Histone Tail Interactions and Functions in Chromatin

描述(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压缩到其长度的数千倍，同时还允许进行有效的过程，如复制基因组和表达其中编码的基因。为了做到这一点，DNA被组装成一个复杂的、多方面的复合体，称为染色质。将DNA包装到染色质的初始水平包括将短小约200bp的片段包裹在由核心组蛋白组成的蛋白卷轴周围，形成称为核小体的结构。极长的、基因组大小的核小体串被组装成更高级别的染色质结构的层次结构，以形成染色体。核小体上方结构层次的形成涉及核小体之间的基本相互作用，这些核小体之间的相互作用是由核小体主体突出的核心组蛋白的尾部结构域提供的。这些结构域的调控是基因表达调控的关键组成部分。虽然人们对核小体结构有了很好的了解，但高阶染色质结构和核小体之间的相互作用仍然不清楚。此外，尾部结构域中的翻译后修饰如何导致染色质高阶结构的调节来调节基因表达还不是很清楚。重要的是，导致核心组蛋白尾部修饰变化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在高阶染色质结构中介导核小体之间的相互作用。本提案中描述的工作的主要目标是：1)通过表征重组的模型复合体和天然染色质中H3和H4尾区的短程和长程核小体间相互作用，了解核心组蛋白尾区如何介导染色质中核小体间的相互作用；2)确定连接器组蛋白与核小体结合的机制，特别是H1C末端结构域如何稳定高阶结构；3)了解核心组蛋白尾区内HMGN‘建筑因子’和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。我们将使用几种新的方法，包括尾-DNA相互作用的位点定向交联、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HMGNs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器组蛋白结构。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.