Chromatin Remodeling and Gene Activation

染色质重塑和基因激活

• 批准号: 10468553
• 负责人: david j clark
• 金额: $ 125.37万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/10468553
• 关键词: ATAC-seq; ATP phosphohydrolase; Affect; Affinity; Binding; Binding Sites; Biotin; CHD1 gene; Cell Nucleus; Cells; ChIP-seq; Chemicals; Childhood; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Code; Collaborations; Complex; Cysteine; DNA; DNA Sequence; Data; Dinucleosome; Disease; Engineering; Ensure; Enzymes; Epigenetic Process; Eukaryotic Cell; Event; Excision; Exhibits; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; HMGN Proteins; Histones; ISWI; Link; Linker DNA; Maleimides; Malignant Neoplasms; Maps; Measures; Mediating; Membrane Proteins; Methods; Methyltransferase; Minor; Modeling; Modification; Molecular Conformation; Mutate; Mutation; Nucleosomes; Null Lymphocytes; Organism; Pharmaceutical Preparations; Phase; Phenocopy; Play; Positioning Attribute; Property; RNA Polymerase II; Reagent; Repression; Resolution; Rhabdoid Tumor; Role; SWI/SNF Family Complex; Saccharomycetales; Sequence-Specific DNA Binding Protein; Signal Transduction; Site; Streptavidin; Structure; Sulfhydryl Compounds; Surface; System; Testing; Transcript; Transcription Initiation Site; Universities; Yeasts; attenuation; autism spectrum disorder; chromatin remodeling; crosslink; design; dimer; genome-wide; high throughput technology; human disease; mutant; nuclease; pandemic disease; prevent; promoter; recruit; response; transcription factor; wound

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 and transcription. These events coincide with the removal of promoter nucleosomes to create a nucleosome-depleted region (NDR). This observation has led to the generally accepted model that promoter nucleosomes physically block transcript initiation, acting as repressors by preventing access to specific transcription factor binding sites. The nucleosome is a very stable structure containing tightly wound DNA that is largely inaccessible to sequence-specific DNA binding proteins. Activation occurs if sequence-specific 'pioneer' transcription factors are present (these proteins bind nucleosomal sites with high affinity), and/or if 'classical' transcription factors, which are normally blocked by nucleosomes, recruit ATP-dependent chromatin remodelers to move or evict promoter nucleosomes, thus facilitating initiation complex formation. The ATP-dependent chromatin remodelers variously move nucleosomes along DNA, or remove the histones altogether, or form arrays of regularly spaced nucleosomes. Examples include the SWI/SNF and RSC complexes, which remodel nucleosomes on genes and at promoters, and the CHD and ISWI complexes, some of which are involved in determining nucleosome spacing. The INO80C complex is unusual because it has both properties. Human diseases have been linked to chromatin remodeling enzymes. For example, mutations in the hSNF5 subunit of the SWI/SNF complex are strongly linked to pediatric rhabdoid tumors, and the CHD remodelers have been linked to cancer and autism. Therapies and drugs aimed at epigenetic targets are being tested. A full understanding of chromatin structure and the mechanisms by which it is manipulated is therefore vital. Our activities this year were severely curtailed by the pandemic, but we were still able to complete two projects. In the first, we focused on understanding the roles of the two forms of the ISW1 complex in yeast (1). Two different ATPdependent nucleosomespacing enzymes are required to establish regular arrays of phased nucleosomes near transcription start sites of yeast genes: Isw1 and Chd1. Cells lacking both Isw1 and Chd1 have extremely disrupted chromatin, such that the nucleosomes are poorly spaced, and poorly positioned relative to the transcription start site. We have shown previously that this chromatin disorganization is at least partly due to a propensity in the double mutant to form closepacked dinucleosomes near the beginnings of genes (i.e., two nucleosomes jammed close together with no intervening linker DNA). Reaching a mechanistic understanding of the roles of the different remodelers is complicated by the fact that the Isw1 ATPase subunit occurs in two different remodeling complexes: ISW1a (composed of Isw1 and Ioc3) and ISW1b (composed of Isw1, Ioc2 and Ioc4). We constructed yeast strains with various combinations of deletions in the Ioc2/3/4 subunits and determined their chromatin organization using MNase-seq, which provides a detailed nucleosome map for each strain. We discovered that ISW1b is primarily responsible for setting nucleosome spacing and resolving closepacked dinucleosomes, whereas ISW1a plays only a minor role. ISW1b and Chd1 make additive contributions to dinucleosome resolution, such that neither enzyme is capable of resolving all dinucleosomes on its own. Since the Ioc4 subunit of ISW1b binds preferentially to the histone H3K36me3 mark, which is mediated by the Set2 H3K36 methyltransferase and associated with active transcription, we tested whether loss of Set2 phenocopies loss of Ioc4. We found that dinucleosome levels are higher in cells lacking Set2, as is true for cells lacking Ioc4 (ISW1b), but, unlike ioc4-null cells, set2-null cells exhibit only a weak effect on nucleosome spacing. We propose that Set2mediated H3K36 trimethylation is important for ISW1bmediated dinucleosome separation which, in turn, may be important for facilitating the passage of RNA polymerase II through the nucleosomes. We conclude that the nucleosome spacing and dinucleosome resolving activities of ISW1b and Chd1 are critical for normal global chromatin organization, whereas ISW1a plays little or no role in chromatin organization at the global level. Thus, ISW1b is responsible for the effects of Isw1 on global chromatin structure; however, the role of ISW1a is still unclear. Almost all methods designed to probe global chromatin structure measure DNA accessibility to nucleases (e.g. MNase-seq and ATAC-seq) or employ chemical crosslinking (e.g. ChIP-seq and HiC). In a collaboration with Jeff Hayes (University of Rochester), we have developed a method to measure genome-wide accessibility of histone protein surfaces within nucleosomes by assessing reactivity of engineered cysteine residues with biotin-maleimide (BM), a thiol-specific reagent (2). We determined the accessibility of three different histone residues by mutating them to cysteine in the appropriate yeast histone gene and then treating purified nuclei with BM. The chromatin is digested to mono-nucleosomes, streptavidin is used to purify modified nucleosomes, and the nucleosomal DNA is subjected to paired-end sequencing. Sequence data for modified nucleosomes are compared with data for input nucleosomes. In the first case, we measured the accessibility of H2B-S116C, which is located near the center of the external flat protein surface of the nucleosome. This residue might be expected to be accessible, unless the chromatin is fully condensed, preventing BM modification. We found that it is generally accessible throughout the genome, suggesting that this histone surface is not usually obscured by tight nucleosome packing. In the second and third cases, we investigated the accessibility of cysteine residues buried inside the nucleosome, which might be exposed if the nucleosome is conformationally altered during transcription, as suggested by studies from other labs: H3-S102C, located at the H2AH2B dimer/H3H4 tetramer interface, and H3-A110C, located at the H3H3 interface. However, we found that neither of these internal sites are significantly exposed. In conclusion, our data are consistent with a global, relatively decondensed chromatin structure, containing nucleosomes that do not generally undergo major conformational changes.

基因激活涉及到一系列因子被募集到启动子中，以响应适当的信号，最终通过RNA聚合酶II和转录形成起始复合物。这些事件与启动子核小体的去除产生核小体耗尽区（NDR）相吻合。这一观察结果导致了普遍接受的模型，即启动子核小体物理上阻断转录起始，通过阻止进入特定的转录因子结合位点而起到抑制因子的作用。核小体是一种非常稳定的结构，含有紧密缠绕的DNA，序列特异性DNA结合蛋白在很大程度上是无法接近的。如果序列特异性的“先锋”转录因子存在（这些蛋白质以高亲和力结合核小体位点），和/或通常被核小体阻断的“经典”转录因子招募依赖atp的染色质重塑子移动或驱逐启动子核小体，从而促进起始复合物的形成，就会发生激活。