Epigenetic transmission in S.cerevisiae: links to DNA replication and cell cycle regulation

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BACKGROUND: Gene silencing in eukaryotes is mediated by compact heterochromatin, which is reassembled after each passage of the DNA replication forks. Orchestrated conversions between the silenced and active states of genes play key roles in metazoan development and in adaptation of single cell organisms. Despite their significance, the mechanisms of such epigenetic conversions are poorly understood. ONGOING RESEARCH: My lab has identified three genes (CAC1, ASF1, RRM3) that alter the rate of epigenetic conversions at the telomeres of S. cerevisiae. CAC1 and ASF1 encode histone chaperones involved in nucleosome reassembly behind the replication forks. RRM3 promotes the resumption of elongation at paused replication forks. Based on these observations by us and others, I propose that the pausing of replication forks causes aberrant chromatin reassembly and predisposes to epigenetic change. Moreover, recently my lab has shown that Cac1p is phosphorylated by two kinases, CDK and DDK. Cells harboring CAC1 mutations at the target sites of these kinases compromise gene silencing at several loci and show cell cycle and growth defects. Based on our data, I propose that chromatin reassembly is linked to the regulation of the cell cycle via CDK and DDK. LONG-TERM GOAL: Using S. cerevisiae as a model organism, we will explore the link between epigenetic conversions and the pausing of DNA replication and will elucidate the roles of CDK and DDK in these processes. SHORT-TERM OBJECTIVES: 1. To understand the molecular mechanisms of the regulation of CAF-1 activity by phosphorylation. Through combining mutations in the CDK and DDK target sites, we will address the following questions: - Do Cac1p mutant proteins associate with chromatin? - Does DDK phosphorylate Cac1p in vivo? - Do mutant Cac1p proteins exacerbate fork pausing at positions of tightly bound proteins? - Do mutant Cac1p proteins associate with stalled replication forks in vivo? 2. To analyze replication fork pausing in CAC1 and ASF1 mutants. We will determine if mutations in these chaperones affect fork processivity in vivo. 3. To analyze fork composition. We will determine if the association of Cac1p (including phosphorylation-deficient mutants) and Asf1p with paused replication forks is altered in cells lacking RRM3. 4. To perform genetic interaction analysis between CAC1, ASF1, RRM3 and genes that regulate the stability of paused forks. Using a novel assay, we will determine if these genes genetically interact in the control of gene silencing and epigenetic conversions. ORIGINALITY and SIGNIFICANCE: My research program is one of the very few in the world that are addressing the link between epigenetic conversions and the pausing of DNA replication. It is therefore expected to make significant contributions to the fields of epigenetics, DNA replication and cell cycle progression. It will also relate to the fields of genome stability and cell differentiation and adaptation.