Brian David Dynlacht, Ph.D.

PNAS Plus: Genome-wide remodeling of the epigenetic landscape during myogenic differentiation
Asp, Patrik; Blum, Roy; Vethantham, Vasupradha; Parisi, Fabio; Micsinai, Mariann; Cheng, Jemmie; Bowman, Christopher; Kluger, Yuval; Dynlacht, Brian David
2011 May 31;108(22):E149-E158, Proceedings of the National Academy of Sciences of the United States of America
We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a 'fail-safe' mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells
— id: 133464, year: 2011, vol: 108, page: E149, stat: Journal Article,

Regulating the transition from centriole to basal body
Kobayashi, Tetsuo; Dynlacht, Brian D
2011 May 2;193(3):435-444, Journal of cell biology
The role of centrioles changes as a function of the cell cycle. Centrioles promote formation of spindle poles in mitosis and act as basal bodies to assemble primary cilia in interphase. Stringent regulations govern conversion between these two states. Although the molecular mechanisms have not been fully elucidated, recent findings have begun to shed light on pathways that regulate the conversion of centrioles to basal bodies and vice versa. Emerging studies also provide insights into how defects in the balance between centrosome and cilia function could promote ciliopathies and cancer
— id: 134200, year: 2011, vol: 193, page: 435, stat: Journal Article,

Centriolar Kinesin Kif24 Interacts with CP110 to Remodel Microtubules and Regulate Ciliogenesis
Kobayashi, Tetsuo; Tsang, William Y; Li, Ji; Lane, William; Dynlacht, Brian David
2011 Jun 10;145(6):914-925, Cell
We have identified a protein, Kif24, that shares homology with the kinesin-13 subfamily of motor proteins and specifically interacts with CP110 and Cep97, centrosomal proteins that play a role in regulating centriolar length and ciliogenesis. Kif24 preferentially localizes to mother centrioles. Loss of Kif24 from cycling cells resulted in aberrant cilia assembly but did not promote growth of abnormally long centrioles, unlike CP110 and Cep97 depletion. We found that loss of Kif24 leads to the disappearance of CP110 from mother centrioles, specifically in cycling cells able to form cilia. Kif24 is able to bind and depolymerize microtubules in vitro. Remarkably, ectopically expressed Kif24 specifically remodels centriolar microtubules without significantly altering cytoplasmic microtubules. Thus, our studies have identified a centriolar kinesin that specifically remodels a subset of microtubules, thereby regulating cilia assembly. These studies also suggest mechanistic differences between the regulation of microtubule elongation associated with centrioles and cilia
— id: 134452, year: 2011, vol: 145, page: 914, stat: Journal Article,

SCF(Cyclin F) controls centrosome homeostasis and mitotic fidelity through CP110 degradation
D'Angiolella, Vincenzo; Donato, Valerio; Vijayakumar, Sangeetha; Saraf, Anita; Florens, Laurence; Washburn, Michael P; Dynlacht, Brian; Pagano, Michele
2010 Jul 1;466(7302):138-142, Nature
Generally, F-box proteins are the substrate recognition subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes, which mediate the timely proteolysis of important eukaryotic regulatory proteins. Mammalian genomes encode roughly 70 F-box proteins, but only a handful have established functions. The F-box protein family obtained its name from Cyclin F (also called Fbxo1), in which the F-box motif (the approximately 40-amino-acid domain required for binding to Skp1) was first described. Cyclin F, which is encoded by an essential gene, also contains a cyclin box domain, but in contrast to most cyclins, it does not bind or activate any cyclin-dependent kinases (CDKs). However, like other cyclins, Cyclin F oscillates during the cell cycle, with protein levels peaking in G2. Despite its essential nature and status as the founding member of the F-box protein family, Cyclin F remains an orphan protein, whose functions are unknown. Starting from an unbiased screen, we identified CP110, a protein that is essential for centrosome duplication, as an interactor and substrate of Cyclin F. Using a mode of substrate binding distinct from other F-box protein-substrate pairs, CP110 and Cyclin F physically associate on the centrioles during the G2 phase of the cell cycle, and CP110 is ubiquitylated by the SCF(Cyclin F) ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal and mitotic abnormalities, such as multipolar spindles and asymmetric, bipolar spindles with lagging chromosomes. These phenotypes were reverted by co-silencing CP110 and were recapitulated by expressing a stable mutant of CP110 that cannot bind Cyclin F. Finally, expression of a stable CP110 mutant in cultured cells also promotes the formation of micronuclei, a hallmark of chromosome instability. We propose that SCF(Cyclin F)-mediated degradation of CP110 is required for the fidelity of mitosis and genome integrity
— id: 110690, year: 2010, vol: 466, page: 138, stat: Journal Article,

Traf7, a MyoD1 transcriptional target, regulates nuclear factor-kappaB activity during myogenesis
Tsikitis, Mary; Acosta-Alvear, Diego; Blais, Alexandre; Campos, Eric I; Lane, William S; Sanchez, Irma; Dynlacht, Brian D
2010 Dec;11(12):969-976, EMBO reports
We have identified the E3 ligase Traf7 as a direct MyoD1 target and show that cell cycle exit-an early event in muscle differentiation-is linked to decreased Traf7 expression. Depletion of Traf7 accelerates myogenesis, in part through downregulation of nuclear factor-kappaB (NF-kappaB) activity. We used a proteomic screen to identify NEMO, the NF-kappaB essential modulator, as a Traf7-interacting protein. Finally, we show that ubiquitylation of NF-kappaB essential modulator is regulated exclusively by Traf7 activity in myoblasts. Our results suggest a new mechanism by which MyoD1 function is coupled to NF-kappaB activity through Traf7, regulating the balance between cell cycle progression and differentiation during myogenesis
— id: 114826, year: 2010, vol: 11, page: 969, stat: Journal Article,

The Mammalian sin3 proteins are required for muscle development and sarcomere specification
van Oevelen, Chris; Bowman, Christopher; Pellegrino, Jessica; Asp, Patrik; Cheng, Jemmie; Parisi, Fabio; Micsinai, Mariann; Kluger, Yuval; Chu, Alphonse; Blais, Alexandre; David, Gregory; Dynlacht, Brian D
2010 Dec;30(24):5686-5697, Molecular & cellular biology
The highly related mammalian Sin3A and Sin3B proteins provide a versatile platform for chromatin-modifying activities. Sin3-containing complexes play a role in gene repression through deacetylation of nucleosomes. Here, we explore a role for Sin3 in myogenesis by examining the phenotypes resulting from acute somatic deletion of both isoforms in vivo and from primary myotubes in vitro. Myotubes ablated for Sin3A alone, but not Sin3B, displayed gross defects in sarcomere structure that were considerably enhanced upon simultaneous ablation of both isoforms. Massively parallel sequencing of Sin3A- and Sin3B-bound genomic loci revealed a subset of target genes directly involved in sarcomere function that are positively regulated by Sin3A and Sin3B proteins. Both proteins were coordinately recruited to a substantial number of genes. Interestingly, depletion of Sin3B led to compensatory increases in Sin3A recruitment at certain target loci, but Sin3B was never found to compensate for Sin3A loss. Thus, our analyses describe a novel transcriptional role for Sin3A and Sin3B proteins associated with maintenance of differentiated muscle cells
— id: 114827, year: 2010, vol: 30, page: 5686, stat: Journal Article,

E2f3b plays an essential role in myogenic differentiation through isoform-specific gene regulation
Asp, Patrik; Acosta-Alvear, Diego; Tsikitis, Mary; van Oevelen, Chris; Dynlacht, Brian David
2009 Jan 1;23(1):37-53, Genes & development
Current models posit that E2F transcription factors can be divided into members that either activate or repress transcription, in part through collaboration with the retinoblastoma (pRb) tumor suppressor family. The E2f3 locus encodes E2f3a and E2f3b proteins, and available data suggest that they regulate cell cycle-dependent gene expression through opposing transcriptional activating and repressing activities in growing and quiescent cells, respectively. However, the role, if any, of E2F proteins, and in particular E2f3, in myogenic differentiation is not well understood. Here, we dissect the contributions of E2f3 isoforms and other activating and repressing E2Fs to cell cycle exit and differentiation by performing genome-wide identification of isoform-specific targets. We show that E2f3a and E2f3b target genes are involved in cell growth, lipid metabolism, and differentiation in an isoform-specific manner. Remarkably, using gene silencing, we show that E2f3b, but not E2f3a or other E2F family members, is required for myogenic differentiation, and that this requirement for E2f3b does not depend on pRb. Our functional studies indicate that E2f3b specifically attenuates expression of genes required to promote differentiation. These data suggest how diverse E2F isoforms encoded by a single locus can play opposing roles in cell cycle exit and differentiation
— id: 92188, year: 2009, vol: 23, page: 37, stat: Journal Article,

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes
Galluzzi, L; Aaronson, SA; Abrams, J; Alnemri, ES; Andrews, DW; Baehrecke, EH; Bazan, NG; Blagosklonny, MV; Blomgren, K; Borner, C; Bredesen, DE; Brenner, C; Castedo, M; Cidlowski, JA; Ciechanover, A; Cohen, GM; De Laurenzi, V; De Maria, R; Deshmukh, M; Dynlacht, BD; El-Deiry, WS; Flavell, RA; Fulda, S; Garrido, C; Golstein, P; Gougeon, ML; Green, DR; Gronemeyer, H; Hajnoczky, G; Hardwick, JM; Hengartner, MO; Ichijo, H; Jaattela, M; Kepp, O; Kimchi, A; Klionsky, DJ; Knight, RA; Kornbluth, S; Kumar, S; Levine, B; Lipton, SA; Lugli, E; Madeo, F; Malorni, W; Marine, JCW; Martin, SJ; Medema, JP; Mehlen, P; Melino, G; Moll, UM; Morselli, E; Nagata, S; Nicholson, DW; Nicotera, P; Nunez, G; Oren, M; Penninger, J; Pervaiz, S; Peter, ME; Piacentini, M; Prehn, JHM; Puthalakath, H; Rabinovich, GA; Rizzuto, R; Rodrigues, CMP; Rubinsztein, DC; Rudel, T; Scorrano, L; Simon, HU; Steller, H; Tschopp, J; Tsujimoto, Y; Vandenabeele, P; Vitale, I; Vousden, KH; Youle, RJ; Yuan, J; Zhivotovsky, B; Kroemer, G
2009 AUG ;16(8):1093-1107, Cell death & differentiation
Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells. Cell Death and Differentiation (2009) 16, 1093-1107; doi:10.1038/cdd.2009.44; published online 17 April 2009
— id: 101090, year: 2009, vol: 16, page: 1093, stat: Journal Article,

Overly Lona Centrioles and Defective Cell Division upon Excess of the SAS-4-Related Protein CPAP
Kohlmaier, G; Loncarek, J; Meng, X; McEwen, BF; Mogensen, MM; Spektor, A; Dynlacht, BD; Khodjakov, A; Gonczy, P
2009 JUN 23 ;19(12):1012-1018, Current biology. CB
The centrosome is the principal microtubule organizing center (MTOC) of animal cells [1]. Accurate centrosome duplication is fundamental for genome integrity and entails the formation of one procentriole next to each existing centriole, once per cell cycle. The procentriole then elongates to eventually reach the same size as the centriole. The mechanisms that govern elongation of the centriolar cylinder and their potential relevance for cell division are not known. Here, we show that the SAS-4-related protein CPAP [2] is required for centrosome duplication in cycling human cells. Furthermore, we demonstrate that CPAP overexpression results in the formation of abnormally long centrioles. This also promotes formation of more than one procentriole in the vicinity of such overly long centrioles, eventually resulting in the presence of supernumerary MTOCs. This in turn leads to multipolar spindle assembly and cytokinesis defects. Overall, our findings suggest that centriole length must be carefully regulated to restrict procentriole number and thus ensure accurate cell division
— id: 100643, year: 2009, vol: 19, page: 1012, stat: Journal Article,

Cep76, a centrosomal protein that specifically restrains centriole reduplication
Tsang, William Y; Spektor, Alexander; Vijayakumar, Sangeetha; Bista, Bigyan R; Li, Ji; Sanchez, Irma; Duensing, Stefan; Dynlacht, Brian D
2009 May;16(5):649-660, Developmental cell
Centrosomes duplicate only once per cell cycle, but the controls that govern this process are largely unknown. We have identified Cep76, a centriolar protein that interacts with CP110. Cep76 is expressed at low levels in G1 and is induced in S and G2 phase, during which point centrioles have already commenced duplication. Interestingly, depletion of Cep76 drives the accumulation of centriolar intermediates in certain types of cancer cells. Enforced Cep76 expression specifically inhibits centriole amplification in cells undergoing multiple rounds of duplication without preventing the formation of extra procentrioles from a parental template. Furthermore, elevated levels of Cep76 do not affect normal centriole duplication. Thus, Cep76 helps limit duplication to once per cell cycle. Our findings also point to mechanistic differences between normal duplication and aberrant centriole amplification, as well as distinctions between diverse modes of amplification
— id: 99216, year: 2009, vol: 16, page: 649, stat: Journal Article,

Genome-wide transcriptional analysis of the human cell cycle identifies genes differentially regulated in normal and cancer cells
Bar-Joseph, Ziv; Siegfried, Zahava; Brandeis, Michael; Brors, Benedikt; Lu, Yong; Eils, Roland; Dynlacht, Brian D; Simon, Itamar
2008 Jan 22;105(3):955-960, Proceedings of the National Academy of Sciences of the United States of America
Characterization of the transcriptional regulatory network of the normal cell cycle is essential for understanding the perturbations that lead to cancer. However, the complete set of cycling genes in primary cells has not yet been identified. Here, we report the results of genome-wide expression profiling experiments on synchronized primary human foreskin fibroblasts across the cell cycle. Using a combined experimental and computational approach to deconvolve measured expression values into 'single-cell' expression profiles, we were able to overcome the limitations inherent in synchronizing nontransformed mammalian cells. This allowed us to identify 480 periodically expressed genes in primary human foreskin fibroblasts. Analysis of the reconstructed primary cell profiles and comparison with published expression datasets from synchronized transformed cells reveals a large number of genes that cycle exclusively in primary cells. This conclusion was supported by both bioinformatic analysis and experiments performed on other cell types. We suggest that this approach will help pinpoint genetic elements contributing to normal cell growth and cellular transformation
— id: 135315, year: 2008, vol: 105, page: 955, stat: Journal Article,

Live or let die: E2F1 and PI3K pathways intersect to make life or death decisions
Dynlacht, Brian David
2008 Jan;13(1):1-2, Cancer cell
In the current issue of Cancer Cell, Hallstrom et al. show that a subset of targets of the growth regulatory transcription factor E2F1 are repressed by a serum-induced PI3K activation, explaining how apoptosis can be suppressed while simultaneously engaging a proliferation program
— id: 75774, year: 2008, vol: 13, page: 1, stat: Journal Article,

Identification and characterization of a novel Mdm2 splice variant acutely induced by the chemotherapeutic agents adriamycin and actinomycin D
Lents, Nathan H; Wheeler, Leroy W; Baldassare, Joseph J; Dynlacht, Brian David
2008 Jun 1;7(11):1580-1586, Cell cycle
Mdm2, as the most important negative regulator of p53, plays an important homeostatic role in regulating cell division and the cellular response to DNA damage, oncogenic insult and other forms of cellular stress. We discovered that the DNA damaging agent adriamycin (doxorubicin) induces a novel aberrantly spliced Mdm2 mRNA which incorporates 108 bp of intronic sequence not normally found in the Mdm2 mature mRNA. Accordingly, we term this Mdm2 splice variant Mdm2(+108). Importantly, this insertion introduces in-frame nonsense codons, thus encoding a profoundly truncated mdm2 protein lacking the C-terminal RING finger domain and the E3 ubiquitin ligase activity. A wide range of pharmacological testing revealed that Mdm2(+108) is induced, in mouse and rat cells, in specific response to Adriamycin and actinomycin D, but not other modes of DNA damage. Meanwhile, antibodies against the N-terminal region of mdm2 reveal a marked reduction in detectable mdm2 protein upon Adriamycin treatment, while p53 accumulates to strikingly high levels. We thus conclude that this alternative spicing of Mdm2 may be an important mechanism to facilitate massive accumulation of p53 in response to genotoxic agents
— id: 96442, year: 2008, vol: 7, page: 1580, 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,

CP110 suppresses primary cilia formation through its interaction with CEP290, a protein deficient in human ciliary disease
Tsang, William Y; Bossard, Carine; Khanna, Hemant; Peranen, Johan; Swaroop, Anand; Malhotra, Vivek; Dynlacht, Brian David
2008 Aug;15(2):187-197, Developmental cell
Primary cilia are nonmotile organelles implicated in signaling and sensory functions. Understanding how primary cilia assemble could shed light on the many human diseases caused by mutations in ciliary proteins. The centrosomal protein CP110 is known to suppress ciliogenesis through an unknown mechanism. Here, we report that CP110 interacts with CEP290--a protein whose deficiency is implicated in human ciliary disease--in a discrete complex separable from other CP110 complexes involved in regulating the centrosome cycle. Ablation of CEP290 prevents ciliogenesis without affecting centrosome function or cell-cycle progression. Interaction with CEP290 is absolutely required for the ability of CP110 to suppress primary cilia formation. Furthermore, CEP290 and CP110 interact with Rab8a, a small GTPase required for cilia assembly. Depletion of CEP290 interferes with localization of Rab8a to centrosomes and cilia. Our results suggest that CEP290 cooperates with Rab8a to promote ciliogenesis and that this function is antagonized by CP110
— id: 83106, year: 2008, vol: 15, page: 187, stat: Journal Article,

Double identity of SCAPER: a substrate and regulator of cyclin A/Cdk2
Tsang, William Y; Dynlacht, Brian David
2008 Mar 15;7(6):702-705, Cell cycle
Cyclin A is a key regulator of DNA replication and mitosis, and cyclin A/Cdk2 activity is critical for progression of cells from G(1)/S to M phase of the cell cycle. There is abundant evidence that cyclin A is predominantly localized to, and functions in, the nucleus, and a number of nuclear cyclin A/Cdk2 substrates have been identified. Evidence supporting the presence of cyclin A and its associated activity in the cytoplasm have also been reported, but the biological significance of this cyclin/Cdk pool during cell cycle progression remains controversial. Recently, we identified and characterized a new cyclin A/Cdk2 substrate named SCAPER which is localized to the endoplasmic reticulum. By sequestering cyclin A/Cdk2, SCAPER is capable of directing the activity of this kinase complex away from the nucleus and regulating cyclin A/Cdk2 equilibrium in distinct subcellular compartments. This work paves new avenues for understanding the role of cytoplasmic cyclin A/Cdk2 and its potential contribution to cancer and tumor formation
— id: 84017, year: 2008, vol: 7, page: 702, stat: Journal Article,

sSgo1, a guardian of centriole cohesion
Tsang, William Y; Dynlacht, Brian David
2008 Mar;14(3):320-322, Developmental cell
Sgo1 plays a key role in protecting sister chromatid cohesion during mitosis. In this issue of Developmental Cell, Wang et al. describe a shorter splice variant of Sgo1 (sSgo1) that functions specifically in centriole cohesion. sSgo1 may be the 'glue' that holds paired centrioles together in an engaged state before their disengagement in late mitosis
— id: 78023, year: 2008, vol: 14, page: 320, stat: Journal Article,

A role for mammalian Sin3 in permanent gene silencing
van Oevelen, Chris; Wang, Jinhua; Asp, Patrik; Yan, Qin; Kaelin, William G Jr; Kluger, Yuval; Dynlacht, Brian David
2008 Nov 7;32(3):359-370, Molecular cell
The multisubunit Sin3 corepressor complex regulates gene transcription through deacetylation of nucleosomes. However, the full range of Sin3 activities and targets is not well understood. Here, we have investigated genome-wide binding of mouse Sin3 and RBP2 as well as histone modifications and nucleosome positioning as a function of myogenic differentiation. Remarkably, we find that Sin3 complexes spread immediately downstream of the transcription start site on repressed and transcribed genes during differentiation. We show that RBP2 is part of a Sin3 complex and that on a subset of E2F4 target genes, the coordinated activity of Sin3 and RBP2 leads to deacetylation, demethylation, and repositioning of nucleosomes. Our work provides evidence for coordinated binding of Sin3, chromatin modifications, and chromatin remodeling within discrete regulatory regions, suggesting a model in which spreading of Sin3 binding is ultimately linked to permanent gene silencing on a subset of E2F4 target genes
— id: 90059, year: 2008, vol: 32, page: 359, stat: Journal Article,

XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks
Acosta-Alvear, Diego; Zhou, Yiming; Blais, Alexandre; Tsikitis, Mary; Lents, Nathan H; Arias, Carolina; Lennon, Christen J; Kluger, Yuval; Dynlacht, Brian David
2007 Jul 6;27(1):53-66, Molecular cell
Using genome-wide approaches, we have elucidated the regulatory circuitry governed by the XBP1 transcription factor, a key effector of the mammalian unfolded protein response (UPR), in skeletal muscle and secretory cells. We identified a core group of genes involved in constitutive maintenance of ER function in all cell types and tissue- and condition-specific targets. In addition, we identified a cadre of unexpected targets that link XBP1 to neurodegenerative and myodegenerative diseases, as well as to DNA damage and repair pathways. Remarkably, we found that XBP1 regulates functionally distinct targets through different sequence motifs. Further, we identified Mist1, a critical regulator of differentiation, as an important target of XBP1, providing an explanation for developmental defects associated with XBP1 loss of function. Our results provide a detailed picture of the regulatory roadmap governed by XBP1 in distinct cell types as well as insight into unexplored functions of XBP1
— id: 73301, year: 2007, vol: 27, page: 53, stat: Journal Article,

E2F-associated chromatin modifiers and cell cycle control
Blais, A; Dynlacht, BD
2007 DEC ;19(6):658-662, Current opinion in cell biology
The E2F family of proteins was identified on the basis of its role in promoting the GO to S phase transition. Research over the past several years has unveiled considerable complexity within the family, with numerous studies pointing to delegation of function for distinct family members. More recent studies highlighted in this review have expanded this picture, suggesting ways in which E2F target gene expression is refined during cell cycle progression by facilitating the acquisition of promoter-specific histone modifications. E2F associated co-activators promote activating histone marks while recruitment of co-repressors associated with E2Fs and the pRB family leads to accretion of inhibitory histone modifications that provoke chromatin compaction
— id: 87206, year: 2007, vol: 19, page: 658, stat: Journal Article,

Retinoblastoma tumor suppressor protein-dependent methylation of histone H3 lysine 27 is associated with irreversible cell cycle exit
Blais, Alexandre; van Oevelen, Chris J C; Margueron, Raphael; Acosta-Alvear, Diego; Dynlacht, Brian David
2007 Dec 31;179(7):1399-1412, Journal of cell biology
The retinoblastoma tumor suppressor protein (pRb) is involved in mitotic exit, promoting the arrest of myoblasts, and myogenic differentiation. However, it is unclear how permanent cell cycle exit is maintained in differentiated muscle. Using RNA interference, expression profiling, and chromatin immunoprecipitations, we show that pRb is essential for cell cycle exit and the differentiation of myoblasts and is also uniquely required to maintain this arrest in myotubes. Remarkably, we also uncover a function for the pRb-related proteins p107 and p130 as enforcers of a G2/M phase checkpoint that prevents progression into mitosis in cells that have lost pRb. We further demonstrate that pRb effects permanent cell cycle exit in part by maintaining trimethylation of histone H3 lysine 27 (H3K27) on cell cycle genes. H3K27 trimethylation silences other genes, including Cyclin D1, in a pRb-independent but polycomb-dependent manner. Thus, our data distinguish two distinct chromatin-based regulatory mechanisms that lead to terminal differentiation
— id: 96443, year: 2007, vol: 179, page: 1399, stat: Journal Article,

Functional genomics via multiscale analysis: application to gene expression and ChIP-on-chip data
Lerman, Gilad; McQuown, Joseph; Blais, Alexandre; Dynlacht, Brian D; Chen, Guangliang; Mishra, Bud
2007 Feb 1;23(3):314-320, Bioinformatics
We present a fast, versatile and adaptive-multiscale algorithm for analyzing a wide-variety of DNA microarray data. Its primary application is in normalization of array data as well as subsequent identification of 'enriched targets', e.g. differentially expressed genes in expression profiling arrays and enriched sites in ChIP-on-chip experimental data. We show how to accommodate the unique characteristics of ChIP-on-chip data, where the set of 'enriched targets' is large, asymmetric and whose proportion to the whole data varies locally. SUPPLEMENTARY INFORMATION: Supplementary figures, related preprint, free software as well as our raw DNA microarray data with PCR validations are available at http://www.math.umn.edu/~lerman/supp/bioinfo06 as well as Bioinformatics online
— id: 71651, year: 2007, vol: 23, page: 314, stat: Journal Article,

Cep97 and CP110 Suppress a Cilia Assembly Program
Spektor, Alexander; Tsang, William Y; Khoo, David; Dynlacht, Brian David
2007 Aug 24;130(4):678-690, Cell
Mammalian centrioles play a dynamic role in centrosome function, but they also have the capacity to nucleate the assembly of cilia. Although controls must exist to specify these different fates, the key regulators remain largely undefined. We have purified complexes associated with CP110, a protein that plays an essential role in centrosome duplication and cytokinesis, and have identified a previously uncharacterized protein, Cep97, that recruits CP110 to centrosomes. Depletion of Cep97 or expression of dominant-negative mutants results in CP110 disappearance from centrosomes, spindle defects, and polyploidy. Remarkably, loss of Cep97 or CP110 promotes primary cilia formation in growing cells, and enforced expression of CP110 in quiescent cells suppresses their ability to assemble cilia, suggesting that Cep97 and CP110 collaborate to inhibit a ciliogenesis program. Identification of Cep97 and other genes involved in regulation of cilia assembly may accelerate our understanding of human ciliary diseases, including renal disease and retinal degeneration
— id: 73828, year: 2007, vol: 130, page: 678, stat: Journal Article,

SCAPER, a novel cyclin A interacting protein that regulates cell cycle progression
Tsang, William Y; Wang, Leyu; Chen, Zhihong; Sanchez, Irma; Dynlacht, Brian David
2007 Aug 13;178(4):621-633, Journal of cell biology
Cyclin A/Cdk2 plays an important role during S and G2/M phases of the eukaryotic cell cycle, but the mechanisms by which it regulates cell cycle events are not fully understood. We have biochemically purified and identified SCAPER, a novel protein that specifically interacts with cyclin A/Cdk2 in vivo. Its expression is cell cycle independent, and it associates with cyclin A/Cdk2 at multiple phases of the cell cycle. SCAPER localizes primarily to the endoplasmic reticulum. Ectopic expression of SCAPER sequesters cyclin A from the nucleus and results specifically in an accumulation of cells in M phase of the cell cycle. RNAi-mediated depletion of SCAPER decreases the cytoplasmic pool of cyclin A and delays the G1/S phase transition upon cell cycle re-entry from quiescence. We propose that SCAPER represents a novel cyclin A/Cdk2 regulatory protein that transiently maintains this kinase in the cytoplasm. SCAPER could play a role in distinguishing S phase- from M phase-specific functions of cyclin A/Cdk2
— id: 73586, year: 2007, vol: 178, page: 621, stat: Journal Article,

CP110 Cooperates with Two Calcium-binding Proteins to Regulate Cytokinesis and Genome Stability
Tsang, William Y; Spektor, Alexander; Luciano, Daniel J; Indjeian, Vahan B; Chen, Zhihong; Salisbury, Jeffery L; Sanchez, Irma; Dynlacht, Brian David
2006 Aug;17(8):3423-3434, Molecular biology of the cell
The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca(2+)-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large ( approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis
— id: 66471, year: 2006, vol: 17, page: 3423, stat: Journal Article,

Pocket protein complexes are recruited to distinct targets in quiescent and proliferating cells
Balciunaite, Egle; Spektor, Alexander; Lents, Nathan H; Cam, Hugh; Te Riele, Hein; Scime, Anthony; Rudnicki, Michael A; Young, Richard; Dynlacht, Brian David
2005 Sep;25(18):8166-8178, Molecular & cellular biology
Biochemical and genetic studies have determined that retinoblastoma protein (pRB) tumor suppressor family members have overlapping functions. However, these studies have largely failed to distinguish functional differences between the highly related p107 and p130 proteins. Moreover, most studies pertaining to the pRB family and its principal target, the E2F transcription factor, have focused on cells that have reinitiated a cell cycle from quiescence, although recent studies suggest that cycling cells exhibit layers of regulation distinct from mitogenically stimulated cells. Using genome-wide chromatin immunoprecipitation, we show that there are distinct classes of genes directly regulated by unique combinations of E2F4, p107, and p130, including a group of genes specifically regulated in cycling cells. These groups exhibit both distinct histone acetylation signatures and patterns of mammalian Sin3B corepressor recruitment. Our findings suggest that cell cycle-dependent repression results from recruitment of an unexpected array of diverse complexes and reveals specific differences between transcriptional regulation in cycling and quiescent cells. In addition, factor location analyses have, for the first time, allowed the identification of novel and specific targets of the highly related transcriptional regulators p107 and p130, suggesting new and distinct regulatory networks engaged by each protein in continuously cycling cells
— id: 58713, year: 2005, vol: 25, page: 8166, stat: Journal Article,

Constructing transcriptional regulatory networks
Blais, Alexandre; Dynlacht, Brian David
2005 Jul 1;19(13):1499-1511, Genes & development
Biological networks are the representation of multiple interactions within a cell, a global view intended to help understand how relationships between molecules dictate cellular behavior. Recent advances in molecular and computational biology have made possible the study of intricate transcriptional regulatory networks that describe gene expression as a function of regulatory inputs specified by interactions between proteins and DNA. Here we review the properties of transcriptional regulatory networks and the rapidly evolving approaches that will enable the elucidation of their structure and dynamic behavior. Several recent studies illustrate how complementary approaches combine chromatin immunoprecipitation (ChIP)-on-chip, gene expression profiling, and computational methods to construct blueprints for the initiation and maintenance of complex cellular processes, including cell cycle progression, growth arrest, and differentiation. These approaches should allow us to elucidate complete transcriptional regulatory codes for yeast as well as mammalian cells
— id: 58064, year: 2005, vol: 19, page: 1499, stat: Journal Article,

CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells
Chen, Zhihong; Indjeian, Vahan B; McManus, Michael; Wang, Leyu; Dynlacht, Brian David
2002 Sep;3(3):339-350, Developmental cell
Centrosome duplication and separation are linked inextricably to certain cell cycle events, in particular activation of cyclin-dependent kinases (CDKs). However, relatively few CDK targets driving these events have been uncovered. Here, we have performed a screen for CDK substrates and have isolated a target, CP110, which is phosphorylated by CDKs in vitro and in vivo. Human CP110 localizes to centrosomes. Its expression is strongly induced at the G1-to-S phase transition, coincident with the initiation of centrosome duplication. RNAi-mediated depletion of CP110 indicates that this protein plays an essential role in centrosome duplication. Long-term disruption of CP110 phosphorylation leads to unscheduled centrosome separation and overt polyploidy. Our data suggest that CP110 is a physiological centrosomal CDK target that promotes centrosome duplication, and its deregulation may contribute to genomic instability
— id: 33140, year: 2002, vol: 3, page: 339, stat: Journal Article,

E2F4 loss suppresses tumorigenesis in Rb mutant mice
Lee, Eunice Y; Cam, Hieu; Ziebold, Ulrike; Rayman, Joseph B; Lees, Jacqueline A; Dynlacht, Brian David
2002 Dec;2(6):463-472, Cancer cell
The E2F transcription factors mediate the activation or repression of key cell cycle regulatory genes under the control of the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. Here we investigate how E2F4, the major 'repressive' E2F, contributes to pRB's tumor-suppressive properties. Remarkably, E2F4 loss suppresses the development of both pituitary and thyroid tumors in Rb(+/-) mice. Importantly, E2F4 loss also suppresses the inappropriate gene expression and proliferation of pRB-deficient cells. Biochemical analyses suggest that this tumor suppression occurs via a novel mechanism: E2F4 loss allows p107 and p130 to regulate the pRB-specific, activator E2Fs. We also detect these novel E2F complexes in pRB-deficient cells, suggesting that they play a significant role in the regulation of tumorigenesis in vivo
— id: 33139, year: 2002, vol: 2, page: 463, stat: Journal Article,

E2F mediates cell cycle-dependent transcriptional repression in vivo by recruitment of an HDAC1/mSin3B corepressor complex
Rayman, Joseph B; Takahashi, Yasuhiko; Indjeian, Vahan B; Dannenberg, Jan-Hermen; Catchpole, Steven; Watson, Roger J; te Riele, Hein; Dynlacht, Brian David
2002 Apr 15;16(8):933-947, Genes & development
Despite biochemical and genetic data suggesting that E2F and pRB (pocket protein) families regulate transcription via chromatin-modifying factors, the precise mechanisms underlying gene regulation by these protein families have not yet been defined in a physiological setting. In this study, we have investigated promoter occupancy in wild-type and pocket protein-deficient primary cells. We show that corepressor complexes consisting of histone deacetylase (HDAC1) and mSin3B were specifically recruited to endogenous E2F-regulated promoters in quiescent cells. These complexes dissociated from promoters once cells reached late G1, coincident with gene activation. Interestingly, recruitment of HDAC1 complexes to promoters depended absolutely on p107 and p130, and required an intact E2F-binding site. In contrast, mSin3B recruitment to certain promoters did not require p107 or p130, suggesting that recruitment of this corepressor can occur via E2F-dependent and -independent mechanisms. Remarkably, loss of pRB had no effect on HDAC1 or mSin3B recruitment. p107/p130 deficiency triggered a dramatic loss of E2F4 nuclear localization as well as transcriptional derepression, which is suggested by nucleosome mapping studies to be the result of localized hyperacetylation of nucleosomes proximal to E2F-binding sites. Taken together, these findings show that p130 escorts E2F4 into the nucleus and, together with corepressor complexes that contain mSin3B and/or HDAC1, directly represses transcription from target genes as cells withdraw from the cell cycle
— id: 33141, year: 2002, vol: 16, page: 933, stat: Journal Article,

E2F integrates cell cycle progression with DNA repair, replication, and G(2)/M checkpoints
Ren, Bing; Cam, Hieu; Takahashi, Yasuhiko; Volkert, Thomas; Terragni, Jolyon; Young, Richard A; Dynlacht, Brian David
2002 Jan 15;16(2):245-256, Genes & development
The E2F transcription factor family is known to play a key role in the timely expression of genes required for cell cycle progression and proliferation, but only a few E2F target genes have been identified. We explored the possibility that E2F regulators play a broader role by identifying additional genes bound by E2F in living human cells. A protocol was developed to identify genomic binding sites for DNA-binding factors in mammalian cells that combines immunoprecipitation of cross-linked protein-DNA complexes with DNA microarray analysis. Among approximately 1200 genes expressed during cell cycle entry, we found that the promoters of 127 were bound by the E2F4 transcription factor in primary fibroblasts. A subset of these targets was also bound by E2F1. Most previously identified target genes known to have roles in DNA replication and cell cycle control and represented on the microarray were confirmed by this analysis. We also identified a remarkable cadre of genes with no previous connection to E2F regulation, including genes that encode components of the DNA damage checkpoint and repair pathways, as well as factors involved in chromatin assembly/condensation, chromosome segregation, and the mitotic spindle checkpoint. Our data indicate that E2F directly links cell cycle progression with the coordinate regulation of genes essential for both the synthesis of DNA as well as its surveillance
— id: 33142, year: 2002, vol: 16, page: 245, stat: Journal Article,

Transcriptional regulation of cell cycle progression
Dynlacht BD
Transcription factors Oxford : BIOS, Academic Press, 2001,
— id: 2840, year: 2001, vol: , page: ?, stat: Chapter,

Active repression and E2F inhibition by pRB are biochemically distinguishable
Ross JF; Naar A; Cam H; Gregory R; Dynlacht BD
2001 Feb 15;15(4):392-397, Genes & development
To understand mechanistically how pRB represses transcription, we used a reconstituted transcription assay and compared pRB activity on naked versus chromatin templates. Surprisingly, when pRB was directly recruited to a naked template, no transcriptional repression was observed. However, we observed active repression when the same promoter was assembled into chromatin. Histone deacetylases do not appear to play a role in this observed repression. Further experiments showed repression could occur after preinitiation complex assembly, in contrast with pRB inhibition of E2F, suggesting discrete mechanisms by which pRB directly inhibits an activator such as E2F or actively represses proximally bound transcription factors
— id: 33143, year: 2001, vol: 15, page: 392, stat: Journal Article,

Human PC4 is a substrate-specific inhibitor of RNA polymerase II phosphorylation
Schang LM; Hwang GJ; Dynlacht BD; Speicher DW; Bantly A; Schaffer PA; Shilatifard A; Ge H; Shiekhattar R
2000 Mar 3;275(9):6071-6074, Journal of biological chemistry
The activity of cyclin-dependent protein kinases (cdks) is physiologically regulated by phosphorylation, association with the specific cyclin subunits, and repression by specific cdk inhibitors. All three physiological regulatory mechanisms are specific for one or more cdks, but none is known to be substrate specific. In contrast, synthetic cdk peptide inhibitors that specifically inhibit cdk phosphorylation of only some substrates, 'aptamers,' have been described. Here, we show that PC4, a naturally occurring transcriptional coactivator, competitively inhibits cdk-1, -2, and -7-mediated phosphorylation of the largest subunit of RNA polymerase II (RNAPII), but it does not inhibit phosphorylation of other substrates of the same kinases. Interestingly, the phosphorylated form of PC4 is devoid of kinase inhibitory activity. We also show that wild-type PC4 but not the kinase inhibitory-deficient mutant of PC4 represses transcription in vivo. Our results point to a novel role for PC4 as a specific inhibitor of RNAPII phosphorylation
— id: 33145, year: 2000, vol: 275, page: 6071, stat: Journal Article,

Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression
Takahashi Y; Rayman JB; Dynlacht BD
2000 Apr 1;14(7):804-816, Genes & development
The E2F transcription factor plays a pivotal role in the timely activation of gene expression during mammalian cell cycle progression, whereas pRB and related proteins control cell growth in part through the ability to block the action of E2F. To identify physiologically important E2F-responsive promoters and to study their occupancy and histone acetylation state in vivo, we have taken advantage of a cross-linking approach in synchronized, living cells. We find that the pattern of E2F and pRB-related polypeptides recruited to these promoters changes in a strikingly dynamic fashion as cells progress from quiescence into G(1) and S phase: Repression of each promoter in quiescent cells is associated with recruitment of E2F-4 and p130 and low levels of histone acetylation, but by late G(1), these proteins are replaced largely by E2F-1 and E2F-3, in concert with acetylation of histones H3 and H4 and gene activation. These findings suggest that repression and activation of E2F-responsive genes may occur through distinct E2F heterodimers that direct the sequential recruitment of enzymes able to deacetylate and then acetylate core histones
— id: 33144, year: 2000, vol: 14, page: 804, stat: Journal Article,

Mechanism of transcriptional repression of E2F by the retinoblastoma tumor suppressor protein
Ross JF; Liu X; Dynlacht BD
1999 Feb;3(2):195-205, Molecular cell
The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes
— id: 33146, year: 1999, vol: 3, page: 195, stat: Journal Article,

Activity and nature of p21(WAF1) complexes during the cell cycle
Cai K; Dynlacht BD
1998 Oct 13;95(21):12254-12259, Proceedings of the National Academy of Sciences of the United States of America
Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. We have characterized a panel of monoclonal antibodies against human p21 in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The use of these reagents has allowed us to address several important, yet unresolved, issues concerning the biological activity of p21, including the potential kinase activity of complexes that associate with this cyclin-dependent kinase inhibitor. We have found that the kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled us to begin the study of the molecular architecture of p21 complexes in vivo. In addition, we found that human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ('free' p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. Our data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21
— id: 33147, year: 1998, vol: 95, page: 12254, stat: Journal Article,

Dual cyclin-binding domains are required for p107 to function as a kinase inhibitor
Castano E; Kleyner Y; Dynlacht BD
1998 Sep;18(9):5380-5391, Molecular & cellular biology
The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks
— id: 33148, year: 1998, vol: 18, page: 5380, stat: Journal Article,

Expression of NPAT, a novel substrate of cyclin E-CDK2, promotes S-phase entry
Zhao J; Dynlacht B; Imai T; Hori T; Harlow E
1998 Feb 15;12(4):456-461, Genes & development
To understand the mechanisms by which CDKs regulate cell cycle progression, it is necessary to identify and characterize the physiological substrates of these kinases. We have developed a screening method to identify novel CDK substrates. One of the cDNAs identified in the screen is identical to the recently isolated NPAT gene. Here we show that NPAT associates with cyclin E-CDK2 in vivo and can be phosphorylated by this CDK. The protein level of NPAT peaks at the G1/S boundary. Overexpression of NPAT accelerates S-phase entry, and this effect is enhanced by coexpression of cyclin E-CDK2. These results suggest that NPAT is a substrate of cyclin E-CDK2 and plays a role in S-phase entry
— id: 33170, year: 1998, vol: 12, page: 456, stat: Journal Article,

Regulation of transcription by proteins that control the cell cycle
Dynlacht BD
1997 Sep 11;389(6647):149-152, Nature
In eukaryotes, progression of a cell through the cell cycle is partly controlled at the level of transcriptional regulation. Yeast and mammalian cells use similar mechanisms to achieve this regulation. Although gaps still remain, progress has been made recently in connecting the links between the cell's cycle and its transcriptional machinery
— id: 33149, year: 1997, vol: 389, page: 149, stat: Journal Article,

Specific regulation of E2F family members by cyclin-dependent kinases
Dynlacht BD; Moberg K; Lees JA; Harlow E; Zhu L
1997 Jul;17(7):3867-3875, Molecular & cellular biology
The transcription factor E2F-1 interacts stably with cyclin A via a small domain near its amino terminus and is negatively regulated by the cyclin A-dependent kinases. Thus, the activities of E2F, a family of transcription factors involved in cell proliferation, are regulated by at least two types of cell growth regulators: the retinoblastoma protein family and the cyclin-dependent kinase family. To investigate further the regulation of E2F by cyclin-dependent kinases, we have extended our studies to include additional cyclins and E2F family members. Using purified components in an in vitro system, we show that the E2F-1-DP-1 heterodimer, the functionally active form of the E2F activity, is not a substrate for the active cyclin D-dependent kinases but is efficiently phosphorylated by the cyclin B-dependent kinases, which do not form stable complexes with the E2F-1-DP-1 heterodimer. Phosphorylation of the E2F-1-DP-1 heterodimer by cyclin B-dependent kinases, however, did not result in down-regulation of its DNA-binding activity, as is readily seen after phosphorylation by cyclin A-dependent kinases, suggesting that phosphorylation per se is not sufficient to regulate E2F DNA-binding activity. Furthermore, heterodimers containing E2F-4, a family member lacking the cyclin A binding domain found in E2F-1, are not efficiently phosphorylated or functionally down-regulated by cyclin A-dependent kinases. However, addition of the E2F-1 cyclin A binding domain to E2F-4 conferred cyclin A-dependent kinase-mediated down-regulation of the E2F-4-DP-1 heterodimer. Thus, both enzymatic phosphorylation and stable physical interaction are necessary for the specific regulation of E2F family members by cyclin-dependent kinases
— id: 33150, year: 1997, vol: 17, page: 3867, stat: Journal Article,

Purification and analysis of CIP/KIP proteins
Dynlacht BD; Ngwu C; Winston J; Swindell EC; Elledge SJ; Harlow E; Harper JW
1997 ;283(7):230-244, Methods in enzymology
— id: 33152, year: 1997, vol: 283, page: 230, stat: Journal Article,

p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity
Woo MS; Sanchez I; Dynlacht BD
1997 Jul;17(7):3566-3579, Molecular & cellular biology
The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases
— id: 33151, year: 1997, vol: 17, page: 3566, stat: Journal Article,

Cyclin-binding motifs are essential for the function of p21CIP1
Chen J; Saha P; Kornbluth S; Dynlacht BD; Dutta A
1996 Sep;16(9):4673-4682, Molecular & cellular biology
The cyclin-dependent kinase (Cdk) inhibitor p21 is induced by the tumor suppressor p53 and is required for the G1-S block in cells with DNA damage. We report that there are two copies of a cyclin-binding motif in p21, Cy1 and Cy2, which interact with the cyclins independently of Cdk2. The cyclin-binding motifs of p21 are required for optimum inhibition of cyclin-Cdk kinases in vitro and for growth suppression in vivo. Peptides containing only the Cy1 or Cy2 motif partially inhibit cyclin-Cdk kinase activity in vitro and DNA replication in Xenopus egg extracts. A monoclonal antibody which recognizes the Cy1 site of p21 specifically disrupts the association of p21 with cyclin E-Cdk2 and with cyclin D1-Cdk4 in cell extracts. Taken together, these observations suggest that the cyclin-binding motif of p21 is important for kinase inhibition and for formation of p21-cyclin-Cdk complexes in the cell. Finally, we show that the cyclin-Cdk complex is partially active if associated with only the cyclin-binding motif of p21, providing an explanation for how p21 is found associated with active cyclin-Cdk complexes in vivo. The Cy sequences may be general motifs used by Cdk inhibitors or substrates to interact with the cyclin in a cyclin-Cdk complex
— id: 33153, year: 1996, vol: 16, page: 4673, stat: Journal Article,

The anti-proliferative effect of sulindac and sulindac sulfide on HT-29 colon cancer cells: alterations in tumor suppressor and cell cycle-regulatory proteins
Goldberg Y; Nassif II; Pittas A; Tsai LL; Dynlacht BD; Rigas B; Shiff SJ
1996 Feb 15;12(4):893-901, Oncogene
Nonsteroidal anti-inflammatory drugs lower the incidence of and mortality from colon cancer. Sulindac reduces the number and size of polyps in patients with familial adenomatous polyposis. We have shown that sulindac and sulindac sulfide reversibly reduce the proliferation rate of HT-29 colon cancer cells, alter their morphology, induce them to accumulate in the G0/G1 phase of the cell cycle, and sulindac sulfide induces cell death by apoptosis. In this study we confirmed that sulindac and sulindac sulfide prevent HT-29 cells from progressing from the G0/G1 into the S phase. This block in cell cycle progression is associated with an initial rise, then an abrupt decrease in the levels of p34cdc2 protein. Sulindac and sulindac sulfide decrease the levels of mitotic cyclins, induce the levels of p21WAF-1/cip1, and reduce the total levels of pRB, with a relative increase in the amount of the underphosphorylated form of pRB in a time- and concentration-dependent manner. In addition, these compounds reduce the levels of mutant p53. These responses are not associated with intestinal cell differentiation and occur independent of the ability of these compounds to induce apoptosis. We conclude that sulindac and sulindac sulfide reduce the levels of major components of the molecular cell cycle machinery and alter the levels of several tumor suppressor proteins in a manner consistent with cell cycle quiescence. These mechanisms may be operative in vivo to account, in part, for the anti-neoplastic effects of these compounds
— id: 33155, year: 1996, vol: 12, page: 893, stat: Journal Article,

Transcriptional control of the cell cycle
Sanchez I; Dynlacht BD
1996 Jun;8(3):318-324, Current opinion in cell biology
Although a significant amount of evidence has demonstrated that there are intimate connections between transcriptional controls and cell cycle regulation, the precise mechanisms underlying these connections remain largely obscure. A number of recent advances have helped to define how critical cell cycle regulators, such as the retinoblastoma family of tumor suppressor proteins and the cyclin-dependent kinases, might function on a biochemical level and how such mechanisms of action have been conserved not only in the regulation of transcription by all three RNA polymerases but also across species lines. In addition, the use of in vivo techniques has begun to explain how the activity of the E2F transcription factor family is tied to the cell cycle dependent expression of target genes
— id: 33154, year: 1996, vol: 8, page: 318, stat: Journal Article,

Retinoblastoma protein. Pol I gets repressed
Dynlacht BD
1995 Mar 9;374(6518):114-114, Nature
— id: 33158, year: 1995, vol: 374, page: 114, stat: Journal Article,

Inhibition of cyclin-dependent kinases by p21
Harper JW; Elledge SJ; Keyomarsi K; Dynlacht B; Tsai LH; Zhang P; Dobrowolski S; Bai C; Connell-Crowley L; Swindell E; et al.
1995 Apr;6(4):387-400, Molecular biology of the cell
p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression
— id: 33169, year: 1995, vol: 6, page: 387, stat: Journal Article,

Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition
Koh J; Enders GH; Dynlacht BD; Harlow E
1995 Jun 8;375(6531):506-510, Nature
The cyclin-dependent kinase inhibitor p16 is a candidate tumour-suppressor protein that maps to a genomic locus strongly associated with familial melanoma and other tumour types. Screening of primary tumours and linkage analysis of familial melanoma pedigrees have identified many potential mutations in p16, but the functional significance of these sequence variants has remained unclear. We report here that p16 can act as a potent and specific inhibitor of progression through the G1 phase of the cell cycle, and we demonstrate that several tumour-derived alleles of p16 encode functionally compromised proteins. The ability of p16 to arrest cell-cycle progression generally correlates with inhibition of cyclin D1/Cdk4 kinase activity in vitro, with two exceptions among the alleles tested. In vivo, the presence of functional retinoblastoma protein appears to be necessary but may not be sufficient to confer full sensitivity to p16-mediated growth arrest. Our results provide support for the notion that p16 is an important cell-cycle regulator whose inactivation contributes to the outgrowth of human tumours
— id: 33157, year: 1995, vol: 375, page: 506, 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,

p107 uses a p21CIP1-related domain to bind cyclin/cdk2 and regulate interactions with E2F
Zhu L; Harlow E; Dynlacht BD
1995 Jul 15;9(14):1740-1752, Genes & development
The kinase activities of the cyclin/cdk complexes can be regulated in a number of ways. The most recently discovered mechanism of regulation is the association of cdk inhibitors (CKIs), such as p21, p27, and p57, with these complexes. In this report we demonstrate that the pRB-related protein p107, like the p21 family of cdk inhibitors, can inhibit the phosphorylation of target substrates by cyclin A/cdk2 and cyclin E/cdk2 complexes, and the associations of p107 and p21 with cyclin/cdk2 rely on a structurally and functionally related interaction domain. Furthermore, interactions between p107 or p21 with cyclin/cdk2 complexes are mutually exclusive. In cells treated with DNA-damaging agents elevated levels of p21 cause a dissociation of p107/cyclin/cdk2 complexes to yield p21/cyclin/cdk2 complexes. Finally, the consequences of cyclin/cdk2 interactions with p107 have been examined. The activation of the p107-bound cyclin/cdk kinases leads to dissociation of p107 from the transcription factor E2F. Together, these results suggest that cyclin/cdk complexes can be regulated by protein molecules from different families in a mutually exclusive manner in response to certain signals and that these inhibitory proteins may have a potential role in regulating macromolecular assembly
— id: 33156, year: 1995, vol: 9, page: 1740, stat: Journal Article,

DNA-binding and trans-activation properties of Drosophila E2F and DP proteins
Dynlacht BD; Brook A; Dembski M; Yenush L; Dyson N
1994 Jul 5;91(14):6359-6363, Proceedings of the National Academy of Sciences of the United States of America
The temporal activation of E2F transcriptional activity appears to be an important component of the mechanisms that prepare mammalian cells for DNA replication. Regulation of E2F activity appears to be a highly complex process, and the dissection of the E2F pathway will be greatly facilitated by the ability to use genetic approaches. We report the isolation of two Drosophila genes that can stimulate E2F-dependent transcription in Drosophila cells. One of these genes, dE2F, contains three domains that are highly conserved in the human homologs E2F-1, E2F-2, and E2F-3. Interestingly, one of these domains is highly homologous to the retinoblastoma protein (RB)-binding sequences of human E2F genes. The other gene, dDP, is closely related to the human DP-1 and DP-2 genes. We demonstrate that dDP and dE2F interact and cooperate to give sequence-specific DNA binding and optimal trans-activation. These features suggest that endogenous Drosophila E2F, like human E2F, may be composed of heterodimers and may be regulated by RB-like proteins. The isolation of these genes will provide important reagents for the genetic analysis of the E2F pathway
— id: 33160, year: 1994, vol: 91, page: 6359, stat: Journal Article,

Differential regulation of E2F transactivation by cyclin/cdk2 complexes
Dynlacht BD; Flores O; Lees JA; Harlow E
1994 Aug 1;8(15):1772-1786, Genes & development
The mammalian transcription factor E2F plays a critical role in the expression of genes required for cellular proliferation. To understand how E2F is regulated, we have developed a reconstituted in vitro transcription assay. Using this E2F-responsive assay, we can demonstrate that E2F-mediated transcription can be directly repressed by the tumor suppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cyclin/kinase complexes exhibit differences in the ability to phosphorylate E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-activation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate how transcriptional regulation of E2F can be linked to cell cycle-dependent activation of kinases
— id: 33159, year: 1994, vol: 8, page: 1772, stat: Journal Article,

The dTAFII80 subunit of Drosophila TFIID contains beta-transducin repeats
Dynlacht BD; Weinzierl RO; Admon A; Tjian R
1993 May 13;363(6425):176-179, Nature
A key component of the RNA polymerase II transcriptional apparatus, TFIID, is a multi-protein complex containing the TATA box-binding protein (TBP) and at least seven tightly associated factors (TAFs). Although the functions of most TFIID subunits are unknown, it is clear that TAFs are not necessary for basal activity but that one or more are required for regulated transcription, and so behave as coactivators. The presence of multiple subunits indicates that there is an intricate assembly process and that TAFs may be responsible for other activities. We have described the properties of the subunit dTAFII110, which can interact directly with the transcriptional activator Sp1 (ref. 5). In addition, the largest subunit, dTAFII250, binds directly to TBP and links other TAFs to the complex. Here we describe the cloning, expression and partial characterization of the Drosophila TAF of M(r) 80,000, dTAFII80. Sequence analysis reveals that dTAFII80 contains several copies of the WD40 (beta-transducin) repeat. Moreover, dTAFII80 shares extended sequence similarity with an Arabidopsis gene, COP1, which encodes a putative transcription factor that is though to regulate development. We have expressed recombinant dTAFII80 and begun to characterize its interaction with other members of the TFIID complex. Purified recombinant dTAFII80 is unable to bind TBP directly or to interact strongly with the C-terminal domain of dTAFII250 (delta N250). Instead, dTAFII80 is only able to recognize and interact with a higher-order complex containing TBP, delta N250, 110 and 60. These findings suggest the formation of TFIID may require an ordered assembly of the TAFs, some of which bind directly to TBP and others that are tethered to the complex as a result of specific TAF/TAF interactions
— id: 33162, year: 1993, vol: 363, page: 176, stat: Journal Article,

Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators
Hoey T; Weinzierl RO; Gill G; Chen JL; Dynlacht BD; Tjian R
1993 Jan 29;72(2):247-260, Cell
The general transcription factor TFIID is a multiprotein complex containing the TATA-binding protein and several associated factors (TAFs), some of which may function as coactivators that are essential for activated, but not basal, transcription. Here we describe the isolation and characterization of the first gene encoding a TAF protein. The deduced amino acid sequence of TAF110 revealed the presence of several glutamine- and serine/threonine-rich regions reminiscent of the protein-protein interaction domains of the regulatory transcription factor Sp1 that are involved in transcription activation and multimerization. In both Drosophila cells and yeast, TAF110 specifically interacts with the glutamine-rich activation domains of Sp1. Moreover, purified Sp1 selectively binds recombinant TAF110 in vitro. These findings taken together suggest that TAF110 may function as a coactivator by serving as a site of protein-protein contact between activators like Sp1 and the TFIID complex
— id: 33164, year: 1993, vol: 72, page: 247, stat: Journal Article,

Largest subunit of Drosophila transcription factor IID directs assembly of a complex containing TBP and a coactivator
Weinzierl RO; Dynlacht BD; Tjian R
1993 Apr 8;362(6420):511-517, Nature
The TFIID complex consists of the TATA-binding protein (TBP) and associated factors (TAFs) serving to mediate transcriptional activation by promoter-specific regulators. Here we report the cloning of Drosophila TAFII250 and the assembly of a partial complex containing recombinant TBP, TAFII110 and the C-terminal domain of TAFII250. This triple complex supports Sp1 activation and reveals specific interactions between TAFII250, TBP and TAFII110
— id: 33163, year: 1993, vol: 362, page: 511, stat: Journal Article,

Cloning and expression of Drosophila TAFII60 and human TAFII70 reveal conserved interactions with other subunits of TFIID
Weinzierl RO; Ruppert S; Dynlacht BD; Tanese N; Tjian R
1993 Dec 15;12(13):5303-5309, EMBO journal
Regulation of transcription initiation by RNA polymerase II requires TFIID, a multisubunit complex composed of the TATA binding protein (TBP) and at least seven tightly associated factors (TAFs). Some TAFs act as direct targets or coactivators for promoter-specific activators while others serve as interfaces for TAF-TAF interactions. Here, we report the molecular cloning, expression and characterization of Drosophila dTAFII60 and its human homolog, hTAFII70. Recombinant TAFII60/70 binds weakly to TBP and tightly to the largest subunit of TFIID, TAFII250. In the presence of TAFII60/70, TBP and TAFII250, a stable ternary complex is formed. Both the human and Drosophila proteins directly interact with another TFIID subunit, dTAFII40. Our findings reveal that Drosophila TAFII60 and human TAFII70 share a high degree of structural similarity and that their interactions with other subunits of TFIID are conserved
— id: 33161, year: 1993, vol: 12, page: 5303, stat: Journal Article,

Biochemical characterization of the Drosophila transcription factor NTF-1 and the TFIID complex
Dynlacht, Brian David
[S.l. : s.n.], 1992,
— id: 1109, year: 1992, vol: , page: , stat: ,

Isolation and characterization of the Drosophila gene encoding the TATA box binding protein, TFIID
Hoey T; Dynlacht BD; Peterson MG; Pugh BF; Tjian R
1990 Jun 29;61(7):1179-1186, Cell
To investigate the biochemical mechanisms involved in interactions between regulatory factors and the general transcription complex, we have cloned, expressed, and characterized the Drosophila gene encoding the TATA binding protein, dTFIID. Comparison of the protein sequences of the Drosophila and yeast TATA binding proteins reveals a bipartite organization consisting of a highly conserved, basic carboxy-terminal domain and a nonconserved amino-terminal region rich in Gln, Gly, Ser, and Met residues. Purified dTFIID protein binds specifically to the TATA sequence and activates basal-level transcription, and the conserved carboxy-terminal half of the molecule is sufficient for both activities. Partially purified TFIID from Drosophila cells mediates activation by the transcription factor Sp1. In contrast, purified dTFIID expressed from the cloned gene is unable to support Sp1-dependent activation, suggesting that other factors may be required to mediate interactions between upstream activators like Sp1 and the TATA binding protein
— id: 33166, year: 1990, vol: 61, page: 1179, stat: Journal Article,

Functional analysis of NTF-1, a developmentally regulated Drosophila transcription factor that binds neuronal cis elements
Dynlacht BD; Attardi LD; Admon A; Freeman M; Tjian R
1989 Nov;3(11):1677-1688, Genes & development
In an effort to characterize sequence-specific transcription factors that regulate gene expression during Drosophila development, we identified and purified a novel DNA-binding activity (NTF-1). The purified protein consists of several polypeptides that bind selectively to a functionally important cis-control element of the Ultrabithorax (Ubx) promoter and to the neurogenic elements of both the dopa decarboxylase (Ddc) and fushi tarazu (ftz) promoter/enhancer regions. Purified NTF-1 activates transcription in vitro in a binding site-dependent manner through upstream sequences of the Ubx promoter. A cDNA clone encoding the open reading frame of NTF-1 was isolated, and the deduced primary amino acid sequence of NTF-1 includes a glutamine-rich region reminiscent of the transcriptional activation domains found in Sp1 but no recognizable DNA-binding domain. NTF-1 expression is temporally regulated during embryonic development. In addition, in situ hybridization experiments revealed that NTF-1 is transcribed in a spatially restricted pattern in the embryo, with the highest level of expression observed in the epidermis and a subset of cells in the CNS. Expression of the NTF-1 cDNA in mammalian cells yields a protein that displays DNA-binding and transcriptional activities indistinguishable from that of the collection of proteins isolated from Drosophila embryos. These findings suggest that NTF-1 is a member of a family of developmentally regulated transcription factors that may be involved in directing the expression of genes such as Ubx, Ddc, and ftz in neuronal cells
— id: 33167, year: 1989, vol: 3, page: 1677, stat: Journal Article,