Defining the molecular interactions that drive histone locus body formation and function

项目总结 真核细胞的核是拥挤的隔间，不仅紧密，而且还组织了几米长的DNA。 这一壮举是通过将DNA包裹在组蛋白周围形成核小体来实现的，核小体是 染色质。细胞中的组蛋白水平是精确平衡的：缺乏组蛋白表达会导致细胞周期停滞， 而组蛋白的过度表达会导致基因组的不稳定。尽管组蛋白基因调控对 适当的细胞活性和功能，调节组蛋白基因表达的机制还不清楚。 组蛋白基因通常聚集在后生动物基因组中。在优秀模型系统的基因组中 果蝇，一个单一的簇包含了所有典型的组蛋白基因。一套抄写 加工因子占据组蛋白基因座，形成一种被称为组蛋白的保守结构。 轨迹体(HLb)。已知HLb成员突变导致HLb形成和/或组蛋白基因缺陷 表达，通常导致动物死亡或不育。我们不知道占据组蛋白的所有因素 并参与组蛋白基因的调控，但我们对HLB的理解还存在很大差距 这些因子专门针对组蛋白基因位点。这项提议的总体目标是定义分子 组蛋白基因的相互作用指定了组蛋白基因以进行独特的调控。 最近的研究发现了果蝇蛋白钳，它能与GA-Repeat特异结合 组蛋白3/组蛋白4启动子内的序列，打开组蛋白基因座上的染色质，促进 所有组蛋白基因的表达。然而，目前还不清楚是否需要在组蛋白基因座上进行钳制 HLb特异性因子的定位，这将牵涉到钳制在指定HLb形成的位置。在……里面 目的1，我将定义DNA-蛋白质相互作用导致HLB的形成和组蛋白基因的表达。这就做 利用已建立的转基因组蛋白基因座系统(A)探讨组蛋白基因的位置要求 夹子-GA-重复相互作用和(B)决定夹子在发育层次中的位置 HLB型地层。同样重要的是，我们要定义HLB组成，因为这是朝着定义 HLB的形成机制和组蛋白基因调控。在目标2中，我将使用以下方法发现新的HLB因子 这既是一种无偏见的蛋白质组筛选，也是一种涉及挖掘现有组学数据集的候选方法。 这种候选方法也将是基于课程的本科生研究体验(CURE)的基础 我将在未来的独立实验室和教室中开发和使用的模块。总而言之，这些 实验将确定HLB的形成机制和组蛋白基因的表达，同时扩展My 实验曲目，并为我未来的实验室和教室创造新的方向。

PROJECT SUMMARY Eukaryotic nuclei are crowded compartments that not only compact but also organize meters of DNA. This feat is achieved by wrapping DNA around histone proteins to form nucleosomes, the basic units of chromatin. Histone levels in the cell are precisely balanced: a lack of histone expression causes cell cycle arrest, whereas histone overexpression leads to genomic instability. Although histone gene regulation is crucial for proper cell viability and function, mechanisms that regulate histone gene expression are poorly defined. Histone genes are often clustered in metazoan genomes. In the genome of the excellent model system Drosophila melanogaster, a single cluster encompasses all the canonical histone genes. A suite of transcription and processing factors occupy the histone gene locus and form a conserved structure known as the histone locus body (HLB). Mutations in known HLB members lead to defects in HLB formation and/or histone gene expression, often causing animal lethality or infertility. We do not know all the factors that occupy the histone locus and contribute to histone gene regulation, and there are large gaps in our understanding of how HLB factors specifically target the histone gene locus. The overall goal of this proposal is to define the molecular interactions at the histone locus that specify histone genes for unique regulation. Recent studies identified the Drosophila protein CLAMP, which binds specifically to GA-repeat sequences within the histone3/histone4 promoter, opens chromatin across the histone locus, and promotes expression of all histone genes. However, it is unclear if CLAMP is required at the histone locus prior to the localization of HLB-specific factors, which would implicate CLAMP in specifying the locus for HLB formation. In Aim 1, I will define the DNA-protein interactions that lead to HLB formation and histone gene expression. I will take advantage of an established transgenic histone locus system to (A) probe the positional requirements of the CLAMP-GA-repeat interaction and (B) determine the placement of CLAMP in the developmental hierarchy of HLB formation. It is also critical that we define HLB composition, as this is an important step towards defining mechanisms of HLB formation and histone gene regulation. In Aim 2, I will discover novel HLB factors using both an unbiased proteomic screen and a candidate approach that involves mining existing -omics datasets. This candidate approach will also be the basis of a Course-Based Undergraduate Research Experience (CURE) module that I will develop and utilize in both my future independent laboratory and classroom. Collectively, these experiments will define mechanisms of HLB formation and histone gene expression while expanding my experimental repertoire and generating new directions for my future laboratory and classroom.