Structure of nucleosomes in complex with transcriptional regulators

描述（由申请人提供）：单个真核细胞的DNA长度超过两米，但通过包装成核蛋白组装体（"染色质"）的分层方案在细胞核中压缩约100，000倍，所述核蛋白组装体具有增加的复杂性和很大程度上未知的结构。连接体组蛋白H1促进了核小体（染色质的基本重复单位）以外的压缩，对底层DNA的转录能力具有深远的影响。接头组蛋白与核小体相互作用的位置和模式是一个激烈争论的主题，序列特异性DNA结合蛋白如何在核小体中识别其结合位点的问题也是如此。这两种类型的相互作用都缺乏结构模型。 本提案的目的是确定两个转录调节因子（全球调节因子H1和特异性转录激活因子FoxA）与核小体复合的结构，使用X射线晶体学和生物物理学方法。建议调查的作用，连接器组蛋白的尾巴在组织的连接器DNA，并测试的假设，组蛋白变体已演变为调节与连接器组蛋白的相互作用。这些研究将在核小体-H1复合物的一个期待已久的结构中达到高潮，这将揭示染色质结构和功能的这个关键组织者的相互作用的位置和模式（具体目标1）。在具体目标2中，将研究转录激活因子FoxA与高度限定的核小体的相互作用。将测试组蛋白八聚体有助于FoxA结合以及组蛋白尾部调节核小体DNA可及性的假设。结构研究将阐明FoxA参与和打开染色质的机制，其结果将代表普遍的Fox（Forkhead）因子在不同生物调控环境中发挥作用的机制的范例。总之，拟议的研究将提供对天然染色质背景下染色质高阶结构DNA识别的深入了解，因此对于理解存储在基因组中的信息是如何组织和访问的至关重要。具体而言，提出了以下具体目标： 1)测试连接组蛋白与核心组蛋白相互作用并组织连接DNA路径的假设。 1.a.连接体组蛋白与核小体结合的生物物理学研究;检验组蛋白变体已进化为调节与连接体组蛋白的相互作用的假设。 1.b.确定H1结合核小体的晶体结构。 2)检验FoxA结合导致核小体结构改变的假设。 2.a. FoxA与具有确定结合位点的单核小体结合的生物物理学分析。 2.b.确定FoxA-核小体复合物的晶体结构。

DESCRIPTION (provided by applicant): The DNA of a single eukaryotic cell is over two meters in length, but compacts in the cell nucleus approximately 100,000 fold by a hierarchical scheme of packaging into nucleoprotein assemblies ("chromatin") of increasing complexity and largely unknown architecture. Compaction beyond the nucleosome, the basic repeating unit of chromatin, is promoted by linker histone H1, with profound implications for the transcriptional competence of the underlying DNA. The location and mode of interaction of linker histones with the nucleosome is a subject of intense debate, as is the question of how sequence-specific DNA binding proteins recognize their binding site in the context of a nucleosome. Structural models are lacking for both types of interaction. The objective of this proposal is to determine the structure of two transcriptional regulators (the global regulator H1 and the specific transcription activator FoxA) in complex with the nucleosome, using x-ray crystallography and biophysical methods. It is proposed to investigate the role of linker histone tails in the organization of linker DNA, and to test the hypothesis that histone variants have evolved to modulate interaction with linker histones. These studies will culminate in a long-overdue structure of the nucleosome-H1 complex, which will reveal the location and mode of interaction of this key organizer of chromatin structure and function (specific aim 1). In specific aim 2, the interaction of the transcription activator FoxA with highly defined nucleosomes will be studied. The hypotheses that the histone octamer contributes to FoxA binding, and that the histone tails modulate accessibility of nucleosomal DNA will be tested. Structural studies will illuminate the mechanism by which FoxA engages and opens chromatin, and the results will represent a paradigm for the mechanism by which the pervasive Fox (Forkhead) factors function in diverse biological regulatory contexts. Together, the proposed studies will provide insight into chromatin higher order structure DNA recognition in a natural chromatin context, and are thus essential to understand how the information stored in the genome is organized and accessed. In particular, the following specific aims are proposed: 1) Test the hypothesis that linker histone interacts with core histones and organizes the path of linker DNA. 1.A. Biophysical investigation of linker histone binding to nucleosomes; test the hypothesis that histone variants have evolved to modulate the interaction with linker histones. 1.B. Determine the crystal structure of H1-bound nucleosomes. 2) Test the hypothesis that FoxA binding results in an altered nucleosome structure. 2.A. Biophysical analysis of the binding of FoxA to mono-nucleosomes with defined binding sites. 2.B. Determine the crystal structure of the FoxA-nucleosome complex.