Abstract
Proliferating cells coordinate histone and DNA synthesis to maintain correct stoichiometry for chromatin assembly. Histone mRNA levels must be repressed when DNA replication is inhibited to prevent toxicity and genome instability due to free non-chromatinized histone proteins. In mammalian cells, replication stress triggers degradation of histone mRNAs, but it is unclear if this mechanism is conserved from other species. The aim of this study was to identify the histone mRNA decay pathway in the yeast Saccharomyces cerevisiae and determine the mechanism by which DNA replication stress represses histone mRNAs. Using reverse transcription-quantitative PCR and chromatin immunoprecipitation–quantitative PCR, we show here that histone mRNAs can be degraded by both 5′ → 3′ and 3′ → 5′ pathways; however, replication stress does not trigger decay of histone mRNA in yeast. Rather, replication stress inhibits transcription of histone genes by removing the histone gene–specific transcription factors Spt10p and Spt21p from histone promoters, leading to disassembly of the preinitiation complexes and eviction of RNA Pol II from histone genes by a mechanism facilitated by checkpoint kinase Rad53p and histone chaperone Asf1p. In contrast, replication stress does not remove SCB-binding factor transcription complex, another activator of histone genes, from the histone promoters, suggesting that Spt10p and Spt21p have unique roles in the transcriptional downregulation of histone genes during replication stress. Together, our data show that, unlike in mammalian cells, replication stress in yeast does not trigger decay of histone mRNAs but inhibits histone transcription.
Highlights
Proliferating cells need to maintain a delicate balance between histone and DNA synthesis to ensure correct stoichiometric amounts for chromatin assembly and avoid genome instability [1, 2]
At the end of the S phase, or when the DNA replication is inhibited during the S phase, histone mRNAs are degraded in a stem loop–dependent manner by 50 to 30 and 30 to 50 pathways [54, 55]
Subsequent studies utilized fusion of histone HTA1 gene with lacZ [21]. When this fusion was expressed from histone promoter, it behaved identically to the endogenous histones, and the corresponding HTA1-lacZ mRNA was repressed when DNA synthesis was inhibited using the cdc8 mutation or HU. When this fusion was expressed from the GAL10 promoter, the fusion mRNA was insensitive to HU, suggesting that the coupling between DNA synthesis and histone mRNAs is due to transcriptional regulation
Summary
Proliferating cells need to maintain a delicate balance between histone and DNA synthesis to ensure correct stoichiometric amounts for chromatin assembly and avoid genome instability [1, 2]. The expression of histone genes is activated by Spt10p, Spt21p, SCB-binding factor (SBF), and MCB-binding factor (MBF) and repressed by the HIR complex [1, 2, 6,7,8]. The protein level of Spt21p is cell cycle regulated; Spt21p is degraded during the G1 and G2/M phases and accumulates only during the S phase, when it binds to histone gene promoters and recruits Gcn5p histone acetyltransferase [8]. The transcriptional repression of histone genes at the end of the S phase or during replication stress induced by hydroxyurea (HU) treatment operates at all 4 histone loci and, except for HTA2–HTB2, requires the HIR complex, Rtt106p, and Asf1p [15,16,17]. The replication stress– induced disassembly of the preinitiation complex (PIC) at the histone promoters is facilitated by Rad53p and Asf1p
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