Regulation of replication initiation and HML silencing in Saccharomyces cerevisiae

The Saccharomyces cerevisiae origin recognition complex (ORC) displays a dual role in silencing and initiation of DNA replication, but requires the contribution of auxiliary factors such as Rap1 or Sum1 for full initiation capacity. In this study, the influence of ORC and factors binding in the vicinity of ORC on origin activity and HML silencing was analysed. The silent mating-type loci HML and HMR of S. cerevisiae contain mating-type information that is permanently repressed. This silencing is mediated by flanking sequence elements, the E- and I-silencers. They contain combinations of binding sites for Rap1, Abf1 and Sum1 as well as for ORC. Together, they recruit other silencing factors, foremost the repressive Sir2/Sir3/Sir4 complex, to establish heterochromatin-like structures at the HM loci. However, the HM silencers exhibit considerable functional redundancy, which has hampered the identification of further silencing factors. Therefore, a synthetic, minimal HML-E silencer (HML-SS ΔI) that lacked this redundancy was constructed during the course of this study. It consisted solely of Rap1 and ORC binding sites and the D2 element, a Sum1 binding site, and all three elements were crucial for minimal HML silencing. This silencer was sensitive to a mutation in RAP1, rap1-12, but less sensitive to orc mutations or sum1Δ. Moreover, deletions of SIR1 and DOT1 led to complete derepression of the HML-SS ΔI silencer. This fully functional, minimal HML-E silencer will therefore be useful to identify novel factors involved in HML silencing. Replication initiation at origins of replication in the yeast genome takes place on chromatin as a template, raising the question how histone modifications, for instance histone acetylation, influence origin firing. Initiation requires binding of ORC to a consensus sequence within origins and of other proteins, for instance Sum1, to recognition sites in the vicinity of ORC to support initiation. Sum1 is part of the Sum1/Rfm1/Hst1 complex that represses meiotic genes during vegetative growth via histone deacetylation by the histone deacetylase (HDAC) Hst1. In this study, it was found that Sum1 functioned in initiation as a component of this complex, implying a role for histone deacetylation in origin activity. Several origins were identified in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1Δ or hst1Δ caused a significant increase in histone H4 lysine 5 (H4 K5) acetylation levels, but not other H4 acetylation sites, at those origins. Furthermore, mutation of lysines to glutamines in the H4 tail, which imitates the constantly acetylated state, resulted in a reduction of origin activity comparable to that in the absence of Hst1, showing that deacetylation of H4 was important for full initiation capacity of these origins.



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