A systematic approach assigning new candidates to functional pathways in gene silencing and unraveling the role of replisome progression complex in heterochromatin inheritance

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How transcriptionally silent chromatin is replicated and epigenetically maintained is poorly understood. This proposal seeks to unravel the mechanisms by which replisome components and other candidates identified in genetics screens contribute to heterochromatin inheritance. A variety of factors and protein complexes assemble into the replisome progression complex that has multiple functions during DNA synthesis and chromatin replication. Several of them are also critical for heterochromatin maintenance. However, while their functions during replication have been extensively studied in Saccharomyces cerevisiae, this model system lacks canonical repressive histone modifications and little is known about their functions with respect to heterochromatin.Through genome-wide quantitative reporter screens, my lab has identified a large number of mutants with heterochromatin defects in the fission yeast Schizosaccharomyces pombe. In this proposal, we focus on a protein of unknown function, Dhm2, and several replisome-associated factors involved in replication fork stability and the transfer of histone proteins to the daughter DNA strands. Our unpublished results indicate that Dhm2 acts redundantly with known heterochromatin pathways, whereas its absence causes DNA damage and phenotypes associated with replication stress. Through a combination of genetics and proteomics, we will test the hypothesis that Dhm2 is a new replication factor and unveil the mechanism that links its molecular function to heterochromatin silencing. By implementing function-specific mutants characterized in S. cerevisiae, we will further investigate how replisome-associated factors contribute to heterochromatin maintenance in S. pombe. To this end, we will employ state-of-the-art approaches that allow us to monitor precisely the inheritance of parental nucleosomes and histone modifications during replication. Finally, through functional genomics and assessing systematically pairwise genetic interactions, we seek to assign other candidates to molecular pathways and regulatory networks in heterochromatin silencing using the powerful E-MAP (epistasis mini-array profiling) approach. S. pombe is an ideal model system to study this process, as it is genetically tractable and comprises conserved hallmarks of heterochromatin that are absent in S. cerevisiae. The proposed work will shed light on the spatial and temporal regulation of heterochromatin and provide critical insights into how heterochromatin is replicated, which has been scarcely studied in the past.