Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins

Abstract

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin - SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 - correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly bind euchromatic DNA replication origins and modify their local chromatin environment.