Regulation of Histone Locus Body (HLB) Organization and Function

描述(由申请人提供)：基因调控需要在细胞核内广泛组织基因组DNA和蛋白质复合体。一种这样的组织形式是称为核体的动态结构的集合。人们对核体如何形成以及对基因表达的贡献知之甚少。我们建议通过研究果蝇的组蛋白基因座(HLB)来研究这些问题。HLB在组蛋白位点组装，它的几个组分被认为参与了组蛋白mRNA的生物合成(即转录和前mRNA加工)。我们最近发现，果蝇多性梳子(Mxc)通过闪光作用共定位于组蛋白基因座，这些蛋白共同启动了HLB的分级组装。此外，HLB的调节依赖于细胞周期，因为Mxc在S期受到依赖于CycE/CDK2的磷酸化，就像它的人类同源基因NPAT一样。然而，Mxc如何定位于组蛋白位点，以及Mxc及其依赖的CycE/CDK2磷酸化在HLb的形成和功能中扮演着什么角色，这些都是关键的未解答的问题。我们假设Mxc定位于组蛋白位置，并核HLB组装，以促进组蛋白mRNA的生物合成。当细胞进入S期时，染色体相关的Mxc被CycE/CDK2磷酸化，这使得它能够招募额外的HLB因子来促进组蛋白基因在整个S期的表达。我正在用果蝇培养的细胞来确定Mxc的结构域，以定位到组蛋白位点和HLb成核，以及Mxc蛋白上CycE/CDK2磷酸化位点的位置。我将探索Mxc和Mxc的CycE/CDK2依赖的磷酸化如何通过产生携带不同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因子招募到组蛋白基因座，以及这如何影响组蛋白mRNA的生物合成。

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.