Mechanistic Studies of silent chromatin spreading

描述(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防止基因表达。无声染色质形成的一个保守特征是阻遏复合体通过一种涉及组蛋白结合蛋白自结合的机制沿着染色质纤维扩散。Sir3是发芽酵母中沉默信息调节(SIR)复合体的一种成分，它与去乙酰化的组蛋白尾巴结合，在体外可以形成寡聚体。我建议通过重点研究Sir3自关联的作用来研究沉默染色质传播的驱动力。我将确定在体外有助于自关联的Sir3结构域，并研究其他SIR蛋白、组蛋白和小分子辅助因子如何调节自关联。同时，我将描述对体内沉默有显性-负面影响的Sir3突变体的特征。作为一个较长期的目标，我还将致力于在体外重建核小体阵列上无声染色质的逐步形成。对这一系统的全面重建将对检验这一提案早期目标的结论以及基于基因研究的许多其他理论具有无价的价值。推动染色质静默传播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.