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 suppressed by deletion of either SIR2 or SIR3. While these 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 MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 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 modify the local chromatin environment of euchromatic DNA replication origins.
Highlights
In eukaryotic cells, efficient genome duplication requires the function of multiple DNA replication origins distributed over the length of each chromosome [1,2,3,4,5]
The prevalent view has been that the heterochromatin proteins required to inhibit origins are confined to heterochromatin
Sir2 and Sir3 were physically associated with euchromatic origins but not non-origin control loci. The levels of these three distinct molecular features of yeast heterochromatin—hypoacetylation of H4K16, Sir2 and Sir3 binding—correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Based on these results we propose that the dhMCM loading reaction has evolved to work within a potentially repressive Sir2,3-chromatin environment that forms around most euchromatic origins
Summary
Efficient genome duplication requires the function of multiple DNA replication origins distributed over the length of each chromosome [1,2,3,4,5]. Origins are selected by a series of steps in late M- to G1-phase during which the origin recognition complex (ORC) binds directly to DNA and recruits the Cdc protein [6,7,8]. The ORC-Cdc6-DNA complex recruits Cdt1-MCM to form an MCM double hexamer (dhMCM) encircling double-stranded DNA [9,10]. DhMCM loading is the event that ‘licenses’ the DNA to function as an origin of replication in S-phase (for recent comprehensive review of yeast replication see [12]). All replication origins exist in the context of chromatin, and origin function is significantly affected by local chromatin structure (reviewed in [13,14]). While there is intense interest in the impact of chromatin structure on origin function, the relevant molecular features of origin-adjacent chromatin and the steps in origin function that they control remain incompletely understood
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