Epigenetic silencing by histone methylation

该实验室最近的一份报告表明，组蛋白H3的组蛋白甲基化，特别是在赖氨酸9上，与异染色质的转录沉默区域有关。在这个建议中，我们测试了组蛋白H3上赖氨酸9的甲基化是由包含SET结构域的一组保守的染色质修饰蛋白家族决定的假设。此外，我们认为异染色质相关蛋白通过其保守的染色质结构域优先结合组蛋白H3上的甲基赖氨酸9，这种结合最终导致异染色质和基因沉默。为了在体外和体内验证这些假说，我们将研究庞氏葡萄球菌可能的组蛋白甲基转移酶CLR4和异染色质相关蛋白Swi6。由CLR4甲基化的特异性组蛋白和残基将在组蛋白甲基转移酶检测中被鉴定。将创建CLR4集合域的缺失和突变，以确定导致甲基化的确切区域和/或残基。我们将使用Biacore技术来确定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，还将产生Swi6染色域的突变体并进行分析，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来确定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，我们还将确定CLR4突变对Swi6定位的影响。这项研究的长期目标是建立和了解组蛋白甲基化和异染色质相关的机制联系。蛋白质的错误调节或错误定位与人类疾病之间的关系。

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.