Characterization of Four Histone H3 Modifications in the DNA-Histone Interface

描述（由申请人提供）：我们实验室的长期目标是确定组蛋白翻译后修饰调节基因表达和 DNA 修复的分子机制。这两个细胞过程对于所有生物体的生长和健康至关重要。翻译后组蛋白修饰的改变会影响基因表达和 DNA 修复的调节，并可能导致 ICF、Rett 和 ATRX 综合征等疾病，最重要的是癌症。 该研究项目将确定组蛋白 H3 残基 K56、K115、T118 和 K122 的四个关键翻译后修饰发挥作用的分子机制。最近在核小体的结构区域中发现了这些修饰。它们在体内单独或一起发生，对于转录调控和 DNA 修复至关重要。这些修饰发挥重要生物学功能的机制仍然知之甚少。然而，每个修饰都隐藏在核小体两个关键区域之一的组蛋白-DNA 界面中的 DNA 下方：二元对称轴和 DNA 入口/出口区域。这意味着这些修饰起作用的机制必须需要核小体构象和/或动力学的显着变化。因此，结合生物化学和生物物理研究是理解这些修饰作用的关键。将通过使用通过表达蛋白连接和连续化学连接构建的组蛋白重建均匀修饰的半合成核小体来研究这四种修饰。将使用多种方法来量化修饰引起的染色质构象和动力学变化：荧光共振能量转移、限制性酶消化、核小体作图、核小体竞争性重构、停流荧光测定和荧光相关光谱。这些实验将确定 DNA-组蛋白界面中的这四种组蛋白 H3 修饰是否以及如何调节，目标 1 为核小体结构和 DNA 位点暴露，目标 2 为核小体定位和稳定性，目标 3 为核小体动力学。 该研究项目的成功完成将从两个方面对科学知识和对疾病的分子理解产生重大影响。首先，它将确定埋藏在核小体 DNA-组蛋白界面内的组蛋白 H3 的四个修饰如何促进 DNA 修复和转录调控。其次，它将深入了解 20 多种翻译后组蛋白修饰的可能机制，这些修饰已知埋藏在整个核小体 DNA-组蛋白界面中。

DESCRIPTION (provided by applicant): The long-term goal of our labs is to determine the molecular mechanisms by which post-translational modifications of histones regulate gene expression and DNA repair. These two cellular processes are vital for the growth and health of all organisms. Alterations in post-translational histone modifications affect the regulation of gene expression and DNA repair and can lead to diseases such as ICF, Rett and ATRX syndromes and, most predominantly, cancer. This research project will determine the molecular mechanisms by which four key post-translational modifications at histone H3 residues K56, K115, T118 and K122 function. These modifications were recently identified in structured regions of the nucleosome. They occur both individually and together in vivo, and are critical for transcriptional regulation and DNA repair. The mechanisms by which these modifications carry out vital biological functions remain poorly understood. However, each modification is buried beneath DNA in the histone-DNA interface at one of two critical regions of the nucleosome: the dyad symmetry axis and the DNA entry-exit region. This implies that the mechanisms by which these modifications function must require significant changes in nucleosome conformation and/or dynamics. Therefore, combined biochemical and biophysical studies are key to understanding the role of these modifications. The four modifications will be studied by reconstituting uniformly modified semi-synthetic nucleosomes using histone proteins that are constructed by expressed protein ligation and by sequential chemical ligation. Multiple approaches will be used to quantify modification-induced changes in chromatin conformation and dynamics: fluorescence resonance energy transfer, restriction enzyme digestions, nucleosome mapping, nucleosome competitive reconstitutions, stopped flow fluorometry and fluorescence correlation spectroscopy. These experiments will determine whether and how these four histone H3 modifications in the DNA-histone interface regulate, in Aim 1, Nucleosome Structure and DNA site Exposure, in Aim 2, Nucleosome Positioning and Stability and, in Aim 3, Nucleosome Dynamics. The successful completion of this research project will make a major impact on scientific knowledge and the molecular understanding of disease in two ways. First, it will determine how four modifications in histone H3 buried within the nucleosome DNA-histone interface facilitate both DNA repair and transcriptional regulation. Second, it will provide insight into the possible mechanisms of over 20 post-translational histone modifications that are known to be buried throughout the nucleosome DNA-histone interface.