Chromatin Structure and Stability in the Solution-State

描述（由申请人提供）：从一开始，该基金就率先使用定义的模型系统来研究核小体阵列和接头含组蛋白的染色质纤维在体外凝聚的机制和大分子决定因素。近年来，我们已经开始关注核心组蛋白N-末端尾部结构域、核小体表面和连接体组蛋白的功能（例如，H1）染色质凝聚中的C-末端结构域。该基金的一个长期目标一直是了解染色质纤维的构象转变如何影响核功能，如转录。通过使用重组组蛋白，我们目前定位于进行许多新颖和创新的基于诱变的实验，这些实验解决了真核基因组如何包装和组织，以及染色质纤维结构如何与转录调控相关。为了继续推进这一独特而富有成效的研究计划，在下一个资助周期内，我建议使用模型重组核小体阵列和含有组蛋白的连接体染色质纤维：（1）表征合理设计的核心组蛋白N-末端“尾结构域”突变体的凝聚，并分析核小体表面对核小体阵列凝聚的贡献，（2）通过研究连接体组蛋白N-末端结构域的功能作用和通过确定内在蛋白质紊乱和特定氨基酸组成如何影响连接体组蛋白C-末端结构域功能，剖析连接体组蛋白如何作为核小体结合和染色质结构蛋白发挥功能，和（3）通过在新的体外模型系统中将核小体阵列和染色质纤维凝聚与它们对RNA聚合酶II依赖性转录的影响相关联，建立涉及染色质结构和转录的结构/功能关系。所提出的研究是创新的，无论是在所提出的问题方面，还是在纯重组系统中研究的组装体的大小和规模方面（例如，核小体超过250 kDa，寡聚染色质纤维可具有数百兆道尔顿的质量）。诱变实验由许多特定假设驱动。所有拟议的研究都得到了已发表或初步数据的支持，但提出了全新的问题。总的来说，拟议的实验将对控制基因组浓缩和解浓缩的分子机制以及它们如何与转录等功能的调节相关联产生前所未有的洞察力。公共卫生相关性：真核生物的基因组被包装成染色质-DNA和组蛋白的重复复合物。染色质同时负责浓缩染色体DNA以适应细胞核和调节功能，如转录。拟议的实验将使用体外染色质模型系统，以大大增加我们对控制基因组结构的分子因素的理解，以及基因组结构如何影响RNA聚合酶II的转录。

DESCRIPTION (provided by applicant): From its inception this grant has pioneered the use of defined model systems to investigate the mechanism and macromolecular determinants of nucleosomal array and linker histone-containing chromatin fiber condensation in vitro. In recent years we have come to focus on the functions of the core histone N-terminal tail domains, the nucleosome surface, and the functions of the linker histone (e.g., H1) C-terminal domain in chromatin condensation. A long term goal of the grant has always been to understand how the conformational transitions of chromatin fibers affect nuclear functions such as transcription. Through the use of recombinant histones, we are currently positioned to perform many novel and innovative mutagenesis-based experiments that address how the eukaryotic genome is packaged and organized, and how chromatin fiber architecture is linked to regulation of transcription. To continue to advance this unique and productive research program, over the next funding interval I propose to use model recombinant nucleosomal arrays and linker histone-containing chromatin fibers to: (1) characterize the condensation of rationally designed core histone N-terminal "tail domain" mutants, and analyze the contributions of the nucleosome surface to nucleosomal array condensation, (2) dissect how linker histones function as nucleosome binding and chromatin architectural proteins, by studying the functional role of the linker histone N-terminal domain and by determining how intrinsic protein disorder and specific amino acid composition influence linker histone C-terminal domain function, and (3) establish structure/function relationships involving chromatin architecture and transcription by correlating nucleosomal array and chromatin fiber condensation with their effects on RNA polymerase II-dependent transcription in a novel in vitro model system. The proposed research is innovative, both in terms of the questions being asked, and the magnitude and scale of the assemblages that will be studied in purely recombinant systems (e.g., the nucleosome exceeds 250 kDa, and oligomeric chromatin fibers can have a mass of hundreds of megadaltons). The mutagenesis experiments are driven by numerous specific hypotheses. All of the proposed studies are supported by published or preliminary data, and yet ask entirely new questions. Taken together, the proposed experiments will yield unprecedented insight into the molecular mechanisms that control condensation and decondensation of the genome, and how they are linked to regulation of functions such as transcription. PUBLIC HEALTH RELEVANCE: The genomes of eukaryotic organisms are packaged into chromatin - a repetitive complex of DNA and histone proteins. Chromatin is simultaneously responsible for condensing chromosomal DNA to fit into the nucleus and regulating function such as transcription. The proposed experiments will use in vitro chromatin model systems to greatly increase our understanding of the molecular factors that control genome architecture, and in turn how genome architecture influences transcription by RNA polymerase II.