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，特别是赖氨酸9，与异染色质的转录沉默区域有关。在该提案中，我们测试了这样的假设：组蛋白H3上的赖氨酸9的甲基化是由包含SET结构域的染色质修饰蛋白的保守家族决定的。此外，我们提出，异染色质相关蛋白优先结合甲基赖氨酸9组蛋白H3的保守染色体结构域，其结合最终导致异染色质化和基因沉默。为了在体外和体内验证这些假设，我们将研究S.粟酒将在组蛋白甲基转移酶试验中鉴定特定的组蛋白和Clr 4甲基化的残基。将产生Clr 4 SET结构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技术来确定Swi 6与组蛋白H3的甲基赖氨酸9结合的特异性相互作用和动力学。此外，将产生并分析Swi 6染色体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确定Swi 6是否在体内与甲基赖氨酸9组蛋白H3选择性共定位。此外，我们将确定Clr 4突变对Swi 6定位的影响。本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性之间的机制联系。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。

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.