The role of histone locus body (HLB) assembly and the cell cycle in histone mRNA biosynthesis

The compartmentalization of nuclear processes such as transcription and histone mRNA biosynthesis into discrete territories is essential for orchestrating diverse gene expression programs. Genomes organize structures called nuclear bodies (NBs) that concentrate factors (proteins and RNA) required for gene expression regulation. The levels of NB components fluctuate continuously in response to diverse physiological inputs. Therefore, gaining insight into how NBs assemble and function is essential for understanding their contribution to genome function. We used the Drosophila histone locus body (HLB) to ask how HLB assembly and function contribute to replication-dependent histone gene expression. HLBs form at the histone locus and concentrate factors required for histone mRNA biosynthesis. In addition, CycE/Cdk2 is known to be required for cell cycle-dependent histone mRNA biosynthesis. We previously demonstrated that the Multi Sex Combs (Mxc) protein is required for HLB assembly and efficient histone mRNA biosynthesis and is also phosphorylated by CycE/Cdk2. However, the role that Mxc and its CycE/Cdk2-dependent phosphorylation play in HLB assembly and histone gene expression was not known. We explored the molecular mechanism by which Mxc assembles an HLB and contributes to histone gene expression, by combining biochemical assays, microscopy, and live imaging. We show that Mxc is a multivalent protein that plays an essential role in HLB assembly. We demonstrate that Mxc self-interacts, via the N-terminal LisH and the novel SIF domains, which is crucial for its accumulation at the histone locus. A region between amino acids 721 and 1481 is essential for HLB assembly independent of the LisH and SIF domains. The last 195 amino acids of Mxc are required for the recruitment of FLASH, a histone pre-mRNA processing factor, to the HLB. In addition, preliminary data suggest that Mxc harbors multiple CycE/Cdk2 phosphoepitopes required for HLB assembly. Combining the present work with our previous findings, we propose that Mxc is capable of forming a three-dimensional scaffold, the HLB, by employing multiple domains, and presumably multiple CycE/Cdk2 phosphoepitopes, to accumulate at the histone locus and to concentrate the required histone mRNA biosynthetic components to assemble a mature HLB that promotes and maintains histone gene expression throughout S phase.Doctor of Philosoph

Concentrating pre-mRNA processing factors in the histone locus body facilitates efficient histone mRNA biogenesis

The histone locus body (HLB) assembles at replication-dependent histone genes and concentrates factors required for histone messenger RNA (mRNA) biosynthesis. FLASH (Flice-associated huge protein) and U7 small nuclear RNP (snRNP) are HLB components that participate in 3′ processing of the nonpolyadenylated histone mRNAs by recruiting the endonuclease CPSF-73 to histone pre-mRNA. Using transgenes to complement a FLASH mutant, we show that distinct domains of FLASH involved in U7 snRNP binding, histone pre-mRNA cleavage, and HLB localization are all required for proper FLASH function in vivo. By genetically manipulating HLB composition using mutations in FLASH, mutations in the HLB assembly factor Mxc, or depletion of the variant histone H2aV, we find that failure to concentrate FLASH and/or U7 snRNP in the HLB impairs histone pre-mRNA processing. This failure results in accumulation of small amounts of polyadenylated histone mRNA and nascent read-through transcripts at the histone locus. Thus, the HLB concentrates FLASH and U7 snRNP, promoting efficient histone mRNA biosynthesis and coupling 3′ end processing with transcription termination

Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body

Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis