Connecting Nuclear Organization and Gene Expression: The Role of the Histone Locus Body in Histone mRNA Biosynthesis

Execution of gene expression involves multiple reactions, many of which are mediated through cis elements in DNA or RNA. Organization within the nucleus, both in the arrangement of DNA and the concentration of trans factors in discrete nuclear environments called nuclear bodies, facilitates aspects of gene expression such as transcription, RNP metabolism and pre-mRNA processing. These substructures, which are visible under the light microscope, potentially promote the efficiency and fidelity of a reaction. To investigate this proposed function of nuclear bodies (NBs), I studied the relationship between the Drosophila replicative histone genes and their associated nuclear body, the histone locus body (HLB). The genes encoding the five histone proteins are tandemly arrayed in 100 copies. The replicative histone genes are highly expressed during S phase of the cell cycle, and encode the only known mRNAs that do not end in a poly (A) tail. We defined a 297 bp sequence that encompassed the H3-H4 bidirectional promoter and demonstrated that activity from this promoter was necessary and sufficient for HLB maturation as well as for activation of the neighboring H2a-H2b gene pair. Thus the HLB plays a role in coordinating gene activation within the histone repeat. I then evaluated the in vivo consequence of preventing accumulation of a critical histone pre-mRNA processing trans factor, FLASH, in the HLB. When FLASH was present at wild type levels in the nucleus, but not localized to the HLB, longer, unprocessed nascent transcripts accumulated at the histone locus. I suggest that the presence of these pre-mRNAs indicates a slower rate of histone pre-mRNA processing. Finally, I characterized the phenotypes of mutants in a pre-mRNA processing factor, Symplekin (Sym), involved in histone pre-mRNA processing, cleavage and polyadenylation and cytoplasmic polyadenylation. I established an in vivo system for testing candidate separation of function Sym mutations. Sym was first discovered at tight junctions, and I provide evidence that this localization indicates likely participation in cytoplasmic polyadenylation. Together this work provides insight into how specific regulatory complexes containing common factors assemble and the role of the HLB in the metabolism and regulation of histone mRNAs.Doctor of Philosoph