Functional Analysis of Histone Regulatory Proteins

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

DNA in all organisms exists in large molecules known as chromosomes. In order to fit huge chromosome molecules into cells, the DNA is repeatedly wrapped in tight spools around chromatin proteins. The major type of chromatin proteins is the histones. In many organisms, a strong peak of histone mRNA and protein synthesis occurs at the G1-S transition of the cell cycle, just prior to DNA synthesis. This ensures that histones are synthesized just as they are needed for packaging newly synthesized DNA. This regulation occurs in many organisms, from humans to yeasts; the ease of genetic manipulation of yeast makes them an attractive model organism for study. In the budding yeast Saccharomyces cerevisiae, four Histone Regulator genes (HIR1, HIR2, HIR3, and HPC2, referred to collectively as HIR genes) are required to repress histone gene transcription outside of the G1-S period. Progress in understanding Hir protein function requires differentiation between their direct contribution to chromosome structure and their regulation of histone synthesis. In this project, these roles will be genetically separated by creating strains in which histone synthesis is no longer regulated by Hir proteins. A genetic screen to define which genes become essential for viability in yeast cells lacking the HIR1 gene will be pursued. Immunoprecipitation, velocity sedimentation and gel filtration experiments will test whether Hir proteins are physically associated with each other or with histones in yeast cell extracts. Using epitope-tagged Hir proteins, these experiments may also identify novel Hir-associated proteins. Hir protein complexes will be isolated and will be used to test hypotheses generated by previous genetic data: are the Hir proteins a site-specific DNA-binding complex? Do they act as a histone deposition factor? If the Hir complex is indeed found to possess histone deposition activity, subsequent experiments will address whether the activity is replication-dependent or synergistic with CAF-I activity. This work will improve our understanding of how cells coordinate chromosome formation with cell growth.