Nucleosome assembly pathways, Rrm3p and phosphorylation of CAF-I contribute to the maintenance of silent chromatin at the FLO genes in Saccharomyces cerevisiae

Abstract

Predominantly, studies of gene repression in S. cerevisiae focus on Sir2p-dependent silencing at the telomeres and the mating type loci. The FLO genes are heavily repressed in laboratory yeasts by a Sir2p-independent mechanism that is not well understood. Chromatin Assembly Factor I (CAF-I) plays a central role in the reassembly of H3/H4 histones during DNA replication. Two kinases, Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), are known to phosphorylate the Cac1p subunit of CAF-I, but their role in the regulation of CAF-I activity is not well understood. In this thesis, I analysed the FLO genes, which encode cell-surface proteins and participate in the formation of floccules and biofilm. I report that the mutations in histone chaperones, the helicase RRM3 and Histone Deacetylases cause de-repression of the FLO genes and reconstitute flocculation. This is linked to increased histone acetylation at the promoter of FLO1, but not the promoter of FLO11. Additionally the histone deacetylase inhibitor Nicotinamide or growth under starvation conditions enhances flocculation in strains compromised in chromatin maintenance. These effects are linked to variegated expression FLO11 and elevated expression of all FLO genes. Simultaneous mutations of both CDK and DDK target sites of CAF-I lead to growth retardation, significant cell cycle defects, altered cell morphology and increased sensitivity to DNA damage. Two of these mutants also show the reconstitution of flocculation and expression of FLO genes, but do not re-capitulate the transient losses in HML silencing or losses in sub-telomeric silencing observed in cac1Δ mutants. An additional mutant causes defects in silencing at the sub-telomere, but not at HML or the FLO genes. I provide detailed analysis of the repression of FLO genes and propose that a gain in epigenetic silencing is a major contributing factor for the loss of flocculation in laboratory budding yeast strains. I have also shown that the role of Rrm3p in silencing extends beyond interaction with CAF-I at sub-telomeric replication forks. In addition, dysfunctional CAF-I produces severe phenotypes, and locus-specific loss of silencing phenotypes which reveal a possible role of CAF-I in the coordination of DNA replication, chromatin reassembly and cell cycle progression.