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Chromatin Remodeling and Gene Activation

染色质重塑和基因激活

基本信息

批准号：
10001292
负责人：
david j clark
金额：
$ 143.31万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10001292
关键词：
Affect Antineoplastic Agents Architecture Binding Binding Sites CHD1 gene California Cell Nucleus Cells Centromere Childhood Chromatin Chromatin Fiber Chromatin Remodeling Factor Chromatin Structure Chromosome Structures Chromosomes Code Collaborations Complex Copper DNA DNA Binding DNA Polymerase II DNA Sequence DNA biosynthesis DNA-Directed RNA Polymerase Data Deacetylase Defect Disease Dissociation Elements Ensure Enzymes Epigenetic Process Eukaryota Eukaryotic Cell Event Exhibits Frequencies Gene Activation Gene Expression Gene Expression Regulation Gene Silencing Genes Genetic Transcription Genome Goals Heterochromatin Histone Code Histone Deacetylase Histone H1 Histone H2A Histones ISWI Ions Link Linker DNA Malignant Neoplasms Mammals Maps Mediating Mediation Messenger RNA Metallothionein Methyltransferase Modeling Modification Molecular Chaperones Mus Mutate Mutation National Cancer Institute Nature Noise Nuclear Nucleosome Core Particle Nucleosomes Phase Phosphotransferases Physiological Play Polymerase Population Positioning Attribute Property Proteins Reaction Regulation Repression Rhabdoid Tumor Role SWI/SNF Family Complex Saccharomycetales Signal Transduction Site Sodium Chloride Stress Structure System Testing Time Toxic effect Transcription Initiation Site Transfer RNA Universities Variant Yeasts attenuation autism spectrum disorder cancer therapy cell type chromatin remodeling combinatorial genome-wide high throughput technology histone acetyltransferase human disease in vivo promoter recruit response single molecule transcription factor tumor heterogeneity

项目摘要

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. For transcription to occur, the promoter must be cleared of nucleosomes to allow transcription complex formation and then Pol II must negotiate the nucleosomes on the gene. Nucleosomes are compact structures capable of blocking transcription at every step. To circumvent and regulate these chromatin blocks, 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 the DNA methylating and de-methylating enzymes present in higher eukaryotes, 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 is 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 remodeling complexes to chromatin organization in vivo. During this Fiscal Year, we have made significant progress towards understanding the roles of the RSC ATP-dependent remodeling complex in gene regulation. RSC is an essential SWI/SNF-like remodeler that is similar to the mammalian PBAF complex and plays an important role in determining the size of the nucleosome-depleted region (NDR) typically found at potentially active gene promoters. In both mammals and yeast, promoter NDRs are flanked by arrays of regularly spaced nucleosomes, which are phased relative to the transcription start site. We examined the interplay between RSC and the ISW1, CHD1 and ISW2 ATP-dependent nucleosome spacing enzymes in chromatin organization and transcription, using isogenic yeast strains lacking all combinations of these enzymes (1). The contributions of these remodelers to chromatin organization are largely combinatorial, distinct, and nonredundant, supporting a model in which the first (+1) nucleosome on the gene is positioned by RSC and then used as a reference nucleosome by the spacing enzymes to create phased nucleosomal arrays. We observed that defective chromatin organization correlates with altered Pol II distribution: RSC-depleted cells exhibit lower levels of elongating Pol II and higher levels of terminating Pol II, consistent with defects in both termination and initiation, suggesting that RSC facilitates both. Cells lacking both ISW1 and CHD1 show the opposite Pol II distribution, suggesting opposite elongation and termination defects. These cells have extremely disrupted chromatin, with high levels of closely packed di-nucleosomes (i.e. two nucleosomes with no intervening linker DNA), primarily involving the second (+2) nucleosome on the gene (i.e., di-nucleosomes composed of either the +1 and +2 nucleosomes or the +2 and +3 nucleosomes). We propose that ISW1 and CHD1 facilitate Pol II elongation by separating nucleosomes that are too close together. In a collaboration with the Karpova Lab (National Cancer Institute), we investigated the role of RSC in transcription factor dynamics (2). The binding of sequence-specific transcription factors to cognate sites is generally highly dynamic. However, how such binding is linked to chromatin remodeling and transcription is unclear. The CUP1 gene encodes a metallothionein responsible for protecting cells against the toxic effects of copper ions; it is induced when excess copper ions bind to the Ace1p transcription factor, which then binds to upstream activating sequences (UAS elements) in the CUP1 promoter to activate transcription. Using single-molecule tracking (SMT), we showed that RSC reduces the time it takes for the Ace1p transcription factor to locate and bind to the CUP1 promoter. We quantified smFISH mRNA data using a gene bursting model and demonstrated that RSC regulates CUP1 transcription bursts by modulating Ace1p transcription factor occupancy, rather than by affecting initiation and elongation rates. The SMT data show that RSC binds to the activated CUP1 promoter transiently. We also observed that RSC does not affect nucleosome occupancy at CUP1. We propose that transient binding of Ace1p and rapid bursts of CUP1 transcription may depend on short repetitive cycles of RSC-mediated nucleosome mobilization. This type of regulation would reduce transcriptional noise and ensure a homogeneous response of the cell population to copper stress. We also collaborated with the Kamakaka Lab (University of California at Santa Cruz) to investigate the role of tRNA genes in chromosomal organization in the nucleus (3). The genome is packaged and organized in an ordered, nonrandom manner within the nucleus, involving contacts between specific chromatin segments and nuclear substructures. tRNA genes are binding sites for transcription factors as well as architectural proteins and are thought to play an important role in this organization. We tested this hypothesis by removing all tRNA genes from chromosome III, either by deletion or by transfer to another chromosome. Surprisingly, loss of all tRNA genes does not grossly affect chromatin architecture or chromosome tethering and mobility. Loss of tRNA genes does affect local chromatin structure by altering both nucleosome positioning and the binding of SMC proteins, as expected. The absence of tRNA genes also alters centromere clustering and reduces the frequency of long-range heterochromatin clustering with concomitant effects on gene silencing.