Our primary goal is to understand how chromatin structure influences gene regulation. Chromatin is generally repressive in nature but its structure is manipulated by cells in a regulated way to determine which genes are potentially transcriptionally active and which genes remain repressed in a given cell type. This regulation depends on interactions between DNA sequence-specific transcription factors, chromatin enzymes and chromatin. The structural subunit of chromatin is the nucleosome core, which contains 147 bp of DNA wrapped 1.7 times around a central histone octamer composed of two molecules each of the four core histones (H2A, H2B, H3 and H4). Generally, nucleosomes are regularly spaced along the DNA, like beads on a string. At physiological salt concentrations, the beads-on-a-string structure folds spontaneously to form a fiber 30 nm wide, assisted by the linker histone (H1), which binds to the nucleosome core and to the linker DNA. Thus, collectively, the histones determine DNA accessibility. Gene activation involves the recruitment of a set of factors to a promoter in response to appropriate signals, ultimately resulting in the formation of an initiation complex by RNA polymerase II (Pol II) and transcription. However, Pol II must negotiate nucleosomes, which are compact structures capable of blocking transcription at every step. To circumvent and regulate this chromatin block, eukaryotic cells possess dedicated enzymes, including ATP-dependent chromatin remodeling machines, histone modifying complexes and histone chaperones. The remodeling machines use ATP to move nucleosomes along or off DNA (e.g. the SWI/SNF, RSC, CHD and ISWI complexes), or to exchange histone variants between nucleosomes (e.g. the SWR complex). The histone modifying complexes contain enzymes which modify the histones post-translationally to alter their DNA-binding properties and to mark them for recognition by other complexes, which have activating or repressive roles (the histone code hypothesis). Histone-modifying enzymes include histone acetylases (HATs), deacetylases (HDACs), methylases and kinases. Histone chaperones mediate histone transfer reactions that occur during transcription and DNA replication (e.g. FACT, Asf1 and the CAF-1 complex). These enzymes, together with DNA methylating and de-methylating enzymes, are central to epigenetics. Many human diseases have been linked to chromatin remodeling enzymes and epigenetic modifications. For example, mutations in the hSNF5 subunit of the SWI/SNF complex are strongly linked to pediatric rhabdoid tumors. The CHD class of ATP-dependent remodelers has also been linked to cancer and to autism. Cancer therapies and drugs aimed at epigenetic targets are being tested. Recent studies have revealed a correlation between a linker histone variant and tumor heterogeneity. A full understanding of chromatin structure and the mechanisms by which it is manipulated is therefore vital.
Our aim i s to dissect chromatin remodeling mechanisms in vivo and to understand their contributions to gene regulation. Our current efforts are focused on elucidating the contributions of the various ATP-dependent chromatin remodeling complexes to chromatin organization in vivo. During this Fiscal Year, we have made significant progress towards understanding the contribution of promoter and enhancer chromatin architecture to gene regulation using both mouse and budding yeast (Saccharomyces cerevisiae) as model organisms. The mechanism by which glucocorticoid hormone controls gene expression is central to an understanding of its role in human metabolism and disease. When the hormone enters a cell, it binds to the glucocorticoid receptor (GR), inducing its translocation from the cytosol to the nucleus, where it binds to glucocorticoid response elements (GREs) to activate or repress target genes. Importantly, GR binds to different GREs in different types of cell, resulting in different patterns of gene expression. This observation raises the question of what determines which sites are bound and which are not. Although the answer is unclear, chromatin structure certainly plays a major role. We collaborated with Dr. Gordon Hager's lab in the National Cancer Institute to study the interaction of glucocorticoid receptor with enhancer chromatin in mouse adenocarcinoma cells (1). We found that enhancers containing GREs occur in one of three major chromatin architectures: they can be nucleosome-depleted, marked by the histone variant H2A.Z in the flanking nucleosomes and associated with the Brg1 SWI/SNF remodeler, or they can be covered by a nucleosome containing H2A.Z and associated with Brg1, or they can be nucleosomal and lack both H2A.Z and Brg1. Hormone-induced GR binding results in nucleosome shifts and increased levels of Brg1 at all three types of GR-enhancer. We propose that nucleosome-depleted GR-enhancers are created and maintained by hormone-independent transcription factors which recruit Brg1, allowing GR to bind to non-nucleosomal DNA (a conventional mode of transcription factor binding). In contrast, at nucleosomal GR-enhancers, GR binds like a pioneer factor (i.e., a transcription factor which binds to protein-free and nucleosomal cognate sites with similar affinities) and then recruits Brg1 to remodel the local chromatin structure, promoting downstream events in gene regulation. Transcription through chromatin by RNA polymerase II is facilitated by various factors, including the SPT6 histone chaperone. We have collaborated with Dr. Keiko Ozato's lab (NICHD) to study the role of SPT6 in interferon-induced transcription in mouse cells (2). We found that SPT6 recruitment to interferon-induced genes depends on an interaction with the histone H3-K36 methyltransferase NSD2, which boosts transcription of these genes. We continued our yeast studies in a collaboration with Dr. Alan Hinnebusch's lab (NICHD) and Dr. Chhabi Govind's lab (Oakland University, Michigan). Firstly, we found that the majority of sequence-specific DNA binding sites for the Gcn4 transcription factor are not in gene promoters as expected, but inside coding regions in vivo. Moreover, Gcn4-bound sites within coding regions are not associated with the NDRs typically observed at promoter sites. Nevertheless, many of these internal Gcn4 sites activate cryptic internal promoters and, in some cases, activate neighboring promoters (3). Secondly, we explored the roles of the related RSC and SWI/SNF remodelers in gene activation (4). We discovered that RSC and SWI/SNF cooperate to remodel promoter chromatin architecture at highly expressed genes, which typically have much wider NDRs than relatively inactive genes. At less active promoters, RSC is more important for chromatin remodeling than SWI/SNF. Finally, we wrote a review assessing our current understanding of the determinants of nucleosome positioning (5).

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14
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2018
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U.S. National Inst/Child Hlth/Human Dev
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Rawal, Yashpal; Chereji, R?zvan V; Valabhoju, Vishalini et al. (2018) Gcn4 Binding in Coding Regions Can Activate Internal and Canonical 5' Promoters in Yeast. Mol Cell 70:297-311.e4
Chereji, R?zvan V; Clark, David J (2018) Major Determinants of Nucleosome Positioning. Biophys J 114:2279-2289
Rawal, Yashpal; Chereji, R?zvan V; Qiu, Hongfang et al. (2018) SWI/SNF and RSC cooperate to reposition and evict promoter nucleosomes at highly expressed genes in yeast. Genes Dev 32:695-710
Johnson, Thomas A; Chereji, Razvan V; Stavreva, Diana A et al. (2018) Conventional and pioneer modes of glucocorticoid receptor interaction with enhancer chromatin in vivo. Nucleic Acids Res 46:203-214
Ouda, Ryota; Sarai, Naoyuki; Nehru, Vishal et al. (2018) SPT6 interacts with NSD2 and facilitates interferon-induced transcription. FEBS Lett 592:1681-1692
Maree, Johannes Petrus; Povelones, Megan Lindsay; Clark, David Johannes et al. (2017) Well-positioned nucleosomes punctuate polycistronic pol II transcription units and flank silent VSG gene arrays in Trypanosoma brucei. Epigenetics Chromatin 10:14
Chereji, R?zvan V; Ocampo, Josefina; Clark, David J (2017) MNase-Sensitive Complexes in Yeast: Nucleosomes and Non-histone Barriers. Mol Cell 65:565-577.e3
Ocampo, Josefina; Chereji, R?zvan V; Eriksson, Peter R et al. (2016) The ISW1 and CHD1 ATP-dependent chromatin remodelers compete to set nucleosome spacing in vivo. Nucleic Acids Res 44:4625-35
Qiu, Hongfang; Chereji, R?zvan V; Hu, Cuihua et al. (2016) Genome-wide cooperation by HAT Gcn5, remodeler SWI/SNF, and chaperone Ydj1 in promoter nucleosome eviction and transcriptional activation. Genome Res 26:211-25
Ocampo, Josefina; Cui, Feng; Zhurkin, Victor B et al. (2016) The proto-chromatosome: A fundamental subunit of chromatin? Nucleus 7:382-7

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