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• W2151294425 abstract "Gene expression shows a significant variation (noise) between genetically identical cells. Noise depends on the gene expression process regulated by the chromatin environment. We screened for chromatin factors that modulate noise in S. cerevisiae and analyzed the results using a theoretical model that infers regulatory mechanisms from the noise versus mean relationship. Distinct activities of the Rpd3(L) and Set3 histone deacetylase complexes were predicted. Both HDACs repressed expression. Yet, Rpd3(L)C decreased the frequency of transcriptional bursts, while Set3C decreased the burst size, as did H2B monoubiquitination (ubH2B). We mapped the acetylation of H3 lysine 9 (H3K9ac) upon deletion of multiple subunits of Set3C and Rpd3(L)C and of ubH2B effectors. ubH2B and Set3C appear to function in the same pathway to reduce the probability that an elongating PolII produces a functional transcript (PolII processivity), while Rpd3(L)C likely represses gene expression at a step preceding elongation. Gene expression shows a significant variation (noise) between genetically identical cells. Noise depends on the gene expression process regulated by the chromatin environment. We screened for chromatin factors that modulate noise in S. cerevisiae and analyzed the results using a theoretical model that infers regulatory mechanisms from the noise versus mean relationship. Distinct activities of the Rpd3(L) and Set3 histone deacetylase complexes were predicted. Both HDACs repressed expression. Yet, Rpd3(L)C decreased the frequency of transcriptional bursts, while Set3C decreased the burst size, as did H2B monoubiquitination (ubH2B). We mapped the acetylation of H3 lysine 9 (H3K9ac) upon deletion of multiple subunits of Set3C and Rpd3(L)C and of ubH2B effectors. ubH2B and Set3C appear to function in the same pathway to reduce the probability that an elongating PolII produces a functional transcript (PolII processivity), while Rpd3(L)C likely represses gene expression at a step preceding elongation. Gene expression noise can distinguish the role of transcription regulators Noise-based predictions are verified by genome-wide profiling of H3K9 acetylation Evidence is provided that Set3C and ubH2B repress PolII processivity Cells that are genetically identical may still behave differently under identical conditions (Barkai and Shilo, 2007Barkai N. Shilo B.Z. Variability and robustness in biomolecular systems.Mol. Cell. 2007; 28: 755-760Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, Raser and O'Shea, 2005Raser J.M. O'Shea E.K. Noise in gene expression: origins, consequences, and control.Science. 2005; 309: 2010-2013Crossref PubMed Scopus (1287) Google Scholar). This nongenetic variability is largely due to noise in gene expression (Bar-Even et al., 2006Bar-Even A. Paulsson J. Maheshri N. Carmi M. O'Shea E. Pilpel Y. Barkai N. Noise in protein expression scales with natural protein abundance.Nat. Genet. 2006; 38: 636-643Crossref PubMed Scopus (603) Google Scholar, Elowitz et al., 2002Elowitz M.B. Levine A.J. Siggia E.D. Swain P.S. Stochastic gene expression in a single cell.Science. 2002; 297: 1183-1186Crossref PubMed Scopus (3838) Google Scholar, Ozbudak et al., 2002Ozbudak E.M. Thattai M. Kurtser I. Grossman A.D. van Oudenaarden A. Regulation of noise in the expression of a single gene.Nat. Genet. 2002; 31: 69-73Crossref PubMed Scopus (1212) Google Scholar). Noise varies between genes, and to a first approximation, it decreases with mean abundance. Yet, many genes deviate from this general trend (Bar-Even et al., 2006Bar-Even A. Paulsson J. Maheshri N. Carmi M. O'Shea E. Pilpel Y. Barkai N. Noise in protein expression scales with natural protein abundance.Nat. Genet. 2006; 38: 636-643Crossref PubMed Scopus (603) Google Scholar, Newman et al., 2006Newman J.R. Ghaemmaghami S. Ihmels J. Breslow D.K. Noble M. DeRisi J.L. Weissman J.S. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.Nature. 2006; 441: 840-846Crossref PubMed Scopus (1162) Google Scholar). For example, the low noise of essential genes and the high noise of stress-related genes are not explained by differences in mean abundance, but instead may depend on differences in the underlying gene expression mechanisms. The prevailing model of gene expression noise assumes that proteins are made in “bursts”: short time intervals in which proteins are produced, interspaced by periods of negligible production (Blake et al., 2003Blake W.J. KAErn M. Cantor C.R. Collins J.J. Noise in eukaryotic gene expression.Nature. 2003; 422: 633-637Crossref PubMed Scopus (1280) Google Scholar, Cai et al., 2006Cai L. Friedman N. Xie X.S. Stochastic protein expression in individual cells at the single molecule level.Nature. 2006; 440: 358-362Crossref PubMed Scopus (885) Google Scholar, Tan and van Oudenaarden, 2010Tan R.Z. van Oudenaarden A. Transcript counting in single cells reveals dynamics of rDNA transcription.Mol. Syst. Biol. 2010; 6: 358Crossref PubMed Scopus (42) Google Scholar, Zenklusen et al., 2008Zenklusen D. Larson D.R. Singer R.H. Single-RNA counting reveals alternative modes of gene expression in yeast.Nat. Struct. Mol. Biol. 2008; 15: 1263-1271Crossref PubMed Scopus (502) Google Scholar). The main stochastic event is burst initiation. Noise is amplified by the burst size (number of proteins made per burst), such that for a given level of mean expression, variability increases in proportion to burst size (Paulsson, 2004Paulsson J. Summing up the noise in gene networks.Nature. 2004; 427: 415-418Crossref PubMed Scopus (1001) Google Scholar, Tan and van Oudenaarden, 2010Tan R.Z. van Oudenaarden A. Transcript counting in single cells reveals dynamics of rDNA transcription.Mol. Syst. Biol. 2010; 6: 358Crossref PubMed Scopus (42) Google Scholar). Burst frequency and burst size can be estimated from the distribution of expression levels: Let μ and η2 denote the respective mean and coefficient of variation (noise) of the expression distribution. The predicted burst size and burst frequency are estimated by η2∗μ and η−2, respectively. Burst size is therefore the normalized variance, accounting for the inherent link between the noise and mean expression (Friedman et al., 2006Friedman N. Cai L. Xie X.S. Linking stochastic dynamics to population distribution: an analytical framework of gene expression.Phys. Rev. Lett. 2006; 97: 168302Crossref PubMed Scopus (468) Google Scholar, Raj et al., 2006Raj A. Peskin C.S. Tranchina D. Vargas D.Y. Tyagi S. Stochastic mRNA synthesis in mammalian cells.PLoS Biol. 2006; 4: e309Crossref PubMed Scopus (1171) Google Scholar, Tan and van Oudenaarden, 2010Tan R.Z. van Oudenaarden A. Transcript counting in single cells reveals dynamics of rDNA transcription.Mol. Syst. Biol. 2010; 6: 358Crossref PubMed Scopus (42) Google Scholar). A key implication of this model is that gene expression can be regulated in two principally different ways. Regulation of burst frequency will coordinately modify mean expression and noise. By contrast, regulation of burst size will change mean expression, but will not alter the coefficient of variation. Therefore, when studying the effect of a regulator of interest on gene expression, it may be beneficial to examine both mean expression and noise. Mean expression will distinguish between activators and repressors while noise may distinguish between regulators of burst frequency versus regulators of burst size. Identifying regulators of burst size is of a particular interest, as it provides insight to noise control. We used the model organism S. cerevisiae to search for regulators of burst size among chromatin-associated factors. Chromatin affects gene expression directly, by restricting DNA accessibility, and indirectly, by recruiting other factors (Henikoff and Shilatifard, 2011Henikoff S. Shilatifard A. Histone modification: cause or cog?.Trends Genet. 2011; PubMed Google Scholar). Previous reports implicated chromatin in noise regulation. First, genes of high noise are associated with promoters that lack the typical nucleosome-free region (NFR). These promoters, termed Occupied Proximal Nucleosome (OPN), show increased sensitivity to regulation by multiple chromatin factors (Blake et al., 2006Blake W.J. Balázsi G. Kohanski M.A. Isaacs F.J. Murphy K.F. Kuang Y. Cantor C.R. Walt D.R. Collins J.J. Phenotypic consequences of promoter-mediated transcriptional noise.Mol. Cell. 2006; 24: 853-865Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar, Cairns, 2009Cairns B.R. The logic of chromatin architecture and remodelling at promoters.Nature. 2009; 461: 193-198Crossref PubMed Scopus (342) Google Scholar, Field et al., 2009Field Y. Fondufe-Mittendorf Y. Moore I.K. Mieczkowski P. Kaplan N. Lubling Y. Lieb J.D. Widom J. Segal E. Gene expression divergence in yeast is coupled to evolution of DNA-encoded nucleosome organization.Nat. Genet. 2009; 41: 438-445Crossref PubMed Scopus (116) Google Scholar, Tirosh and Barkai, 2008Tirosh I. Barkai N. Two strategies for gene regulation by promoter nucleosomes.Genome Res. 2008; 18: 1084-1091Crossref PubMed Scopus (310) Google Scholar). Furthermore, individual deletions of three chromatin factors, the acetyl-transferase GCN5 and the chromatin remodelers SNF6 and ARP8, increased expression noise driven by the inducible PHO5 promoter (Raser and O'Shea, 2004Raser J.M. O'Shea E.K. Control of stochasticity in eukaryotic gene expression.Science. 2004; 304: 1811-1814Crossref PubMed Scopus (1115) Google Scholar). Recent systematic assays for noise regulators using a specific reporter also pointed to chromatin-associated factors (Rinott et al., 2011Rinott R. Jaimovich A. Friedman N. Exploring transcription regulation through cell-to-cell variability.Proc. Natl. Acad. Sci. USA. 2011; 108: 6329-6334Crossref PubMed Scopus (39) Google Scholar). Motivated by this data, we screened 137 nonessential chromatin factors for modifiers of the normalized noise (the predicted burst size). We initially hypothesized that burst size is regulated primarily at the level of burst duration, likely depending on the promoter or 5′ end of genes. Surprisingly, the modification that had the strongest predicted (repressive) effect on burst size was H2B monoubiquitination (ubH2B), which is generally associated with transcription elongation (Fleming et al., 2008Fleming A.B. Kao C.F. Hillyer C. Pikaart M. Osley M.A. H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation.Mol. Cell. 2008; 31: 57-66Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, Pavri et al., 2006Pavri R. Zhu B. Li G. Trojer P. Mandal S. Shilatifard A. Reinberg D. Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II.Cell. 2006; 125: 703-717Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, Shilatifard, 2006Shilatifard A. Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression.Annu. Rev. Biochem. 2006; 75: 243-269Crossref PubMed Scopus (890) Google Scholar) and is found primarily within the coding region (Schulze et al., 2009Schulze J.M. Jackson J. Nakanishi S. Gardner J.M. Hentrich T. Haug J. Johnston M. Jaspersen S.L. Kobor M.S. Shilatifard A. Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation.Mol. Cell. 2009; 35: 626-641Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The second process identified was Set3C-dependent deacetylation. Similar to ubH2B, Set3C was also associated with transcription elongation. Further, at least in certain cases, its recruitment depends on H3K4 dimethylation, which is promoted by ubH2B (Kim and Buratowski, 2009Kim T. Buratowski S. Dimethylation of H3K4 by Set1 recruits the Set3 histone deacetylase complex to 5′ transcribed regions.Cell. 2009; 137: 259-272Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Wang et al., 2002Wang A. Kurdistani S.K. Grunstein M. Requirement of Hos2 histone deacetylase for gene activity in yeast.Science. 2002; 298: 1412-1414Crossref PubMed Scopus (212) Google Scholar). PolII processivity is an elongation-related process affecting burst size. This measure defines the probability that an elongating PolII will produce a functional transcript, rather than terminate prematurely (Mason and Struhl, 2005Mason P.B. Struhl K. Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.Mol. Cell. 2005; 17: 831-840Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Theoretically, when burst events are well separated in time, burst size increases linearly with PolII processivity. Notably, since burst size is the total number of proteins made per burst, other aspects affecting elongation (e.g., elongation velocity) will modulate burst size only through their effect on PolII processivity. We therefore hypothesized that both ubH2B and Set3C-dependent deacetylation repress PolII processivity and examined this hypothesis using several high-throughput data sets. Set3C is one of multiple histone deacetylation complexes (HDACs) expressed in S. cerevisiae. HDAC complexes are extensively studied, yet their individual functions are only partially understood (Kurdistani and Grunstein, 2003Kurdistani S.K. Grunstein M. Histone acetylation and deacetylation in yeast.Nat. Rev. Mol. Cell Biol. 2003; 4: 276-284Crossref PubMed Scopus (550) Google Scholar). The predicted role of Set3C in decreasing burst size was of particular interest to us, as it differed from the predicted function of other HDACs. The well-studied Rpd3(L) complex, for example, repressed the predicted burst frequency and not the burst size. Furthermore, while Rpd3(L)C is known to act as a repressor of gene expression (Kurdistani and Grunstein, 2003Kurdistani S.K. Grunstein M. Histone acetylation and deacetylation in yeast.Nat. Rev. Mol. Cell Biol. 2003; 4: 276-284Crossref PubMed Scopus (550) Google Scholar, Robyr et al., 2002Robyr D. Suka Y. Xenarios I. Kurdistani S.K. Wang A. Suka N. Grunstein M. Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases.Cell. 2002; 109: 437-446Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar), Set3C is required for the rapid induction of the Gal1 gene (Kim and Buratowski, 2009Kim T. Buratowski S. Dimethylation of H3K4 by Set1 recruits the Set3 histone deacetylase complex to 5′ transcribed regions.Cell. 2009; 137: 259-272Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Wang et al., 2002Wang A. Kurdistani S.K. Grunstein M. Requirement of Hos2 histone deacetylase for gene activity in yeast.Science. 2002; 298: 1412-1414Crossref PubMed Scopus (212) Google Scholar). Our data, on the other hand, suggest that Set3C, like Rpd3(L)C, acts primarily as a repressor of gene expression, albeit through different means. We therefore explored further the distinct activities of these two complexes. HDAC activities can be distinguished by their effect on the genome-wide histone acetylation. Such mapping revealed, for example, a “division of labor” between HDACs acting on different gene promoters (Robyr et al., 2002Robyr D. Suka Y. Xenarios I. Kurdistani S.K. Wang A. Suka N. Grunstein M. Microarray deacetylation maps determine genome-wide functions for yeast histone deacetylases.Cell. 2002; 109: 437-446Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, Wang et al., 2002Wang A. Kurdistani S.K. Grunstein M. Requirement of Hos2 histone deacetylase for gene activity in yeast.Science. 2002; 298: 1412-1414Crossref PubMed Scopus (212) Google Scholar). Existing data reporting acetylation profiles in Rpd3(L)C and Set3C mutant background, however, mapped acetylation at promoter only, and are of a low spatial resolution. To examine the role of Set3C in transcription elongation and PolII processivity, and to test whether ubH2B and Set3C function in the same pathway, we wished to examine acetylation profiles in mutants affecting histone acetylation and ubH2B. We therefore generated a high-resolution map of H3K9 acetylation in five mutants deleted of Set3C and Rpd3(L)C components and in four mutants deleted of ubH2B effectors. These data were analyzed in combination with existing functional genomic data sets. Based on this, we now provide evidence that ubH2B and Set3-dependent deacetylation function in the same pathway to reduce PolII processivity, while Rpd3(L)C's key role in gene repression precedes elongation. We selected 137 chromatin factors and tested how their individual deletions modulate the expression (mean and variance) of a fluorescence reporter driven by one of 11 representative promoters (Figure 1A ). Our screen covered most of the nonessential chromatin modifiers, including regulators of histone acetylation, methylation, phosphorylation or ubiquitination, chromatin remodelers, histone variant, exchange factors, elements of the general transcription machinery, and chromatin silencing genes (Table S1). The 11 promoters used as reporters spanned a range of intermediate expression levels that are high enough to be detected by flow cytometry, yet not too high to ensure a significant contribution of noise intrinsic to the transcription process (Figure 1B). All promoters were inserted into the HIS3 locus upstream of the reporter and were combined with the deletion mutants using Synthetic Genetic Array (SGA) (Tong and Boone, 2006Tong A.H. Boone C. Synthetic genetic array analysis in Saccharomyces cerevisiae.Methods Mol. Biol. 2006; 313: 171-192PubMed Google Scholar). Altogether, we generated a set of 137X11 strains, each deleted of one chromatin regulator and carrying one YFP-driving promoter. We used flow cytometry to measure the single-cell reporter expression in each strain. Promoters changed their expression in ∼20%–30% of the deletions (Figure S1). The expression changes were moderate (∼30%) and did not correlate with promoters' mean abundance or noise. This is consistent with a recent study showing that individual deletions of most chromatin regulators have minor effect on the transcription profile (Lenstra et al., 2011Lenstra T.L. Benschop J.J. Kim T. Schulze J.M. Brabers N.A. Margaritis T. van de Pasch L.A. van Heesch S.A. Brok M.O. Groot Koerkamp M.J. et al.The specificity and topology of chromatin interaction pathways in yeast.Mol. Cell. 2011; 42: 536-549Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Burst-size is regulated by H2B ubiquitination and Set3-dependent deacetylation: From the distribution of expression levels, we calculated the predicted burst size and burst frequency. For this, we measured the coefficient of variation while gating for cell population of similar cell-cycle phase and size. Burst size was calculated by multiplying the coefficient of variation by the mean expression, while burst frequency was calculated as the inverse of the coefficient of variation, as noted above (Figure S2A). Significant effects of the deletions on burst size versus burst frequency were consistent between the different promoters. We classified the genes into known complexes and functional groups (Table S1) and examined for consistent behavior (Figure 2A ). Burst size was increased by deletions of LGE1 and RAD6, genes required for H2B monoubiquitination at lysine residues 123 (ubH2B) (Robzyk et al., 2000Robzyk K. Recht J. Osley M.A. Rad6-dependent ubiquitination of histone H2B in yeast.Science. 2000; 287: 501-504Crossref PubMed Scopus (526) Google Scholar). The predicted burst frequency was most strongly affected by histone acetylation: decreasing when acetyltrasferases were deleted (e.g., SAGA complex) and increasing in cells deleted of Rpd3(L)C components (SAP30 and PHO23, although not RPD3 itself) (Keogh et al., 2005Keogh M.C. Kurdistani S.K. Morris S.A. Ahn S.H. Podolny V. Collins S.R. Schuldiner M. Chin K. Punna T. Thompson N.J. et al.Cotranscriptional set2 methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex.Cell. 2005; 123: 593-605Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). Surprisingly, the deletion of Set3C HDAC components (SET3 and HOS2) increased the predicted burst size, but not burst frequency (Figures 1C, 1D, and 2A). To further verify these results, we chose nine regulators, including components of the Set3C and Rpd3(L)C, as well as genes involved in ubiquitination, deubiquitination, methylation, and histone remodeling, and deleted them individually in ∼200 additional reporter strains carrying different GFP-fused proteins (Table S2). Consistent with the results of the initial screen, burst frequency increased when deleting the SAP30 component of Rpd3(L)C, while deletion of SET3 or genes required for ubH2B (RAD6, LGE1), increased the predicted burst size. Further, preventing H2B ubiquitination via the H2B-K123R mutation (Robzyk et al., 2000Robzyk K. Recht J. Osley M.A. Rad6-dependent ubiquitination of histone H2B in yeast.Science. 2000; 287: 501-504Crossref PubMed Scopus (526) Google Scholar) increased the predicted burst size of most GFP-fused genes (Figures 2B and S2B). Taken together, this analysis suggests that ubH2B and Set3C-dependent deacetylation repress burst size in multiple genes. Our analysis assigned ubH2B a role as a repressor of burst size. If the activity of ubH2B in reducing burst size is general, then highly ubiquitinated genes will be of low noise (per mean expression). To examine this prediction, we compared the genome-wide profile of ubH2B (Schulze et al., 2009Schulze J.M. Jackson J. Nakanishi S. Gardner J.M. Hentrich T. Haug J. Johnston M. Jaspersen S.L. Kobor M.S. Shilatifard A. Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation.Mol. Cell. 2009; 35: 626-641Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) with the predicted burst size (normalized noise) of ∼2,000 yeast GFP-fused proteins (Newman et al., 2006Newman J.R. Ghaemmaghami S. Ihmels J. Breslow D.K. Noble M. DeRisi J.L. Weissman J.S. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise.Nature. 2006; 441: 840-846Crossref PubMed Scopus (1162) Google Scholar). Indeed, ubH2B levels were inversely correlated with the predicted burst size (c = −0.37, Figure 2C), supporting a general role of ubH2B in reducing burst size. We further noticed elevated levels of ubH2B at highly expressed genes (c = 0.44). Together, our results suggest that ubH2B is targeted to genes of high expression, where it acts to reduce burst size. ubH2B can repress burst size by increasing the transition from permissive to nonpermissive chromatin state, thereby reducing burst duration. Such regulation would imply a role at gene promoter or 5′ gene end. ubH2B, however, is found primarily within the coding region and is relatively uniform there (Schulze et al., 2009Schulze J.M. Jackson J. Nakanishi S. Gardner J.M. Hentrich T. Haug J. Johnston M. Jaspersen S.L. Kobor M.S. Shilatifard A. Linking cell cycle to histone modifications: SBF and H2B monoubiquitination machinery and cell-cycle regulation of H3K79 dimethylation.Mol. Cell. 2009; 35: 626-641Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) (Figure S2C). Furthermore, previous studies assigned ubH2B roles in transcription elongation: it stabilizes nucleosomes, promotes their reassembly (Chandrasekharan et al., 2009Chandrasekharan M.B. Huang F. Sun Z.W. Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability.Proc. Natl. Acad. Sci. USA. 2009; 106: 16686-16691Crossref PubMed Scopus (146) Google Scholar, Fleming et al., 2008Fleming A.B. Kao C.F. Hillyer C. Pikaart M. Osley M.A. H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation.Mol. Cell. 2008; 31: 57-66Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), and is required for the elongation-promoting phosphorylation of PolII by Ctk1 (Wyce et al., 2007Wyce A. Xiao T. Whelan K.A. Kosman C. Walter W. Eick D. Hughes T.R. Krogan N.J. Strahl B.D. Berger S.L. H2B ubiquitylation acts as a barrier to Ctk1 nucleosomal recruitment prior to removal by Ubp8 within a SAGA-related complex.Mol. Cell. 2007; 27: 275-288Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Further, limiting H2B ubiquitination increases sensitivity to drugs interfering with transcriptional elongation (Kim and Buratowski, 2009Kim T. Buratowski S. Dimethylation of H3K4 by Set1 recruits the Set3 histone deacetylase complex to 5′ transcribed regions.Cell. 2009; 137: 259-272Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, Wyce et al., 2007Wyce A. Xiao T. Whelan K.A. Kosman C. Walter W. Eick D. Hughes T.R. Krogan N.J. Strahl B.D. Berger S.L. H2B ubiquitylation acts as a barrier to Ctk1 nucleosomal recruitment prior to removal by Ubp8 within a SAGA-related complex.Mol. Cell. 2007; 27: 275-288Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Thus, ubH2B is more likely to affect burst size through its role in transcription elongation. Burst size depends on PolII processivity, namely the probability that an elongating PolII will produce a functional transcript rather than terminating prematurely. PolII processivity is regulated at the level of transcription elongation and not transcription initiation (Mason and Struhl, 2005Mason P.B. Struhl K. Distinction and relationship between elongation rate and processivity of RNA polymerase II in vivo.Mol. Cell. 2005; 17: 831-840Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, Struhl, 2005Struhl K. Transcriptional activation: mediator can act after preinitiation complex formation.Mol. Cell. 2005; 17: 752-754Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, Zhang et al., 2007Zhang Z. Klatt A. Henderson A.J. Gilmour D.S. Transcription termination factor Pcf11 limits the processivity of Pol II on an HIV provirus to repress gene expression.Genes Dev. 2007; 21: 1609-1614Crossref PubMed Scopus (29) Google Scholar). We hypothesized that ubH2B reduces burst size by repressing PolII processivity. In support of that, preventing H2B ubiquitination by the htb-K123R mutation led to PolII processivity defects at the GAL1 gene (Chandrasekharan et al., 2009Chandrasekharan M.B. Huang F. Sun Z.W. Ubiquitination of histone H2B regulates chromatin dynamics by enhancing nucleosome stability.Proc. Natl. Acad. Sci. USA. 2009; 106: 16686-16691Crossref PubMed Scopus (146) Google Scholar, Fleming et al., 2008Fleming A.B. Kao C.F. Hillyer C. Pikaart M. Osley M.A. H2B ubiquitylation plays a role in nucleosome dynamics during transcription elongation.Mol. Cell. 2008; 31: 57-66Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). A measure that depends on PolII processivity, but is easier to measure, is PolII efficiency, which we define as the number of mRNA transcripts produced per gene-bound PolII (normalized to gene length). We therefore predicted that PolII efficiency will be low at genes that are of high ubH2B. To examine this, we defined PolII efficiency using the genome-wide binding data of the PolII Rpb3 subunit or the group of elongation factors (Mayer et al., 2010Mayer A. Lidschreiber M. Siebert M. Leike K. Söding J. Cramer P. Uniform transitions of the general RNA polymerase II transcription complex.Nat. Struct. Mol. Biol. 2010; 17: 1272-1278Crossref PubMed Scopus (329) Google Scholar). mRNA levels were quantified using the data of Yassour et al., 2009Yassour M. Kaplan T. Fraser H.B. Levin J.Z. Pfiffner J. Adiconis X. Schroth G. Luo S. Khrebtukova I. Gnirke A. et al.Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing.Proc. Natl. Acad. Sci. USA. 2009; 106: 3264-3269Crossref PubMed Scopus (173) Google Scholar (Supplemental Experimental Procedures). As predicted, PolII efficiency correlates with burst size (c = 0.25; Figure S2F) and is inversely correlated with ubH2B (c = −0.39; Figures 2C and S2F). An independent measure for ubH2B levels is provided by the binding profile of Paf1, an elongation factor facilitating ubH2B (Kim and Roeder, 2009Kim J. Roeder R.G. Direct Bre1-Paf1 complex interactions and RING finger-independent Bre1-Rad6 interactions mediate histone H2B ubiquitylation in yeast.J. Biol. Chem. 2009; 284: 20582-20592Crossref PubMed Scopus (83) Google Scholar, Warner et al., 2007Warner M.H. Roinick K.L. Arndt K.M. Rtf1 is a multifunctional component of the Paf1 complex that regulates gene expression by directing cotranscriptional histone modification.Mol. Cell. Biol. 2007; 27: 6103-6115Crossref PubMed Scopus (71) Google Scholar). Paf1 binding profile was strongly correlated with ubH2B level (c = 0.69) and was inversely correlated with PolII efficiency and burst size (Figure S2D). PolII efficiency depends on PolII processivity, but may also be regulated by additional processes. For example, slowing elongation will decrease processivity (and burst size) only if increasing the probability of premature PolII termination, but will reduce efficiency even if not effecting processivity. As an additional, more direct measure of PolII processivity, we examined the decrease in PolII density along the gene, using the high-resolution data recently published (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). Indeed, consistent with ubH2B repressing PolII processivity, the decrease in PolII density along the gene was inversely correlated with ubH2B levels (c = −0.28) and also with Paf1 binding (c = −0.39) (Figures S2" @default.
• W2151294425 created "2016-06-24" @default.
• W2151294425 creator A5001182042 @default.
• W2151294425 creator A5005251958 @default.
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• W2151294425 creator A5035315840 @default.
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• W2151294425 date "2012-07-01" @default.
• W2151294425 modified "2023-10-12" @default.
• W2151294425 title "Expression Noise and Acetylation Profiles Distinguish HDAC Functions" @default.
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