Mechanistic Analysis of Chromatin Remodeling

描述（由申请人提供）： 将DNA包装到染色质中使其在很大程度上无法进入转录、复制、重组和修复的核心核过程。ATP依赖的染色质重塑马达在增加染色质内的DNA访问以及产生促进转录抑制的高阶染色质结构中起关键作用。然而，染色质重塑马达的功能机制在很大程度上是未知的。该提案的总体目标是使用从充分研究的马达如驱动蛋白和解旋酶中学到的概念来理解主要的ATP依赖性染色质重塑复合物（人ACF）如何起作用。ACF产生均匀间隔的核小体，使更高阶的染色质折叠和基因沉默。我们在上一个资助期的工作表明，ACF的功能是作为一个二聚体马达，其中每个ATP酶亚基轮流参与核小体的任何一侧。在此，我们将在这些发现的基础上解决以下问题：（1）ACF中两个ATP酶亚基的活性是如何协调的？(2)ACF在其反应机制中如何识别和使用特定的核小体特征？(3)ACF活性如何受相邻核小体的调节？ 公共卫生相关性： ACF复合物在介导遗传性基因沉默中起着至关重要的作用。与这些作用一致，ACF复合物组分的突变与严重的发育缺陷和特定癌症相关。对ACF机制的详细了解将有助于了解其活性如何调节以及如何发生故障。

DESCRIPTION (provided by applicant): PROJECT SUMMARY The packaging of DNA into chromatin makes it largely inaccessible for the central nuclear processes of transcription, replication, recombination and repair. ATP-dependent chromatin remodeling motors play key roles in both increasing DNA access within chromatin as well in generating higher-order chromatin structures that promote transcriptional repression. However, the mechanisms by which chromatin remodeling motors function are largely unknown. The overall goal of this proposal is to use concepts learnt from well-studied motors such as kinesin and helicases to understand the how a major ATP- dependent chromatin-remodeling complex, human ACF functions. ACF generates evenly spaced nucleosomes to enable higher-order chromatin folding and gene silencing. Our work over the last grant period has shown that ACF functions as a dimeric motor in which each ATPase subunit takes turns engaging either side of a nucleosome. Here we will build on these discoveries to address the following questions: (1) How are the activities of the two ATPase subunits in ACF coordinated? (2) How does ACF recognize and use specific nucleosomal features in its reaction mechanism? (3) How is ACF activity regulated by the presence of adjacent nucleosomes? PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE ACF complexes play crucial roles in mediating heritable gene silencing. Consistent with these roles, mutations in the components of ACF complexes are associated with severe developmental defects and specific cancers. A detailed understanding of ACF mechanism will provide insights into how its activity is regulated and how it malfunctions.