Mutations in the DNA processivity factor PCNA (POL30) predispose to epigenetic instability at the FLO11 locus.

The FLO genes in S. cerevisiae are repressed by heterochromatin formation involving histone deacetylases, transcription factors, and non-coding RNAs. Here we report that mutations in processivity factor POL30 (PCNA) that show transient de-repression at the sub-telomeres and the mating-type loci, do not de-repress the FLO loci. However, deletions of the replisome-stability factors RRM3 and TOF1 along with pol30 mutations induce flocculation phenotypes. The phenotypes correlate with increased expression of reporter proteins driven by FLO11 promoter, the frequency of silent→active conversions of FLO11, and reduced expression of the regulatory lncRNAs ICR1 and PWR1. Alterations in the local replication landscape of FLO11 indicate a link between replication fork pausing and the stability of gene silencing. Analyses of these mutants at the sub-telomeres and HMLα show a similar de-repression phenotype and suggest transient instability of both active and silent states of FLO11. We conclude that RRM3 and TOF1 interact differentially with the pol30 mutations to promote transient de-repression or complete epigenetic conversions of FLO11. We suggest that interaction between POL30, RRM3, and TOF1 is essential to maintain epigenetic stability at the studied loci.