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: 9153821
• 负责人: shivinder s grewal
• 金额: $ 190.49万
• 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/9153821
• 关键词: Acetylation; Amino Acids; Antineoplastic Agents; Binding; Biochemical; Biological Assay; 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; Euchromatin; Eukaryota; Eukaryotic Cell; Event; Family; Fission Yeast; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genetic Screening; Genetic Transcription; Genome; 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; genome integrity; histone methyltransferase; histone modification; human disease; inhibitor/antagonist; insight; malignant breast neoplasm; methylation pattern; 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 Clr3, Clr4 and Clr6) are involved in modification 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. Identification 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. We also gained insight into the significance of the role of HDAC in regulating histone turnover. By using a newly developed assay, we were able to detect differential turnover rates at heterochromatin and euchromatin domains. Interestingly, we found that defects in RNAi machinery, which is required to establish the H3K9me mark for HP1 recruitment, cause increased histone turnover. Similarly, we found that defects in HP1, or the associated histone deacetylase (HDAC) activity, also cause increased histone turnover. This work has yielded a novel insight into the role of HDACs, which are recruited by HP1 or other factors, in precluding histone turnover to promote silencing and inheritance of heterochromatin. These findings have implications for our understanding of heterochromatin assembly in higher eukaryotes, as the machinery and activities that operate in fission yeast are often conserved.

我们在S. pombe的研究强调了组蛋白修饰在高阶染色质组装中的作用，表明在异染色质沉默缺陷突变（如Clr3， Clr4和Clr6）的遗传筛选中发现的几个因素参与了组蛋白尾部的修饰。其中，Clr4属于高度保守的组蛋白甲基转移酶Suv39家族，该家族在与重复DNA元件相关的异色结构域上特异性地将赖氨酸9 （H3- k9）上的组蛋白H3甲基化。生化分析表明，Clr4是一个多亚基复合物的组成部分，该复合物含有cullin家族蛋白Cul4，作为组装泛素连接酶的支架，以及WD蛋白Rik1，通过RNA聚合酶II转录偶联过程介导Clr4活性募集到目标重复位点。Clr3和Clr6是组蛋白十乙酰化酶，与人类II类和I类hdac具有很强的同源性。我们已经证明Clr6存在于至少两个不同的核心复合物中。其中一种复合物（Clr6-C1）主要针对基因启动子，并负责通过组蛋白的局部去乙酰化调节基因表达。第二个Clr6复合体（Clr6- cii）靶向转录的染色体区域和着丝点位点，负责组蛋白的全局去乙酰化。我们的分析表明，Clr6-CII的缺陷取消了染色质的全局保护功能，如抑制反义转录物、特异性抑制异染色质重复序列和保护DNA免受基因毒性物质损伤。我们还对Clr3进行了生化表征。Clr3存在于称为SHREC的多酶效应复合体中，除了与Clr3相关的组蛋白十乙酰化酶活性外，还含有Snf2家族染色质重塑因子Mit1。我们已经证明，SHREC是所有主要异染色质结构域的靶标，其活性对于核小体的正确定位以组装高阶染色质结构至关重要，而高阶染色质结构对异染色质功能至关重要。我们正在继续研究这些和其他组蛋白修饰活性的功能。考虑到组蛋白修饰因子在物种间是保守的，并控制着基本的染色体过程，包括在发育过程中基因表达模式的稳定维持和基因组完整性的维持，对它们的深入了解对于开发治疗癌症和其他人类疾病的有效治疗措施非常重要。异染色质在特定位点成核，其扩散方式取决于组蛋白十乙酰化酶、异染色质蛋白的活性和Clr4甲基化H3- k9的能力，以及通过其色域与甲基化的H3尾部结合的能力。此外，H3-K9的甲基化对于HP1蛋白如Swi6、Chp2和Chp1的募集是必不可少的。我们的研究揭示了一个新的主题，即与甲基化H3-K9结合的HP1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适当分离的蛋白。Chp1是RITS复合体的一个组成部分，将RNAi机制连接到异染色质位点上，促进顺式中重复序列的转录后沉默。然而，Chp2和Swi6在异染色质组装中的确切功能及其与其他因素的关联尚不清楚。我们最近发现Swi6和Chp2与Clr6和SHREC组蛋白去乙酰化酶复合物结合，这对于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Swi6和Chp2蛋白及其相关的HDAC复合物在限制RNA聚合酶II占用周围中心异染色质结构域方面具有重叠功能。有趣的是，纯化的Swi6片段还含有参与多种染色体过程的因子，如染色质重塑和DNA复制。此外，Swi6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内聚所必需的内聚因子，该变体以异染色质依赖的方式结合到染色质中。这些分析表明，HP1蛋白与多种因素相关，包括抑制染色质组装所必需的组蛋白修饰因子。确定HP1相关因子及其在染色质组装中的作用可能有助于我们了解与HP1表达改变相关的乳腺癌的原因。尽管HP1蛋白对于组蛋白去乙酰化酶在异染色质结构域中优先招募重复元件至关重要，但存在其他机制将这些活性靶向于分散在整个基因组中的重复序列。具体来说，我们发现了一种新的反转录转座子的基因组监测机制，该机制由转座子衍生的CENP-B同源物家族组成。我们发现，CENP-Bs定位并募集组蛋白去乙酰化酶来沉默反转录转座子。这种机制也抑制分布在S. pombe基因组中的反转录转座子残基。cenp - b介导的监测是主动的，能够阻止已灭绝的反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的反转录转座子监视途径，并表明真核细胞具有一系列抑制因子活性，这些抑制因子要么通过HP1蛋白跨大结构域靶向，要么以特定位点的方式被CENP-B和其他DNA结合因子靶向。我们还深入了解了HDAC在调节组蛋白转换中的重要作用。通过使用新开发的分析，我们能够检测异染色质和常染色质结构域的差异周转率。有趣的是，我们发现建立H3K9me标记以募集HP1所需的RNAi机制缺陷导致组蛋白周转增加。同样，我们发现HP1或相关组蛋白去乙酰化酶（HDAC）活性的缺陷也会导致组蛋白周转增加。这项工作对hdac的作用产生了新的见解，hdac由HP1或其他因子募集，在阻止组蛋白转换以促进异染色质的沉默和遗传中发挥作用。这些发现对我们理解高等真核生物中的异染色质组装具有重要意义，因为在裂变酵母中运作的机制和活动通常是保守的。