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• W2948294589 abstract "•Yeast sirtuins, Hst3 and Hst4, globally repress coding and noncoding transcription•Transcriptional regulation by sirtuins prevents formation of excessive DNA-RNA hybrids•Increased R loops due to sirtuin loss are associated with more DNA breaks•Overexpression of RNaseH1 alleviates genomic instability due to sirtuin loss The mammalian sirtuin, SIRT6, is a key tumor suppressor that maintains genome stability and regulates transcription, though how SIRT6 family members control genome stability is unclear. Here, we use multiple genome-wide approaches to demonstrate that the yeast SIRT6 homologs, Hst3 and Hst4, prevent genome instability by tuning levels of both coding and noncoding transcription. While nascent RNAs are elevated in the absence of Hst3 and Hst4, a global impact on steady-state mRNAs is masked by the nuclear exosome, indicating that sirtuins and the exosome provide two levels of regulation to maintain transcription homeostasis. We find that, in the absence of Hst3 and Hst4, increased transcription is associated with excessive DNA-RNA hybrids (R-loops) that appear to lead to new DNA double-strand breaks. Importantly, dissolution of R-loops suppresses the genome instability phenotypes of hst3 hst4 mutants, suggesting that the sirtuins maintain genome stability by acting as a rheostat to prevent promiscuous transcription. The mammalian sirtuin, SIRT6, is a key tumor suppressor that maintains genome stability and regulates transcription, though how SIRT6 family members control genome stability is unclear. Here, we use multiple genome-wide approaches to demonstrate that the yeast SIRT6 homologs, Hst3 and Hst4, prevent genome instability by tuning levels of both coding and noncoding transcription. While nascent RNAs are elevated in the absence of Hst3 and Hst4, a global impact on steady-state mRNAs is masked by the nuclear exosome, indicating that sirtuins and the exosome provide two levels of regulation to maintain transcription homeostasis. We find that, in the absence of Hst3 and Hst4, increased transcription is associated with excessive DNA-RNA hybrids (R-loops) that appear to lead to new DNA double-strand breaks. Importantly, dissolution of R-loops suppresses the genome instability phenotypes of hst3 hst4 mutants, suggesting that the sirtuins maintain genome stability by acting as a rheostat to prevent promiscuous transcription. The epigenetic control of gene expression and genome stability plays a central role in ensuring normal cellular function. Dysregulation has been associated with numerous human malignancies, and chromatin factors have emerged as some of the most frequently affected proteins in cancer (Morgan and Shilatifard, 2015Morgan M.A. Shilatifard A. Chromatin signatures of cancer.Genes Dev. 2015; 29: 238-249Crossref PubMed Scopus (144) Google Scholar, Shah et al., 2014Shah M.A. Denton E.L. Arrowsmith C.H. Lupien M. Schapira M. A global assessment of cancer genomic alterations in epigenetic mechanisms.Epigenetics Chromatin. 2014; 7: 29Crossref PubMed Scopus (51) Google Scholar). SIRT6 is a mammalian member of the Sirtuin family of nicotinamide adenine dinucleotide (NAD+) dependent lysine deacetylases that are conserved across all species (Frye, 2000Frye R.A. Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins.Biochem. Biophys. Res. Commun. 2000; 273: 793-798Crossref PubMed Scopus (1153) Google Scholar). SIRT6 functions primarily as a lysine 56 (H3-K56Ac) (Michishita et al., 2009Michishita E. McCord R.A. Boxer L.D. Barber M.F. Hong T. Gozani O. Chua K.F. Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6.Cell Cycle. 2009; 8: 2664-2666Crossref PubMed Scopus (299) Google Scholar, Yang et al., 2009Yang B. Zwaans B.M. Eckersdorff M. Lombard D.B. The sirtuin SIRT6 deacetylates H3 K56Ac in vivo to promote genomic stability.Cell Cycle. 2009; 8: 2662-2663Crossref PubMed Scopus (211) Google Scholar) and lysine 9 (H3-K9Ac) (Michishita et al., 2008Michishita E. McCord R.A. Berber E. Kioi M. Padilla-Nash H. Damian M. Cheung P. Kusumoto R. Kawahara T.L. Barrett J.C. et al.SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin.Nature. 2008; 452: 492-496Crossref PubMed Scopus (841) Google Scholar) histone H3 deacetylase at promoters to regulate the expression of genes involved in various pathways, including metabolism, pluripotency, inflammation, and ribosome biogenesis (Etchegaray et al., 2015Etchegaray J.P. Chavez L. Huang Y. Ross K.N. Choi J. Martinez-Pastor B. Walsh R.M. Sommer C.A. Lienhard M. Gladden A. et al.The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine.Nat. Cell Biol. 2015; 17: 545-557Crossref PubMed Scopus (118) Google Scholar, Kugel and Mostoslavsky, 2014Kugel S. Mostoslavsky R. Chromatin and beyond: the multitasking roles for SIRT6.Trends Biochem. Sci. 2014; 39: 72-81Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, Kugel et al., 2016Kugel S. Sebastián C. Fitamant J. Ross K.N. Saha S.K. Jain E. Gladden A. Arora K.S. Kato Y. Rivera M.N. et al.SIRT6 Suppresses Pancreatic Cancer through Control of Lin28b.Cell. 2016; 165: 1401-1415Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Global changes in transcription in the absence of SIRT6 have not been reported. Deletion of Sirt6 causes major genomic and metabolic instability (Mostoslavsky et al., 2006Mostoslavsky R. Chua K.F. Lombard D.B. Pang W.W. Fischer M.R. Gellon L. Liu P. Mostoslavsky G. Franco S. Murphy M.M. et al.Genomic instability and aging-like phenotype in the absence of mammalian SIRT6.Cell. 2006; 124: 315-329Abstract Full Text Full Text PDF PubMed Scopus (1227) Google Scholar), and loss of SIRT6 is sufficient to drive tumorigenesis in mice independent of oncogene activation (Sebastián et al., 2012Sebastián C. Zwaans B.M. Silberman D.M. Gymrek M. Goren A. Zhong L. Ram O. Truelove J. Guimaraes A.R. Toiber D. et al.The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.Cell. 2012; 151: 1185-1199Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Mutations of Sirt6 that affect activity have been identified in human cancers (Kugel et al., 2015Kugel S. Feldman J.L. Klein M.A. Silberman D.M. Sebastián C. Mermel C. Dobersch S. Clark A.R. Getz G. Denu J.M. Mostoslavsky R. Identification of and Molecular Basis for SIRT6 Loss-of-Function Point Mutations in Cancer.Cell Rep. 2015; 13: 479-488Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), and, strikingly, Sirt6 is deleted in ∼60% and ∼30% of pancreatic and colorectal cancer cell lines, respectively (Sebastián et al., 2012Sebastián C. Zwaans B.M. Silberman D.M. Gymrek M. Goren A. Zhong L. Ram O. Truelove J. Guimaraes A.R. Toiber D. et al.The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.Cell. 2012; 151: 1185-1199Abstract Full Text Full Text PDF PubMed Scopus (509) Google Scholar). Together, the results point to an important role for SIRT6 as a tumor suppressor that regulates transcription and maintains genome stability. Hst3 and Hst4 are the two SIRT6 homologs in yeast that regulate H3-K56Ac levels (Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, Maas et al., 2006Maas N.L. Miller K.M. DeFazio L.G. Toczyski D.P. Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4.Mol. Cell. 2006; 23: 109-119Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), with the highest deacetylation activity observed during the S/G2 phase transition (Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, Maas et al., 2006Maas N.L. Miller K.M. DeFazio L.G. Toczyski D.P. Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4.Mol. Cell. 2006; 23: 109-119Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Similar to Sirt6, deletion of HST3 and HST4 induces a host of genome instability phenotypes, including spontaneous DNA double-strand breaks, replication fork collapse, increased chromosomal loss, impairment of break-induced replication, and heightened susceptibility to genotoxic agents (Brachmann et al., 1995Brachmann C.B. Sherman J.M. Devine S.E. Cameron E.E. Pillus L. Boeke J.D. The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability.Genes Dev. 1995; 9: 2888-2902Crossref PubMed Scopus (524) Google Scholar, Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, Che et al., 2015Che J. Smith S. Kim Y.J. Shim E.Y. Myung K. Lee S.E. Hyper-Acetylation of Histone H3K56 Limits Break-Induced Replication by Inhibiting Extensive Repair Synthesis.PLoS Genet. 2015; 11: e1004990Crossref PubMed Scopus (25) Google Scholar). Notably, these phenotypes are alleviated by inactivation of the Asf1 subunit of the Rtt109 histone acetyltransferase (HAT) complex or by a non-acetylatable H3-K56R mutant, suggesting that persistent H3-K56 hyperacetylation promotes genomic instability (Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, Celic et al., 2008Celic I. Verreault A. Boeke J.D. Histone H3 K56 hyperacetylation perturbs replisomes and causes DNA damage.Genetics. 2008; 179: 1769-1784Crossref PubMed Scopus (62) Google Scholar, Maas et al., 2006Maas N.L. Miller K.M. DeFazio L.G. Toczyski D.P. Cell cycle and checkpoint regulation of histone H3 K56 acetylation by Hst3 and Hst4.Mol. Cell. 2006; 23: 109-119Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). A prevailing model proposes that DNA damage is caused by the presence of hyperacetylated nucleosomes due to the lack of Hst3 and Hst4 that either impede replication fork progression or destabilize stalled forks (Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Here, we provide evidence of a functional link between increased transcription driven by loss of the sirtuins and the genomic instability phenotype observed in a hst3 hst4 mutant. Using a combination of native elongating transcript sequencing (NET-seq) (Churchman and Weissman, 2011Churchman L.S. Weissman J.S. Nascent transcript sequencing visualizes transcription at nucleotide resolution.Nature. 2011; 469: 368-373Crossref PubMed Scopus (550) Google Scholar) and RNA sequencing (RNA-seq) analyses, we show that Hst3 and Hst4 are required to repress transcription of coding and non-coding RNAs. Nascent RNAs are increased throughout coding regions in the absence of Hst3 and Hst4, and we also observe a shift in RNA polymerase II (Pol II) occupancy toward transcription start sites (TSSs). In addition, divergent antisense transcription is increased around the −1 nucleosome, similar to what was observed previously at several promoters (Marquardt et al., 2014Marquardt S. Escalante-Chong R. Pho N. Wang J. Churchman L.S. Springer M. Buratowski S. A chromatin-based mechanism for limiting divergent noncoding transcription.Cell. 2014; 157: 1712-1723Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Together, the results are consistent with increased transcription initiation at divergent promoters in a hst3 hst4 mutant, providing an additional mechanism utilized by cells to limit divergent ncRNA abundance. Interestingly, we find that increased nascent RNA is not reflected in the steady-state mRNA pool due to activity of the nuclear exosome. This impact of the exosome was also seen previously in a rtt109Δ mutant that lacked H3-K56Ac. We further use DNA-RNA immunoprecipitation with deep sequencing (DRIP-seq) (Ginno et al., 2012Ginno P.A. Lott P.L. Christensen H.C. Korf I. Chédin F. R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters.Mol. Cell. 2012; 45: 814-825Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar) analyses to identify loci with increased R-loop levels in the absence of Hst3 and Hst4. We show that a subset of regions with increased R-loops are also prone to the formation of DNA double-strand breaks, and we find that overexpression of human RNase-H1 suppresses the sensitivity of a hst3 hst4 mutant to genotoxic stress. Together, the results indicate that the sirtuins function to regulate transcription in order to prevent pervasive R-loop formation and subsequent genomic instability. Deletion of HST3 and HST4 leads to genomic instability (Brachmann et al., 1995Brachmann C.B. Sherman J.M. Devine S.E. Cameron E.E. Pillus L. Boeke J.D. The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability.Genes Dev. 1995; 9: 2888-2902Crossref PubMed Scopus (524) Google Scholar, Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), making such strains susceptible to second-site suppressor mutations. Notably, previous studies showed that the loss of both Hst3 and Hst4 is needed in order to observe measurable phenotypes (Celic et al., 2006Celic I. Masumoto H. Griffith W.P. Meluh P. Cotter R.J. Boeke J.D. Verreault A. The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation.Curr. Biol. 2006; 16: 1280-1289Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), indicating that they perform redundant roles. Therefore, we wanted to establish an alternative approach to characterize the impact of Hst3 and Hst4 on transcription. To this end, the anchor away system (Haruki et al., 2008Haruki H. Nishikawa J. Laemmli U.K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.Mol. Cell. 2008; 31: 925-932Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) was used to conditionally deplete Hst3 from the nucleus in a hst4Δ strain by tagging the C terminus of the HST3 locus with the FKBP12-rapamycin-binding (FRB) domain (hst4Δ HST3-FRB). The parent strain harbors a FK506 binding protein (FKBP12) fused to the C terminus of RPL13A, which is a highly abundant ribosomal protein that shuttles from the nucleus to the cytoplasm during ribosome assembly. A ternary complex between the FRB and FKBP12 domains is formed in the presence of rapamycin and, thus, rapidly depletes Hst3 from the nucleus. In addition, anchor away strains contain a rapamycin resistant tor1-1 allele to ensure rapamycin is not toxic to the wild-type (WT) strain (Haruki et al., 2008Haruki H. Nishikawa J. Laemmli U.K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes.Mol. Cell. 2008; 31: 925-932Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). We first confirmed that depletion of Hst3 in a hst4Δ displays a similar phenotype to a hst3Δ hst4Δ strain by spot dilution assay. The hst4Δ HST3-FRB strain is sensitive to 0.01% methyl methanesulfonate (MMS) and 0.1 M hydroxyurea (HU) only in the presence of rapamycin, similar to the hst3Δ hst4Δ strain in the presence of MMS and HU on DMSO (Figure S1A). Consistent with redundant roles for Hst3 and Hst4, the individual HST3-FRB mutant is not sensitive to genotoxic agents in the presence of rapamycin (Figure S1A). Given that mammalian Sirt6 plays key roles in transcription, we sought to determine the impact of yeast Hst3 and Hst4 on nascent RNA production. We performed NET-seq (Churchman and Weissman, 2011Churchman L.S. Weissman J.S. Nascent transcript sequencing visualizes transcription at nucleotide resolution.Nature. 2011; 469: 368-373Crossref PubMed Scopus (550) Google Scholar) in WT and the hst4Δ HST3-FRB mutant, using asynchronous cells treated with rapamycin for 3 h. Since we anticipated a potential for global changes in transcription, S. pombe cells were used as a spike-in control for library normalization. Loss of Hst3 and Hst4 led to a global shift in the nascent RNA transcriptome, with an average fold increase of ∼1.4 (p < 2 × 10−16, Mann-Whitney U test) (Figure 1A), and approximately a quarter of the genome (1,092 genes) increased by 1.5-fold or greater (false discovery rate [FDR] ≤ 0.1) in the hst4Δ HST3-FRB mutant. Loss of Hst3 and Hst4 has a somewhat larger impact on poorly expressed genes, as the log2 fold change (LFC) between mutant and WT cells is greater for the bottom 25% and 50% of genes transcribed in WT, compared to the top quatriles (p < 2.2 × 10−16, Mann-Whitney U test) (Figure 1B). Metagene plots of mean nascent transcript levels, representative genome browser views of NET-seq data, and a heatmap of the log2-fold change between the hst4Δ HST3-FRB mutant and WT confirmed higher levels of transcription throughout genic regions (Figures 1C, S1B, and S1C) in the mutant compared to the WT, especially for genes within the lowest quartile of expression levels (Figure 1C). Genes within the top 25% of the WT expression level also showed increases in nascent RNA, though these increases were greater near the TSS compared to the gene body and transcription termination site (TTS) (Figure 1C, bottom; Figure S1C). In addition, there was an increase in the 5′ to 3′ ratio of RNA transcripts genome wide (p < 2.2 × 10−16, Mann-Whitney U test) (Figure S1D). As further evidence that loss of Hst3 and Hst4 causes a shift in the Pol II distribution toward the TSS, we analyzed the distribution of Pol II after normalizing for differences in overall transcription. In agreement with the 5′ to 3′ ratios (Figure S1D), we observed a shift in Pol II distribution toward the TSS and a corresponding decrease near the TTS (Figure 1D). Taken together, our analyses indicate that Hst3 and Hst4 repress transcription initiation and, furthermore, that the absence of these sirtuins leads to the accumulation of Pol II near the TSS, which may be indicative of increased Pol II pausing. Studies in recent years have demonstrated that many eukaryotic promoters are inherently bidirectional (Scruggs et al., 2015Scruggs B.S. Gilchrist D.A. Nechaev S. Muse G.W. Burkholder A. Fargo D.C. Adelman K. Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin.Mol. Cell. 2015; 58: 1101-1112Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, Wei et al., 2011Wei W. Pelechano V. Järvelin A.I. Steinmetz L.M. Functional consequences of bidirectional promoters.Trends Genet. 2011; 27: 267-276Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), and transcription termination sequences, RNA degradation complexes, and chromatin modifying factors function to limit the abundance of divergent non-coding RNAs (ncRNAs) relative to mRNAs (Hainer et al., 2015Hainer S.J. Gu W. Carone B.R. Landry B.D. Rando O.J. Mello C.C. Fazzio T.G. Suppression of pervasive noncoding transcription in embryonic stem cells by esBAF.Genes Dev. 2015; 29: 362-378Crossref PubMed Scopus (51) Google Scholar, Huang and Workman, 2013Huang F. Workman J.L. Directing transcription to the right way.Cell Res. 2013; 23: 1153-1154Crossref PubMed Scopus (2) Google Scholar, Marquardt et al., 2014Marquardt S. Escalante-Chong R. Pho N. Wang J. Churchman L.S. Springer M. Buratowski S. A chromatin-based mechanism for limiting divergent noncoding transcription.Cell. 2014; 157: 1712-1723Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Wei et al., 2011Wei W. Pelechano V. Järvelin A.I. Steinmetz L.M. Functional consequences of bidirectional promoters.Trends Genet. 2011; 27: 267-276Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Whitehouse et al., 2007Whitehouse I. Rando O.J. Delrow J. Tsukiyama T. Chromatin remodelling at promoters suppresses antisense transcription.Nature. 2007; 450: 1031-1035Crossref PubMed Scopus (331) Google Scholar). We compared the abundance of divergent antisense nascent transcripts by NET-seq in WT and hst4Δ HST3-FRB mutant cells at tandem genes (2,716) by analyzing antisense reads in the region from −600 to −100 bp from the TSS. We observed a global increase in nascent transcripts upstream of genes in the absence of Hst3 and Hst4, with an average fold increase of ∼1.6 (p < 2 × 10−16, Mann-Whitney U test) (1,051 LFC ≥ 0.59, FDR ≤ 0.1) (Figure 2A). The increase in transcription maps around the −1 and −2 nucleosomes, with little change in the nucleosome depleted region (NDR; Figure 2B). These data are in agreement with previous results that showed increased divergent transcription by northern blot in the absence of Hst3 and Hst4 at several promoters (Marquardt et al., 2014Marquardt S. Escalante-Chong R. Pho N. Wang J. Churchman L.S. Springer M. Buratowski S. A chromatin-based mechanism for limiting divergent noncoding transcription.Cell. 2014; 157: 1712-1723Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In addition to divergent antisense transcripts, we investigated the role of Hst3 and Hst4 on cryptic unstable transcript (CUT) levels. CUTs are 5′ capped and polyadenylated ∼400 bp transcripts that are rapidly degraded due a high abundance of binding motifs for the Nrd1-Nab1-Sen1 (NNS) termination machinery and subsequent targeting by the nuclear exosome (Arigo et al., 2006Arigo J.T. Eyler D.E. Carroll K.L. Corden J.L. Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3.Mol. Cell. 2006; 23: 841-851Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, Schulz et al., 2013Schulz D. Schwalb B. Kiesel A. Baejen C. Torkler P. Gagneur J. Soeding J. Cramer P. Transcriptome surveillance by selective termination of noncoding RNA synthesis.Cell. 2013; 155: 1075-1087Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Thiebaut et al., 2006Thiebaut M. Kisseleva-Romanova E. Rougemaille M. Boulay J. Libri D. Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the nrd1-nab3 pathway in genome surveillance.Mol. Cell. 2006; 23: 853-864Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Nascent CUT RNAs are also increased in the hst4 HST3-FRB mutant compared to WT (1.6-fold, p = 6 × 10−13, Mann-Whitney U test) (245 LFC ≥ 0.59, FDR ≤ 0.1) (Figures 2C and 2D). Taken together, our analyses point to an important role for Hst3 and Hst4 in limiting non-coding RNA production. Our NET-seq analyses indicated that Hst3 and Hst4 repress the transcription initiation of genes. Therefore, we investigated whether the increase in nascent transcription translated to increased steady-state mRNA levels by analyzing RNA profiles by stranded RNA-seq. Similar to NET-seq analyses, S. pombe cells were used as a spike-in control for library normalization. Unexpectedly, and in contrast to the global increase in nascent transcription, steady-state mRNA levels remained relatively unchanged in the absence of Hst3 and Hst4 (0.981-fold, p = 0.02, Mann-Whitney U test) (Figure 3A). Hst3 and Hst4 negatively regulate the steady-state RNA level of 225 genes (FDR ≤ 0.1, LFC ≥ 0.59, edgeR) and positively regulate 85 genes (FDR ≤ 0.1, LFC ≤ −0.59, edgeR) (Figure S2A). Consistent with what was observed at the nascent RNA level, only very poorly transcribed genes in WT were increased to a greater extent in the mutant compared to the highly transcribed genes (p < 2.2 × 10−16, Mann-Whitney U test) (Figure 3B). Taken together, these results are similar to our previous analyses of the H3-K56 acetyltransferase Rtt109, in which we observed little change in the steady-state mRNA pool despite a global decrease in Pol II occupancy in a rtt109Δ strain (Rege et al., 2015Rege M. Subramanian V. Zhu C. Hsieh T.H. Weiner A. Friedman N. Clauder-Münster S. Steinmetz L.M. Rando O.J. Boyer L.A. Peterson C.L. Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis.Cell Rep. 2015; 13: 1610-1622Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The observation that the steady-state mRNA pool remains relatively unchanged even though there is a global increase in nascent RNA production led us to investigate the similarities and differences between the NET-seq and RNA-seq datasets. A k-means clustering approach was used to identify subsets of genes that are differentially regulated at the nascent and steady-state levels (Figure 3C; Table S1; see also Figures S2B and S2C for genome browser views). Group A genes, which are highly transcribed in WT cells (Figure 3D), show small increases at the nascent RNA level, whereas many show an opposite, decreased level in the steady-state RNA pool (Figure 3C). Nascent RNA transcription of the other three groups of genes (groups B–D) was increased in the absence of Hst3 and Hst4, but variable effects are observed at the steady-state level (Figure 3C). With the exception of group B genes, which are the most poorly transcribed genes in WT cells (Figure 3D) and are upregulated in the hst4Δ HST3-FRB mutant by RNA-seq analyses, steady-state mRNA levels are minimally affected (group D) or are decreased (group C) in the hst4Δ HST3-FRB mutant (Figure 3C). The results reveal that many of the increased transcripts observed at the nascent RNA level in the absence of Hst3 and Hst4 are post-transcriptionally regulated and, thus, are not observed in the steady-state RNA pool. In addition to a role in regulating ncRNA transcription (Schneider et al., 2012Schneider C. Kudla G. Wlotzka W. Tuck A. Tollervey D. Transcriptome-wide analysis of exosome targets.Mol. Cell. 2012; 48: 422-433Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and processing small nuclear and nucleolar RNAs (snRNAs and snoRNAs) (Gudipati et al., 2012Gudipati R.K. Xu Z. Lebreton A. Séraphin B. Steinmetz L.M. Jacquier A. Libri D. Extensive degradation of RNA precursors by the exosome in wild-type cells.Mol. Cell. 2012; 48: 409-421Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), the nuclear exosome plays a more general role in the surveillance of nuclear mRNAs (Rege et al., 2015Rege M. Subramanian V. Zhu C. Hsieh T.H. Weiner A. Friedman N. Clauder-Münster S. Steinmetz L.M. Rando O.J. Boyer L.A. Peterson C.L. Chromatin Dynamics and the RNA Exosome Function in Concert to Regulate Transcriptional Homeostasis.Cell Rep. 2015; 13: 1610-1622Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, Schmid et al., 2012Schmid M. Poulsen M.B. Olszewski P. Pelechano V. Saguez C. Gupta I. Steinmetz L.M. Moore C. Jensen T.H. Rrp6p controls mRNA poly(A) tail length and its decoration with poly(A) binding proteins.Mol. Cell. 2012; 47: 267-280Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To investigate whether the nuclear exosome might be responsible for masking the impact of Hst3 and Hst4 loss on the steady-state RNA pool, the anchor away system was used to deplete the 3′ to 5′ exonuclease subunit, Rrp6, from the nucleus for 3 h, alone or in combination with the hst4Δ HST3-FRB mutant. Interestingly, growth assays revealed an additive effect of depleting Rrp6 in the absence of both Hst3 and Hst4, as the cells become more sensitive to HU compared to either the RRP6-FRB single mutant or the hst4Δ HST3-FRB double mutant (Figure S3A). RNA-seq was performed in the hst4Δ HST3-FRB RRP6-FRB triple mutant and RRP6-FRB single mutant, and these datasets were compared to the NET-seq and RNA-seq datasets from the hst4Δ HST3-FRB double mutant, using the same gene groups identified in Figure 3C (Figure 4A; Table S2). Inactivation of the nuclear exosome increased steady-state RNA levels to those more closely resembling what was observed by NET-seq in the absence of Hst3 and Hst4 (Figure 4A). Remarkably, many of the RNAs that increased due to depletion of Hst3 and Hst4 were also increased by the single depletion of the RNA exosome (Figures 4A–C, S3B, and S3C), indicating that Hst3 and Hst4 and the nuclear exosome regulate many of the same target genes. However, there are many genes in groups B–D whose expression is increased to a greater extent in the hst4Δ HST3-FRB RRP6-FRB triple mutant compared to the RRP6-FRB single mutant (Figures 4A, 4C, S3B, and S3C), and there are an additional ∼800 genes that are increased ≥ 1.5-fold over WT (FDR ≤ 0.1, edgeR) only in the hst4Δ HST3-FRB RRP6-FRB triple mutant (Figures 4C and S3D). Taken together, the RNA-seq analyses in the absence of the nuclear exosome confirm the observations made by NET-seq. Transcription is elevated in the absence of Hst3 and Hst4, and at many loci the nuclear exosome functions to degrade the increased nascent transcripts. In addition, there are genes that a" @default.
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• W2948294589 title "Yeast Sirtuin Family Members Maintain Transcription Homeostasis to Ensure Genome Stability" @default.
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