' and 5' of IIB, which comprise a helix-turn-helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID
— id: 69928, year: 1998, vol: 12, page: 34, stat: Journal Article,

Immunoaffinity purification of the human multisubunit transcription factor IIH
LeRoy, G; Drapkin, R; Weis, L; Reinberg, D
1998 Mar 20;273(12):7134-7140, Journal of biological chemistry
A procedure to immunoaffinity purify the human transcription factor IIH (TFIIH) was developed using a monoclonal antibody that recognizes an epitope in ERCC3 (XPB), the largest subunit of TFIIH. The epitope recognized by the monoclonal antibody was mapped to 20 amino acids. A peptide containing the epitope was capable of displacing TFIIH from an immunoaffinity column containing the monoclonal antibody. The immunoaffinity purification procedure described allows a simple and efficient method to purify both the 'core' and 'holo' TFIIH complexes
— id: 69926, year: 1998, vol: 273, page: 7134, stat: Journal Article,

Requirement of RSF and FACT for transcription of chromatin templates in vitro
LeRoy, G; Orphanides, G; Lane, W S; Reinberg, D
1998 Dec 4;282(5395):1900-1904, Science
Transcription of naked DNA in vitro requires the general transcription factors and RNA polymerase II. However, this minimal set of factors is not sufficient for transcription when the DNA template is packaged into chromatin. Here, a factor that facilitates activator-dependent transcription initiation on chromatin templates was purified. This factor, remodeling and spacing factor (RSF), has adenosine triphosphate-dependent nucleosome-remodeling and spacing activities. Polymerases that initiate transcription with RSF can only extend their transcripts in the presence of FACT (facilitates chromatin transcription). Thus, the minimal factor requirements for activator-dependent transcription on chromatin templates in vitro have been defined
— id: 69918, year: 1998, vol: 282, page: 1900, stat: Journal Article,

The mammalian transcriptional repressor RBP (CBF1) targets TFIID and TFIIA to prevent activated transcription
Olave, I; Reinberg, D; Vales, L D
1998 Jun 1;12(11):1621-1637, Genes & development
RBP is a cellular protein that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest lately because of its established roles in regulating gene expression, in Drosophila and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, promoter. The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators: dTAFII110, a subunit of TFIID, and TFIIA to repress transcription. The domain of dTAFII110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAFII110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription
— id: 68518, year: 1998, vol: 12, page: 1621, stat: Journal Article,

FACT, a factor that facilitates transcript elongation through nucleosomes
Orphanides, G; LeRoy, G; Chang, C H; Luse, D S; Reinberg, D
1998 Jan 9;92(1):105-116, Cell
The requirements for transcriptional activation by RNA polymerase II were examined using chromatin templates assembled in vitro and a transcription system composed of the human general transcription factors and RNA polymerase II. Activator-induced, energy-dependent chromatin remodeling promoted efficient preinitiation complex formation and transcription initiation, but was not sufficient for productive transcription. Polymerases that initiated transcription on remodeled chromatin templates encountered a block to transcription proximal to the promoter. Entry into productive transcription required an accessory factor present in HeLa cell nuclear extract, FACT (facilitates chromatin transcription), which we have purified. FACT acts subsequent to transcription initiation to release RNA polymerase II from a nucleosome-induced block to productive transcription. The biochemical properties and polypeptide composition of FACT suggest that it is a novel protein factor that facilitates transcript elongation through nucleosomes
— id: 69927, year: 1998, vol: 92, page: 105, stat: Journal Article,

The RNA polymerase II general transcription factors: past, present, and future
Reinberg, D; Orphanides, G; Ebright, R; Akoulitchev, S; Carcamo, J; Cho, H; Cortes, P; Drapkin, R; Flores, O; Ha, I; Inostroza, J A; Kim, S; Kim, T K; Kumar, P; Lagrange, T; LeRoy, G; Lu, H; Ma, D M; Maldonado, E; Merino, A; Mermelstein, F; Olave, I; Sheldon, M; Shiekhattar, R; Zawel, L
1998 ;63:83-103, Cold Spring Harbor symposia on quantitative biology
— id: 69915, year: 1998, vol: 63, page: 83, stat: Journal Article,

NAT, a human complex containing Srb polypeptides that functions as a negative regulator of activated transcription
Sun, X; Zhang, Y; Cho, H; Rickert, P; Lees, E; Lane, W; Reinberg, D
1998 Aug;2(2):213-222, Molecular cell
A complex that represses activated transcription and contains the human homologs of the yeast Srb7, Srb10, Srb11, Rgr1, and Med6 proteins was isolated. The complex is devoid of the Srb polypeptides previously shown to be components of the yeast Mediator complex that functions in transcriptional activation. The complex phosphorylates the CTD of RNA polymerase II (RNAPII) at residues other than those phosphorylated by the kinase of TFIIH. Moreover, the complex specifically interacts with RNAPII. The interaction is not mediated by the CTD of RNAPII, but is precluded by phosphorylation of the CTD. Our results indicate that the complex is a subcomplex of the human RNAPII holoenzyme. We suggest that the RNAPII holoenzyme is a transcriptional control panel, integrating and responding to specific signals to activate or repress transcription
— id: 69921, year: 1998, vol: 2, page: 213, stat: Journal Article,

The dermatomyositis-specific autoantigen Mi2 is a component of a complex containing histone deacetylase and nucleosome remodeling activities
Zhang, Y; LeRoy, G; Seelig, H P; Lane, W S; Reinberg, D
1998 Oct 16;95(2):279-289, Cell
Histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. ATP-dependent nucleosome remodeling, mediated by different multisubunit complexes, was thought to be involved only in transcription activation. Here we report the isolation of a protein complex that contains both histone deacetylation and ATP-dependent nucleosome remodeling activities. The complex contains the histone deacetylases HDAC1/2, histone-binding proteins, the dermatomyositis-specific autoantigen Mi2beta, a polypeptide related to the metastasis-associated protein 1, and a novel polypeptide of 32 kDa. Patients with dermatomyositis have a high rate of malignancy. The finding that Mi2beta exists in a complex containing histone deacetylase and nucleosome remodeling activities suggests a role for chromatin reorganization in cancer metastasis
— id: 69920, year: 1998, vol: 95, page: 279, stat: Journal Article,

SAP30, a novel protein conserved between human and yeast, is a component of a histone deacetylase complex
Zhang, Y; Sun, Z W; Iratni, R; Erdjument-Bromage, H; Tempst, P; Hampsey, M; Reinberg, D
1998 Jun;1(7):1021-1031, Molecular cell
Histone acetylation plays a key role in the regulation of eukaryotic gene expression. Recently, histone acetylation and deacetylation were found to be catalyzed by structurally distinct, multisubunit complexes that mediate, respectively, activation and repression of transcription. Here, we identify SAP30 as a novel component of the human histone deacetylase complex that includes Sin3, the histone deacetylases HDAC1 and HDAC2, histone binding proteins RbAp46 and RbAp48, as well as other polypeptides. Moreover, we describe a SAP30 homolog in yeast that is functionally related to Sin3 and the histone deacetylase Rpd3. The human SAP30 complex is active in deacetylating core histone octamers, but inactive in deacetylating nucleosomal histones due to the inability of the histone binding proteins RbAp46 and RbAp48 to gain access to nucleosomal histones. These results define SAP30 as a component of a histone deacetylase complex conserved among eukaryotic organisms
— id: 69924, year: 1998, vol: 1, page: 1021, stat: Journal Article,

Affinity purification of a human RNA polymerase II complex using monoclonal antibodies against transcription factor IIF
Cho, H; Maldonado, E; Reinberg, D
1997 Apr 25;272(17):11495-11502, Journal of biological chemistry
Five different monoclonal antibodies that immunoreact with RAP74, the large subunit of general transcription factor (TF) IIF, were produced and characterized. Using one of these antibodies, an affinity purification procedure was devised to isolate a human RNA polymerase II complex. This procedure is fast, simple, and reproducible and does not require extensive purification. The RNA polymerase II complex isolated using this procedure contains SRB (suppressor of RNA polymerase B) polypeptides, transcription factors IIE and IIF, limiting amounts of TFIIH, and the TATA-binding protein, but was devoid of TFIIB
— id: 69933, year: 1997, vol: 272, page: 11495, stat: Journal Article,

The human immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme
Cujec, T P; Cho, H; Maldonado, E; Meyer, J; Reinberg, D; Peterlin, B M
1997 Apr;17(4):1817-1823, Molecular & cellular biology
The human immunodeficiency virus (HIV) encodes a transcriptional transactivator (Tat), which binds to an RNA hairpin called the transactivation response element (TAR) that is located downstream of the site of initiation of viral transcription. Tat stimulates the production of full-length viral transcripts by RNA polymerase II (pol II). In this study, we demonstrate that Tat coimmunoprecipitates with the pol II holoenzyme in cells and that it binds to the purified holoenzyme in vitro. Furthermore, Tat affinity chromatography purifies a holoenzyme from HeLa nuclear extracts which, upon addition of TBP and TFIIB, supports Tat transactivation in vitro, indicating that it contains all the cellular proteins required for the function of Tat. By demonstrating that Tat interacts with the holoenzyme in the absence of TAR, our data suggest a single-step assembly of Tat and the transcription complex on the long terminal repeat of HIV
— id: 69934, year: 1997, vol: 17, page: 1817, stat: Journal Article,

Transcription: why are TAFs essential?
Hampsey, M; Reinberg, D
1997 Jan 1;7(1):R44-R46, Current biology. CB
The TAFs are transcription factors associated with the TATA-binding protein, and until recently they were assumed to link specific activators to the general transcription machinery. Recent results suggest that the essential functions of TAFs are not as coactivators of transcription but as determinants of promoter selectivity
— id: 69937, year: 1997, vol: 7, page: R44, stat: Journal Article,

The Dr1/DRAP1 heterodimer is a global repressor of transcription in vivo
Kim, S; Na, J G; Hampsey, M; Reinberg, D
1997 Feb 4;94(3):820-825, Proceedings of the National Academy of Sciences of the United States of America
A general repressor extensively studied in vitro is the human Dr1/DRAP1 heterodimeric complex. To elucidate the function of Dr1 and DRAP1 in vivo, the yeast Saccharomyces cerevisiae Dr1/DRAP1 repressor complex was identified. The repressor complex is encoded by two essential genes, designated YDR1 and BUR6. The inviability associated with deletion of the yeast genes can be overcome by expressing the human genes. However, the human corepressor DRAP1 functions in yeast only when human Dr1 is coexpressed. The yDr1/Bur6 complex represses transcription in vitro in a reconstituted RNA polymerase II transcription system. Repression of transcription could be overcome by increasing the concentration of TATA-element binding protein (TBP). Consistent with the in vitro results, overexpression of YDR1 in vivo resulted in decreased mRNA accumulation. Furthermore, YDR1 overexpression impaired cell growth, an effect that could be rescued by overexpression of TBP. In agreement with our previous studies in vitro, we found that overexpression of Dr1 in vivo also affected the accumulation of RNA polymerase III transcripts, but not of RNA polymerase I transcripts. Our results demonstrate that Dr1 functions as a repressor of transcription in vivo and, moreover, directly targets TBP, a global regulator of transcription
— id: 69936, year: 1997, vol: 94, page: 820, stat: Journal Article,

Trajectory of DNA in the RNA polymerase II transcription preinitiation complex
Kim, T K; Lagrange, T; Wang, Y H; Griffith, J D; Reinberg, D; Ebright, R H
1997 Nov 11;94(23):12268-12273, Proceedings of the National Academy of Sciences of the United States of America
By using site-specific protein-DNA photocrosslinking, we define the positions of TATA-binding protein, transcription factor IIB, transcription factor IIF, and subunits of RNA polymerase II (RNAPII) relative to promoter DNA within the human transcription preinitiation complex. The results indicate that the interface between the largest and second-largest subunits of RNAPII forms an extended, approximately 240 A channel that interacts with promoter DNA both upstream and downstream of the transcription start. By using electron microscopy, we show that RNAPII compacts promoter DNA by the equivalent of approximately 50 bp. Together with the published structure of RNAPII, the results indicate that RNAPII wraps DNA around its surface and suggest a specific model for the trajectory of the wrapped DNA
— id: 69930, year: 1997, vol: 94, page: 12268, stat: Journal Article,

RNA polymerase II stalled at a thymine dimer: footprint and effect on excision repair
Selby, C P; Drapkin, R; Reinberg, D; Sancar, A
1997 Feb 15;25(4):787-793, Nucleic acids research
Bulky lesions in the template strand block the progression of RNA polymerase II (RNAP II) and are repaired more rapidly than lesions in the non-transcribed strand, which do not block transcription. In order to better understand the basis of this transcription-coupled repair we developed an in vitro system with purified transcription and nucleotide excision repair proteins and a plasmid containing the adenovirus major late promoter and a thymine dimer in the template strand downstream of the transcription start site. The footprint of RNAP II stalled at the thymine dimer, obtained using DNase I, lambda exonuclease and T4 polymerase 3'-->5'exonuclease, covers approximately 40 nt and is nearly symmetrical around the dimer. The ternary complex formed at the lesion site is rather stable, with a half-life of approximately 20 h. Surprisingly, addition of human repair proteins results in repair of transcription-blocking dimers in the ternary complex. The blocked polymerase neither inhibits nor stimulates repair and repair is observed in the absence of CSB protein, the putative human transcription-repair coupling factor
— id: 69935, year: 1997, vol: 25, page: 787, stat: Journal Article,

Accurate positioning of RNA polymerase II on a natural TATA-less promoter is independent of TATA-binding-protein-associated factors and initiator-binding proteins
Weis, L; Reinberg, D
1997 Jun;17(6):2973-2984, Molecular & cellular biology
Two promoter elements, the TATA element and initiator (Inr), are capable of directing specific transcription initiation of protein-encoding genes by RNA polymerase II (RNAPII). Although binding to the TATA element by the TATA-binding protein (TBP) has been shown to be the initial recognition step in transcription complex formation in vitro, the mechanism through which the basal machinery assembles into a functional complex on TATA-less promoters is controversial. Evidence supporting numerous models of Inr-mediated transcription complex formation exists, including the nucleation of a complex by Inr-binding proteins, a component of the TFIID complex, or a specific upstream activator common to many TATA-less promoters, Sp1. Using various techniques, we have undertaken a systematic analysis of the natural TATA-less human DNA polymerase beta (beta-pol) gene promoter. Although the beta-pol promoter contains upstream Sp1 elements and a functional Inr that binds YY1, neither of these factors is essential for Inr-mediated transcription complex formation. A complex containing TBP, TFIIB, TFIIF, and RNAPII (DBPolF complex) is capable of forming on the promoter in an Inr-dependent manner. A single point mutation within the Inr that affects DBPolF complex formation diminishes beta-pol transcriptional activity
— id: 69931, year: 1997, vol: 17, page: 2973, stat: Journal Article,

Functional dissection of a human Dr1-DRAP1 repressor complex
Yeung, K; Kim, S; Reinberg, D
1997 Jan;17(1):36-45, Molecular & cellular biology
The heterotetrameric Dr1-DRAP1 transcriptional repressor complex was functionally dissected. Dr1 was found to contain two domains required for repression of transcription. The tethering domain interacts with the TATA box binding protein and directs the repressor complex to the promoter. This tethering domain can be replaced by a domain conferring sequence-specific recognition to the repressor complex. In the absence of the tethering domain, Dr1 interacts with its corepressor DRAP1, but this interaction is not functional. The enhancement of Dr1-mediated repression of transcription by DRAP1 requires the tethering domain. The second domain of Dr1 is the repression domain, which is glutamine-alanine rich. A 65-amino-acid polypeptide containing the repression domain fused to the Ga14 DNA binding domain repressed transcription when directed to TATA-containing and TATA-less promoters. This repression domain was also found to functionally and directly interact with the TATA box binding protein
— id: 69938, year: 1997, vol: 17, page: 36, stat: Journal Article,

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II
Yue, Z; Maldonado, E; Pillutla, R; Cho, H; Reinberg, D; Shatkin, A J
1997 Nov 25;94(24):12898-12903, Proceedings of the National Academy of Sciences of the United States of America
5'-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5'-triphosphatase activity and catalyzed formation of RNA 5'-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5'-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts
— id: 69929, year: 1997, vol: 94, page: 12898, stat: Journal Article,

Histone deacetylases and SAP18, a novel polypeptide, are components of a human Sin3 complex
Zhang, Y; Iratni, R; Erdjument-Bromage, H; Tempst, P; Reinberg, D
1997 May 2;89(3):357-364, Cell
An important event in gene expression is the covalent modification of histone proteins. We have found that the mammalian transcriptional repressor Sin3 (mSin3) exists in a complex with histone deacetylases HDAC1 and HDAC2. Consistent with the observation that mSin3-mediated repression of transcription involves the modification of histone polypeptides, we found that the mSin3-containing complex includes polypeptides that tether the mSin3 complex to core histone proteins. In addition, two novel mSin3-associated polypeptides, SAP18 and SAP30, were identified. We isolated a cDNA encoding human SAP18 and found that SAP18 is a component of an mSin3-containing complex in vivo. Moreover, we demonstrate a direct interaction between SAP18 and mSin3. SAP18 represses transcription in vivo when tethered to the promoter, consistent with the ability of SAP18 to interact with mSin3
— id: 69932, year: 1997, vol: 89, page: 357, stat: Journal Article,

Human cyclin-dependent kinase-activating kinase exists in three distinct complexes
Drapkin, R; Le Roy, G; Cho, H; Akoulitchev, S; Reinberg, D
1996 Jun 25;93(13):6488-6493, Proceedings of the National Academy of Sciences of the United States of America
Transcription factor IIH (TFIIH) is a multisubunit complex required for transcription and for DNA nucleotide excision repair. TFIIH possesses three enzymatic activities: (i) an ATP-dependent DNA helicase, (ii) a DNA-dependent ATPase, and (iii) a kinase with specificity for the carboxyl-terminal domain of RNA polymerase II. The kinase activity was recently identified as the cdk (cyclin-dependent kinase) activating kinase, CAK, composed of cdk7, cyclin H, and MAT-1. Here we report the isolation and characterization of three distinct CAK-containing complexes from HeLa nuclear extracts: CAK, a novel CAK-ERCC2 complex, and TFIIH. CAK-ERCC2 can efficiently associate with core-TFIIH to reconstitute holo-TFIIH transcription activity. We present evidence proposing a critical role for ERCC2 in mediating the association of CAK with core TFIIH subunits
— id: 69942, year: 1996, vol: 93, page: 6488, stat: Journal Article,

High-resolution mapping of nucleoprotein complexes by site-specific protein-DNA photocrosslinking: organization of the human TBP-TFIIA-TFIIB-DNA quaternary complex
Lagrange, T; Kim, T K; Orphanides, G; Ebright, Y W; Ebright, R H; Reinberg, D
1996 Oct 1;93(20):10620-10625, Proceedings of the National Academy of Sciences of the United States of America
We have used a novel site-specific protein-DNA photocrosslinking procedure to define the positions of polypeptide chains relative to promoter DNA in binary, ternary, and quaternary complexes containing human TATA-binding protein, human or yeast transcription factor IIA (TFIIA), human transcription factor IIB (TFIIB), and promoter DNA. The results indicate that TFIIA and TFIIB make more extensive interactions with promoter DNA than previously anticipated. TATA-binding protein, TFIIA, and TFIIB surround promoter DNA for two turns of DNA helix and thus may form a 'cylindrical clamp' effectively topologically linked to promoter DNA. Our results have implications for the energetics, DNA-sequence-specificity, and pathway of assembly of eukaryotic transcription complexes
— id: 69940, year: 1996, vol: 93, page: 10620, stat: Journal Article,

Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA
MA, D; Olave, I; Merino, A; Reinberg, D
1996 Jun 25;93(13):6583-6588, Proceedings of the National Academy of Sciences of the United States of America
Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This 'mini-TFIIA' interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function
— id: 69941, year: 1996, vol: 93, page: 6583, stat: Journal Article,

Purification of human RNA polymerase II and general transcription factors
Maldonado, E; Drapkin, R; Reinberg, D
1996 ;274:72-100, Methods in enzymology
— id: 69946, year: 1996, vol: 274, page: 72, stat: Journal Article,

A human RNA polymerase II complex associated with SRB and DNA-repair proteins
Maldonado, E; Shiekhattar, R; Sheldon, M; Cho, H; Drapkin, R; Rickert, P; Lees, E; Anderson, C W; Linn, S; Reinberg, D
1996 May 2;381(6577):86-89, Nature
We report here the isolation of a human RNA polymerase II complex containing a subset of the basal transcription factors and the human homologues of the yeast SRB (for suppressors of RNA polymerase B) proteins. The complex contains transcriptional coactivators and increases the activation of transcription. In addition, some components of the RNA polymerase II complex participate in DNA repair
— id: 69943, year: 1996, vol: 381, page: 86, stat: Journal Article,

Requirement of a corepressor for Dr1-mediated repression of transcription
Mermelstein, F; Yeung, K; Cao, J; Inostroza, J A; Erdjument-Bromage, H; Eagelson, K; Landsman, D; Levitt, P; Tempst, P; Reinberg, D
1996 Apr 15;10(8):1033-1048, Genes & development
A Dr1-associated polypeptide (DRAP1) was isolated from HeLa cells and found to function as a corepressor of transcription. Corepressor function requires an interaction between DRAP1 and Dr1. Heterodimer formation was dependent on a histone fold motif present at the amino terminus of both polypeptides. Association of DRAP1 with Dr1 results in higher stability of the Dr1-TBP-TATA motif complex and precluded the entry of TFIIA and/or TFIIB to preinitiation complexes. DRAP1 was found to be expressed in all tissues analyzed with higher levels in tissues with a low mitotic index. Analysis of DRAP1 in the developing brain of rat demonstrated undetectable levels of DRAP1 in actively dividing cells but high levels of DRAP1 expression in differentiated non dividing cells. Dr1 was immunodetected in all cells analyzed. A model for DRAP1-dependent, Dr1-mediated repression of transcription is proposed
— id: 69944, year: 1996, vol: 10, page: 1033, stat: Journal Article,

The general transcription factors of RNA polymerase II
Orphanides, G; Lagrange, T; Reinberg, D
1996 Nov 1;10(21):2657-2683, Genes & development
— id: 69939, year: 1996, vol: 10, page: 2657, stat: Journal Article,

Protein-protein interactions in eukaryotic transcription initiation: structure of the preinitiation complex
Tang, H; Sun, X; Reinberg, D; Ebright, R H
1996 Feb 6;93(3):1119-1124, Proceedings of the National Academy of Sciences of the United States of America
We have used alanine scanning to analyze protein-protein interactions by human TATA-element binding protein (TBP) within the transcription preinitiation complex. The results indicate that TBP interacts with RNA polymerase II and general transcription factors IIA, IIB, and IIF within the functional transcription preinitiation complex and define the determinants of TBP for each of these interactions. The results permit construction of a model for the structure of the preinitiation complex
— id: 69945, year: 1996, vol: 93, page: 1119, stat: Journal Article,

Requirement for TFIIH kinase activity in transcription by RNA polymerase II
Akoulitchev, S; Makela, T P; Weinberg, R A; Reinberg, D
1995 Oct 12;377(6549):557-560, Nature
An array of tandem heptapeptide repeats at the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II constitute a highly conserved structure essential for viability. Studies have established that the CTD is phosphorylated at different stages of the transcription cycle, and that it may be involved in transcriptional regulation. The exact role of the CTD remains elusive, as in vitro reconstituted transcription using the adenovirus major late promoter does not require the CTD. Previous studies showed that transcription from the murine dihydrofolate reductase (DHFR) promoter can be only accomplished by the form of RNA polymerase II that contains the hypophosphorylated CTD (RNAPIIA), but not by the form that lacks it (RNAPIIB). Here we show that the CTD, but not its phosphorylation, is required for initiation of transcription. We also show that transcription requires CTD kinase activity provided by the CDK subunit of TFIIH
— id: 69947, year: 1995, vol: 377, page: 557, stat: Journal Article,

Solution structure of the C-terminal core domain of human TFIIB: similarity to cyclin A and interaction with TATA-binding protein
Bagby, S; Kim, S; Maldonado, E; Tong, K I; Reinberg, D; Ikura, M
1995 Sep 8;82(5):857-867, Cell
TFIIB is an essential component of the machinery that transcribes protein-coding genes. The three-dimensional structure of the human TFIIB core domain (TFIIBc) has been determined using multidimensional heteronuclear magnetic resonance spectroscopy. The molecule consists of two direct repeats that adopt similar alpha-helical folds, conferring pseudo-twofold symmetry. An extensive, central basic surface including an amphipathic alpha helix is critical to the function of TFIIB as a bridge between the TBP-promoter complex and RNA polymerase II and associated general and regulatory transcription factors. Similarities between the TFIIBc and cyclin A folds indicate that elements of the eukaryotic cell cycle control apparatus evolved from more fundamental transcriptional control components, demonstrating a link between the transcription and cell cycle molecular machineries
— id: 69948, year: 1995, vol: 82, page: 857, stat: Journal Article,

Visualization of TBP oligomers binding and bending the HIV-1 and adeno promoters
Griffith, J D; Makhov, A; Zawel, L; Reinberg, D
1995 Mar 10;246(5):576-584, Journal of molecular biology
The binding of the 28 kDa yeast TATA binding protein (yTBP) to the HIV and adeno major late promoters has been examined by electron microscopy (EM). Three different EM preparative methods were employed: direct mounting and shadowcasting of fixed samples, cryofixation and freeze-drying followed by shadowcasting, and negative staining of unfixed samples. Excellent agreement among the three methods was obtained. With ten yTBP monomers/DNA fragment, up to 25% of the DNA molecules contained easily distinguished protein particles at the TATA box and, less frequently, smaller particles were observed. Non-specific binding to DNA ends was common. The mass of the easily distinguished particles measured 63(+/- 5) kDa (cryofixation and shadowcasting) and 48(+/- 6) kDa (negative staining) indicating TBP dimerization. With 22 and 44 yTBP monomers/DNA, yTBP polymerization produced DNA-protein rods 9 nm wide and 20 to 30 nm long, frequently with two DNA strands exiting one end. Bending analysis revealed that yTBP dimers bend the DNA about the TATA box by 80 to 90 degrees. Although these protein ratios are relatively high, the structures formed demonstrate the propensity of yTBP to engage in protein-protein interactions
— id: 69952, year: 1995, vol: 246, page: 576, stat: Journal Article,

News on initiation and elongation of transcription by RNA polymerase II
Maldonado, E; Reinberg, D
1995 Jun;7(3):352-361, Current opinion in cell biology
Transcription by RNA polymerase II is a complex process that requires additional factors to initiate transcription at the promoters. New developments in the past year have furthered our understanding of the functions of the transcription factors and provided more insights into the mechanisms involved in the regulation of initiation and elongation of transcription. One of the most significant advances of the past year was the discovery of the involvement of the general transcription factor TFIIH in DNA excision repair. Surprisingly, studies aimed at identifying the kinase activity within TFIIH responsible for phosphorylating the carboxy-terminal domain of RNA polymerase II revealed it to be the MO15/Cdk7 kinase and its partner, cyclin H. These exciting observations suggest a paradigm for linking transcription, DNA excision repair and cell cycle progression through one pivotal factor
— id: 69950, year: 1995, vol: 7, page: 352, stat: Journal Article,

Transcriptional activation. Tuning-up transcription
Sheldon, M; Reinberg, D
1995 Jan 1;5(1):43-46, Current biology. CB
Eukaryotic transcription is a complex process, and recent results identify multiple steps that need to be stimulated to activate transcription, one of which is a change in conformation of the general transcription factor TFIIB
— id: 69953, year: 1995, vol: 5, page: 43, stat: Journal Article,

Cdk-activating kinase complex is a component of human transcription factor TFIIH
Shiekhattar R; Mermelstein F; Fisher RP; Drapkin R; Dynlacht B; Wessling HC; Morgan DO; Reinberg D
1995 Mar 16;374(6519):283-287, Nature
Transcription factor IIH (TFIIH) contains a kinase capable of phosphorylating the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII). Here we report the identification of the Cdk-activating kinase (Cak) complex (Cdk7 and cyclin H) as a component of TFIIH after extensive purification of TFIIH by chromatography. We find that affinity-purified antibodies directed against cyclin H inhibit TFIIH-dependent transcription and that both cyclin H and Cdk7 antibodies inhibit phosphorylation of the CTD of the largest subunit of the RNAPII in the preinitiation complex. Cak is present in at least two distinct complexes, TFIIH and a smaller complex that is unable to phosphorylate RNAPII in the preinitiation complex. Both Cak complexes, as well as recombinant Cak, phosphorylate a CTD peptide. Finally, TFIIH was shown to phosphorylate both Cdc2 and Cdk2, suggesting that there could be a link between transcription and the cell cycle machinery
— id: 33168, year: 1995, vol: 374, page: 283, stat: Journal Article,

The 62- and 80-kDa subunits of transcription factor IIH mediate the interaction with Epstein-Barr virus nuclear protein 2
Tong, X; Drapkin, R; Reinberg, D; Kieff, E
1995 Apr 11;92(8):3259-3263, Proceedings of the National Academy of Sciences of the United States of America
EBNA 2 (Epstein-Barr virus nuclear antigen 2) is an acidic transactivator essential for EBV transformation of B lymphocytes. We show that EBNA 2 directly interacts with general transcription factor IIH. Glutathione S-transferase (GST)-EBNA 2 acidic domain fusion protein depleted transcription factor IIH activity from a TFIIH nuclear fraction. The p89 (ERCC3), p80 (ERCC2), and p62 subunits of TFIIH were among the proteins retained by GST-EBNA 2. Eluates from the GST-EBNA 2 beads reconstituted activity in a TFIIH-dependent in vitro transcription assay. The p62 and p80 subunits of TFIIH independently bound to GST-EBNA 2, whereas the p34 subunit of TFIIH only bound in the presence of p62. A Trp-->Thr mutation in the EBNA 2 acidic domain abolishes EBNA 2 transactivation in vivo and greatly compromised EBNA 2 association with TFIIH activity and with the p62 and p80 subunits, providing a link between EBNA 2 transactivation and these interactions. Antibodies directed against the p62 subunit of TFIIH coimmunoprecipitated EBNA 2 from EBV-transformed B lymphocytes, indicating that EBNA 2 associates with TFIIH in vivo
— id: 69951, year: 1995, vol: 92, page: 3259, stat: Journal Article,

Recycling of the general transcription factors during RNA polymerase II transcription
Zawel, L; Kumar, K P; Reinberg, D
1995 Jun 15;9(12):1479-1490, Genes & development
We have analyzed the fate of the RNA polymerase II (RNAPII) general transcription factors during the transition from initiation to elongation using multiple approaches. We demonstrate that all of the basal factors coexist in mature initiation complexes but that following nucleotide addition, this complex becomes disrupted. During this transition, TFIID remains promoter-bound whereas TFIIB, TFIIE, TFIIF, and TFIIH are released. Upon release, TFIIB reassociates with TFIID, reforming the RNAPII docking site, the DB complex. TFIIE is released before formation of the tenth phosphodiester bond. This precedes TFIIH release, which occurrs after the transcription complex reaches +30. TFIIF is unique in that it is the only basal factor detected in the RNAPII elongation complex. Following its release from the initiation complex, TFIIF has the ability to reassociate with a stalled RNAPII
— id: 69949, year: 1995, vol: 9, page: 1479, stat: Journal Article,

Common themes in assembly and function of eukaryotic transcription complexes
Zawel, L; Reinberg, D
1995 ;64:533-561, Annual review of biochemistry
Eukaryotes contain three distinct RNA polymerase enzymes, each responsible for the transcription of a subclass of nuclear genes. Despite this division of labor, each RNA polymerase system follows a common blueprint to execute the loading of the polymerase onto the relevant promoter region. The RNA polymerase II system appears unique in that after RNA polymerase II has loaded onto the DNA, two auxiliary factors, TFIIE and TFIIH, are necessary for its escape from the promoter region. The complexity of the RNA polymerase II initiation pathway provides a multitude of potential targets for transcriptional activators. Tight control over transcription initiation levels is afforded by multiple cofactors that both enhance and repress
— id: 69954, year: 1995, vol: 64, page: 533, stat: Journal Article,

Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II
Drapkin, R; Reardon, J T; Ansari, A; Huang, J C; Zawel, L; Ahn, K; Sancar, A; Reinberg, D
1994 Apr 21;368(6473):769-772, Nature
The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH
— id: 69962, year: 1994, vol: 368, page: 769, stat: Journal Article,

The multifunctional TFIIH complex and transcriptional control
Drapkin, R; Reinberg, D
1994 Nov;19(11):504-508, Trends in biochemical sciences
RNA polymerase II (Pol II) requires seven general transcription factors (GTFs) and ATP for transcription initiation. Transcription factor IIH (TFIIH) has emerged as the sole GTF with enzymatic activity. In addition to its essential role in transcription initiation, recent studies have demonstrated a direct involvement of TFIIH in DNA excision repair processes. The enzymatic properties and functional duality of TFIIH make it a prime target for regulation by viral and cellular factors
— id: 69955, year: 1994, vol: 19, page: 504, stat: Journal Article,

Transcription. The essential twist
Drapkin, R; Reinberg, D
1994 Jun 16;369(6481):523-524, Nature
— id: 69961, year: 1994, vol: 369, page: 523, stat: Journal Article,

Where transcription meets repair
Drapkin, R; Sancar, A; Reinberg, D
1994 Apr 8;77(1):9-12, Cell
— id: 69963, year: 1994, vol: 77, page: 9, stat: Journal Article,

Interaction of the Dr1 inhibitory factor with the TATA binding protein is disrupted by adenovirus E1A
Kraus, V B; Inostroza, J A; Yeung, K; Reinberg, D; Nevins, J R
1994 Jul 5;91(14):6279-6282, Proceedings of the National Academy of Sciences of the United States of America
Past experiments have shown that the adenovirus E1A12S product activates the hsp70 promoter, dependent on the TATA element and dependent on N-terminal E1A sequences. Other experiments have identified a factor termed Dr1 that interacts with and inhibits the transcriptional activity of the TATA-binding protein (TBP). We now find that the E1A12S protein can disrupt the interaction of the Dr1 factor with the TATA-specific TBP factor, allowing the productive interaction of TBP with TFIIA. This E1A-mediated disruption is dependent on N-terminal sequences that are also essential for the TATA-dependent trans-activation of the hsp70 promoter. Moreover, we also find that Dr1 expression in transfected cells can inhibit transcription from the hsp70 promoter and that this can be overcome by coexpression of the wild-type E1A protein, dependent on N-terminal sequences. We conclude that the activation of hsp70 through the TATA element may be mechanistically similar to the activation of the E2 promoter via E2F, in each case involving a release of a transcription factor from an inactive complex
— id: 69960, year: 1994, vol: 91, page: 6279, stat: Journal Article,

Reconstitution of human TFIIA activity from recombinant polypeptides: a role in TFIID-mediated transcription
Sun, X; Ma, D; Sheldon, M; Yeung, K; Reinberg, D
1994 Oct 1;8(19):2336-2348, Genes & development
Human TFIIA activity is composed of three subunits (alpha, beta, gamma). Here we report the isolation of a human cDNA clone encoding the gamma-subunit and the reconstitution of TFIIA activity from recombinant polypeptides (holo-TFIIA). Protein-protein interaction analysis established that the beta and gamma subunits of TFIIA interact with the TBP component of TFIID. The alpha-subunit is recruited into the complex by association with the gamma-subunit. Functional studies indicate that recombinant TFIIA stimulates basal TFIID-dependent transcription but is without effect on TBP-dependent transcription. Our studies indicate that TFIIA not only functions by physically removing negative components present in TFIID (antirepression), as demonstrated previously, but that it can stimulate basal transcription through components of the TFIID complex. Holo-TFIIA also stimulated activation of transcription in vitro as well as in vivo in transfected HeLa cells
— id: 69957, year: 1994, vol: 8, page: 2336, stat: Journal Article,

Differential regulation of RNA polymerases I, II, and III by the TBP-binding repressor Dr1
White, R J; Khoo, B C; Inostroza, J A; Reinberg, D; Jackson, S P
1994 Oct 21;266(5184):448-450, Science
RNA polymerases I, II, and III each use the TATA-binding protein (TBP). Regulators that target this shared factor may therefore provide a means to coordinate the activities of the three nuclear RNA polymerases. The repressor Dr1 binds to TBP and blocks the interaction of TBP with polymerase II- and polymerase III-specific factors. This enables Dr1 to coordinately regulate transcription by RNA polymerases II and III. Under the same conditions, Dr1 does not inhibit polymerase I transcription. By selectively repressing polymerases II and III, Dr1 may shift the physiological balance of transcriptional output in favor of polymerase I
— id: 69956, year: 1994, vol: 266, page: 448, stat: Journal Article,

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53
Xiao, H; Pearson, A; Coulombe, B; Truant, R; Zhang, S; Regier, J L; Triezenberg, S J; Reinberg, D; Flores, O; Ingles, C J
1994 Oct;14(10):7013-7024, Molecular & cellular biology
Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation
— id: 69958, year: 1994, vol: 14, page: 7013, stat: Journal Article,

Structure-function analysis of the TBP-binding protein Dr1 reveals a mechanism for repression of class II gene transcription
Yeung, K C; Inostroza, J A; Mermelstein, F H; Kannabiran, C; Reinberg, D
1994 Sep 1;8(17):2097-2109, Genes & development
Dr1, a repressor of class II genes, regulates transcription by a novel mechanism. Biochemical analyses reveal that Dr1 directly interacts with the multiprotein TFIID complex. By use of the yeast two-hybrid system, we demonstrate that the association of Dr1 with the TATA-binding protein (TBP) subunit of TFIID occurs in vivo. In addition, Dr1 can repress transcription from TATA-containing as well as TATA-less promoters in transient transfection assays. Importantly, Dr1-mediated repression can be reversed by overexpression of TBP in vivo. By use of diverse approaches, we mapped two distinct domains in Dr1 required for repression. One domain is essential for the Dr1-TBP interaction, and the second is rich in alanine residues. The TBP-binding domain of Dr1 cannot be replaced by a heterologous DNA-binding domain in mediating repression. We demonstrate that some, but not all, transcriptional activators can reverse Dr1-mediated repression in vivo
— id: 69959, year: 1994, vol: 8, page: 2097, stat: Journal Article,

Oct-2 facilitates functional preinitiation complex assembly and is continuously required at the promoter for multiple rounds of transcription
Arnosti, D N; Merino, A; Reinberg, D; Schaffner, W
1993 Jan;12(1):157-166, EMBO journal
Octamer factor 2 (Oct-2, OTF-2, NF-A2) is an 'upstream' promoter factor that binds to the octamer motif (ATGCAAAT) implicated in control of immunoglobulin gene transcription in B-lymphocytes. We have studied the role of Oct-2 in the process of transcription initiation in vitro using both nuclear extracts and purified basal transcription factors. Oct-2 specifically stimulates transcription from octamer-containing promoters in both systems. Thus, Oct-2 is a 'true activator', rather than merely an 'anti-repressor' counteracting the effect of histones. In order-of-addition experiments, Oct-2 is required early, together with TFIID, to allow formation of a preinitiation complex. Oct-2 cannot functionally interact with cloned TATA binding protein (TBP) but rather requires 'coactivators' found in the TFIID fraction. In single-round transcription experiments, early competition for Oct-2 by an octamer oligonucleotide is deleterious, but no effect is seen after assembly of a complete preinitiation complex. However, for multiple rounds of transcription, Oct-2 is continuously required at the promoter; this result argues against a 'hit-and-run' mechanism whereby the activator becomes dispensible after organizing a TFIID-promoter complex. In agreement with our previous studies in vivo, the N-terminal glutamine-rich activation domain of Oct-2 is required for full activity in vitro, indicating that this domain directly interacts with basal transcription factors
— id: 69971, year: 1993, vol: 12, page: 157, stat: Journal Article,

Interaction of human thyroid hormone receptor beta with transcription factor TFIIB may mediate target gene derepression and activation by thyroid hormone
Baniahmad, A; Ha, I; Reinberg, D; Tsai, S; Tsai, M J; O'Malley, B W
1993 Oct 1;90(19):8832-8836, Proceedings of the National Academy of Sciences of the United States of America
The human thyroid hormone receptor beta (hTR beta) is capable of both transcriptional silencing and hormone-dependent activation. However, the detailed mechanism of this transcriptional regulation remains to be elucidated. One possibility is that hTR beta interacts directly with factors of the basal transcriptional machinery, thereby modulating basal promoter activity in a direct manner, as has been shown for other transcription factors. Here, we show that hTR beta interacts specifically with the human basal transcription factor TFIIB. Deletion analysis revealed two contact sites in the receptor: one is located in the N terminus, while the other is part of the ligand-binding domain (LBD) and is located at the C terminus. Interestingly, each receptor contact site interacts with different sites in TFIIB. Cotransfection experiments revealed that, when fused to the DNA-binding domain of yeast transcription factor GAL4, the C-terminal interaction site of hTR beta was transcriptionally inactive; however, when it was cotransfected with the remaining part of the LBD on a separate molecule, silencing function was restored. In agreement with that, we show that thyroid hormone is able to significantly decrease the interaction of its receptor LBD with TFIIB. Our data suggest that hTR beta acts as a transcriptional silencer by interacting with TFIIB and that thyroid hormone may act in part by preventing transcriptional repression at this level
— id: 69965, year: 1993, vol: 90, page: 8832, stat: Journal Article,

Regulation of RNA polymerase II transcription
Drapkin, R; Merino, A; Reinberg, D
1993 Jun;5(3):469-476, Current opinion in cell biology
Transcription initiation plays a central role in the regulation of gene expression. Exciting developments in the last year have furthered our understanding of the interactions between general transcription factors and how these factors respond to modulators of transcription
— id: 69969, year: 1993, vol: 5, page: 469, stat: Journal Article,

Multiple functional domains of human transcription factor IIB: distinct interactions with two general transcription factors and RNA polymerase II
Ha, I; Roberts, S; Maldonado, E; Sun, X; Kim, L U; Green, M; Reinberg, D
1993 Jun;7(6):1021-1032, Genes & development
Transcription factor IIB (TFIIB) plays a pivotal role in the formation of transcription-competent initiation complexes. TFIIB was found to interact with the TATA-binding protein, the small subunit of TFIIF, and RNA polymerase II. These interactions require distinct domains in TFIIB. Using the gel mobility-shift assay, it was found that the amino terminus of TFIIB was necessary for the formation of complexes containing RNA polymerase II and TFIIF, whereas the carboxy-terminal domain, which is composed of two imperfect direct repeats and includes a putative amphipathic alpha-helix, was sufficient for the formation of complexes containing the TATA-binding protein and TFIIB (DB complex). Protein-protein interaction analyses demonstrate that the amphipathic alpha-helix in TFIIB is important for the interaction with the TATA-binding protein. Specific residues mapping to the carboxyl terminus of the second direct repeat were found to be crucial for the interaction of TFIIB and RNA polymerase II. The interaction with the small subunit of TFIIF was mapped to the amino terminus of TFIIB, which includes a zinc finger
— id: 69968, year: 1993, vol: 7, page: 1021, stat: Journal Article,

Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription
Ma, D; Watanabe, H; Mermelstein, F; Admon, A; Oguri, K; Sun, X; Wada, T; Imai, T; Shiroya, T; Reinberg, D
1993 Nov;7(11):2246-2257, Genes & development
Transcription factor IIA has been shown to interact with the TATA-binding protein and to act early during preinitiation complex formation. The human factor is composed of three subunits (alpha, beta, gamma). A human cDNA clone encoding the largest subunit of TFIIA (alpha) was isolated. The recombinant alpha polypeptide, together with the beta and gamma subunits, was capable of reconstituting TFIIA activity. Studies using antibodies raised against recombinant alpha polypeptide demonstrate that TFIIA can be an integral component of the preinitiation complex. We demonstrate that TFIIA not only interacts with TBP but also can associate with the TFIID complex. Functional assays establish that TFIIA has no apparent role in basal transcription but plays an important role in activation of transcription. Interestingly, amino acid sequence analyses of the beta-subunit demonstrate these residues to be entirely contained within the carboxyl terminus of the cDNA clone encoding the alpha-subunit
— id: 69964, year: 1993, vol: 7, page: 2246, stat: Journal Article,

DNA topoisomerase I is involved in both repression and activation of transcription
Merino, A; Madden, K R; Lane, W S; Champoux, J J; Reinberg, D
1993 Sep 16;365(6443):227-232, Nature
Reconstituted transcription reactions containing the seven general transcription factors, in addition to RNA polymerase II, respond poorly to transcriptional activators. Two factors, Dr2 and ACF, necessary for high levels of transcription in response to an activator have been identified. ACF can enhance basal and activated transcription. Dr2 represses basal transcription, but this can be overcome by transcriptional activators or TFIIA. Dr2 is human DNA topoisomerase I. The DNA relaxation activity of topoisomerase I is dispensable for transcriptional repression. The effect of Dr2 is specific for TATA-box-containing promoters and is mediated by the TATA-binding protein
— id: 69966, year: 1993, vol: 365, page: 227, stat: Journal Article,

Interaction between an acidic activator and transcription factor TFIIB is required for transcriptional activation
Roberts, S G; Ha, I; Maldonado, E; Reinberg, D; Green, M R
1993 Jun 24;363(6431):741-744, Nature
How eukaryotic promoter-specific activator proteins (activators) stimulate transcription is a central question. We have previously shown that an acidic activator can directly interact with the general transcription factor TFIIB and increase its stable assembly into a preinitiation complex. We have proposed that this increase in TFIIB assembly is at least part of the mechanism by which an acidic activator functions. A prediction of this hypothesis is that a TFIIB mutant unable to interact with an acidic activator could not support activated transcription, and here we present experiments that verify this prediction. In conjunction with previous studies, our results argue that interaction between an acidic activator and TFIIB is necessary for transcriptional activation
— id: 69967, year: 1993, vol: 363, page: 741, stat: Journal Article,

The cycling of RNA polymerase II during transcription
Zawel, L; Lu, H; Cisek, L J; Corden, J L; Reinberg, D
1993 ;58:187-198, Cold Spring Harbor symposia on quantitative biology
— id: 69972, year: 1993, vol: 58, page: 187, stat: Journal Article,

Initiation of transcription by RNA polymerase II: a multi-step process
Zawel, L; Reinberg, D
1993 ;44:67-108, Progress in nucleic acid research & molecular biology
— id: 69970, year: 1993, vol: 44, page: 67, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II: purification and analysis of transcription factor IIA and identification of transcription factor IIJ
Cortes, P; Flores, O; Reinberg, D
1992 Jan;12(1):413-421, Molecular & cellular biology
The previously described transcription factor IIA (TFIIA) protein fraction was separated into two factors that affect transcription, TFIIA and TFIIJ. TFIIA was found to have a stimulatory effect, and TFIIJ was found to be required for transcription. The requirement of TFIIJ was observed when bacterially produced purified human or yeast (Saccharomyces cerevisiae) TATA-binding protein (TBP) was used in lieu of the endogenous HeLa cell TFIID complex, suggesting that TFIIJ may be part of the TFIID complex. The stimulatory activity of TFIIA was found also to be dependent on the source of the TBP. Transcription reactions reconstituted with TFIID were stimulated by TFIIA; however, when human or yeast TBP was used instead of TFIID, TFIIA had no effect. TFIIA was found to interact with the TBP and was extensively purified by the use of affinity chromatography on columns containing immobilized recombinant yeast TBP. TFIIA is a heterotrimer composed of polypeptides of 34, 19, and 14 kDa. These three polypeptides were required to isolate, by using the gel mobility shift assay, a stable complex between TBP and the TATA box sequence
— id: 69980, year: 1992, vol: 12, page: 413, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of factor IIH
Flores, O; Lu, H; Reinberg, D
1992 Feb 5;267(4):2786-2793, Journal of biological chemistry
Two new factors required for transcription of class II genes have been identified. These factors, TFIIH and TFIIJ, were required together with the previously described general factors (TFIIA, TFIIB, TFIID, TFIIE, and TFIIF) and RNA polymerase II for transcription of different class II genes. TFIIH was extensively purified, and the activity appeared to coelute with polypeptides of 33 and 95 kDa. The role of TFIIH and TFIIJ in preinitiation complex assembly was analyzed using mobility shift assays. It was found that TFIIH and TFIIJ association with the preinitiation complex was ordered and required the previous assembly of a preinitiation complex intermediate containing factors IID, IIB, IIF, IIE, and RNA polymerase II. A model for the ordered assembly of the general factors and RNA polymerase II is presented
— id: 69979, year: 1992, vol: 267, page: 2786, stat: Journal Article,

Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription
Inostroza, J A; Mermelstein, F H; Ha, I; Lane, W S; Reinberg, D
1992 Aug 7;70(3):477-489, Cell
We have discovered a protein termed Dr1 that interacts with the TATA-binding protein, TBP. The association of Dr1 with TBP results in repression of both basal and activated levels of transcription. The interaction of Dr1 with TBP precludes the formation of a transcription-competent complex by inhibiting the association of TFIIA and/or TFIIB with TBP. Dr1 activity is associated with a 19 kd protein. A cDNA clone encoding Dr1 was isolated. Dr1 is phosphorylated in vivo and phosphorylation of Dr1 affected its interaction with TBP. Our results suggest a regulatory role for Dr1 in repression of transcription mediated via phosphorylation
— id: 69975, year: 1992, vol: 70, page: 477, stat: Journal Article,

Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II
Lu, H; Zawel, L; Fisher, L; Egly, J M; Reinberg, D
1992 Aug 20;358(6388):641-645, Nature
Phosphorylation of the carboxy-terminal domain of the largest subunit of RNA polymerase II is believed to control the transition from transcription initiation to elongation. The general transcription factor IIH (TFIIH) contains a kinase activity capable of phosphorylating this domain. Factors that promote the association of RNA polymerase II with the preinitiation complex stimulate this activity. The transcription factor IIE, which is required for the stable association of TFIIH with the preinitiation complex, affects the processivity of TFIIH kinase
— id: 69974, year: 1992, vol: 358, page: 641, stat: Journal Article,

Protein kinases from Aspergillus nidulans that phosphorylate the carboxyl-terminal domain of the largest subunit of RNA polymerase II
Stone, N; Reinberg, D
1992 Mar 25;267(9):6353-6360, Journal of biological chemistry
Three serine kinases which phosphorylate the CTD of RNA polymerase II have been identified in Aspergillus nidulans. The kinases (KI, KII, KIII) were identified using a synthetic peptide containing four copies of the CTD consensus heptamer repeat, and differ in chromatographic behavior, and apparent molecular mass (KI approximately 60kDa; KII approximately 82kDa; KIII approximately 43 kDa). KIII utilized, in addition to peptide, histone H1 as substrate, whereas casein was not phosphorylated by any of the three kinases. The kinases appear to be unrelated to the p34cdc2 kinase, as judged by Western blot analysis and the position of serine phosphorylation of the synthetic CTD peptide. KI was highly purified and renaturation experiments have shown that it consists of a single polypeptide of 57 kDa. KI also phosphorylated RNA polymerase II associated in a preinitiation complex
— id: 69978, year: 1992, vol: 267, page: 6353, stat: Journal Article,

Specific interaction between the nonphosphorylated form of RNA polymerase II and the TATA-binding protein
Usheva, A; Maldonado, E; Goldring, A; Lu, H; Houbavi, C; Reinberg, D; Aloni, Y
1992 May 29;69(5):871-881, Cell
Fractionation of a transcription-competent HeLa cell extract on a column containing one copy of the heptamer repeat (YSPTSPS) present in the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II resulted in the loss of transcriptional activity. Fractionation of the extracts on columns containing mutations of the heptamer repeat was without effect. Such transcriptionally inactive extracts regained their ability to specifically transcribe different class II promoters upon the addition of human TFIID, recombinant yeast TATA-binding protein (TBP), or proteins bound to the column. Fractionation of RNA polymerase II on columns containing human or yeast TBP resulted in the specific retention of the nonphosphorylated form of RNA polymerase II. The phosphorylated form of the enzyme was unable to interact with TBP. The specific interaction of RNA polymerase II with TBP was mediated by the CTD of RNA polymerase II
— id: 69977, year: 1992, vol: 69, page: 871, stat: Journal Article,

Transcription by RNA polymerase II: initiator-directed formation of transcription-competent complexes
Weis, L; Reinberg, D
1992 Nov;6(14):3300-3309, FASEB journal
Studies of transcription by RNA polymerase II have revealed two promoter elements, the TATA motif and the initiator (Inr), capable of directing specific transcription initiation. Although binding to the TATA motif by one of the components of the transcription machinery has been shown to be the initial recognition step in transcription complex formation, many promoters that lack a traditional TATA motif have recently been described. In such TATA-less promoters, the Inr element is critical in positioning RNA polymerase II. Various Inr elements have been described and classified according to sequence homology. These Inr elements are recognized specifically by Inr-binding proteins. Interaction between these Inr-binding proteins and components of the basal transcription machinery provides a means through which a transcription competent complex can be formed
— id: 69973, year: 1992, vol: 6, page: 3300, stat: Journal Article,

Advances in RNA polymerase II transcription
Zawel, L; Reinberg, D
1992 Jun;4(3):488-495, Current opinion in cell biology
Multiple protein factors are necessary to mediate transcription by RNA polymerase II. Recently, a number of advances have been made in our understanding of how general transcription factors collectively modulate basal transcription in the context of different promoter environments and how this process is activated and repressed by accessory components
— id: 69976, year: 1992, vol: 4, page: 488, stat: Journal Article,

Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II
Bengal, E; Flores, O; Krauskopf, A; Reinberg, D; Aloni, Y
1991 Mar;11(3):1195-1206, Molecular & cellular biology
We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II
— id: 69990, year: 1991, vol: 11, page: 1195, stat: Journal Article,

The initiator directs the assembly of a transcription factor IID-dependent transcription complex
Carcamo, J; Buckbinder, L; Reinberg, D
1991 Sep 15;88(18):8052-8056, Proceedings of the National Academy of Sciences of the United States of America
Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or 'TATA' motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif
— id: 69985, year: 1991, vol: 88, page: 8052, stat: Journal Article,

The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex
Flores, O; Lu, H; Killeen, M; Greenblatt, J; Burton, Z F; Reinberg, D
1991 Nov 15;88(22):9999-10003, Proceedings of the National Academy of Sciences of the United States of America
We found that transcription factor IIF mediates the association of RNA polymerase II with promoter sequences containing transcription factors IID, IIB, and IIA (DAB complex). The resulting DNA-protein complex contained RNA polymerase II and the two subunits of transcription factor IIF (RAP 30 and RAP 74). Cloned human RAP 30 was sufficient for the recruitment of RNA polymerase II to the DAB complex. This ability of RAP 30 to recruit RNA polymerase to a promoter is also a characteristic of sigma factors in prokaryotes
— id: 69982, year: 1991, vol: 88, page: 9999, stat: Journal Article,

Cloning of a human gene encoding the general transcription initiation factor IIB
Ha, I; Lane, W S; Reinberg, D
1991 Aug 22;352(6337):689-695, Nature
Transcription factor IIB (TFIIB) has a central role in transcription of class II genes. The purification of the human TFIIB protein and isolation of a complementary DNA encoding TFIIB activity is reported here. The sequence of TFIIB, which seems to be encoded by a single gene, contains a repeated motif, in addition to a motif with similarity to the prokaryotic sigma-factors. The recombinant protein expressed in bacteria substituted for all the functions attributed to the human TFIIB protein
— id: 69986, year: 1991, vol: 352, page: 689, stat: Journal Article,

Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID
Horikoshi, N; Maguire, K; Kralli, A; Maldonado, E; Reinberg, D; Weinmann, R
1991 Jun 15;88(12):5124-5128, Proceedings of the National Academy of Sciences of the United States of America
Adenovirus E1A has long been known to activate/repress cellular and viral transcription. The transcriptional activity of nuclear extracts was depleted after chromatography on immobilized E1A protein columns that specifically retained the transcription factor (TF) IID. Stronger direct interactions between E1A and human TFIID than between E1A and yeast TFIID suggest that the unique sequences of the human protein may be involved. We have demonstrated that this interaction occurs directly between bacterially produced E1A and bacterially produced human TFIID in a protein blot assay. We propose that E1A protein may transduce regulatory signals from upstream activators to basal elements of the transcriptional machinery by contacting TFIID
— id: 69988, year: 1991, vol: 88, page: 5124, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of general transcription factor IIE
Inostroza, J; Flores, O; Reinberg, D
1991 May 15;266(14):9304-9308, Journal of biological chemistry
Mammalian RNA polymerase II transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase II. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase II, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase II
— id: 69989, year: 1991, vol: 266, page: 9304, stat: Journal Article,

Binding of general transcription factor TFIIB to an acidic activating region
Lin, Y S; Ha, I; Maldonado, E; Reinberg, D; Green, M R
1991 Oct 10;353(6344):569-571, Nature
A central issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription. Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions. First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator. Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region. But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors, coactivators or mediators. A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000. Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein
— id: 69984, year: 1991, vol: 353, page: 569, stat: Journal Article,

The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex
Lu, H; Flores, O; Weinmann, R; Reinberg, D
1991 Nov 15;88(22):10004-10008, Proceedings of the National Academy of Sciences of the United States of America
The two forms of RNA polymerase II that exist in vivo, phosphorylated (IIO) and nonphosphorylated (IIA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase II in the complex was converted by a cellular protein kinase to the phosphorylated form
— id: 69983, year: 1991, vol: 88, page: 10004, stat: Journal Article,

Structure and functional properties of human general transcription factor IIE
Peterson, M G; Inostroza, J; Maxon, M E; Flores, O; Admon, A; Reinberg, D; Tjian, R
1991 Dec 5;354(6352):369-373, Nature
The general transcription factor IIE (TFIIE) is an essential component of the eukaryotic RNA polymerase II initiation complex. We have isolated human complementary DNA clones for both the subunits of TFIIE. Using purified recombinant proteins we find that both subunits are essential to form a stable preinitiation complex and to reconstitute basal-level and Sp1-activated transcription in vitro. Analysis of their predicted amino-acid sequences reveals several intriguing structural motifs that could provide insight into the role of TFIIE in transcription initiation
— id: 69981, year: 1991, vol: 354, page: 369, stat: Journal Article,

Interaction of CAP sequence site binding factor and transcription factor IID preceding and following binding to the adenovirus 2 major late promoter
Safer, B; Reinberg, D; Jacob, W F; Maldonado, E; Carcamo, J; Garfinkel, S; Cohen, R
1991 Jun 15;266(17):10989-10994, Journal of biological chemistry
Interaction of cloned yeast, drosophila, and human transcription factor IID (yTFIID, dTFIID, and hTFIID, respectively) with the adenovirus 2 major late promoter (Ad2 MLP) confers a more limited pattern of DNase I protection than that obtained using highly purified native hTFIID (Hahn, S., Buratowski, S., Sharp, P. A. and Guarente, L. (1989) EMBO J. 8, 3379-3382; Van Dyke, M. W., and Sawadogo, M. (1990) Mol. Cell. Biol. 10, 3415-3420; Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, P.A., and Roeder, R.G. (1989) Nature 341, 299-303; Peterson, M. G., Tanese, N., Pugh, B.F., and Tjian, R. (1990) Science 248, 1625-1630; Hoey, T., Dynlacht, B. D., Peterson, M.G., Pugh, B.F., and Tjian, R. (1990) Cell 61, 1179-1186). Since the mass of the cloned TFIIDs is considerably less than that of native hTFIID (27-38 kDa versus 120-140 kDa), it is considered likely that native hTFIID exists as a mixed heterodimer. We have recently identified, purified, and characterized a novel transcription factor that binds to the CAP site region (+1 to +23) of the Ad2 MLP. This CAP site binding factor, designated CBF, is required for optimal transcriptional activity. We now show that when bound to the Ad2 MLP, yTFIID and CBF interact to generate the extended pattern of DNase I protection conferred by native hTFIID. In addition, bound yTFIID and CBF interact such that the stability of the complex exceeds that of each factor bound alone. We also demonstrate the existence in nuclear extracts of a hTFIID and CBF heterodimer by the electrophoretic mobility shift analysis. CBF, therefore, may represent the first identified member of a large family of gene-specific TFIID-associated factors that are required for the regulated gene-specific expression of TFIID activity
— id: 69987, year: 1991, vol: 266, page: 10989, stat: Journal Article,

A TATA-like sequence located downstream of the transcription initiation site is required for expression of an RNA polymerase II transcribed gene
Carcamo, J; Maldonado, E; Cortes, P; Ahn, M H; Ha, I; Kasai, Y; Flint, J; Reinberg, D
1990 Sep;4(9):1611-1622, Genes & development
TFIID, the TATA-binding protein, was found to stimulate transcription from the adenovirus IVa2 promoter, a promoter considered to lack the TATA motif. Remarkably, a TATA-like sequence element located downstream of the transcription start site binds TFIID and is required for TFIID-dependent transcription from the IVa2 promoter. Transcription from the IVa2 and the adjacent adenovirus major late promoter (Ad-MLP) is divergent, and the cap sites are separated by 212 nucleotides. Nevertheless, the TATA motifs of the IVa2 promoter and Ad-MLP were found to be oriented in the same direction. An initiator motif around the transcription start site is located in the IVa2 promoter, and in contrast to the TATA motifs, the IVa2-initiator is in the opposite orientation with respect to the initiator of the Ad-MLP. A model is presented in which the polar nature of the initiator governs the direction of transcription. We propose that RNA polymerase II and accessory factors recognize the initiator in an orientation-dependent fashion. The recognition of the IVa2 initiator by RNA polymerase is enhanced by the binding of TFIID to the downstream TATA motif
— id: 69992, year: 1990, vol: 4, page: 1611, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and subunit composition of transcription factor IIF
Flores, O; Ha, I; Reinberg, D
1990 Apr 5;265(10):5629-5634, Journal of biological chemistry
Transcription factor IIF (TFIIF), a protein factor required for transcription of class II genes, has been purified to near homogeneity. TFIIF is a heterodimer of 30- and 78-kDa polypeptides. Both the 30- and 78-kDa polypeptides were necessary to reconstitute TFIIF activity. TFIIF activity eluted from a gel filtration column with an apparent molecular mass of 220 kDa, suggesting that the native factor exists as a dimer of the heterodimer
— id: 69993, year: 1990, vol: 265, page: 5629, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex
Maldonado, E; Ha, I; Cortes, P; Weis, L; Reinberg, D
1990 Dec;10(12):6335-6347, Molecular & cellular biology
Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities
— id: 69991, year: 1990, vol: 10, page: 6335, stat: Journal Article,

TPA can overcome the requirement for EIa and together act synergistically in stimulating expression of the adenovirus EIII promoter
Buckbinder, L; Miralles, V J; Reinberg, D
1989 Dec 20;8(13):4239-4250, EMBO journal
We have examined the control of gene expression from the adenovirus early region III (Ad-EIII) promoter, which contains two previously defined elements, the AP1 and ATF sites. We found that the AP1 element is capable of mediating activation by the adenovirus immediate early (EIa) gene products. Consistent with studies demonstrating that the AP1 site mediates signal transduction in response to 12-O-tetradecanoylphorbol 13-acetate (TPA) we have shown that TPA can activate Ad-EIII expression and overcome the requirement for EIa. Together TPA and EIa elicited a synergistic response in expression from the Ad-EIII promoter during both transient expression assays and viral infections. This synergistic effect required the AP1 element. An EIII promoter construct, in which sequences upstream of the TATA box had been replaced with four AP1 sites, was responsive to TPA and EIa and in combination promoted the synergistic effect. The analysis of specific factors involved in transcription from the Ad-EIII indicated that proteins recognizing the ATF and AP1 sites were important in expression from this promoter in vitro. Purification of protein factors that specifically stimulated EIII expression resulted in the isolation of a set of factors of the AP1 family. Affinity purified AP1 recognized and activated transcription through both the AP1 and ATF elements. In addition, a protein fraction was identified with DNA binding activity specific for the ATF element. This fraction was dependent on the ATF site for transcriptional activity
— id: 69994, year: 1989, vol: 8, page: 4239, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Role of factors IID and MLTF in transcription from the adenovirus major late and IVa2 promoters
Carcamo, J; Lobos, S; Merino, A; Buckbinder, L; Weinmann, R; Natarajan, V; Reinberg, D
1989 May 5;264(13):7704-7714, Journal of biological chemistry
The role of the adenovirus major late upstream transcription factor (MLTF) in transcription from the adenovirus major late and the IVa2 promoters was studied. The transcription initiation site of the IVa2 promoter is located 210 nucleotides upstream from the CAP site of the major late promoter. Transcription from these two promoters occurs on different DNA strands. Thus, this divergent transcription suggests that the same factor could simultaneously regulate the expression of two different genes. This was investigated utilizing a reconstituted transcription system in vitro. The addition of MLTF to reaction mixtures containing the purified general transcription factors and the major late promoter resulted in a 10-12-fold stimulation of transcription. This stimulation was because of an increase of the stability of the preinitiation complex. MLTF allowed DNA template molecules to undergo multiple rounds of transcription. MLTF also stimulated transcription from the adenovirus-encoded IVa2 promoter. Surprisingly, reconstitution experiments indicated that transcription from the IVa2 promoter which does not have a TATA sequence required all the previously described general transcription factors, including TFIID, the TATA binding protein. The requirement for TFIID was demonstrated by reconstitution experiments as well as by oligonucleotide competition experiments. The implications of this observation are discussed
— id: 69999, year: 1989, vol: 264, page: 7704, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Factors IIE and IIF independently interact with RNA polymerase II
Flores, O; Maldonado, E; Reinberg, D
1989 May 25;264(15):8913-8921, Journal of biological chemistry
The purification and characterization of transcription factor IIF (TFIIF), a factor required for transcription by the RNA polymerase II machinery, is described. TFIIF was isolated from the previously described IIE protein fraction. TFIIF enters into the transcription cycle via a preinitiation complex, and it is required for the formation of a complex capable of initiating transcription in the presence of heparin concentrations that inhibit the action of a free factor. TFIIF and TFIIE independently interacted with purified RNA polymerase II. TFIIF and TFIIE were both required for transcription of several class II promoters, including a promoter that lacks the conserved TATA box. Interestingly TFIIF was absolutely required for the formation of a preinitiation complex; however, it also affected the elongation phase of the transcription cycle. TFIIF, together with the previously described elongation factor TFIIS, was required for efficient elongation
— id: 69998, year: 1989, vol: 264, page: 8913, stat: Journal Article,

Phosphorylation of cellular proteins regulates their binding to the cAMP response element
Merino, A; Buckbinder, L; Mermelstein, F H; Reinberg, D
1989 Dec 15;264(35):21266-21276, Journal of biological chemistry
We have studied the protein factors that promote transcription via binding to the cAMP response element (CRE) present in the adenovirus early region III (EIII) and early region IV (EIV) promoters. Three sets of CRE-binding phosphoproteins, ranging in molecular mass from 65-72, 38-43, and 31-37 kDa, were identified in vivo from HeLa cells. Western blot analysis revealed that all three sets of proteins identified were immunologically related to the transcription factor AP1. We found that binding of these proteins to the CRE could be regulated by phosphorylation in vitro. EivF, a 65-72-kDa protein was found to bind specifically to the adenovirus EIV promoter. We have also shown that the smaller molecular mass proteins of 31-37 and 38-43 kDa were able to bind to the CRE present in the adenovirus EIV promoter, as well as to two related DNA elements present in the adenovirus EIII promoter, the ATF and AP1 sites. Phosphorylation of these proteins with the cAMP-dependent protein kinase, affected their transcriptional activity and binding affinity to the three sites. Furthermore, the binding specificity of the 31-37-kDa polypeptides was mediated by cAMP-dependent protein kinase in vitro. Our data suggests that phosphorylation of factors that bind to the CRE may, in part, underlie the cellular response to the adenovirus-encoded Ela protein
— id: 69995, year: 1989, vol: 264, page: 21266, stat: Journal Article,

Initiation of transcription by RNA polymerase II
Mermelstein, F H; Flores, O; Reinberg, D
1989 Sep 21;1009(1):1-10, Biochimica & biophysica acta
— id: 69996, year: 1989, vol: 1009, page: 1, stat: Journal Article,

The adenovirus inverted terminal repeat functions as an enhancer in a cell-free system
Miralles, V J; Cortes, P; Stone, N; Reinberg, D
1989 Jun 25;264(18):10763-10772, Journal of biological chemistry
Two binding sites for EivF, a factor involved in transcription from the adenovirus early promoter iv (Eiv), were mapped within the adenovirus inverted terminal repeats (ITR). Consistent with the observation that EivF was required to initiate transcription from the Eiv promoter and with the demonstration that two EivF binding sites were present in the ITR, we show that the inverted terminal repeat region was able to promote transcription from the CAP site of the Eiv promoter in vitro and in an EIa-dependent fashion in vivo. The minimum sequence within the ITR capable of directing EIa-dependent transcription consists of forty nine nucleotides comprising two EivF binding sites and at least one Sp1 binding site. This 49-base pair fragment possesses the characteristics of an enhancer which is induced by EIa. The enhancer is active in HeLa cell nuclear extracts. Transcription directed by the ITR required EivF and the general transcription factors. The addition of purified Sp1 factor specifically stimulated transcription which correlates with the presence of Sp1 binding sites between the two EivF recognition sites
— id: 69997, year: 1989, vol: 264, page: 10763, stat: Journal Article,

EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction
Cortes, P; Buckbinder, L; Leza, M A; Rak, N; Hearing, P; Merino, A; Reinberg, D
1988 Aug;2(8):975-990, Genes & development
The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP
— id: 70001, year: 1988, vol: 2, page: 975, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. RNA polymerase II-associating protein 30 is an essential component of transcription factor IIF
Flores, O; Maldonado, E; Burton, Z; Greenblatt, J; Reinberg, D
1988 Aug 5;263(22):10812-10816, Journal of biological chemistry
Transcription from class II promoters requires five general factors, IIA, IIB, IID, IIE, and IIF, in addition to RNA polymerase II for basal levels of transcription (Reinberg, D., Flores, O., and Buckbinder, L. (1987) in Molecular Biology of RNA: New Perspectives (Inouye, M., and Dudock, B., eds) pp. 423-439, Academic Press, Orlando, FL). A protein fraction containing transcription factors (TF) IIE and IIF was able to reconstitute transcription from the adenovirus major late promoter when added to extracts depleted of the RNA polymerase II-associating proteins RAP 30 and RAP 74 (Sopta, M., Carthew, R.W., and Greenblatt, J. (1985) J. Biol. Chem. 260, 10353-10360). Studies with monoaffinity-purified antibodies directed against RAP 30 demonstrated, by Western blot analysis, that RAP 30 copurifies on five columns with transcription factor IIF. That RAP 30 is a functional component of TFIIF was also demonstrated; preincubation of anti-RAP 30 antibodies with purified TFIIF inhibited transcription. Inhibition of transcription was overcome by the addition of purified TFIIF. RAP 30 is an integral part of a preinitiation complex; the incubation of all the general transcription factors with a promoter-containing DNA, prior to the addition of the anti-RAP 30 antibodies, resulted in the formation of a DNA-protein complex that was not inhibited by the antibodies. Incubation of the transcription factors in the absence of a promoter-containing DNA resulted in a complex that was partially resistant to the antibodies
— id: 70000, year: 1988, vol: 263, page: 10812, stat: Journal Article,

Purification and functional characterization of transcription factor SII from calf thymus. Role in RNA polymerase II elongation
Rappaport, J; Reinberg, D; Zandomeni, R; Weinmann, R
1987 Apr 15;262(11):5227-5232, Journal of biological chemistry
SII was purified from calf thymus tissue to apparent homogeneity by a rapid procedure. The 38-kDa protein stimulated RNA synthesis by purified calf thymus RNA polymerase II 4-fold. The calf thymus SII had similar chromatographic properties and molecular size and cross-reacted immunologically with antibodies to mouse SII (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588). We have substituted the purified calf thymus SII for the partially purified HeLa transcription factor IIS fraction in a HeLa (human) transcription system reconstituted with purified factors and RNA polymerase II. The purified protein stimulated specific transcription from the adenovirus 2 major late promoter by increasing the efficiency of the elongation reaction
— id: 70002, year: 1987, vol: 262, page: 5227, stat: Journal Article,

Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site
Reinberg, D; Horikoshi, M; Roeder, R G
1987 Mar 5;262(7):3322-3330, Journal of biological chemistry
Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported
— id: 70004, year: 1987, vol: 262, page: 3322, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of initiation factors IIB and IIE
Reinberg, D; Roeder, R G
1987 Mar 5;262(7):3310-3321, Journal of biological chemistry
Two general transcription factors (IIE and IIB) (TF) were purified from HeLa cell nuclear extracts and shown to be absolutely required, along with two additional factors (IIA and IID) and RNA polymerase II, for specific transcription initiation at the adenovirus major late promoter. TFIIB and TFIIE were also required, in addition to TFIIA, TFIID, RNA polymerase II, and the adenovirus 2 major late promoter, for the formation of a (preinitiation) complex that could initiate transcription (upon addition of nucleoside triphosphates) in the presence of heparin concentrations which inhibited the action of unbound factors. Glycerol gradient analyses indicated independent interactions of TFIIE with TFIIB and with the purified RNA polymerase II, but not with RNA polymerase III. Transcription factors IIB and IIE were also shown to be required for specific initiation of transcription from several cellular and viral class II promoters
— id: 70005, year: 1987, vol: 262, page: 3310, stat: Journal Article,

Factors involved in specific transcription by mammalian RNA polymerase II. Transcription factor IIS stimulates elongation of RNA chains
Reinberg, D; Roeder, R G
1987 Mar 5;262(7):3331-3337, Journal of biological chemistry
A factor that stimulates random transcription of purified DNAs by RNA polymerase II has been partially purified and analyzed with respect to its possible role in specific transcription from class II promoters. Studies of the effect of this factor (transcription factor IIS) on transcription from the adenovirus major late promoter in a system reconstituted with RNA polymerase II and purified factors (IIA, IIB, IIE, and IID) indicated that it acted subsequent to the initiation step and that it stimulated the rate of elongation. Kinetic experiments indicated that the factor affected the efficiency with which the RNA polymerase II passed through pausing sites. The relationship of transcription factor IIS to a protein previously purified from Erlich ascites tumor cells (Sekimizu, K., Nakanishi, Y., Mizuno, D., and Natori, S. (1979) Biochemistry 18, 1582-1588) was also studied
— id: 70003, year: 1987, vol: 262, page: 3331, stat: Journal Article,

The enzymatic conversion of 3'-phosphate terminated RNA chains to 2',3'-cyclic phosphate derivatives
Reinberg, D; Arenas, J; Hurwitz, J
1985 May 25;260(10):6088-6097, Journal of biological chemistry
The enzyme, RNA cyclase, has been purified from cell-free extracts of HeLa cells approximately 6000-fold. The enzyme catalyzes the conversion of 3'-phosphate ends of RNA chains to the 2',3'-cyclic phosphate derivative in the presence of ATP or adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and Mg2+. The formation of 1 mol of 2',3'-cyclic phosphate ends is associated with the disappearance of 1 mol of 3'-phosphate termini and the hydrolysis of 1 mol of ATP gamma S to AMP and thiopyrophosphate. No other nucleotides could substitute for ATP or ATP gamma S in the reaction. The reaction catalyzed by RNA cyclase was not reversible and exchange reactions between [32P]pyrophosphate and ATP were not detected. However, an enzyme-AMP intermediate could be identified that was hydrolyzed by the addition of inorganic pyrophosphate or 3'-phosphate terminated RNA chains but not by 3'-OH terminated chains or inorganic phosphate. 3'-[32P](Up)10Gp* could be converted to a form that yielded, (Formula: see text) after degradation with nuclease P1, by the addition of wheat germ RNA ligase, 5'-hydroxylpolynucleotide kinase, RNA cyclase, and ATP. This indicates that the RNA cyclase had catalyzed the formation of the 2',3'-cyclic phosphate derivative, the kinase had phosphorylated the 5'-hydroxyl end of the RNA, and the wheat germ RNA ligase had catalyzed the formation of a 3',5'-phosphodiester linkage concomitant with the conversion of the 2',3'-cyclic end to a 2'-phosphate terminated residue
— id: 70006, year: 1985, vol: 260, page: 6088, stat: Journal Article,

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. I. Characterization of the termination and reinitiation reactions
Brown, D R; Roth, M J; Reinberg, D; Hurwitz, J
1984 Aug 25;259(16):10545-10555, Journal of biological chemistry
The phi X174 (phi X) gene A protein-mediated termination and reinitiation of single-stranded circular (SS(c] phi X viral DNA synthesis in vitro were directly and independently analyzed. Following incubation together with purified DNA replication enzymes from Escherichia coli, ATP, [alpha-32P]dNTPs, and either the phi X A protein and phi X replicative form I (RF I) DNA, or the purified RF II X A complex, the phi X A protein was detected covalently linked to newly synthesized 32P-labeled DNA. Formation of the phi X A protein-[32P]DNA covalent complex required all the factors necessary for phi X (+) SS(c) DNA synthesis in vitro. Thus, it was a product of the reinitiation reaction and an intermediate of the replication cycle. Identification of this complex provided direct evidence that reinitiation of phi X (+) strand DNA synthesis involved regeneration of the RF II X A complex. Substitution of 2',3'-dideoxyguanosine triphosphate (ddGTP) for dGTP in reaction mixtures resulted in the formation of covalent phi X A protein 32P-oligonucleotide complexes; these complexes were trapped analogues of the regenerated RF II X A complex. They could not act catalytically due to the presence of ddGMP residues at the 3'-termini of the oligonucleotide moieties. Reaction mixtures containing ddGTP also yielded nonradioactive (+) SS(c) DNA products derived from circularization of the displaced (+) strand of the input parental template DNA. The formation of the phi X A protein-32P-oligonucleotide complexes and nonradioactive (+) SS(c) DNA were used to assay both reinitiation and termination reactions, respectively. Both reactions required DNA synthesis from the 3'-hydroxyl primer at nucleotide residue 4305 which was formed by cleavage of phi X RF I DNA by the phi X A protein. Elongation of this primer by 18, but not 11 nucleotides was sufficient to support each reaction. Reinitiation reactions proceeded rapidly and were essentially complete after 90 s. In contrast, when ddGTP was replaced with dGTP in reaction mixtures, DNA synthesis proceeded with linear kinetics for up to 10 min. These results suggested that in the presence of all four dNTPs, active templates supported more than 40 rounds of DNA synthesis
— id: 70007, year: 1984, vol: 259, page: 10545, stat: Journal Article,

DNA structures required for phi X174 A-protein-directed initiation and termination of DNA replication
Brown, D R; Reinberg, D; Schmidt-Glenewinkel, T; Roth, M; Zipursky, S L; Hurwitz, J
1983 ;47 Pt 2:701-715, Cold Spring Harbor symposia on quantitative biology
— id: 70011, year: 1983, vol: 47 Pt 2, page: 701, stat: Journal Article,

Analysis of the phiX174 gene A protein using in vitro DNA replication systems
Brown, D R; Reinberg, D; Schmidt-Glenewinkel, T; Zipursky, S L; Hurwitz, J
1983 ;100:217-239, Methods in enzymology
— id: 70010, year: 1983, vol: 100, page: 217, stat: Journal Article,

DNA sequences which support activities of the bacteriophage phi X174 gene A protein
Brown, D R; Schmidt-Glenewinkel, T; Reinberg, D; Hurwitz, J
1983 Jul 10;258(13):8402-8412, Journal of biological chemistry
The DNA sequence of 30 nucleotides which surrounds the origin of viral strand DNA replication is highly conserved amongst the icosahedral single-stranded DNA bacteriophages. The A gene of these phages encodes a protein which is required for initiation and termination of viral strand DNA synthesis and acts as a nicking-closing activity specifically within this 30-nucleotide sequence. A system of purified Escherichia coli host proteins and phi X174 gene A protein has been developed which specifically replicates in vitro the viral strand of phi X174 from RF (replicative form) I template DNA and yields single-stranded circular DNA products (RF leads to SS(c) DNA replication system). Recombinant plasmids carrying inserts derived from phage phi X174 or G4 DNA which range in length from 49 to 1175 base pairs and contain the 30-nucleotide conserved sequence have been shown to support phi X A protein-dependent DNA synthesis in vitro in this replication system. We report here that insertion of the 30-nucleotide sequence alone into pBR322 allows the resulting recombinant plasmids to support phi X A protein-dependent in vitro DNA synthesis as efficiently as phi X174 template DNA in the RF leads to SS(c) replication system. The 30-nucleotide sequence functions as a fully wild type DNA replication origin as determined by the rate of DNA synthesis and the structure of resulting DNA products. Furthermore, the DNA sequence requirements for nicking of RF I DNA by the phi X A protein and for supporting replication origin function have been partially separated. Homology to positions 1, 29, and 30 of the 30-nucleotide conserved sequence are not required for cleavage of RF I DNA by the A protein; homology to position 1 but not 29 or 30 is required for efficient DNA replication
— id: 70008, year: 1983, vol: 258, page: 8402, stat: Journal Article,

Studies on the phi X174 gene A protein-mediated termination of leading strand DNA synthesis
Reinberg, D; Zipursky, S L; Weisbeek, P; Brown, D; Hurwitz, J
1983 Jan 10;258(1):529-537, Journal of biological chemistry
Recombinant RF (replicate form) I DNAs containing the bacteriophage phi X174 gene A protein-recognition sequence are cleaved by the phi X A protein yielding a phi X RF II X A protein complex (Zipursky, S.L., Reinberg, D., and Hurwitz, J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5182-5186). Such complexes support DNA synthesis in both RF I leads to SS(c) and RF I leads to RF I phi X DNA replication reactions in vitro. Two phi X A protein-recognition sequences were inserted into plasmid pBR322. Both sequences were contiguous with the same strand of the vector DNA and separated by 667 and 4275 base pairs. This recombinant plasmid (G27-4) was cleaved by the phi X A protein at either insert and both inserts support the initiation of RF leads to SS(c) DNA synthesis. This was verified by the finding that replication products were circular molecules of 667 and 4275 nucleotides. This finding is in keeping with the multifunctional activities associated with the phi X A protein; these include the site-specific nicking of RF I DNA which initiates DNA synthesis and site-specific termination resulting in the circularization of the displaced DNA strand. The phi X A protein and the Escherichia coli rep and SSb proteins catalyze the unwinding of phi X RF I DNA in vitro (Scott, J.F., Eisenberg, S., Bertsch, L.L., and Kornberg, A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 193-197). Recombinant plasmid G27-4 RF I DNA was also unwound in vitro by this enzyme system; in this case, both circular and linear single-stranded DNA molecules of 667 and 4275 nucleotides, as well as full length circular single-stranded DNA were formed. Full length linear DNA was not detected. The two single-stranded circular DNA products formed as leading strands in RF leads to SS(c) reaction mixtures containing G27-4 RF I DNA differed in their ability to support lagging strand DNA synthesis. It was shown that the large single-stranded circular product included DNA sequences homologous to a replication factor Y effector sequence required for RF leads to RF and SS(c) leads to RF replication (Zipursky, S.L., and Marians, K.J. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6521-6525). The 4275-nucleotide, but not the 667-nucleotide, single-stranded circular DNA product was converted to a duplex structure
— id: 70009, year: 1983, vol: 258, page: 529, stat: Journal Article,

Separate requirements for leading and lagging strand DNA synthesis during phi X A protein-dependent RF goes to RF DNA replication in vitro
Reinberg, D; Zipursky, S L; Hurwitz, J
1981 Dec 25;256(24):13143-13151, Journal of biological chemistry
— id: 70012, year: 1981, vol: 256, page: 13143, stat: Journal Article,

Initiation of DNA replication by the dnaG protein
Benz, E W Jr; Reinberg, D; Vicuna, R; Hurwitz, J
1980 Feb 10;255(3):1096-1106, Journal of biological chemistry
Highly purified preparations of dnaG protein from Escherichia coli prime minus strand synthesis of phage alpha 3 DNA in vitro. This protein synthesizes primer oligonucleotides which may be composed of ribonucleotide or deoxyribonucleotide moieties or both. The presence of deoxyribonucleotide moieties in the chain limits primer chain length; this effect occurs even when ribonucleoside triphosphates are included in the priming reaction. The dnaG protein can use ADP in place of ATP. Primer formation by dnaG protein is strictly stoiochiometric in vitro; one molecule of dnaG protein is required to prime one molecule of alpha 3 DNA. All of these primers are equally efficient in the subsequent elongation reaction with DNA elongation factors I and III, dnaZ gene product, and DNA polymerase III to form RFII. The site recognized by dnaG protein on alpha 3 DNA in vitro is within the same region of the alpha 3 chromosome as the origin of replication in vivo. Structural properties of this site are crucial to dnaG action in vitro. No other enzymatic activity for dnaG protein has been detected
— id: 70014, year: 1980, vol: 255, page: 1096, stat: Journal Article,

In vitro DNA replication of recombinant plasmid DNAs containing the origin of progeny replicative form DNA synthesis of phage phi X174
Zipursky, S L; Reinberg, D; Hurwitz, J
1980 Sep;77(9):5182-5186, Proceedings of the National Academy of Sciences of the United States of America
The origin of phage phi X174 progeny replicative form (RF) DNA synthesis has been inserted into the plasmid vector pBR322 and cloned. In direct contrast to pBR322, the recombinant superhelical plasmids can substitute for phi X174 RFI DNA as template in phi X174-specific reactions in vitro. We have shown that the recombinant plasmids: (i) are cleaved by the phi X174 A protein; (ii) support net synthesis of unit-length single-stranded circular DNA in the presence of the phi X174 A protein and Escherichia coli rep protein, DNA-binding protein, and DNA polymerase III elongation system; (iii) support replication of duplexes catalyzed by the phi X174 A protein and extracts of E. coli
— id: 70013, year: 1980, vol: 77, page: 5182, stat: Journal Article,