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.

该实验室最近的一份报告表明，组蛋白 H3 的组蛋白甲基化（特别是赖氨酸 9）与异染色质的转录沉默区域相关。在本提案中，我们测试了以下假设：组蛋白 H3 上赖氨酸 9 的甲基化是由包含 SET 结构域的染色质修饰蛋白保守家族决定的。此外，我们提出异染色质相关蛋白通过其保守的染色结构域优先结合组蛋白 H3 上的甲基赖氨酸 9；其结合最终导致异染色质化和基因沉默。为了在体外和体内测试这些假设，我们将研究粟酒裂殖酵母的假定组蛋白甲基转移酶 Clr4 和异染色质相关蛋白 Swi6。 Clr4 甲基化的特定组蛋白和残基将在组蛋白甲基转移酶测定中进行鉴定。将创建 Clr4 SET 结构域的删除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。此外，将生成并分析 Swi6 染色结构域的突变体，以确定甲基赖氨酸 9 与组蛋白 H3 的结合。我们将通过免疫沉淀和免疫荧光确定 Swi6 是否在体内选择性地与甲基赖氨酸 9 组蛋白 H3 共定位。此外，我们将确定 Clr4 突变对 Swi6 定位的影响。这项研究的长期目标是建立和理解组蛋白甲基化和异染色质相关之间的机制联系。蛋白质以及两者的错误调节或错误定位如何与人类疾病相关。