Mechanistic Studies of silent chromatin spreading

描述（由申请人提供）：表观遗传染色质沉默被真核生物用于稳定染色体的结构区域或阻止基因表达。沉默染色质形成的一个保守特征是抑制因子复合物沿着染色质纤维的扩散，其机制涉及组蛋白结合蛋白的自结合。Sir3是出芽酵母中沉默信息调节器（SIR）复合体的一个组成部分，与去乙酰化的组蛋白尾部结合，并在体外形成低聚物。我建议通过关注Sir3自结合的作用来研究沉默染色质扩散的驱动力。我将确定Sir3在体外有助于自结合的结构域，并研究自结合如何被其他Sir蛋白、组蛋白和小分子辅助因子调节。同时，我将描述Sir3突变体在体内对沉默有显性负作用。作为一个长期目标，我还将致力于在体外重建核小体阵列上沉默染色质的逐步形成。对这一系统的全面重建对于检验本建议的早期目标得出的结论以及基于遗传研究的许多其他理论将是非常宝贵的。驱动沉默染色质扩散的物理相互作用也可能有助于这些区域的维持和表观遗传。

DESCRIPTION (provided by applicant): Epigenetic chromatin silencing is used by eukaryotic organisms to stabilize structural regions of the chromosome or prevent gene expression. A conserved feature of silent chromatin formation is the spreading of repressor complexes along the chromatin fiber by a mechanism that involves the self-association of histone- binding proteins. Sir3, a component of the silent information regulator (SIR) complex in budding yeast, binds to deacetylated histone tails and can form oligomers in vitro. I propose to study the driving forces of silent chromatin spreading by focusing on the role of Sir3 self-association. I will determine the domains of Sir3 that contribute to self-association in vitro and examine how self-association is modulated by other Sir proteins, histones, and small-molecule cofactors. In parallel, I will characterize Sir3 mutants that have a dominant- negative effect on silencing in vivo. As a longer-term goal, I will also work towards reconstituting the step-wise formation of silent chromatin on nucleosomal arrays in vitro. A full reconstitution of this system will be invaluable for testing the conclusions from the earlier aims of this proposal, as well as many other theories based on genetic studies. The physical interactions that drive silent chromatin spreading are also likely to contribute to maintenance and epigenetic inheritance of these regions.