The Role of Histone Chaperones in Histone Acetylation and Nucleosome Dynamics

描述(申请人提供)：在每个真核细胞的核内都有一个根本的悖论：遗传物质必须被组织和压缩，但仍然可以被转录机器读出。在维持这种平衡的众多因素中，有两个是组蛋白和组蛋白结合蛋白。组蛋白最终负责将染色体DNA压缩近50万倍，以适应细胞核。虽然基因组的可及性部分受组蛋白乙酰转移酶(KATS)的作用调节，但组蛋白伴侣蛋白直接与组蛋白相互作用，并可以在DNA上组装和/或分解它们。KATS共价修饰组蛋白，因此有可能改变染色质结构。令人兴奋的新证据在结构和功能上将KATS和组蛋白伴侣蛋白联系在一起。然而，关于这些蛋白质合作管理紧凑和基因组可及性的机制，人们几乎一无所知。为了开始理解这一重要的生物学问题，本项目建议以组蛋白乙酰转移酶(KAT)Rtt109为模型系统进行研究。Rtt109使用两个结构上不相关的组蛋白伴侣蛋白，Vps75和ASF1。在体内，Vps75已被证明与Rtt109直接相互作用，但Rtt109的功能只需要ASF1。这两个伴侣在体外都激活了Rtt109乙酰转移酶的活性，但Rtt109在多个位置对组蛋白进行乙酰化，Vps75和ASF1似乎改变了Rtt109的特异性。如何实现特异性和选择性的生化和分子理解是目前染色质领域的一个主要挑战。这个项目将采用和扩展新的方法来研究复杂的蛋白质-蛋白质网络，这些网络需要调节染色质的动力学和翻译后的特异性。

DESCRIPTION (provided by applicant): There is a fundamental paradox within the nucleus of every eukaryotic cell: The genetic material must be organized and compacted yet remain accessible for readout by transcription machinery. Two of the many factors that retain this balance are histones and histone binding proteins. Histones are ultimately responsible for compacting the chromosomal DNA almost 500,000-fold to fit into the nucleus. While genome accessibility is regulated in part by the actions of histone acetyltransferases (KATs), histone chaperones interact directly with histones and can assemble and/or disassemble them on DNA. KATs covalently modify the histones and therefore have the potential to alter chromatin structure. Exciting new evidence structurally and functionally link KATs and histone chaperones. However, virtually nothing is known about the mechanisms by which these proteins cooperate to manage compaction and genome accessibility. To begin to understand this important biological question, this project proposes to study the histone acetyltransferase (KAT) Rtt109 as a model system. Rtt109 employs two structurally unrelated histone chaperones, Vps75 and Asf1. In vivo, Vps75 has been shown to directly interact with Rtt109, but only Asf1 is required for Rtt109 function. Both chaperones activate Rtt109 acetyltransferase activity in vitro, but Rtt109 acetylates histones in multiple locations, and Vps75 and Asf1 appear to alter Rtt109 specificity. A biochemical and molecular understanding of how specificity and selectivity is achieved is currently a major challenge in the chromatin field. This project will employ and expand on new methodologies for studying complex protein-protein networks needed to regulate chromatin dynamics and post-translational specificity.