Roles of Chromatin-modifying Factors in Epigenetic Control of the Genome

染色质修饰因子在基因组表观遗传控制中的作用

• 批准号: 8349397
• 负责人: shivinder s grewal
• 金额: $ 109.25万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8349397
• 关键词: Acetylation; Amino Acids; Antineoplastic Agents; Binding; Biochemical; Cancer Biology; Cell physiology; Centromere; Chromatin; Chromatin Modeling; Chromatin Remodeling Factor; Chromatin Structure; Chromosome Segregation; Chromosome Structures; Complex; Coupled; Cullin Family Protein; DNA; DNA Binding; DNA Damage; DNA biosynthesis; Defect; Development; Elements; Enzymes; Epigenetic Process; Eukaryotic Cell; Event; Family; Fission Yeast; Gene Expression; Gene Expression Regulation; Gene Targeting; Genetic Screening; Genetic Transcription; Genome; Genomics; Heterochromatin; Higher Order Chromatin Structure; Histone Deacetylase; Histone Deacetylation; Histone H3; Histones; Homologous Gene; Human; Link; Lysine; Maintenance; Malignant Neoplasms; Measures; Mediating; Methylation; Modification; Molecular; Mutagens; Nucleosomes; Pathway interactions; Pattern; Phosphorylation; Positioning Attribute; Post-Translational Protein Processing; Process; Protein Binding; Proteins; RNA Interference; RNA Polymerase II; Recruitment Activity; Repression; Research; Retrotransposon; Role; Sister Chromatid; Site; Structure; Tail; Therapeutic; Transcript; Transposase; Variant; Work; cancer therapy; cell type; centromere autoantigen 80K; centromere protein A; chromatin remodeling; cohesin; cohesion; histone methyltransferase; histone modification; human disease; inhibitor/antagonist; malignant breast neoplasm; mutant; novel; prevent; promoter; scaffold; ubiquitin ligase

The involvement of histone modifications in higher-order chromatin assembly has been highlighted by our studies in S. pombe showing that several factors identified in genetic screen for mutants defective in heterochromatic silencing (such as such as Clr3, Clr4 and Clr6) are involved in modifications of histone tails. Among these, Clr4 belongs to a highly conserved Suv39 family of histone methyltransferases, which specifically methylate histone H3 at lysine 9 (H3-K9) across heterochromatic domains associated with repetitive DNA elements. Biochemical analysis has shown that Clr4 is a component of multisubunit complex containing a cullin family protein Cul4 that serves as scaffold to assemble ubiquitin ligases, and a WD protein Rik1 which mediates recruitment of Clr4 activity to the target repeat loci via a RNA polymerase II transcription coupled process. Clr3 and Clr6 are histone decaetylases with strong homologies to class II and class I HDACs from humans. We have shown that Clr6 exists in at least two distinct core complexes. One of these complexes (Clr6-C1) predominantly targets gene promoters and is responsible for regulation of gene expression through local deacetylation of histones. The second Clr6 complex (Clr6-CII) that targets transcribed chromosomal regions and centromeric loci is responsible for global deacetylation of histones. Our analyses suggest that defects in Clr6-CII abrogate global protective functions of chromatin such as suppression of antisense transcripts, strand-specific repression of heterochromatic repeats and protection of DNA from damage by genotoxic agents. We have also performed biochemical characterization of Clr3. Clr3 exists in a multienzyme effector complex termed SHREC that in addition to histone decaetylase activity associated with Clr3 contains a Snf2 family chromatin remodeling factor Mit1. We have shown that SHREC is targeted across all major heterochromatic domains and its activities are essential for proper positioning of nucleosomes to assemble higher-order chromatin structures, critical for heterochromatin functions. We are continuing to investigate the functions of these and other histone modifying activities. Given that histone modifiers are conserved among species and control fundamental chromosomal processes including stable maintenance of gene expression patterns during development and maintenance of genomic integrity, their deeper understanding is important for the development of effective therapeutic measures for treatment of cancer and other human diseases. Heterochromatin nucleated at specific sites spread in a manner that depends upon the activities of histone decaetylases, heterochromatin proteins and the ability of Clr4 to both methylate H3-K9 as well as bind to methylated H3 tail via its chromodomain. Moreover, methylation of H3-K9 is essential for recruitment of HP1 proteins such as Swi6, Chp2 and Chp1. Our research has unraveled a new theme wherein HP1 proteins bound to methylated H3-K9 provide a dynamic platform for factors involved in many cellular processes, including proteins involved in cell-type switching and proper segregation of chromosomes. Chp1, a component of the RITS complex tethers RNAi machinery to heterochromatic loci, facilitating post-transcriptional silencing of repeats in cis. However, the exact functions of Chp2 and Swi6 in heterochromatin assembly and their associations with other factors were poorly understood. We recently showed that Swi6 and Chp2 associate with Clr6 and SHREC histone deacetylase complexes, which are critical for transcriptional silencing of the heterochromatic centromeric repeats. This work further revealed that Swi6 and Chp2 proteins and their associated HDAC complexes have overlapping functions in limiting RNA polymerase II occupancy across pericentromeric heterochromatin domains. Interestingly, purified Swi6 fraction also contains factors involved in a variety of chromosomal processes such as chromatin remodeling and DNA replication. In addition, Swi6 co-purifies a cohesin loading factor essential for sister chromatid cohesion, and with centromere-specific histone H3 variant CENP-A, which is incorporated into chromatin in a heterochromatin-dependent manner. These analyses suggest that HP1 proteins associate with a variety of factors including histone-modifying factors essential for the assembly of repressive chromatin. Identifications of HP1 associated factors and their role in chromatin assembly may help us understand the causes of breast cancer associated with altered HP1 expression. Although HP1 proteins are critical for the preferential recruitment of histone deacetylases to repeat elements within heterochromatin domains, alternative mechanisms exist to target these activities to repeats dispersed across the genome. Specifically, we have uncovered a novel genome surveillance mechanism for retrotransposons by a family of transposase-derived CENP-B homologs. We found that CENP-Bs localize at and recruit histone deacetylases to silence retrotransposons. This mechanism also represses retrotransposon relics scattered throughout the S. pombe genome. CENP-B-mediated surveillance is proactive, capable of preventing an extinct retrotransposon from reentering the host genome. These results reveal a likely ancient retrotransposon surveillance pathway and suggest that eukaryotic cells have a toolkit of repressor activities that are either targeted across large domains via HP1 proteins or in a site-specific manner by CENP-B and other DNA binding factors.

组蛋白修饰参与高级染色质组装已经在我们对S。粟酒裂殖酵母的研究表明，在异染色质沉默缺陷突变体的遗传筛选中鉴定的几个因子（例如Clr 3、Clr 4和Clr 6）参与组蛋白尾部的修饰。其中，Clr 4属于组蛋白甲基转移酶的高度保守的Suv 39家族，其特异性地在与重复DNA元件相关的异染色质结构域中在赖氨酸9（H3-K9）处甲基化组蛋白H3。生物化学分析表明，Clr 4是多亚基复合物的一个组成部分，该复合物含有cullin家族蛋白Cul 4和WD蛋白Rik 1，cullin家族蛋白Cul 4作为组装泛素连接酶的支架，WD蛋白Rik 1通过RNA聚合酶II转录偶联过程介导Clr 4活性向靶重复位点的募集。Clr 3和Clr 6是与来自人类的II类和I类HDAC具有强同源性的组蛋白十乙酰基化酶。我们已经表明，Clr 6存在于至少两个不同的核心复合物。这些复合物之一（Clr 6-C1）主要靶向基因启动子，并负责通过组蛋白的局部去乙酰化来调节基因表达。第二个Clr 6复合物（Clr 6-CII）靶向转录的染色体区域和着丝粒位点，负责组蛋白的整体脱乙酰化。我们的分析表明，在Clr 6-CII的缺陷废除全球保护功能的染色质，如抑制反义转录，链特异性抑制异染色质重复和保护DNA免受遗传毒性剂的损害。我们还进行了Clr 3的生化表征。Clr 3存在于一种称为SHREC的多酶效应复合物中，除了与Clr 3相关的组蛋白脱乙酰酶活性外，该复合物还含有Snf 2家族染色质重塑因子Mit 1。我们已经表明，SHREC是针对所有主要的异染色质结构域，其活动是必要的正确定位的核小体组装高阶染色质结构，异染色质功能的关键。我们正在继续研究这些和其他组蛋白修饰活动的功能。鉴于组蛋白修饰剂在物种中是保守的，并且控制基本的染色体过程，包括在发育期间稳定维持基因表达模式和维持基因组完整性，因此对其的更深入理解对于开发用于治疗癌症和其他人类疾病的有效治疗措施是重要的。在特定位点成核的异染色质以依赖于组蛋白十乙酰基化酶、异染色质蛋白的活性和Clr 4甲基化H3-K9以及通过其染色体结构域结合甲基化H3尾的能力的方式扩散。此外，H3-K9的甲基化对于HP 1蛋白如Swi 6、Chp 2和Chp 1的募集是必需的。我们的研究揭示了一个新的主题，其中与甲基化H3-K9结合的HP 1蛋白为参与许多细胞过程的因子提供了一个动态平台，包括参与细胞类型转换和染色体正确分离的蛋白质。Chp 1是RITS复合体的一个组成部分，它将RNAi机制与异染色质基因座连接在一起，促进顺式重复序列的转录后沉默。然而，Chp 2和Swi 6在异染色质组装中的确切功能及其与其他因素的关系知之甚少。我们最近发现Swi 6和Chp 2与Clr 6和SHREC组蛋白脱乙酰酶复合物相关，这对异染色质着丝粒重复序列的转录沉默至关重要。这项工作进一步揭示了Swi 6和Chp 2蛋白及其相关的HDAC复合物在限制RNA聚合酶II在近着丝粒异染色质结构域中的占据方面具有重叠功能。有趣的是，纯化的Swi 6组分还含有参与各种染色体过程的因子，如染色质重塑和DNA复制。此外，Swi 6还与着丝粒特异性组蛋白H3变体CENP-A共同纯化了姐妹染色单体凝聚所必需的凝聚素加载因子，后者以异染色质依赖性方式掺入染色质中。这些分析表明，HP 1蛋白与多种因素，包括组蛋白修饰因子的组装抑制染色质必不可少的。HP 1相关因子的鉴定及其在染色质组装中的作用可能有助于我们了解与HP 1表达改变相关的乳腺癌的原因。虽然HP 1蛋白对于组蛋白脱乙酰基酶优先募集到异染色质结构域内的重复元件是至关重要的，但是存在替代机制来将这些活性靶向分散在基因组中的重复。具体来说，我们已经发现了一种新的基因组监控机制，逆转录转座子转座酶衍生的CENP-B同源家族。我们发现CENP-Bs定位于组蛋白去乙酰化酶并募集组蛋白去乙酰化酶来沉默反转录转座子。这种机制也抑制了散布在整个S.粟酒基因组CENP-B介导的监视是主动的，能够防止灭绝的逆转录转座子重新进入宿主基因组。这些结果揭示了一种可能的古老逆转录转座子监视途径，并表明真核细胞具有一套阻遏物活性，这些阻遏物活性通过HP 1蛋白跨越大结构域靶向，或通过CENP-B和其他DNA结合因子以位点特异性方式靶向。