Epigenetic silencing by histone methylation

组蛋白甲基化导致的表观遗传沉默

• 批准号: 6520587
• 负责人: JUDD C RICE
• 金额: $ 4.42万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-25 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6520587
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; Schizosaccharomyces pombe; animal tissue; autoradiography; fluorescence microscopy; gene induction /repression; gene mutation; heterochromatin; histones; immunofluorescence technique; immunoprecipitation; methylation; methyltransferase; polymerase chain reaction; posttranslational modifications; protein binding; protein structure function; scintillation counter

A recent report from this laboratory suggests that histone methylation of histone H3, specifically on lysine 9, is associated with transcriptionally silent regions of heterochromatin. In this proposal, we test the hypothesis that the methylation of lysine 9 on histone H3 is dictated by a conserved family of chromatin modifying proteins containing the SET domain. In addition, we propose that heterochromatin-associated proteins preferentially bind methyl-lysine 9 on histone H3 by their conserved chromo domain; the binding of which ultimately leads to heterochromatinization and gene silencing. To test these hypotheses in vitro and in vivo, we will be investigating the putative histone methyltransferase, Clr4, and heterochromatin-associated protein, Swi6, of S. pombe. The specific histone and residue methylated by Clr4 will be identified in the histone methyltransferase assay. Deletions and mutations of the Clr4 SET domain will be created to determine the exact region and/or residue responsible for methylation. We will employ BIAcore technology to define the specific interaction and kinetics of Swi6 binding to methyl-lysine 9 of histone H3. In addition, mutants of the Swi6 chromo domain will be generated and analyzed to determine binding of methyl-lysine 9 on histone H3. We will determine if Swi6 co- localizes selectively with methyl-lysine 9 histone H3 in vivo, by immunoprecipitations and immunofluorescence. In addition, we will determine the effects of Clr4 mutations on Swi6 localization. The long range goal of this research is to establish and understand a mechanistic link between histone methylation and heterochromatin-associated. proteins and how the misregulation or mistargeting of either is associated with human disease.

该实验室的最新报告表明，组蛋白H3的组蛋白甲基化，特别是在赖氨酸9上，与异染色质的转录沉默区域有关。在此提案中，我们检验了一个假设，即组蛋白H3上赖氨酸9的甲基化由保守的染色质家族修饰含有固定结构域的蛋白质。此外，我们建议异染色质相关蛋白优先结合甲基赖氨酸9在组蛋白H3上通过其保守的Chromo结构域结合。其结合最终导致异染色性化和基因沉默。为了在体外和体内检验这些假设，我们将研究POMBE S. POMBE的推定组蛋白甲基转移酶，CLR4和异染色质相关蛋白Swi6。在组蛋白甲基转移酶测定中将鉴定由CLR4甲基化的特异性组蛋白和残基。将创建CLR4集域的缺失和突变，以确定负责甲基化的确切区域和/或残基。我们将采用BIACORE技术来定义SWI6与组蛋白H3的甲基赖氨酸9结合的特定相互作用和动力学。另外，将生成并分析SWI6 Chromo结构域的突变体，以确定甲基赖氨酸9对组蛋白H3的结合。我们将通过免疫沉淀和免疫荧光来确定SWI6是否在体内选择性地与甲基赖氨酸9组蛋白H3合作。此外，我们将确定CLR4突变对SWI6定位的影响。这项研究的远距离目标是建立和理解组蛋白甲基化与异染色质相关之间的机械联系。蛋白质以及任何一种与人类疾病有关的错误调节或误导。