Regulation of Histone Locus Body (HLB) Organization and Function

描述（由申请人提供）：基因调控需要细胞核内基因组DNA和蛋白质复合物的广泛组织。一种这样的组织形式是被称为核体的动态结构的集合。关于核小体如何形成以及如何促进基因表达，我们知之甚少。我们建议通过研究果蝇组蛋白基因座体（HLB）来调查这些问题。HLB在组蛋白基因座处组装，并且已知其几种组分参与组蛋白mRNA生物合成（即，转录和前mRNA加工）。我们最近发现，果蝇多性梳（Mxc）co-localizes到组蛋白基因座与FLASH，这些蛋白质一起启动的HLB的分级组装。此外，HLB以细胞周期依赖性方式调节，因为Mxc在S期期间经历CycE/Cdk 2依赖性磷酸化，其人直向同源物NPAT也是如此。然而，Mxc如何定位于组蛋白位点以及Mxc及其CycE/Cdk 2依赖性磷酸化在HLB的形成和功能中发挥什么作用是关键的未回答的问题。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的生物合成。当细胞进入S期时，染色体相关的Mxc被CycE/Cdk 2磷酸化，这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。我使用果蝇培养的细胞来确定Mxc的结构域需要定位到组蛋白位点和HLB成核以及CycE/Cdk 2磷酸化位点的Mxc蛋白的位置。我将探讨如何Mxc和Mxc的CycE/Cdk 2依赖磷酸化有助于HLB的形成和功能，通过产生转基因苍蝇窝藏不同的Mxc突变。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.