Proper regulation of histone biosynthesis during the cell cycle is critical for coordinating chromatin assembly and DNA replication. Overproduction or underproduction of histone protein results in replication stress and genome instability that contributes to the development of cancer. In this proposal, we undertake a comprehensive analysis of replication-dependent (RD) histone mRNA biosynthesis and how this process is regulated during the cell cycle in the context of animal development. RD-histone mRNAs are the only eukaryotic mRNAs in animal cells that lack a polyA tail, ending instead in a conserved stem loop structure. In contrast, mRNAs for histone variants such as H3.3 and H2A.Z are encoded by polyadenylated mRNAs that are not cell cycle regulated. Generation of the unique RD-histone mRNA 3? end results from the activity of an evolutionary conserved set of pre-RNA processing factors. The genes encoding all five RD-histone proteins are clustered in metazoan genomes, and transcription and pre- mRNA processing factors required for histone mRNA biosynthesis are organized into a nuclear body (the histone locus body or HLB) that assembles at these gene clusters. We will determine the requirements for the coordinate synthesis of the RD-histone mRNAs using both biochemical and genetic approaches in Drosophila, with a particular focus on the role that the HLB plays in histone transcription and pre-mRNA processing. Histone gene clusters provide a system in which one can readily study the expression of a tightly regulated set of genes at all levels of mRNA biosynthesis, from the organization of genes within the nucleus through activation of transcription, pre-mRNA processing and transcription termination. Our Drosophila experimental paradigm permits the in vivo interrogation of these fundamental processes in gene expression in ways that are unavailable in other experimental systems.
Each time DNA is replicated during the process of cell proliferation, histone proteins are synthesized in large amounts to package newly made DNA into chromosomes. We will combine biochemical and genetic studies in the fruit fly Drosophila to study the mechanisms and regulation of the synthesis of histones proteins. Factors controlling histone synthesis are critical for stable inheritance of genetic information during organism development, and thus for the suppression of human disease. Because of high evolutionary conservation, our studies in flies will allow us to understand the function of these factors in humans.
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