Structural and Functional Characterization of the Chd1 Chromatin Remodeler

摘要 染色质重塑蛋白是ATP依赖性DNA移位酶，其催化分解、重组和降解。 核小体在真核基因组中的重新定位。从多种类型的癌症和 与重塑突变相关的发育障碍，染色质重塑是正常发育所必需的。 增长和发展。该提案旨在解决重塑行动的核心机制问题， 调节，使用Chd1染色质重塑作为模型系统。最近的研究表明， 重塑剂使用扭曲缺陷机制移动核小体。在这个过程中，重塑者将不同的 ATP酶马达的核苷酸依赖性构象产生然后消除DNA扭曲（扭曲 缺陷），其刺激核小体瞬时吸收，然后在重塑物处释放额外的BP 结合位点目前，尚不清楚重塑如何产生扭曲缺陷。本提案的目标1旨在 确定Chd 1的关键残基和元件，这些残基和元件是将DNA扭曲成扭曲缺陷所必需的， 对这个中心过程的机械理解。该提案所涉及的另一个问题是， 重塑ATP酶受到调节。对于Chd 1，核小体滑动活性与Chd外的DNA偶联。 核小体，要求在"入口"侧的侧翼DNA和在"入口"侧的很少或没有DNA的偏好。 "出口"那一边。对于Chd 1，侧翼DNA通过与Chd 1偶联的DNA结合结构域控制滑动活性。 自身抑制成分本提案目标2所解决的一个中心问题是， 的自抑制元件的控制核小体上的不同结构域的安排。实验 旨在揭示核小体上重塑重排的动力学转变。最后，一个主要 尚未回答的问题是，两个与同一核小体结合的重塑体如何影响彼此的活性。Chd1 已显示以2：1的比例与核小体结合，其中DNA结合结构域与 色素ATP酶，但这些相互作用的意义是未知的。目标3测试假设，两个 与同一核小体结合的相反的重塑物相互拮抗。这些研究将 为重塑者如何改变核小体结构、核小体的自动调节作用提供了新的机制见解。 中央ATP酶马达，并协调和潜在地控制核小体上的其他重塑。

ABSTRACT Chromatin remodelers are ATP-dependent DNA translocases that catalyze disassembly, reassembly, and repositioning of nucleosomes throughout eukaryotic genomes. As evidenced from multiple types of cancer and developmental disorders associated with remodeler mutations, chromatin remodeling is essential for normal growth and development. This proposal aims to address core mechanistic questions of remodeler action and regulation, using the Chd1 chromatin remodeler as a model system. Recent studies revealed that chromatin remodelers shift nucleosomes using a twist defect mechanism. In this process, remodelers couple distinct nucleotide-dependent conformations of the ATPase motor to create and then eliminate DNA distortions (twist defects) that stimulate the nucleosome to transiently absorb and then release an extra bp at the remodeler binding site. Currently, it is unknown how remodelers create twist defects. Aim 1 of this proposal seeks to identify key residues and elements of Chd1 necessary for distorting DNA into twist defects, which should allow for a mechanistic understanding of this central process. Another question addressed by this proposal is how remodeler ATPases are regulated. For Chd1, nucleosome sliding activity is coupled to DNA outside the nucleosome, with a requirement for flanking DNA on the “entry” side and preference for little or no DNA on the “exit” side. For Chd1, flanking DNA controls sliding activity through a DNA-binding domain that is coupled to autoinhibitory elements. A central question addressed by Aim 2 of this proposal is how stability and interactions of autoinhibitory elements are controlled by distinct domain arrangements on the nucleosome. Experiments are designed to reveal kinetic transitions of remodeler rearrangements on the nucleosome. Finally, a major unanswered question is how two remodelers bound to the same nucleosome affect activity of each other. Chd1 has been shown to bind to nucleosomes in a 2:1 ratio, with the DNA-binding domain interacting in trans with the chromo-ATPase, yet the significance of these interactions is unknown. Aim 3 tests the hypothesis that two opposing remodelers bound to the same nucleosome antagonize each other. Together, these studies will provide new mechanistic insights into how remodelers alter nucleosome structure, autoregulate action of their central ATPase motor, and coordinate and potentially control other remodelers on the nucleosome.