Genome-wide mechanisms and dynamics of ATP-dependent chromatin remodeling complexes

项目摘要/摘要 SWI/SNF家族依赖于ATP的染色质重构体是多亚单位蛋白质复合体， 动态定位染色体核小体以调节DNA可及性、转录因子结合、 和特定细胞类型的基因表达。SWI/SNF功能在哺乳动物的几个阶段中都是至关重要的 最近的人类全基因组和外显子组测序研究揭示了惊人的突变 编码SWI/SNF亚单位的基因在一系列疾病中的频率-从神经障碍到 超过20%的癌症。疾病相关的SWI/SNF突变通常会导致蛋白质亚单位的丢失 进一步破坏复合体的稳定，并导致亚基组成和功能的改变。值得注意的是， SWI/SNF亚基组成的异质性也在细胞类型内和细胞类型之间自然观察到， 假设导致复杂的和组织特异性的SWI/SNF功能。尽管它们很重要，但它们如何 亚基决定底物的选择性，SWI/SNF复合体的机械功能不佳 理解，很大程度上是因为缺乏强有力的方法来表征它们在基因组中的结合位置 以及它们如何与全基因组染色质相互作用。因此，本提案的主要目标就是解决这一关键问题 知识鸿沟。由于染色质重构体直接与核小体相互作用，因此澄清是很重要的 与重构体结合的核小体组合物和结构。可溶物的标准生化特性 从细胞提取液中提纯的成分不能确定与 染色质结合的复合体。了解重构体-染色质的动力学参数也很重要 互动，例如重建者被招募到目标站点的速度有多快，以及它在多长时间内保持绑定 它的基因组位置。候选人建议通过使用结构和功能来解决这些问题 表观基因组学方法和活细胞单分子成像，以表征重构体染色质 在复杂和动态的细胞内染色质环境中的相互作用。目标1是 确定具有不同亚基组成的SWI/SNF复合体在全基因组中的占有率。目标2是 确定全基因组重构体结合核小体的结构和组蛋白组成，以及b) 活细胞中重构体-染色质相互作用的动力学参数。目标3是研究 带有抑制性染色质的SWI/SNF。该项目的指导阶段将在 弗雷德·哈钦森癌症研究中心史蒂文·亨尼科夫博士的赞助。候选人将会 在大学希拉·特维斯博士的指导下接受活细胞单分子成像培训 不列颠哥伦比亚省。拟议的研究和培训将为候选人提供坚实的基础 作为独立的调查者，研究染色质重塑的机制和动力学 对基本细胞过程的调节。

PROJECT SUMMARY/ABSTRACT The SWI/SNF family ATP-dependent chromatin remodelers are multi-subunit protein complexes that dynamically position chromosomal nucleosomes to modulate DNA accessibility, transcription-factor binding, and cell-type-specific gene expression. SWI/SNF function is crucial at several stages of mammalian development, and recent human whole-genome and exome sequencing studies revealed striking mutational frequencies in genes encoding SWI/SNF subunits across a range of diseases - from neurologic disorders to over 20% of all cancers. Disease-associated SWI/SNF mutations often cause the loss of a protein subunit that further destabilizes the complex and results in altered subunit composition and functions. Of note, heterogeneity in SWI/SNF subunit composition is also observed naturally within and across cell types, hypothesized to result in complex- and tissue-specific SWI/SNF functions. Despite their importance, how these subunits determine the substrate preference and mechanistic functions of SWI/SNF complexes are not well understood, largely due to the lack of robust approaches to characterize where they bind within the genome and how they interact with chromatin genome-wide. Thus, the main goal of this proposal is to address this key knowledge gap. Since chromatin remodelers directly interact with nucleosomes, it is important to clarify remodeler-bound nucleosome compositions and structures. Standard biochemical characterization of soluble components purified from cellular extracts cannot determine the nucleosome structures associated with chromatin-bound complexes. It is also important to understand the kinetic parameters of remodeler-chromatin interactions, such as how fast a remodeler is recruited to its target sites, and how long does it remain bound at its genomic sites. The candidate proposes to address these questions by using structural and functional epigenomics approaches and live-cell single-molecule imaging, to characterize remodeler-chromatin interactions in the context of the complex and dynamic chromatin environment inside cells. Aim 1 is to determine the genome-wide occupancy of SWI/SNF complexes with distinct subunit compositions. Aim 2 is to determine a) the structures and histone composition of remodeler-bound nucleosomes genome-wide, and b) the kinetic parameters of remodeler-chromatin interactions in live cells. Aim 3 is to study the interaction of SWI/SNF with repressive chromatin. The mentored phase of this project will be completed under the sponsorship of Dr. Steven Henikoff at the Fred Hutchinson Cancer Research Center. The candidate will acquire training in live-cell single-molecule imaging under the supervision of Dr. Sheila Teves at the University of British Columbia. The proposed research and training will provide a strong foundation for the candidate to develop as an independent investigator, studying chromatin remodeling mechanisms and dynamics in the regulation of fundamental cellular processes.