Structural and Functional Characterization of the Chd1 Chromatin Remodeler

摘要 染色质重构体是依赖于ATP的DNA转位酶，它催化分解、重组和 核小体在整个真核基因组中的重新定位。从多种类型的癌症和 与重构体突变相关的发育障碍，染色质重塑对正常至关重要 成长和发展。该提案旨在解决重建器行动的核心机械问题和 调节，使用Chd1染色质重构体作为模型系统。最近的研究表明，染色质 重构体使用扭曲缺陷机制来移动核小体。在此过程中，重构器耦合不同 依赖核苷酸的ATPase马达构象以产生并消除DNA扭曲(扭曲 缺陷)，刺激核小体瞬间吸收，然后在重整器处释放额外的BP 结合部位。目前，尚不清楚改装器如何产生扭曲缺陷。这项提案的目标1旨在 确定将DNA扭曲成扭曲缺陷所必需的关键残基和Chd1元件，这应该允许 对这一中心过程的机械性理解。这项提议解决的另一个问题是如何 重构体ATPase受到调控。对于Chd1，核小体滑动活动与DNA偶联在 核小体，要求DNA在进入侧的侧翼，并倾向于在入口侧很少或没有DNA “出口”那一边。对于chd1，侧翼的dna通过连接到的dna结合域控制滑动活动。 自身抑制因子。这项提议的目标2解决的一个中心问题是，稳定性和相互作用如何 自身抑制元件的数目由核小体上不同的结构域排列控制。实验是 旨在揭示核小体上重塑重排的动力学转变。最后，一个主要的 悬而未决的问题是，两个结合在同一核小体上的重构体如何影响彼此的活性。CHD1 已被证明以2：1的比例与核小体结合，dna结合域与反式作用。 染色质-ATPase，但这些相互作用的意义尚不清楚。AIM 3测试了两个 结合在同一核小体上的相互对立的重构体相互对抗。总而言之，这些研究将 提供了关于重构体如何改变核小体结构、自动调节其行为的新的机械性见解 中央ATPase马达，协调和潜在地控制核小体上的其他重构体。

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.