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

项目总结/摘要 SWI/SNF家族ATP依赖性染色质重塑物是多亚基蛋白复合物， 动态定位染色体核小体以调节DNA可及性，转录因子结合， 和细胞类型特异性基因表达。SWI/SNF功能在哺乳动物的几个阶段至关重要， 最近的人类全基因组和外显子组测序研究显示， 在一系列疾病中编码SWI/SNF亚基的基因的频率-从神经系统疾病到 超过20%的癌症疾病相关的SWI/SNF突变通常会导致蛋白亚基的丢失， 进一步使复合物不稳定并导致亚基组成和功能改变。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间自然观察到， 假设导致复杂和组织特异性SWI/SNF功能。尽管他们的重要性，如何这些 SWI/SNF复合物的底物选择性和作用机制尚不清楚 这主要是由于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的染色质相互作用。因此，本提案的主要目标是解决这一关键问题， 知识差距。由于染色质重塑直接与核小体相互作用，因此澄清 重塑剂结合的核小体组成和结构。可溶性蛋白的标准生化表征 从细胞提取物中纯化的成分无法确定与之相关的核小体结构 染色质结合的复合物。了解重塑体-染色质的动力学参数也很重要 相互作用，例如重塑剂被招募到其靶位点的速度，以及它在靶位点保持结合的时间 它的基因组位点。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，以表征重塑染色质 在细胞内复杂而动态的染色质环境中，目标1： 确定具有不同亚基组成的SWI/SNF复合物的全基因组占有率。目标二是 确定a）全基因组的重塑体结合的核小体的结构和组蛋白组成，和B） 活细胞中重塑体-染色质相互作用的动力学参数。目的3是研究 SWI/SNF与抑制性染色质。该项目的辅导阶段将在 弗雷德哈钦森癌症研究中心的史蒂文·亨尼科夫博士的赞助。候选人将 在大学的Sheila Teves博士的监督下获得活细胞单分子成像的培训 是不列颠哥伦比亚省的一部分。拟议的研究和培训将为候选人提供坚实的基础， 作为一个独立的研究者，研究染色质重塑机制和动力学， 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.