Histone Tail Interactions and Functions in Chromatin

描述（由申请人提供）：人类和其他真核生物的染色体提供了看似截然不同的功能，将基因组DNA压缩到其长度的几千倍，同时也允许有效的过程，如复制基因组和表达其中编码的基因。为了完成这一过程，DNA被组装成一个复杂的、多方面的复合体，即染色质。将DNA包装成染色质的初始水平包括在由核心组蛋白组成的蛋白卷周围包裹约200 bp的短片段，形成称为核小体的结构。非常长的、基因组大小的核小体串被组装成一个层次的高阶染色质结构，形成染色体。核小体以上结构水平的形成涉及核心组蛋白的“尾部”结构域提供的核小体之间的基本相互作用，这些结构域从核小体的主体中伸出。这些结构域的调控是基因表达调控的关键组成部分。虽然核小体的结构已经被很好地理解，但高阶染色质结构和核小体间的相互作用仍然不清楚。此外，尾结构域内的翻译后修饰如何导致染色质高阶结构的调节以调节基因表达尚不清楚。重要的是，导致核心组蛋白尾部修饰改变的突变与包括人类癌症在内的疾病有关。在先前的工作中，我们证明了核心组蛋白尾部结构域介导高阶染色质结构中的核小体间相互作用。本建议所述工作的主要目标是：1)通过表征重组模型复合物和天然染色质中H3和H4尾结构域的短程和远程核小体间相互作用，了解核心组蛋白尾结构域如何介导染色质中的核小体间相互作用；2)定义连接体组蛋白与核小体结合的机制；特别是H1 c末端结构域如何稳定高阶结构；3)了解HMGN“结构因子”和核心组蛋白尾部结构域内特定赖氨酸的乙酰化如何改变染色质结构以允许基因表达的机制。我们将使用几种新方法，包括尾部dna相互作用的定向交联、基于荧光的方法（FRET）和其他化学探测方法来研究尾部结构域、H1的结构和相互作用，以及hmgn和乙酰化对这些结构域的影响。此外，我们将开发一种体内FRET方法来评估活细胞中的核心组蛋白和连接组蛋白结构。这些结果将为理解转录、复制、DNA修复和其他过程如何在染色质环境中发生提供基础。

DESCRIPTION (provided by applicant): The chromosomes of humans and other eukaryotes provide the seemingly contrasting functions of compacting the genomic DNA several thousand times its length while also allowing for efficient processes such as replicating the genome and expressing genes encoded within it. To accomplish this, the DNA is assembled into a complicated, multifaceted complex known as chromatin. The initial level of packaging DNA into chromatin involves wrapping short ~200 bp segments around protein spools comprised of the core histone proteins into structures known as nucleosomes. Immensely long, genome-sized strings of nucleosomes are assembled into a hierarchy of higher-order chromatin structures, to form chromosomes. Formation of levels of structure above the nucleosome involves essential inter-nucleosome interactions provided by the 'tail' domains of the core histone proteins, which protrude out from the main body of nucleosomes. Regulation of these domains is a key component of the regulation of gene expression. While nucleosome structure is fairly well understood, higher- order chromatin structures and inter-nucleosome interactions remain poorly defined. Moreover, how posttranslational modifications within the tail domains result in modulation of chromatin higher order structures to regulate gene expression is not well understood. Importantly, mutations that result in changes to core histone tail modifications have been linked to diseases including cancers in humans. In prior work we demonstrated that the core histone tail domains mediate inter-nucleosome interactions in higher-order chromatin structures. The primary goals of the work described in this proposal are to: 1) understand how the core histone tail domains mediate inter-nucleosome interactions in chromatin by characterizing short-range and long-range inter-nucleosomal interactions of the H3 and H4 tail domains in both reconstituted model complexes and in native chromatin, 2) to define the mechanisms by which linker histones bind to nucleosomes, and in particular how the H1 C-terminal domain stabilizes higher order structures and 3) understand how the mechanisms by which the HMGN 'architectural factors' and acetylation of specific lysines within the core histone tail domains alter chromatin structure to allow for gene expression. We will use several novel approaches including site-directed crosslinking of tail-DNA interactions, fluorescence-based methods (FRET) and other chemical probing approaches to investigate structures and interactions of the tail domains, H1, and the effect of HMGNs and acetylation on these domains. In addition, we will develop an in vivo FRET approach to assess core histone and linker histone structures in live cells. These results will provide a basis for understanding how transcription, replication, DNA repair, and other processes occur in a chromatin environment.