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.
An important aspect of genome function is the organization of proteins and RNAs into microscopically visible structures within the nucleus called nuclear bodies, whose normal size, number, and distribution have been recently shown to undergo numerous cancer-related alterations. Although important progress has been made associating nuclear bodies with several nuclear processes, how they function and whether nuclear body assembly is the cause or a consequence of gene expression are questions that remain unanswered. Understanding how nuclear bodies assemble and the roles they play in the regulation of gene expression will enable us to diagnose tumor formation and to identify targets for therapeutic treatments.