Breaking Nucleosomal Symmetry

打破核小体对称性

• 批准号: 8892203
• 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/8892203
• 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; Genetic study; 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; public health relevance; 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 /摘要我们已经开发了第一次操纵核小体的自然对称性的方法，以测试该对称性在功能上重要的程度。这些问题在具有天然组蛋白的细胞中无法提出。因此，我们设计了具有强大异二聚体相互作用的组蛋白H3S，并且排除了与野生型H3分子的相互作用。现在，我们将使用这些改变的H3S来测量核小体不对称如何影响基因表达和组蛋白修饰模式，如下：AIM 1。确定组蛋白尾巴之间上皮相互作用的机械基础。在我们的初步研究中，我们观察到可修改残基突变的不同表型：在一种情况下，与野生型伴侣配对的单个不对称的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误导。我们将将这些研究扩展到大量组蛋白突变，以了解组蛋白突变体之间观察到的上毒的机械基础。 AIM 2。确定组蛋白串扰是在顺式中还是在Trans中起作用。大量的组蛋白修饰酶优先对带有第二个修饰的核小体作用，这种现象通常称为“交叉对话”。 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组蛋白突变。总之，这些研究将揭示先前未开发的生化依赖性途径，这些途径改变了组蛋白的修饰模式，并区分了依赖于一个与两个组蛋白H3 N-末端的基因表达调节事件。值得注意的是，由于真核生物中核心组蛋白的极端保护，这项工作将为探索后生动物的相关问题打开道路。由于组蛋白的修饰对于从酵母到人的基因表达的各个方面都是至关重要的，并且在包括癌症在内的人类疾病中起着重要作用，因此这些研究将揭示不受影响的调节机制，这些机制控制了人类健康和生长控制。