Breaking Nucleosomal Symmetry

打破核小体对称性

• 批准号: 8695935
• 负责人: PAUL D. KAUFMAN
• 金额: $ 31.39万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-15 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8695935
• 关键词: Affect; Biochemical; Biochemistry; Biological Process; Cells; Chromatin; Chromosomes; Defect; Dependency; Enzymes; Eukaryota; Event; Exhibits; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Epistasis; Genomics; Growth; Health; Histone H3; Histones; Human; Life; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Messenger RNA; Methods; Modification; Molecular Genetics; Mutate; Mutation; Nucleosomes; Pathway interactions; Pattern; Phenotype; Play; Point Mutation; Proteins; Role; Saccharomycetales; Site; System; Tail; Testing; Time; Work; Yeasts; abstracting; base; design; genome-wide; histone modification; human disease; in vivo; insight; interdisciplinary approach; man; mutant; novel; protein structure prediction; research study; response; stoichiometry

DESCRIPTION (provided by applicant): Project Summary / Abstract We have developed methods to manipulate for the first time the natural symmetry of nucleosomes, in order to test the extent to which this symmetry is functionally important. These questions cannot be pursued in cells with natural histones. Therefore, we have designed altered histone H3s that have obligate heterodimeric interactions, and which preclude interaction with wild-type H3 molecules. We will now use these altered H3s to measure how nucleosomal asymmetry affects gene expression and histone modification patterns, as follows: Aim 1. Identify the mechanistic basis for epistatic interactions between histone tails. In our preliminary studies, we observed distinct classes of phenotypes upon mutation of modifiable residues: in one case, a single asymmetric H3 point mutation paired with a wild-type partner exhibited all the transcriptional defects of a double point mutant. In another case, genes were only misregulated in symmetric double mutants. We will extend these studies to a large set of histone mutations to understand the mechanistic basis for the epistasis observed between pairs of histone mutants. Aim 2. Determine whether histone crosstalk functions in cis or in trans. A great number of histone modifying enzymes preferentially act on nucleosomes carrying some second modification, a phenomenon often referred to as "cross-talk". We will use genetic and biochemical approaches to assess whether crosstalk occurs in cis, on the same tail, or in trans, on opposite tails: we will identify the quantitative difference in gene expression between cells with cis and trans double K->R mutations in the H3 tail, and perform mass spectrometric analysis of purified asymmetric nucleosomes to determine whether second site modifications are lost in cis, in trans, or are unaffected by monomeric histone mutations. Together, these studies will reveal previously unexplored biochemical dependency pathways that alter histone modification patterns, and distinguish gene expression regulatory events that are dependent on one versus two histone H3 N-termini. Notably, because of the extreme conservation of core histones among eukaryotes, this work will open the way to exploring related questions in metazoans. Because histone modifications are central to all aspects of gene expression from yeast to man, and play major roles in human diseases including cancer, these studies will reveal unappreciated regulatory mechanisms that govern human health and growth control.

描述（由申请人提供）： 项目摘要/摘要我们首次开发了操纵核小体自然对称性的方法，以测试这种对称性在功能上的重要性。这些问题无法在具有天然组蛋白的细胞中探究。因此，我们设计了改变的组蛋白 H3，其具有专性异二聚体相互作用，并且排除与野生型 H3 分子的相互作用。现在，我们将使用这些改变的 H3 来测量核小体不对称性如何影响基因表达和组蛋白修饰模式，如下所示： 目标 1. 确定组蛋白尾部之间上位相互作用的机制基础。在我们的初步研究中，我们观察到可修饰残基突变后出现不同类型的表型：在一种情况下，与野生型伴侣配对的单个不对称 H3 点突变表现出双点突变体的所有转录缺陷。在另一种情况下，基因仅在对称双突变体中被错误调节。我们将把这些研究扩展到大量组蛋白突变，以了解在组蛋白突变体对之间观察到的上位性的机制基础。目标 2. 确定组蛋白串扰是顺式还是反式起作用。大量组蛋白修饰酶优先作用于携带某些第二修饰的核小体，这种现象通常被称为“串扰”。我们将使用遗传和生化方法来评估串扰是否发生在顺式（同一尾部）或反式（相反尾部）：我们将确定具有顺式和反式双 K->R 突变的细胞之间基因表达的定量差异H3 尾部，并对纯化的不对称核小体进行质谱分析，以确定第二个位点修饰是否在顺式、反式中丢失，或者是否不受单体组蛋白突变的影响。总之，这些研究将揭示以前未探索过的改变组蛋白修饰模式的生化依赖性途径，并区分依赖于一个和两个组蛋白 H3 N 末端的基因表达调控事件。值得注意的是，由于真核生物中核心组蛋白的极端保守性，这项工作将为探索后生动物中的相关问题开辟道路。由于组蛋白修饰对于从酵母到人类的基因表达的各个方面都至关重要，并且在包括癌症在内的人类疾病中发挥着重要作用，因此这些研究将揭示控制人类健康和生长控制的未受重视的调节机制。