Abstract: ABSTRACT: BACKGROUND: 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 the replication initiator, the Origin Recognition Complex (ORC), to a consensus sequence within origins. In addition, other proteins bind to recognition sites in the vicinity of ORC and support initiation. In previous work, we identified Sum1 as an origin-binding protein that contributes to efficient replication 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. RESULTS: In this study, we investigated how Sum1 affected replication initiation. We found that it functioned in initiation as a component of the Sum1/Rfm1/Hst1 complex, implying a role for histone deacetylation in origin activity. We identified several origins in the yeast genome whose activity depended on both Sum1 and Hst1. Importantly, sum1 or hst1 deletion 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. CONCLUSIONS: Taken together, our results demonstrate a role for histone deacetylation in origin activity and reveal a novel aspect of origin regulation by chromatin. These results suggest recruitment of the Sum1/Rfm1/Hst1 complex to a number of yeast origins, where Hst1 deacetylated H4 K5.