Mechanistic Studies of silent chromatin spreading

描述（由申请人提供）：真核生物使用表观遗传染色质沉默来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阻遏物复合物沿着染色质纤维通过涉及组蛋白结合蛋白的自缔合的机制扩散。Sir3是芽殖酵母中沉默信息调节因子（SIR）复合物的一种组分，与脱乙酰基组蛋白尾部结合，并可在体外形成寡聚体。我建议研究沉默的染色质扩散的驱动力，专注于Sir3自我协会的作用。我将确定领域的Sir3，有助于在体外的自我协会，并研究自我协会是如何调制的其他爵士蛋白，组蛋白和小分子辅因子。同时，我将表征对体内沉默具有显性负效应的Sir3突变体。作为一个长期的目标，我也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。这个系统的完全重建对于检验这个建议的早期目标的结论以及许多其他基于遗传学研究的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.