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

Abstract

25Most active DNA replication origins are found within euchromatin, while origins within 26 heterochromatin are often inactive or inhibited. In yeast, origin activity within 27 heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at 28 some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing 29 view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. 30However, growth defects in yeast mutants compromised for loading the MCM helicase, 31 such as cdc6-4, are robustly suppressed by deletion of either SIR2 or SIR3. While this 32 and other observations indicate that SIR2,3 can have a negative impact on at least some 33 euchromatic origins, the genomic scale of this effect was unknown. It was also unknown 34 whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or 35 was an indirect effect of their previously established roles within heterochromatin. Using 36 both MCM ChIP-Seq and MNase-H4K16ac ChIP-Seq data, we show that a SIR2 37 deletion rescues MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. 38Therefore, Sir2 exhibits a pervasive effect at the majority of euchromatic origins. 39Importantly, in wild type (i.e. CDC6) cells, origin-adjacent nucleosomes were depleted 40 for H4K16 acetylation in a SIR2-dependent manner. In addition, both Sir2 and Sir3 41 directly bound to nucleosomes adjacent to euchromatic origins. The relative levels of 42 each of these molecular hallmarks of yeast heterochromatin -SIR2-dependent H4K16 43 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion 44 suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and 45 H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that 46 map to a surface of the nucleosome important for Sir3 binding. We conclude that 47 heterochromatin proteins directly bind euchromatic DNA replication origins and modify 48 their local chromatin environment. 49 50 3 AUTHOR SUMMARY 51When a cell divides, it must copy or "replicate" its DNA. DNA replication starts at 52 chromosomal regions called origins when a collection of replication proteins gains local 53 access to unwind the two strands. Chromosomal DNA is packaged into a protein-DNA 54 complex called chromatin and there are two major structurally and functionally distinct 55 types. Euchromatin allows DNA replication proteins to access origin DNA, while 56 heterochromatin inhibits their access. The prevalent view has been that the 57 heterochromatin proteins required to inhibit origins are confined to heterochromatin. In 58 this study, the conserved heterochromatin proteins, Sir2 and Sir3, are shown to both 59 physically and functionally associate with the majority of origins in euchromatin. This 60 raises important new questions about the chromosomal targets of heterochromatin 61 proteins, and how and why the majority of origins exist within a potentially repressive 62 chromatin structure. 63 64 replic...

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