Breaking Nucleosomal Symmetry

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程度。这些问题不能在含有天然组蛋白的细胞中解决。因此，我们设计了改变的组蛋白H3s，它具有专有的异二聚体相互作用，并排除了与野生型H3分子的相互作用。我们现在将使用这些改变的H3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：目的1.确定组蛋白尾部之间上位性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的不同类型的表型：在一个案例中，单个不对称H3点突变与野生型配对显示出双点突变的所有转录缺陷。在另一种情况下，基因只在对称的双突变体中被错误调控。我们将把这些研究扩展到一大组组蛋白突变，以了解组蛋白突变对之间观察到的上位性的机制基础。目的2.确定组蛋白串扰是否在顺式或反式中起作用。大量的组蛋白修饰酶优先作用于进行二次修饰的核小体，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是否在顺式、同一条尾巴或反式尾巴上发生串扰：我们将确定H3尾部具有顺式和反式双K-gt；R突变的细胞之间基因表达的定量差异，并对纯化的不对称核小体进行质谱分析，以确定第二位点修饰是在顺式、反式中丢失，还是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的改变组蛋白修饰模式的生化依赖途径，并区分依赖于一个组蛋白H3N末端和两个组蛋白H3N末端的基因表达调控事件。值得注意的是，由于核心组蛋白在真核生物中的极端保守，这项工作将为探索后生动物的相关问题开辟道路。由于组蛋白修饰是从酵母到人类的所有基因表达的核心，并在包括癌症在内的人类疾病中发挥重要作用，这些研究将揭示管理人类健康和生长控制的未被认识的调控机制。

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.