Regulation of Histone Locus Body (HLB) Organization and Function

组蛋白位点体 (HLB) 组织和功能的调控

• 批准号: 8524369
• 负责人: Esteban A. Terzo
• 金额: $ 2.97万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8524369
• 关键词: Address; Affect; Amino Acids; Anabolism; Biological; Biological Assay; Biological Process; Cell Cycle; Cell Cycle Progression; Cell Nucleolus; Cell Nucleus; Cell Proliferation; Cells; Chromosomes; Collection; Comb animal structure; Cultured Cells; DNA; Data; Deletion Mutation; Development; Diagnosis; Dimerization; Drosophila genus; Drosophila melanogaster; Embryo; Engineering; Gene Expression; Gene Expression Regulation; Genetic; Genetic Transcription; Genome; Genomics; Histones; Human; Laboratories; Lead; Light Microscope; Link; Location; Malignant Neoplasms; Messenger RNA; Modeling; Molecular; Mutate; Mutation; Normal Cell; Nuclear; Nuclear Structure; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Proteins; Recruitment Activity; Regulation; Research; Role; S Phase; Site; Structure; Testing; Transcript; Transcription Process; Transgenic Organisms; fly; in vivo; mRNA Precursor; mutant; protein complex; public health relevance; scaffold; sex; therapeutic target; tool; tumor

DESCRIPTION (provided by applicant): Gene regulation requires the extensive organization of genomic DNA and protein complexes within the nucleus. One such form of organization is the collection of dynamic structures known as nuclear bodies. Relatively little is known about how nuclear bodies form and contribute to gene expression. We propose to investigate these issues by studying the Drosophila melanogaster Histone Locus Body (HLB). The HLB assembles at the histone locus and several of its components are known to be involved in histone mRNA biosynthesis (i.e., transcription and pre-mRNA processing). We have recently shown that Drosophila Multi Sex Combs (Mxc) co-localizes to the histone locus with FLASH, and together these proteins initiate the hierarchical assembly of the HLB. In addition, HLBs are regulated in a cell cycle-dependent manner, as Mxc is subjected to CycE/Cdk2-dependent phosphorylation during S phase, as is its human orthologue, NPAT. However, how Mxc localizes to the histone locus and what role Mxc and its CycE/Cdk2-dependent phosphorylation play in the formation and in the function of the HLB are critical unanswered questions. We hypothesize that Mxc localizes to the histone locus and nucleates HLB assembly to facilitate histone mRNA biosynthesis. When cells enter S phase, the chromosome-associated Mxc is phosphorylated by CycE/Cdk2, which enables it to recruit additional HLB factors to promote histone gene expression throughout S phase. I am using Drosophila cultured cells to determine the domains of Mxc required for localization to the histone locus and HLB nucleation as well as the location of the CycE/Cdk2 phosphorylation sites on the Mxc protein. I will explore how Mxc and Mxc's CycE/Cdk2-dependent phosphorylation contribute to HLB formation and function by generating transgenic flies harboring different Mxc mutations. These genetic tools will enable us to determine how Mxc recruits HLB factors to the histone locus and how this affects histone mRNA biosynthesis.

描述（由申请人提供）：基因调控需要基因组 DNA 和蛋白质复合物在细胞核内的广泛组织。其中一种组织形式是称为核体的动态结构的集合。关于核体如何形成以及如何促进基因表达，人们知之甚少。我们建议通过研究果蝇组蛋白基因座体 (HLB) 来研究这些问题。 HLB 在组蛋白基因座处组装，已知其多个组件参与组蛋白 mRNA 生物合成（即转录和前 mRNA 加工）。我们最近表明，果蝇多性梳 (Mxc) 与 FLASH 共定位于组蛋白基因座，并且这些蛋白质一起启动 HLB 的分层组装。此外，HLB 以细胞周期依赖性方式受到调节，因为 Mxc 在 S 期经历 CycE/Cdk2 依赖性磷酸化，与其人类直系同源物 NPAT 一样。然而，Mxc 如何定位到组蛋白位点以及 Mxc 及其 CycE/Cdk2 依赖性磷酸化在 HLB 的形成和功能中发挥什么作用，是尚未解答的关键问题。我们假设 Mxc 定位于组蛋白基因座并使 HLB 组装成核以促进组蛋白 mRNA 生物合成。当细胞进入 S 期时，染色体相关的 Mxc 被 CycE/Cdk2 磷酸化，使其能够招募额外的 HLB 因子以促进整个 S 期的组蛋白基因表达。我使用果蝇培养细胞来确定定位到组蛋白位点和 HLB 成核所需的 Mxc 结构域以及 Mxc 蛋白上 CycE/Cdk2 磷酸化位点的位置。我将探讨 Mxc 和 Mxc 的 CycE/Cdk2 依赖性磷酸化如何通过生成携带不同 Mxc 突变的转基因果蝇来促进 HLB 的形成和功能。这些遗传工具将使我们能够确定 Mxc 如何将 HLB 因子招募到组蛋白位点以及这如何影响组蛋白 mRNA 生物合成。