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

定义驱动组蛋白位点体形成和功能的分子相互作用

• 批准号: 10464621
• 负责人: Casey Alexandra Schmidt
• 金额: $ 6.76万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10464621
• 关键词: Address; Anabolism; Animal Model; Animals; Binding; Binding Sites; Biochemistry; Bioinformatics; Biological Assay; Biological Models; Biotin; Body Composition; Cell Cycle Arrest; Cell Nucleus; Cell Survival; Cell physiology; Cells; ChIP-seq; Chromatin; Chromosomes; Complement; Crowding; Cultured Cells; DNA; DNA Damage; DNA-Protein Interaction; Data Set; Defect; Development; Drosophila Proteins; Drosophila genus; Drosophila melanogaster; Elements; Embryo; Embryonic Development; Ensure; Face; Future; Galaxy; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Models; Genetic Transcription; Genome; Genomic DNA; Genomic Instability; Genomics; Goals; Health; Histones; Infertility; Label; Laboratories; Lead; Ligase; Location; Maps; Mass Spectrum Analysis; Mining; Molecular Biology; Mutation; Nature; Nuclear; Nucleosomes; Phase; Positioning Attribute; Proteins; Proteomics; Quantitative Reverse Transcriptase PCR; RNA Interference; Regulation; Research; Role; Specific qualifier value; Structure; System; Tandem Repeat Sequences; Time; Training; Transgenes; Transgenic Organisms; Work; Zinc Fingers; base; career; cohort; experimental study; genomic locus; in vivo; member; meter; mutant; novel; overexpression; pedagogy; promoter; skills; tool; transcription factor; undergraduate research experience; whole genome

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.

项目摘要 真核生物核是拥挤的隔室，不仅紧凑，而且还组织了DNA米。 通过将DNA围绕组蛋白蛋白形成核小体，可以实现此壮举 染色质。细胞中的组蛋白水平精确平衡：缺乏组蛋白表达会导致细胞周期停滞， 而组蛋白的过表达导致基因组不稳定性。尽管组蛋白基因调节对于 适当的细胞活力和功能，调节组蛋白基因表达的机制的定义很差。 组蛋白基因通常聚集在后生基因组中。在优秀模型系统的基因组中 果蝇Melanogaster，一个群集涵盖了所有规范组蛋白基因。一套转录 和加工因子占据了组蛋白基因座并形成称为组蛋白的保守结构 基因座体（HLB）。已知HLB成员的突变导致HLB形成和/或组蛋白基因的缺陷 表达，通常会导致动物致死性或不育。我们不知道所有占用组蛋白的因素 基因座并有助于组蛋白基因调节，我们对HLB的理解有很大的差距 因素明确靶向组蛋白基因基因座。该提议的总体目标是定义分子 在组蛋白基因座的相互作用，该基因蛋白指定组蛋白基因的独特调节。 最近的研究确定了果蝇蛋白夹，该蛋白质夹与GA-重复专门结合 组蛋白3/组蛋白4启动子内的序列，在整个组蛋白基因座上打开染色质，并促进 所有组蛋白基因的表达。但是，目前尚不清楚在组蛋白基因座之前是否需要夹具 HLB特异性因素的定位，这将暗示夹具指定HLB形成的基因座。在 AIM 1，我将定义导致HLB形成和组蛋白基因表达的DNA蛋白相互作用。我会 利用已建立的转基因组蛋白基因蛋白基因座系统来探测 夹具-GA重复相互作用，（b）确定夹具在发育层次结构中的位置 HLB形成。我们定义HLB组成也是至关重要的，因为这是定义的重要一步 HLB形成和组蛋白基因调节的机制。在AIM 2中，我会发现使用新颖的HLB因素 无偏的蛋白质组学屏幕和候选方法涉及挖掘现有的 - 组数据集。 这种候选方法还将成为基于课程的本科研究经验（CURE）的基础。 我将在未来的独立实验室和课堂上开发和使用的模块。总的来说，这些 实验将定义HLB形成和组蛋白基因表达的机制，同时扩展我 实验性曲目并为我的未来实验室和课堂创造新的方向。