RNAi Screen For Regulators Of The Spatial Clustering Of Pericentric Heterochromatin

The structural and dynamic organization of the mammalian cell nucleus impacts many aspects of chromatin regulation and gene expression, yet little is known about the underlying molecular principles of nuclear organization. We are interested in determining these principles, as a way to understand at the mechanistic level the so far mostly correlative link between nuclear organization and gene regulation. Toward this end, we are performing an RNAi screen coupled to high-throughput microscopy, for regulators of pericentric heterochromatin and its nuclear clustering. We have generated an NIH3T3 cell line stably expressing HP1α-GFP at near endogenous levels (NIH3T3-HP1α-GFP). HP1α-GFP is expressed near homogenously across the cell population and the fluorescent signal is of sufficient intensity to be detected in high-throughput assays.Moreover, we show that HP1&slpha;-GFP is correctly targeted to pericentric heterochromatin clusters where it co-localizes with established markers for these structures. We validated our screening strategy by transiently knocking-down known regulators of pericentric heterochromatin, and observed disruption of HP1α-GFP foci. Furthermore, we have demonstrated that chemical disruption of known regulators of pericentric heterochromatin, such as histone deacetylases and DNA methyltransferases also cause disruption of our reporter HP1α-GFP foci. We expect that genome-wide RNAi on this cell line will unveil several previously unsuspected regulators of the formation and clustering of heterochromatin inside the nucleus.

 

 

 

 

The Role Of Mammalian Sin3 In Gene Activation And Transcriptional Regulation

The Sin3 repressive complex was first identified in budding yeast. There are two isoforms of mammalian Sin3, Sin3A and Sin3B, both of which are highly homologous to yeast Sin3. The Sin3 core complex consists of Sin3, HDAC1, HDAC2, RbAp46, RbAp48, SAP30, SAP18, and SDS3. This core complex serves as a scaffold for enzymatic activities by recruiting other factors such as SWI/SNF and RBP2, a histone demethylase. Sin3 has classically been associated with gene repression, which is mostly likely achieved through HDAC activity. However, our lab has previously indentified a subset of genes that are highly activated through Sin3 binding. Furthermore, many of these genes play a role in muscle development. Using C2C12’s, a mouse skeletal muscle cell line, the goal of this work is to determine what factors and histone modifications may associate with Sin3 in order to achieve gene activation.

APC/C^Cdh1-Dependent Proteolysis Of USP1 Regulates UV DNA Damage Response

Our lab is interested in understanding the molecular pathogenesis of the Fanconi anemia (FA) human genetic disorder. Cells derived from FA patients have abnormal cell cycle distribution, defects in DNA repair, genomic instability and hypersensitivity to DNA damaging agents. In normal cells, FA proteins are involved in a complex DNA repair pathway. Upon DNA damage, two key Fanconi proteins: FANCI and FANCD2 are monoubiquitinated enabling their localization at the site of DNA damage. The deubiquitining enzyme USP1 can antagonize the monoubiquitination of FANCI and FANCD2. However, it is unclear how USP1 activity is regulated during DNA damage, therefore my main research objective is to study the regulation of USP1 activity during DNA damage.

The Effect of miR-33 Antagonism on Reverse Cholesterol Transport, HDL Functionality, and Atherosclerosis

An antagonist for miR33 (a microRNA) is a potential therapy that can be used to increase HDL and play a protective role in atherosclerosis. This miR-33 antagonist has been shown to both block progression and induce regression of atherosclerosis in Ldlr-/- mice. MiR33 blocks the expression of the ABCA1 protein (expressed in the liver and macrophages), which plays a role in cholesterol efflux. The anti-mir33 treatment inhibits the inhibition of ABCA1 resulting in increased HDL levels and decreased atherogenesis. My work focuses on assessing the macrophage specific contribution of ABCA1 on atherosclerosis by performing in vivo regression studies in Ldlr-/- mice transplanted with the bone marrow of ABCA1 knockout mice. I will also work on a progression study in ApoE-/- mice, which develop atherosclerosis but have more normal total cholesterol values than the Ldlr-/- previously studied.

Aberrant Activation of mTOR Promotes Schwann Cell Dedifferentiation

Demyelination can result from Schwann cell dedifferentiation and the consequent breakdown of their myelin sheaths. Although demyelination is a significant source of morbidity in nerve injuries and neuropathies, the mechanisms involved have remained elusive. Our lab has developed an experimental model of demyelination that involves addition of the Neuregulin-1 (NRG1) growth factor to myelinating cocultures. This results in the activation of the erbB receptors and their downstream signaling pathways followed by Schwann cell dedifferentiation and demyelination. Among the pathways activated by NRG1 is the mammalian target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational apparatus and integrates a number of upstream signals. We have found that inhibiting mTOR with rapamycin effectively prevents NRG1-induced demyelination. mTOR activity is also elevated in the PMP22 overexpressing transgenic rat, a murine model of the demyelinating human neuropathy Charcot-Marie-Tooth 1A (CMT1A). This activation inhibits Schwann cell differentiation as inhibition of mTOR with rapamycin rescues myelination in a tissue culture model of CMT1A. These results implicate mTOR as a therapeutic target in demyelinating and dysmyelinating neuropathies.

Reactivation Of Latent HSV-1 Results In A UniquePprogram Of Viral Lytic Gene Expression.

Herpes simplex virus type 1 (HSV-1) is a pathogen infecting approximately 80% of the human population. Like all herpes viruses, HSV-1 shows two modes of infection, lytic and latent. HSV-1 establishes long-term latency in peripheral neurons where viral transcription is limited to the latency-associated transcripts (LATs). During reactivation, the virus initiates a new program of productive cycle gene expression that culminates in the assembly and release of infectious virions. While mechanisms controlling lytic cycle gene expression in acutely-infected cells are clearly known, the molecular details of reactivation remain obscure. Development of a robust primary neuron culture system in which HSV-1 infection enters a semi-quiescent state indistinguishable in molecular terms from in vivo latency, has allowed us to probe the response of the viral transcriptome to defined reactivation signals. Using PI3-kinase inhibitors to mimic interruption of nerve growth factor (NGF) signaling, we find a viral transcription profile strikingly different from the classic lytic profile observed in acutely infected neurons.

Determining The Role Of FBW7 Mutations In T-cell Acute Lympoblastic Leukemia

FBW7, an F-box constituent of the SCF E3 ubiquitin ligase complex, can act as a tumor suppressor by targeting numerous oncoproteins for proteasomal degradation. We and others have previously shown that up to 30% of T-cell acute lymphoblastic leukemias (TALL) harbor single, somatic missense mutations in the substrate-binding domain of FBW7. We have developed novel genetic tools in mice to faithfully mimic the effect of these particular mutations on T-ALL progression. We have found that a single mutant allele of FBW7 significantly accelerates leukemia development in conjunction with activating Notch1 mutants, even with those that are weak initiators of leukemia on their own. Further mechanistic study in these mouse models is potentially valuable for the development of targeted therapies in the wide range of malignancies where FBW7 is found mutated.

Function Of CP110-Interacting Proteins In Centrosome Duplication And Ciliogenesis

Centrosomes are the main microtubule organizing centers in animal cells and serve as basal bodies from which primary cilia emanate and elongate. Centrosome amplification occurs frequently in most types of cancer. Centrosomal and ciliary protein mutations directly cause a variety of ciliopathies such as polycystic kidney disease and Bardet-Biedl syndrome. CP110, a 110KD centrosomal protein that has been identified by our group, along with its interacting partners such as Cep76, Cep97 and Cep290, plays a critical role in centrosome duplication, centriole length control, cytokinesis, and primary cilia formation. By mass spectrometry, we have recently identified several novel CP110- interacting proteins that are crucial for these cellular processes. My major goal is to characterize these proteins, uncover their molecular mechanisms, and examine their roles in centrosome duplication and ciliogenesis.

Cellular And MolecularRresponses Of The Epithelial Cells To Distinct Forms Of Death

During development and throughout life some cells within tissues undergo apoptosis. Apoptotic cells are cleared without compromising the barrier function of the epithelium. Using time-lapse microscopy and quantitative image analysis we show that clearance of apoptotic cells is characterized by coordinated elongation of the neighboring cells. We hypothesized that cell-cell adhesion is required for the process to occur normally. To test the hypothesis we utilized a panel of carcinoma cell lines with various E-cadherin mediated cohesiviness. We find that only cells expressing detectable levels of E-cadherin elongate in response to apoptosis and are therefore able to clear the dead cells. We propose that cell adhesion is required to produce the coordinated cellular behavior that drives the rapid clearance of the dead remnants to maintain epithelial function. Additionally, we carry out a comparative analysis of another form of cell death, necrosis and its impact on the epithelia.

Pyrophosphate Removal In Escherichia coli RNA Degradation

Bacteria live in a constantly changing environment, and therefore have a continuous need to modulate their protein expression. Three processes control protein synthesis in those microorganisms: transcription, translation, and mRNA degradation. It had long been thought that mRNA degradation in Escherichia coli begins with endonucleolytic cleavage by RNase E. However, recent work has shown that RNase E cleavage can be triggered by a prior step: the removal of pyrophosphate from the 5’ terminus of primary transcripts. An enzyme that catalyzes this initial event (RppH) has been discovered in E. coli. E. coli also have exonucleases that can degrade transcripts from the 3’ end. Canonical transcripts bear a protective stem-loop at this end. Previous evidence has suggested that elements at the 5’ end, including the phosphorylation state, affect exonucleolytic degradation through these stem-loops. Using a novel RNA I have shown that pyrophosphate removal by RppH can influence efficient RNA degradation from the 3’ end of transcripts in vivo.

 

 

Regulation of the Mammary Epithelial Hierarchy by TGF-β1

Estrogen signaling through estrogen receptor (ER) plays a role in mammary epithelial cell proliferation. Several studies have shown that the majority of estrogen receptorpositive (ER+) cells do not proliferate, but are required to regulate proliferation in ERnegative (ER-) cells via paracrine mechanisms. A small subpopulation of ER+ cells that maintain proliferative potential, has been postulated to be early progenitor cells that could be the origin of ER+ breast cancer. Our lab found that nearly all of the ER+ cells in mouse mammary gland at estrus co-localize with indices of TGF-β activity, consistent with their non-proliferative status. Proliferation of ER+ cells is increased in Tgfβ1 heterozygote mammary gland and the frequency of ER expressing cells increase in these mice. Therefore, it has been demonstrated that TGF-β restrains proliferation of mammary epithelial cells and it is speculated that it regulates the subpopulation of ER+ cells that are considered to represent lineage-restricted progenitors. Since different ideas motivate the concept that tissue-specific stem cells or early progenitors may be critical cellular targets in carcinogenesis, we speculate that disruption of TGF-β activity is involved in the origins of ER+ breast cancer. In addition to its role in regulating proliferation, we hypothesize that TGF-β regulates the decision of the pluoripotent mammary cell population to give rise to an ER positive or ER negative progenitor.

Interactions between axons and Schwann cells in the peripheral nervous system induce myelination. Nectin-like 4 (Necl-4) is expressed on myelinating Schwann cells and binds specifically to Necl-1 expressed on axons. These proteins mediate heterophilic interactions at the internode. Knock down of Necl-4 inhibits Schwann cell differentiation and subsequent myelination. A key question is how Necl-4 promotes myelination. Recent data has identified Par-3, a PDZ-domain containing protein of the Par-aPKC polarity complex, as required for Schwann cell myelination. Our preliminary results indicate that Necl-4, through its PDZ-binding domain, interacts with Par-3 directly. Necl-4 also interacts with the 4.1G protein via its FERM-binding domain. Together these findings suggest a model where Par-3 is recruited by Necl-4 to the adaxonal membrane of the Schwann cell upon axon-glial contact, generating anterior-posterior polarity and the formation of a leading edge in the Schwann cell that drives spiral wrapping of the axon.

The Role Of Non-coding RNA In Epigenetics

Recently it has been discovered that a large percentage of transcribed RNA does not code for any protein. A subset of these RNA, termed long-intergenic non-coding RNA (lincRNA), have been shown to be involved in epigenetic control by recruiting chromatin modifying proteins to specific genes. We hope to identify protein-lincRNA interactions in a genome-wide fashion, and then understand the mechanism by which this interaction encodes epigenetic information./p>

Posttranslational Modification Of Ras As A Modulator Of Signaling Capacity And Pleiotrophy

Receptor tyrosine kinases (RTK) are essential mediators of signal transmission in response to extracellular cues that regulate fundamental processes such as cell proliferation, differentiation, and survival. A central route by which RTK-initiated signals are transduced involves the activation of the small guanine nucleotide binding protein Ras by the guanine nucleotide exchange factor SoS. Activated Ras in turn induces a broad spectrum of cellular responses through the stimulation of multiple effector pathways. Posttranslational modification of the Ras molecule through its CAAX domain has proven to be essential in directing its sub-cellular localization based signaling capacity. Numerous other signaling pathways have also provided evidence that modification of myriad amino acid side chains can regulate enzymatic function in complex and often surprising ways. Here we test the rationale that Acetylation of a lysine residue located near the SoS binding domain, known as switch 2, of Ras has an effect on its capacity for SoS-mediated nucleotide exchange.

The C-Terminal Domain Of RNA Polymerase Ii Is Modified By Site-Specific Methylation

The Carboxy-terminal domain (CTD) of RNA Polymerase II (RNAPII) in mammals contains 52 pseudo repeats with the consensus sequence YSPTSPS and undergoes extensive post-translational modification. Phosphorylation of the CTD is essential for transcriptional initiation and elongation, providing a binding platform for the recruitment of pre-mRNA processing factors. Here, we show that the CTD of RNAPII is methylated at a single arginine contained within a non- consensus heptad repeat. Using an unbiased biochemical approach, we purified and identified the transcriptional co-activator CARM1 as the only methyltransferase targeting this site. Although methylation at Arg1810 is present on the hyper-phosphorylated form of RNAPII in vivo, Ser-2 or Ser-5 phosphorylation inhibits CARM1 activity towards this site in vitro, suggesting that this modification is placed before transcription initiation. The Tudor domain containing protein 3 (TDRD3) binds specifically to the methylated CTD and may function as an adaptor that links this post-translational modification to downstream events.

 

 

Elucidating The Functional Rrole Of Bcl-2 Phosphorylation

Bcl-2 is the founding member of a family of both pro-apoptotic and anti-apoptotic proteins that regulate the mitochondrial pathway of apoptosis. Bcl-2 activity is regulated through various post-translational modifications such as phosphorylation, ubiquitination, S-nitrosylation, and caspase cleavage. Various studies have shown that phosphorylation is a key modification that affects the anti-apoptotic activity of Bcl-2 when cells undergo stress. Bcl-2 phosphorylation has also been found to alter the stability of the protein. However, it remains controversial as to whether this phosphorylation activates or inactivates Bcl-2, and whether it induces degradation or stabilization of the protein. I aim to resolve the conflicts in the literature and to determine how Bcl-2 phosphorylation affects its stability and activity, with the ultimate goal of identifying the E3 ligase responsible for its degradation.

 

 

 

Role Of A Novel Adhesion GPCR In Mammary Development And Breast Cancer

I have begun to characterize the expression of a novel orphan adhesion G-protein coupled receptor (GPCR) during mammary development. Preliminary data using a GPCR-Gal reporter mouse indicates specific expression of our GPCR during periods of mammary proliferation in both embryonic and adult development. During puberty, expression is restricted to the cap cells of terminal end buds (TEB), thought to be the location of pubertal mammary stem cells. Our GPCR is also upregulated in MMTV-Wnt1 mammary tumors, which are enriched in mammary stem or early progenitor cells. Currently, I am collecting sufficient antigen that will be used for generating a monoclonal antibody to the protein, which I will use to further characterize GPCR+ cells by FACS. Future studies aim to fully evaluate the role of this GPCR in normal mammary development, as well as its contribution to the development of mammary tumors.

Influence Of Bacterial Metabolism As A Feature Of Food Source On C. elegans Physiology

Caenorhabditis elegans are standardly fed on Escherichia coli OP50 that is uracil auxotrophic strain, which provides limited growth of these bacteria on nematode growth medium. Whereas more natural food source for C. elegans such as soil bacteria Bacillus subtilis could exhibits different metabolic functions and doing so influence nematodes’ physiology. The latter may refer better to the natural physiological state of these worms. One of the main features of the undomesticated wild bacterial strain is biofilm formation. We aim to study the influence of B. subtilis biofilm formation on C. elegans life span and stress resistance. .

Determining The Rrole Of Jarid2 And Aebp2 In The PRC2 Kinetics

The polycomb group (PcG) proteins are highly conserved transcriptional repressors that regulate the essential development genes. Polycomb repressive complex 2 (PRC2) functions as the histone methyl transferase placing H3K27me marks which in turn recruit the PRC1 complex for the transcriptional repression. Recent studies have showed that many other factors including Jarid2 and Aebp2 could interact with PRC2 core complex (EED, SUZ12, RbAp46/48 and EZH2) and alter its activity. Here we investigated how Jarid2 and Aebp2 could regulate PRC2 activity using in vitro histone methyltransferase assay. Surprisingly, the significant increase in PRC2/EZH2 activity in the presence of Jarid2 observed at low substrate levels, rapidly declined upon the addition of 500 ng and more substrate observed. However, we did not detect evidence of Jarid2-mediated stimulation of PRC2/Ezh1, in spite of the similar Jarid2 interaction obtained with these Ezh homologues. In addition, we identified the essential domains of AEBP2 and Jarid2 to regulate the PRC2 activity.

 

Elucidating The Role Of The Ubiquitin-proteasome System In Embryonic Stem Cell Fates

Pluripotent embryonic stem (ES) cells, as well as tissue-specific adult stem cells, have the unique ability to both self-renew and differentiate. Transcriptional regulation has been in the center of stem cell self-renewal for the last couple of decades and several transcriptional networks have been identified as key regulators of ES cells. However, accumulating evidence suggests that regulation of gene transcription is not the whole answer to the self-renewal question. Strong data suggest that the UPS may be an important regulator of stem cells. To investigate the role of the UPS in embryonic stem cells, we performed a siRNA-based ubiquitin screen to identify members of the UPS that regulate ES cell self-renewal and differentiation. We also set to characterize their novel substrates using mass-spectrometry-based approaches. The elucidation of the role the UPS in stem cell biology can not only lead to a better understanding of stem cell fate decisions but also the development of new therapeutics that have broad implications for carcinogenesis as well as degenerative diseases.

 

 

Dissecting The Targeting Signals In The Kras4A Hypervariable Domain

Ras signaling requires membrane association. For decades, it had been thought that Ras signals exclusively from the plasma membrane; however, it was discovered that Ras proteins are also found on subcellular compartments such as the Golgi apparatus and vesicles, and that signaling outputs from these compartments differ. Understanding how the four Ras isoforms are trafficked within the cell is thus critical in understanding how they exert their numerous signaling effects, especially in light of their importance in human cancer. KRAS, the most frequently mutated Ras gene in tumors, encodes two splice variants, K-Ras4A and K-Ras4B. The pronounced difference in post-translational processing between K-Ras4A and K-Ras4B may distinguish the function of the two splice variants on a signaling level. The goal of my project is to investigate the signals for K-Ras4A targeting and how its trafficking is regulated, and through this explore how its signaling is distinct from that of K-Ras4B.

 

 

Dynamic Imaging Of Granulocyte Recruitment During Spleen Listeriosis After Activation Of The Inflammasome

As subsets of the Nod-like receptor family, inflammasomes activate caspase-1 inducing apoptosis and the secretion of pro-inflammatory cytokines by the host. Recent studies have found that inflammasome activation leads to diminished long-term protective immunity and decreased T cell priming during Listeriosis of the spleen. We hypothesize that early granulocyte recruitment in response to secreted cytokine leads to the premature clearance of bacteria thereby altering T cell activation within the white pulp of the spleen. By coupling two-photon imaging to live tissue sectioning, we were able to expose the white pulp of the spleen and directly visualize LysM⁺ granulocyte recruitment from the red pulp and marginal zone into the white pulp of the spleen at various time points after infection. Inflammasome activation led to a decrease in the number of granulocytes and early recruitment into the WP. These data suggest that inflammasome activation leads to the early recruitment of granulocytes that prematurely clear Listeria before the priming of T cells can occur.

The Role Of Paf1 Complex In Transcription During Myogenic Differentiation

The Paf1C (RNA Pol II-associated factor1 complex), originally identified in yeast, is an evolutionarily conserved protein complex, consisting of Ctr9, Leo1, Rtf1, Paf1, Cdc73, & the mammalian cell specific Ski8. Paf1C plays roles in various cellular processes including transcription, mRNA quality control and cell-cycle regulation, and specifically in metazoans is involved in Notch and Wnt signaling. My project is concerned with the role of Paf1C in transcription during myogenic differentiation. This process involves the induction of proliferating myoblasts to terminally differentiate into myotubes. Previous work from our lab identified remodeling of chromatin modifications during this process, others’ work showed that the effects of Paf1C on transcription is partly attributed to its involvement in the deposition of various histone marks. Therefore, Paf1C could be an important regulator of spatial and temporal patterns of gene expression during myogenic differentiation, owing to its histone modification abilities and possibly other mechanisms.