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

描述(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，最主要的是癌症。 本研究项目将确定组蛋白H3残基K56、K115、T118和K122四个关键的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。它们既可以单独存在，也可以在体内共同存在，对转录调控和DNA修复至关重要。这些修饰实现重要生物功能的机制仍然知之甚少。然而，每一种修饰都被埋在组蛋白-DNA界面的DNA下面，位于核小体的两个关键区域之一：二分体对称轴和DNA进出区域。这意味着，这些修饰发挥作用的机制必须要求核小体构象和/或动力学发生重大变化。因此，生化和生物物理的联合研究是理解这些修饰作用的关键。这四种修饰将通过使用组蛋白重组统一修饰的半合成核小体进行研究，组蛋白是通过表达的蛋白质连接和顺序化学连接构建的。将使用多种方法来量化修饰引起的染色质构象和动力学的变化：荧光共振能量转移、限制性内切酶消化、核小体图谱、核小体竞争重组、停流荧光法和荧光相关光谱。这些实验将确定DNA-组蛋白界面上的这四个组蛋白H3修饰是否以及如何调节核小体结构和DNA位点暴露，在目标1中，在目标2中，核小体定位和稳定性，以及在目标3中，核小体动力学。 这项研究项目的成功完成将在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，它将确定隐藏在核小体DNA-组蛋白界面中的组蛋白H3中的四个修饰如何促进DNA修复和转录调控。其次，它将提供对20多个翻译后组蛋白修饰的可能机制的洞察，这些修饰已知隐藏在整个核小体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.