Timely and complete chromatin replication is critical to a number of cellular processes, including proper chromosome disjunction at mitosis and the propagation of transcriptionally active or inactive states. In budding yeast, Saccharomyces cerevisiae, new histone synthesis is coupled to the period of DNA replication primarily through the activity of a cohort of transcriptional repressors. Two of these repressors, the products of the evolutionarily conserved HIR1 and HIR2 genes, act in the absence of DNA binding as a putative corepressor complex. Their activity is modulated by a subset of Clb-dependent Cdc28 kinases, which may target Hir1p for inactivation at the G1/S phase boundary. The two proteins appear to repress transcription by remodeling chromatin structure at the genes they control. Hir2p binds to histones H2B, H3, and H4 in vitro, and deletion of HIR1 causes increased accessibility of an H2A-H2B locus to nuclease digestion. This has lead to the hypothesis that the Hir proteins function as novel class of nucleosome assembly or stability factors. The long range goal is to understand how Hir proteins interact with histones and other factors to cause repression by chromatin. Genetic, molecular, and biochemical approaches will be used: i) to relate the interactions of Hir proteins with histones and other proteins to specific chromatin changes i vivo, ii) to understand how the Clb1/2-kinase inactivates Hir proteins in the cell cycle, and to learn whether the Hir proteins are regulated by an S phase checkpoint pathway; and iii) to isolate mutations in the HTB1 gene that relieve transcriptional repression in vivo by altering nucleosome assembly or stability. Because inappropriate transcription at the wrong time or in the wrong place can lead to developmental defects or oncogenesis, factors that mediate repression by chromatin are critical cellular regulators. By studying how two yeast proteins repress transcription through their chromatin interactions, we should gain general insight into this important biological phenomenon.
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