Maintenance of Low Histone Ubiquitylation by Ubp10 Correlates with Telomere-Proximal Sir2 Association and Gene Silencing

Molecular Cell, Vol. 17, 585–594, February 18, 2005, Copyright ©2005 by Elsevier Inc.

DOI 10.1016/j.molcel.2005.01.007

Maintenance of Low Histone Ubiquitylation by Ubp10 Correlates with Telomere-Proximal Sir2 Association and Gene Silencing N.C. Tolga Emre,1 Kristin Ingvarsdottir,1 Anastasia Wyce,1 Adam Wood,2 Nevan J. Krogan,3 Karl W. Henry,1,4 Keqin Li,1 Ronen Marmorstein,1 Jack F. Greenblatt,3 Ali Shilatifard,2 and Shelley L. Berger1,* 1 Gene Expression and Regulation Program The Wistar Institute Philadelphia, Pennsylvania 19024 2 Department of Medical Genetics University of Toronto Toronto, Ontario M5G 1L6 Canada 3 Department of Biochemistry and Molecular Biology Saint Louis University Cancer Center Saint Louis, Missouri 63110

Summary Low levels of histone covalent modifications are associated with gene silencing at telomeres and other regions in the yeast S. cerevisiae. Although the histone deacetylase Sir2 maintains low acetylation, mechanisms responsible for low H2B ubiquitylation and low H3 methylation are unknown. Here, we show that the ubiquitin protease Ubp10 targets H2B for deubiquitylation, helping to localize Sir2 to the telomere. Ubp10 exhibits reciprocal Sir2-dependent preferential localization proximal to telomeres, where Ubp10 serves to maintain low H2B Lys123 ubiquitylation in this region and, through previously characterized crosstalk, maintains low H3 Lys4 and Lys79 methylation in a slightly broader region. Ubp10 is also localized to the rDNA locus, a second silenced domain, where it similarly maintains low histone methylation. We compare Ubp10 to Ubp8, the SAGA-associated H2B deubiquitylase involved in gene activation, and show that telomeric and gene-silencing functions are specific to Ubp10. Our results suggest that these H2B-deubiquitylating enzymes have distinct genomic functions. Introduction Transcriptional silencing in the yeast S. cerevisiae is a genetically accessible model for revealing potential mechanisms underlying heterochromatin function in more complex eukaryotic genomes. In yeast, genes are silenced in telomere-proximal regions of the genome, referred to as telomere position effect (TPE) (Gottschling et al., 1990), at the silent mating type loci (HMR and HML) and within the rDNA locus (Rusche et al., 2003). Genes in these regions are repressed in a nonspecific manner, similar to higher eukaryotic heterochromatic repression. TPE requires the silent information regula*Correspondence: [email protected] 4 Present address: Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129.

tory (Sir) complex composed of Sir3, Sir4, and the histone deacetylase Sir2 (Grunstein, 1997; Rusche et al., 2003). Appropriate delimitation of the regions targeted by the Sir complex is important for genomic regulation. In this regard, the improper spreading of the Sir complex from silenced regions into active regions is counteracted by several chromatin-related mechanisms within euchromatin and at boundary regions. These mechanisms include histone acetylation (Kimura et al., 2002; Suka et al., 2002) and the presence of the histone variant H2A.Z (Meneghini et al., 2003) among other proteins (Ladurner et al., 2003). One of the histone modifications involved in silencing in yeast is histone H3 methylation (me) (Bryk et al., 2002; Krogan et al., 2002; van Leeuwen et al., 2002). meLys79 and meLys4 are low within silenced loci but higher within open reading frames (ORFs) in euchromatin (Bernstein et al., 2002; Bryk et al., 2002; Ng et al., 2003). Substitution mutation at Lys79 results in spreading of Sir proteins into nonsilenced regions (van Leeuwen et al., 2002; Ng et al., 2003). Taken together, these findings lead to a model where meLys79 controls the extent of silencing indirectly by inhibiting Sir binding at nonsilenced regions, thereby localizing limited cellular pools of Sirs to silenced loci (van Leeuwen et al., 2002; van Leeuwen and Gottschling, 2002). The recent demonstrations of Sir3 spreading in Lys4 methylation mutants in vivo and of the binding of a recombinant Sir3 construct preferentially to nonmethylated H3 N-terminal peptide (compared to a Lys4 trimethylated peptide) in vitro (SantosRosa et al., 2004) further extend this model. The addition of ubiquitin, a 76 amino acid-conserved polypeptide, to cellular substrate proteins at specific lysine residues as polyubiquitin chains marks them for degradation by the proteasome. This process is achieved by the concerted action of enzymes called ubiquitinactivating enzymes (E1), ubiquitin conjugases (E2), and ubiquitin ligases (E3) (Hochstrasser, 1996). More recent studies suggest additional roles for ubiquitin conjugation, such as in vesicular trafficking and signaling, where the monoubiquitiyl moiety can act as an interaction surface (Hicke, 2001; Aguilar and Wendland, 2003). Histone H2B is monoubiquitylated (ub) at Lys-123 by the ubiquitin conjugase Rad6/ligase Bre1 (Robzyk et al., 2000; Hwang et al., 2003; Wood et al., 2003). Mutant Bre1, mutant Rad6, or the lysine-to-arginine substitution mutation at residue 123 of histone H2B (H2B-K123R, hereafter referred to as “htb1-KR”) causes derepression of a URA3 telomeric reporter, suggesting that ubH2B is also involved in silencing (Huang et al., 1997; Sun and Allis, 2002; Hwang et al., 2003; Wood et al., 2003). H2B ubiquitylation is required for the methylation of H3 Lys4 (Dover et al., 2002; Sun and Allis, 2002) and Lys79 (Briggs et al., 2002; Ng et al., 2002) assayed in bulk cellular histones, possibly mediated by the subunits of the 19S proteasome, although in a proteolysis-independent manner (Ezhkova and Tansey, 2004). These observations suggest that loss of ubH2B in euchromatin may lead to loss of H3 methylation and, hence, to Sir delocalization from silenced regions, leading to decreased silencing.

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Ubiquitin-specific proteases (UBPs in yeast, USPs in mammals) are thiol proteases specifically involved in cleaving the ubiquitin moiety from protein substrates or processing of polyubiquitin chains (Wilkinson, 1997, 2000). Although the ubiquitin-specific proteases constitute the largest family of proteins in the ubiquitin pathway, physiological roles are assigned to only a few of them, and much less is known about their substrate specificity (Wilkinson, 1997; Soboleva and Baker, 2004). We and others recently reported that Ubp8, a component of the transcriptional coactivator SAGA complex, is an H2B deubiquitylase functioning at promoters of certain SAGA-dependent genes (Henry et al., 2003; Daniel et al., 2004). Both H2B ubiquitylation and Ubp8-mediated deubiquitylation are required for proper transactivation, possibly because sequential addition and removal of ubiquitin sets a correct balance of Lys4 compared to Lys36 methylation on H3 (Henry et al., 2003; Kao et al., 2004). Another ubiquitin protease, UBP10, was isolated in a screen for high copy disruptors of telomeric silencing in yeast and, hence, named disruptor of telomeric silencing 4 (DOT4) (Singer et al., 1998). Both deletion and overexpression of UBP10 were shown to cause silencing defects (Singer et al., 1998; Kahana and Gottschling, 1999), and these silencing defects are probably involved in the apoptotic phenotype observed in UBP10 null mutants (Bettiga et al., 2004; Orlandi et al., 2004). However, neither UBP10’s mechanism of action nor its substrates in silencing have been identified. In this study, we focus on mechanisms by which Ubp10 regulates telomeric gene silencing through histone H2B deubiquitylation. Further, we provide evidence that the two histone H2Bdirected deubiquitylases Ubp10 and Ubp8 are involved in distinct genomic processes. Results Ubp10 Deubiquitylates Histone H2B In Vivo and In Vitro The identification of Ubp8 as an H2B-deubiquitylating enzyme within SAGA led us to test whether certain potential ubiquitin proteases other than Ubp8 also target histone H2B in transcription-related processes. Because of previous observations linking them to transcriptional silencing (Moazed and Johnson, 1996; Singer et al., 1998; Kahana and Gottschling, 1999), we considered Ubp3 and Ubp10. In a strain bearing the UBP10 deletion, increased levels of ubiquitylated H2B (ubH2B) are observed in bulk histones compared to the wild-type (wt) strain, similar to the levels observed upon deletion of UBP8 (Henry et al., 2003), suggesting that Ubp10 also targets the ubiquitin moiety on histone H2B (Figure 1A). In contrast, deletion of UBP3 does not result in such an increase (Figure 1A), consistent with our recent observation (Henry et al., 2003). In control experiments, strains bearing either the htb1-KR substitution or lacking the FLAG epitope on H2B show no detectable ubH2B or no H2B-specific bands, respectively, again as we previously observed (Henry et al., 2003 and data not shown). We then examined whether Ubp10 can deubiquitylate H2B in vitro. Recombinant Ubp10 prepared from bacteria removes ubiquitin from ubH2B, as assessed by dis-

appearance of double tagged-ubiquitylated H2B (HAubH2B-FLAG) and appearance of faster migrating HAub (Figure 1B). As a control, the SAGA complex (which contains Ubp8) is also able to deubiquitylate H2B in vitro (Figure 1B) (Henry et al., 2003). To further test the role of Ubp10 in vivo, we expressed GAL1 promoter-driven GST-Ubp10 in yeast. GST-Ubp10 is detected in galactose, but not in glucose, which represses the GAL1 promoter (Figure 1C). In the presence of GST-Ubp10, the level of ubH2B is strongly reduced but, as a control, there is no effect on the level of histone H3 (Figure 1C). We compared this reduced level of uH2B to the level observed in the absence of the H2B ubiquitin ligase Bre1 and found the levels are similarly lowered (Figure 1C) (Hwang et al., 2003; Wood et al., 2003). Taken together, these results suggest that histone H2B is a direct target for Ubp10-mediated deubiquitylation in vivo. Ubp10 Is Targeted to the Right Arm of Chromosome VI Telomere and to the rDNA Locus to Maintain Low Levels of ubH2B and meH3 The previous observation that UBP10 has a role in gene silencing at the telomere (Singer et al., 1998) raised the possibility that Ubp10 may be functioning directly at the silenced chromatin. To test this hypothesis, we first examined the localization of FLAG epitope-tagged Ubp10 at the right arm of chromosome VI (ChrVI-R) by using chromatin immunoprecipitation (ChIP). ChrVI-R was used as a model in this study because it has been extensively analyzed for silencing (Hecht et al., 1996; Strahl-Bolsinger et al., 1997; Kimura et al., 2002; Suka et al., 2002) and because it is nonhomologous to the rest of the yeast genome, providing unique primer sequences for PCR (Figure 2A). The level of Ubp10-FLAG is 3- to 4-fold higher proximal to the telomere than at more internal regions of ChrVI-R (Figure 2B). The enrichment of Ubp10 at the telomere suggested that ubH2B might be a substrate of Ubp10 at this location. Therefore, we examined the distribution of ubH2B at ChrVI-R (Figures 2C and 2D). A chromatin double immunoprecipitation (ChDIP) assay was used to monitor ubH2B, which involves ␣-FLAG immunoprecipitation of H2B-FLAG, followed by FLAG peptide elution from beads, ␣-HA immunoprecipitation against HA-ub, and real-time PCR analysis of coprecipitated DNA (Henry et al., 2003; Kao et al., 2004). The level of HA-ubH2B-FLAG was compared to the level in a strain bearing a substitution of the htb1-KR that served as the background for the assay. The normalization of the signal to that from htb1-KR ensured the detection of specific enrichment of ubH2B, except for the remote possibility of detecting other ubiquitylated proteins that specifically interact with ubH2B, but not htb1-KR. ubH2B levels are low close to the chromosome end (0.2 kb region) as recently observed (Kao et al., 2004), slightly higher in the ORFless 7.3 kb region, and even higher at the more internal 20 kb region, which is inside the ORF of the RET2 gene (Figure 2C). We investigated whether Ubp10 has a role in maintaining low ubH2B levels near the telomere. To test this possibility, the level of ubH2B was determined by ChDIP from the UBP10 deletion strain and compared to the wt level. The level of ubH2B increases in the UBP10 deletion strain proximal to the telomere (0.2 kb)

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Figure 1. Role of Ubp10 in Histone H2B Deubiquitylation (A) Effect of UBP10 deletion on monoubiquitylation of H2B in vivo. H2B-FLAG from acidextracted cell lysates of wild-type (wt) and various UBP-deletion strains were immunoprecipitated and immunoblotted with ␣-FLAG antibody. The slower migrating band is due to monoubiquitylation of histone H2B (Robzyk et al., 2000; Henry et al., 2003). (B) Deubiquitylating activity of recombinant Ubp10 on ubiquitylated histone H2B in vitro. Recombinant Ubp10 (rUbp10, residues 360– 736), purified SAGA complex containing Ubp8 (positive control), or buffer lacking enzyme (“mock”) were incubated with H2B immunopurified with ␣-FLAG antibody from yeast strains harbouring FLAG-H2B and HAubiquitin. The extent of deubiquitylating activity was assessed by the disappearance of the HA-ubH2B-FLAG band and the appearance of free ubiquitin (HA-ub) on ␣-FLAG and ␣-HA immunoblots, respectively. Asterisk denotes crossreacting bands. (C) Effect of expressing GST-Ubp10 on histone H2B deubiquitylation in vivo. Strains that are either wt, deleted for BRE1 as a negative control for uH2B (Hwang et al., 2003; Wood et al., 2003), or containing GST-Ubp10 under the GAL1 promoter were grown in the presence of glucose (⫺, no induction for GAL1 promoter), or galactose (⫹, inducing condition for GAL1 promoter). Ubiquitylation levels of histone H2B were observed by ␣-FLAG immunoblotting. Induction of GST-Ubp10 was assessed by immunoblotting with ␣-GST, and ␣-histone H3 immunobloting served as a loading control.

but is not significantly altered at the other regions tested (7.3 kb and 20 kb) (Figure 2D), consistent with a role of Ubp10 in maintaining low ubiquitylation levels proximal to the telomere. Because of the established relationship between H2B ubiquitylation and H3 methylation (Briggs et al., 2002; Dover et al., 2002; Ng et al., 2002; Sun and Allis, 2002), we examined whether Ubp10 helps to maintain low

methylation proximal to the telomere. By using ChIP analysis, we found that levels of both meLys4 and meLys79 in histone H3 are inversely correlated with distance from the telomere at ChrV1-R, as previously observed for meLys79 at ChrVI-R (Ng et al., 2003) (Figures 3A and 3B, top). In the absence of Ubp10, the level of meLys4 and meLys79 increases at regions closer to the telomere (0.2 kb and 3.6 kb) (Figures 3A and 3B). The Figure 2. Analysis of Ubp10 at ChrVI-R

(A) Genomic structure of the right arm of chromosome VI (ChrVI-R). Approximate relative levels of Sir2 binding (not to scale) are depicted along the chromosome (Strahl-Bolsinger et al., 1997; Kimura et al., 2002; Suka et al., 2002) (top). Indicated below are the locations of ORFs used for RT-PCR assays (middle) and the approximate locations of primer pairs used for chromatin immunoprecipitation (ChIP) analyses (bottom). (B) Association of Ubp10 with ChrVI-R. ChIP assays were performed with ␣-FLAG on lysates from a strain harboring carboxy-terminal tagged UBP10 and analyzed by quantitative PCR using ChrVI-R primers shown in (A). The values represent the input normalized immunoprecipitates relative to the signal obtained from the GAL1 promoter (Henry et al., 2003). (C) ubH2B levels on ChrVI-R. Chromatin double immunoprecipitation (ChDIP) assays were performed by immunoprecipitation with antiFLAG and anti-HA antibodies in a sequential manner to detect ubiquitylated H2B (HAubH2B-FLAG) levels. The signal from the wt strain is normalized to the signal from the respective input and then to the background signal obtained from the htb1-KR strain. (D) Changes in ubH2B levels in UBP10 deletion mutant at ChrVI-R. ChDIP assays, as performed in (C), show the fold difference in histone H2B ubiquitylation in the deletion mutant ubp10⌬ compared to the wt strain. Signals were 1.5- to 9-fold lower in the htb1-KR strain, which were taken as the background signal (data not shown).

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Figure 4. Analysis of Ubp10 at rDNA Locus (A) Association of Ubp10 with ribosomal DNA loci compared to the 20 kb region of ChrVI-R. ChIP analysis of tagged Ubp10 performed as in Figure 2B. (B) Trimethylated H3 Lys4 levels on ribosomal DNA loci compared to the 20 kb region of ChrVI-R. ChIP analysis of histone H3 K4 methylation performed as in Figure 3A.

Figure 3. Histone H3 Methylation Patterns in UBP10 Deletion Strains (A) Trimethylated H3 Lys4 levels on ChrVI-R. Input-normalized average quantitative PCR signals from ChIP experiments are plotted for the wt strain (top) and for the UBP10 deletion strain relative to the wt (bottom). (B) Trimethylated H3 Lys79 levels on ChrVI-R. Presented as in (A). (C) Effect of expressing GST-Ubp10 on methylation status of histone H3 Lys4 and Lys79. Strains that are either wt or containing GSTUbp10 under the GAL1 promoter were grown in the presence of glucose (Glu) or galactose (Gal). The acetylation status of Lys-12 of histone H4, phosphorylation status of H3 Ser-10 (internal loading controls), the methylation status of histone H3 at Lys4 and Lys79, and expression of GST-Ubp10 were examined by immunoblotting using antibodies recognizing each substrate. Triangles indicate increasing amounts of lysate loaded on the gel.

regions that are affected show low levels of methylation in the wt state (compare Figures 3A and 3B, top panel and bottom panel). The increase in telomeric histone ubiquitylation and methylation caused by ubp10 deletion as assayed by ChIP is comparable in magnitude to the increases previously observed in histone acetylation upon deletion of SIR2 (Robyr et al., 2002).

To further examine the effect of Ubp10 on methylation, we tested whether GAL1-driven expression of GSTUbp10, which decreases ubH2B levels (Figure 1C), also decreases methylation in bulk histones in vivo. Highlevel expression of GST-Ubp10 lowers meLys4 and meLys79 levels but, as controls, causes no significant change in Ser-10 phosphorylation in histone H3 or Lys12 acetylation in histone H4 (Figure 3C). Taken together, we conclude that Ubp10 activity correlates with decreased ubiquitylation and methylation, and these effects preferentially occur at the silenced loci. We tested whether the effects of Ubp10 loss are generally true at silenced regions by analysis of the ribosomal DNA locus. Ribosomal DNA repeats show Sir2and Net1-dependent silencing in the budding yeast (Rusche et al., 2003). First, compared to 20 kb region on ChrVI-R (Figure 3B), FLAG-tagged Ubp10 is enriched at 25 S and 5 S regions of rDNA by ChIP (Figure 4A). In addition, loss of Ubp10 increases the Lys4 trimethylation at these regions compared to wt (Figure 4B), a trend that closely matches Ubp10 localization (Figure 4A). Ubp10 and Sir2 Are Mutually Required for Telomere-Proximal Localization, and Ubp10 Maintains Low Gene Expression It was recently reported that UBP10 contributes to genome-wide reduced expression of subtelomeric genes (Orlandi et al., 2004), consistent with its previously proposed role in telomeric gene silencing (Singer et al., 1998). We tested whether deletion of UBP10 results in increased expression of an endogenous gene near the region of the ChrVI-R telomere harboring low levels of ubiquitylation. The ORF proximal to the telomere end (YFR057W, hereafter referred to as ORF 57) exhibits very low transcript levels compared to the more distal ORFs 54 and 55 (data not shown), as is expected from its telomere-proximal location correlating with high levels of Sir protein binding (Hecht et al., 1996; Strahl-Bolsinger et al., 1997; Kimura et al., 2002; Suka et al., 2002). ORF 57 transcript levels increase in the ubp10⌬ strain, but expression of more telomere-distal ORFs are either un-

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changed (ORF 55) or increased to a significantly lesser extent (ORF 54) (Figure 5A), consistent with a specific and localized role of Ubp10 in telomeric silencing. The magnitude of the ORF 57 expression increase caused by deletion of ubp10 is comparable to increases previously observed by SIR2 deletion (Wyrick et al., 1999). We examined the effect of both substituting the H2B ubiquitylation site and deleting UBP10 on ORF 57 expression, hypothesizing that if ubH2B is an important substrate of Ubp10 in regulating gene silencing, the effect of the double mutation will be similar to loss of the ubiquitylation site. The htb1-KR mutant increases ORF 57 expression more than ubp10⌬ (Figure 5B) does, which is reasonable given the genome-wide effect on histone methylation of the substitution mutation in H2B (Briggs et al., 2002; Dover et al., 2002; Sun and Allis, 2002). Importantly, the double mutation causes similar levels of increased ORF 57 gene expression, as does the single htb1-KR mutant (Figure 5B). These results are consistent with the idea that ubH2B is a key substrate of Ubp10 in telomeric silencing at ChrVI-R. Sir proteins are localized to telomeres and are required for proper silencing (Grunstein, 1997; Gasser and Cockell, 2001; Rusche et al., 2003). We hypothesized that increased H2B ubiquitylation and increased H3 Lys4 and Lys79 methylation resulting from loss of Ubp10 may lead to altered Sir localization. To test this, we monitored the localization of epitope-tagged Sir2 by ChIP. In wt cells, the level of Sir2 is high at the region closest to the telomere (0.2 kb) and drops sharply at more internal regions (Figure 5C), consistent with previous studies (Hecht et al., 1996; Strahl-Bolsinger et al., 1997; Kimura et al., 2002; Suka et al., 2002). UBP10 deletion causes a 2- to 3-fold decrease in Sir2-HA levels at the telomereproximal (0.2 kb) region (Figure 5C). Western blotting shows that the total Sir2-HA levels in the cell are comparable in wt and UBP10 deletion strains (Figure 5C, bottom), arguing against the possibility that decreased Sir2 localization to the telomere is an indirect effect of decreased total Sir2 levels. It was previously demonstrated that UBP10 interacted with SIR4 in a yeast two-hybrid assay (Kahana and Gottschling, 1999). This finding, together with our observations regarding the preferential association of Ubp10 with silenced regions (Figures 2B and 4A), raised the possibility that Sir proteins may, in turn, be involved in the proper localization of Ubp10 to the silent loci. To test this, we analyzed the association of Ubp10 with ChrVI-R in a strain where SIR2 is deleted. In the absence of Sir2, Ubp10’s association with the telomere-proximal 0.2 kb region decreases significantly (Figure 5D), although total cellular Ubp10 levels are comparable in wt and SIR2 deletion strains (Figure 5D, bottom). This suggests that Sir2 and Ubp10 are involved in mutual localization to the silenced regions. Taken together these results establish a correlation between Ubp10’s molecular role in maintaining low histone modifications/ high Sir levels and Ubp10’s physiological role in maintaining low transcription of telomere-proximal genes. Ubp8 Does Not Have Similar Telomeric Functions as Ubp10 Ubp10 and Ubp8 similarly target H2B for deubiquitylation and both contain the C-terminal UCH domain (Pfam

Figure 5. Effect of H2B Ubiquitylation State on Gene Silencing and Sir2 Localization at ChrVI-R (A) The relative transcript levels of genes near the ChrVI-R telomere in UBP10 deletion mutant compared to the wt. Quantitative RT-PCR experiments were performed with total RNA to assess the transcript levels of ORF 57 (YFR057W), ORF 55 (YFR055W), and ORF 54 (YFR054C). The expression levels in the ubp10⌬ mutant are presented relative to those in the wt strain after normalizing each signal to those obtained from 18S ribosomal RNA locus as loading control. (B) Comparison of transcript levels at YFR057W (ORF 57). Wt transcript levels are compared to those from UBP10 deletion, ubiquitylation site mutant htb1-KR, and the double mutant (htb1-KR /ubp10⌬) strains. Signals obtained from ACT1 used for normalizations. (C) Association of Sir2 with the telomere-proximal regions of ChrVIR. Strains are either wt or deleted for UBP10 and harbor Sir2 tagged with three copies of HA epitope in its chromosomal location. Quantitative PCR analyses of ChIP assays with ␣-HA monoclonal antibody are shown (top). Whole-cell extracts from designated strains were immunoblotted and probed with ␣-HA antibody to show total cellular levels of tagged Sir2 (bottom). (D) Association of Ubp10 with the telomere-proximal regions of ChrVI-R. Strains are either wt or deleted for SIR2 and harbor Ubp10 tagged with 13 copies of Myc epitope in its chromosomal location. Analysis performed as in (C) (top). Whole-cell extracts from designated strains were immunoblotted and probed with ␣-Myc antibody to show total cellular levels of tagged Ubp10 (bottom).

accession number: PF00443, [Bateman et al., 2004]), a hallmark of ubiquitin proteases. In addition, Ubp8 has an N-terminal zinc finger domain of type ZnF_UBP (ubiquitin carboxy-terminal hydrolase-like zinc finger; Pfam domain ID: PF02148), which is required for Ubp8’s association with the SAGA complex (Ingvarsdottir et al.,

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Figure 6. Comparison of the Effects of UBP10 and UBP8 (A) Effect of combined UBP8 and UBP10 deletion on monoubiquitylation of H2B in vivo. Acid-extracted lysates of indicated strains were immunoblotted with ␣-FLAG antibody. H2B-FLAG and ubH2B-FLAG are as in Figure 1A. ubp8⌬10⌬ stands for the ubp8⌬/ubp10⌬ double deletion strain. (B) Association of Ubp8 with ChrVI-R. ChIP assays were performed as in Figure 2B but with ␣-HA on lysates from a strain harbouring carboxyterminal tagged UBP8. (C) Comparison of the effects of UBP8 to UBP10 at ChrVI-R. The data for the deletion of UBP10 come from the following (left to right): Figure 2D, Figure 3A, Figure 3B, and Figure 5A. Refer to the respective figure legends for details. (D) Growth of UBP/GCN5 deletion strains on Ethanol/Glycerol (EtOH/Gly) media. Cultures of strains (gcn5⌬; gcn5⌬ubp10⌬; and ⌬gcn5⌬ubp8⌬) were spotted on regular synthetic complete SC (control) and SC-EtOH/Gly plates as 5-fold dilution series. Plates were scanned after 2–3 days growth at 30⬚C. (E) Growth of UBP deletion strains combined with deletions of HTZ1 or SWR1 complex components. Growths of four spores from individual tetrads on rich media are shown as vertical strips. Two representative tetrads are presented side by side for each mating. The genotype of each spore for deletion of a given Swr1 complex component and UBP (as accessed by growth of replica-plated spores on selective media for a particular deletion, data not shown) is indicated under each tetrad. “⫹” stands for the presence, and “⫺” stands for the deletion, of a particular gene (for example, the upper left-most spore, designated as “⫺/⫺ ”, is deleted both for HTZ1 and for UBP10). The spores with double deletion are marked with an asterisk for additional visual guidance.

2005). The N-terminal region of Ubp10, in contrast, does not contain a known conserved domain structure. Thus, the distinct N termini of Ubp8 and Ubp10 may be critical for establishing the appropriate genomic location for H2B deubiquitylation. Therefore, we tested possible functional differences of Ubp10 compared to Ubp8. First, we examined the effect of simultaneous loss of

Ubp8 and Ubp10 on levels of ubH2B in vivo. The level of ubH2B is higher in the double null ubp8⌬ubp10⌬ than in either of the single deletion strains (Figure 6A), consistent with the possibility that Ubp8 and Ubp10 target different subpopulations of ubH2B for deubiquitylation. We examined Ubp8 association with ChrVI-R telomere, and in contrast to Ubp10’s preferential associa-

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Figure 7. A Model for the Role of Histone Deubiquitylation in Gene Silencing The role of Ubp10-dependent histone H2B deubiquitylation in silencing (left) and the mechanisms by which silencing is disrupted when UBP10 is deleted (right, top) and when UBP10 is overexpressed (right, bottom). See text for details. Abbreviations: U, ubiquitylation; M, methylation of histones; and SIR, Sir silencing complex. Only relevant components (not to scale) are depicted.

tion with the telomere-proximal region (Figure 2B), Ubp8 does not show a preference to the 0.2 kb region (Figure 6B). We compared UBP10 deletion to UBP8 deletion on effects at the telomere-proximal regions of ChrVI-R. For each assay employed (i.e., ubH2B, meLys4, and meLys79 levels by ChIP and ORF57 expression by RTPCR), UBP8 deletion is not significantly different from the background or from the wt strain, and the effect is much lower than that of UBP10 deletion (Figure 6C). These results argue against a role for Ubp8 in telomeric silencing, in contrast to Ubp10. We then examined the effects of UBP deletions on growth media that monitors SAGA/Gcn5-dependent activity. Our previous data indicate that combined deletion of UBP8 and GCN5 hinders growth on ethanol/glycerol possibly because of low transcription of the ADH2 gene (Henry et al., 2003). In contrast to poorer growth exhibited by gcn5⌬ubp8⌬, the double mutant gcn5⌬ubp10⌬ grows no worse than gcn5⌬ alone (Figure 6D). These data suggest that Ubp8, but not Ubp10, works with Gcn5 to regulate SAGA-dependent genes. We also compared the genome-wide alterations in gene expression in ubp8⌬ and ubp10⌬ strains and found no significant overlap in the genes that are regulated by the two ubiquitin proteases (data not shown). In S. cerevisiae, the histone H2A variant H2A.Z (Htz1) functions as a boundary element to prevent the spread of Sir-dependent silencing (Meneghini et al., 2003) after its deposition into chromatin by SWR-C, a 13 protein complex that contains Swr1 (Krogan et al., 2003; Kobor et al., 2004; Mizuguchi et al., 2004), a member of the Snf2 family of ATP-dependent remodelling factors. Our data suggest that Ubp10 functions to maintain Sir2 within telomere-proximal regions through low H2B ubiquitylation and H3 methylation. Thus, although hypothesized to work by different mechanisms, both Htz1 and Ubp10 function to control the spread of Sir proteins and silencing. Therefore, based on this similar functional

outcome, but dissimilar proposed mechanisms of action, we examined whether synthetic growth defects would be seen when double mutant strains were generated containing deletions of UBP10 and HTZ1 or members of the SWR-C. We found that any combination of deletion of UBP10 and deletion of HTZ1 or genes encoding six subunits of the SWR-C (SWR1, SWC2, SWC3, SWC5, SWC6, and ARP6) are synthetically sick in growth compared to the single deletions (Figure 6E and data not shown). Moreover, no such genetic interactions are seen for deletion of UBP8 and deletion of HTZ1 or any member of the SWR-C (Figure 6E and data not shown). Taken together, our results indicate that Ubp10 functions to promote telomeric silencing, whereas Ubp8 functions in SAGA-related gene regulation. Thus, although both Ubp8 and Ubp10 have a common substrate in H2B, they function in distinct physiological pathways. Discussion In this study, we show that the process of histone H2B deubiquitylation is important for transcriptional gene silencing. A model for the role of Ubp10 that emerges from our results and previous data is shown in Figure 7. Ubp10 is targeted to the telomere to sustain histone H2B in a state of reduced ubiquitylation. Low ubiquitylation helps to maintain low H3 methylation, and this state is compatible with Sir complex association with chromatin (van Leeuwen et al., 2002; van Leeuwen and Gottschling, 2002; Ng et al., 2003; Santos-Rosa et al., 2004). The Sir complex keeps telomere-proximal gene expression low. Thus, combined with previous observations, there appears to be two mutually reinforcing systems to maintain appropriate histone modifications for telomeric silencing: the targeting of histone ubiquitylation and methylation (through Rad6/Bre1, Set1, and Dot1) to the bulk of the genome to keep it in a “poised” state for transcription

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(Zhang, 2003) and the targeting of histone deubiquitylation to the telomeres to maintain low ubiquitylation/ methylation and high density of Sir proteins and, hence, low gene expression. This model helps to explain why both loss of ubiquitylation and loss of deubiquitylation result in reduced silencing. The absence of ubiquitylation in poised chromatin, such as in the htb1-KR mutant, allows Sir proteins to “leak” out of silenced regions, possibly due to affinity of Sir proteins for nonmethylated (van Leeuwen et al., 2002; van Leeuwen and Gottschling, 2002; Ng et al., 2003; Santos-Rosa et al., 2004) and/or nonubiquitylated (and nonacetylated [Rusche et al., 2003]) histones. In contrast, loss of deubiquitylation at the silenced regions (such as in UBP10 deletion) leads to increased ubH2B/meH3, which may “push” Sirs out of silenced chromatin (Figure 7, top right). In this regard, overexpression of Ubp10 can lead to promiscuous deubiquitylation across the genome (Figure 1C), functionally mimicking the htb1-KR mutant, and thus causing silencing defects (Figure 7, bottom right). Although our results strongly suggest that Ubp10-dependent histone H2B deubiquitylation is implicated in silencing, other deubiquitylation targets of Ubp10 cannot be ruled out. H2B deubiquitylation occurs in both open, transcriptionally competent chromatin and silenced chromatin achieved through distinct ubiquitin proteases (Ubp8 and Ubp10) that appear to be targeted through different mechanisms and apparently deubiquitylate different subpopulations of H2B. H2B ubiquitylation/deubiquitylation is utilized to activate euchromatic gene expression, and the localization/timing of deubiquitylation is regulated via SAGA-associated Ubp8 (Henry et al., 2003; Daniel et al., 2004; Kao et al., 2004). In silenced chromatin, H2B ubiquitylation is constitutively low, leading to low methylation and low gene expression. Ubp10, but not Ubp8, helps to maintain low telomeric ubiquitylation, perhaps through association with Sir4 (Kahana and Gottschling, 1999). There are 16 putative UBPs encoded by the yeast genome, and the substrate specificity of most of them is currently unknown (Soboleva and Baker, 2004). We have characterized two UBPs, Ubp8 (Henry et al., 2003; Ingvarsdottir et al., 2005) and Ubp10 (this study), as histone-deubiquitylating enzymes in distinct physiological processes. It is possible that other yeast UBPs also target histone H2B for deubiquitylation in other processes, such as in ubH2B-mediated double-strand break formation in meiosis (Yamashita et al., 2004). Alternatively, Ubp10 or Ubp8 may also be involved in these processes. How ubH2B exerts its effects at the mechanistic level is currently not well established (Henry and Berger, 2002). Whatever the mechanism, histone H2B ubiquitylation is required for both Lys4 and Lys79 methylation on H3 (Zhang, 2003; Osley, 2004). In vitro, it has been shown that Sir3 binds better to an unmethylated H3 tail peptide, compared to a peptide methylated at Lys4 (Santos-Rosa et al., 2004), providing a link to explain how ubH2B levels (partly controlled by Ubp10) can affect Sir protein association and, thus, silencing. Multiple, possibly mutually reinforcing mechanisms exist to enforce repressive silent chromatin structures essential for genomic stability. Our work suggests two examples of synergistic or collaborative silencing mech-

anisms. Firstly, Ubp10 may work in conjunction with the boundary histone Htz1 to regulate silencing. Other boundary elements, such as active tRNA genes (Donze and Kamakaka, 2001), also exist to limit silencing, and these may also synergize with Ubp10 in promoting silencing. Secondly, similar to deubiquitylation by Ubp10, Sir proteins have a direct role at telomeres to deacetylate histones (Grunstein, 1997; Gasser and Cockell, 2001; Rusche et al., 2003). We have found that Ubp10 and Sir2 are required for each other’s localization to the telomere-proximal silenced region. This mutual dependence of Ubp10 and Sir2 localization suggests that there may be a positive feedback mechanism among Sir proteins and Ubp10 for the establishment (and/or the maintenance) of silenced chromatin structure. Indeed, Ubp10mediated low ubiquitylation likely underlies the positive feedback mechanism previously suggested for low H3 Lys79 methylation and Sir3 binding at the silenced regions (Ng et al., 2003). Thus, because of the apparent critical nature of genomic silencing, Ubp10-mediated deubiquitylation is one of several regulatory functions. One challenge will be to unravel the complex interplay between these varied mechanisms. Experimental Procedures Yeast Strains and Plasmids The S. cerevisiae strains Y131, Y133, YKH007, YKH013, YKH045, YKH047, YKH063, T29, JR5-2A HTB1, JR5-2A FHTB1, JR5-2A Fhtb1-KR, IPY37, and YKH068 have been described elsewhere (Robzyk et al., 2000; Sun and Allis, 2002; Henry et al., 2003). Deletions of ORFs were perfomed by the one-step gene replacement method (Henry et al. [2003] and references therein). Deletion of UBP10 was performed by using the his5⫹ selectable marker to construct the following strains: T62-2 (from Y131), T63-1 (from Y133), T39 (from JR5-2A FHTB1), and T100 (from IPY36T). C-terminal epitope tagging of UBP10 with 3⫻ FLAG (strain T51-3) or with 13⫻ Myc (strain T18) were performed on JR5-2A as described (Henry et al., [2003] and references therein). p41-URA3 (p41U) plasmid was used as the source of HA-tagged ubiquitin (HA-ub) in ChDIP experiments. The TRP1 marker of plasmid p41 ([Robzyk et al., 2000], kindly provided by Mary-Ann Osley, University of New Mexico) was replaced by subcloning with the URA3 marker of pBS1539 to construct the p41U plasmid. The strains T54 (from JR5-2A FHTB1), T55-1 (from JR5-2A Fhtb1-KR), and T57 (from T39) were made by transforming the p41U plasmid. SIR2 gene was epitope tagged with 3⫻ HA originally in JR5-2A FHTB1 as described (Henry et al. [2003] and references therein) to construct the strain T46. The genomic DNA of this strain was used as a template to amplify by PCR the 3⫻ HA-KanMX cassette with ⵑ250 bp-flanking regions, which was then used for transformation to construct the strain T49 (from T39). The strain with SIR2 deletion, T124-1, was constructed from the T18 background strain similarly by transforming a PCR-amplified sir2::KanMX cassette. The strain T102-1s (gcn5⌬ubp10⌬) for the plate growth phenotype assay was constructed from T100 (ubp10⌬) as described (Candau et al., 1996). For the GST-Ubp10 expression studies, yeast strain YAS069 contains the plasmid pEG-KG (URA3, 2␮, UBP10), which expresses GST-tagged Ubp10 from the GAL1 promoter. The parental strain (wt) YAS005 (MATa hta1-htb1⌬::LEU2, hta2-htb2⌬::TRP1, leu2-⌬1, ura3-52, trp1-⌬63, his3-⌬200, and plasmid FB1251 [HIS3 CEN ARS HTA1-HTB1]) expressing FLAG-H2B as the sole source of Histone H2B was obtained from Dr. Kevin Struhl (strain FY406). YAS019 (from YAS005) containing the bre1::KanMX deletion was generated by PCR-based gene deletion of the BRE1 locus. YAS078 (from YAS005) contains the pEG-KG plasmid (URA3, 2␮, and UBP10). Analyses of Histone H2B Ubiquitylation and Deubiquitylation The relative levels of ubH2B in various UBP deletion strains (T39, T29, and YKH007) and controls (JR5-2A FHTB1 and JR5-2A) were

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investigated by ␣-FLAG immunoblotting (with or without ␣-FLAG immunoprecipitation) for FLAG-tagged histone H2B as described (Robzyk et al., 2000; Henry et al., 2003). In vitro deubiquitylation assays were performed as described (Henry et al., 2003). The recombinant Ubp10 (rUbp10; residues 360–736) was overexpressed as an N-terminal 6⫻ His-tagged fusion protein in Escherichia coli and purified. The SAGA complex containing Ubp8 was purified as described (Henry et al., 2003). For demonstrating the effects of GSTUbp10 expression on histone H2B ubiquitylation, strains were grown in raffinose media overnight then in YPD (Glucose) or YPGal (Galactose) at 30⬚C for 8 hr. Nuclear extracts were prepared for GSTUbp10 expression experiments and then subjected to SDS-PAGE and Western analysis. Expression Analysis Yeast cultures from strains Y131, Y133, YKH063, T62-2, and T63-1 were grown in YPD medium at 30⬚C to an A600 of 0.8 to 1.0, and 5 ml cultures were harvested and frozen. Total RNA was isolated with the YeaStar RNA Kit (Zymo Research), DNase I was treated with the DNAfree Kit (Ambion), and cDNA was synthesized with TaqMan RT Reagents (Applied Biosystems) according to the manufacturer recommendations. cDNA was amplified with primer pairs in Figure 2A in an ABI Prism 7000 System (Applied Biosystems) by using a pair of primers for 18S ribosomal RNA or ACT1 locus for normalization in parallel. Amplification data were collected in real time. Error bars denote one SD. ChIP ChIP and ChDIP experiments were performed essentially as described (Burke et al., 2000; Henry et al., 2003) with the following modifications: for ChIP, cultures were grown in 50 ml YPD medium to A600 ⵑ0.8, formaldehyde crosslinked, and harvested; for ChDIP, overnight cultures were grown in SC medium lacking uracil, washed in YP medium, and diluted into 500 ml YP medium with 2% galactose. Cultures were grown to A600 ⫽ 0.5–0.7, formaldehyde crosslinked, and harvested, and immunoprecipitations were performed as described (Henry et al., 2003). The antibodies used for ChIP were ␣-FLAG (M2; Sigma), ␣-HA (12CA5; Roche), ␣-Myc-Agasose (Santa Cruz), ␣-K4-trimethyl (ab8580; Abcam), and ␣-K79-trimethyl (ab2621; Abcam) for histone H3. Inputs and eluates of the immunoprecipitations were amplified with primer pairs described in Figure 2A or with primers pairs for rDNA region with an ABI Prism 7000 Thermal Cycler (Applied Biosystems). Normalized immunoprecipitations were obtained by dividing the average signal from the eluate by the average signal of the respective input, except when further normalizations were indicated. Error bars denote one SD. The primer sequences are available upon request. Synthetic Gene Deletion Analysis Synthetic gene deletion analysis was carried out as previously described (Tong et al., 2001).

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Acknowledgments

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We thank M. Osley for HA-Ub expression plasmid, T. Edlind and K. Struhl for yeast strains, and Jean Dorsey for technical assistance. We acknowledge T. Krishnamoorthy, S. McMahon, and G. Moore for critically reading the manuscript. We thank the Berger lab and especially D. Gottschling for valuable discussions. Research was supported by research grants from the National Institutes of Health (NIH) and National Science Foundation to S.L.B; a National Research Service Award to K.W.H. and NIH training grant awards to K.W.H. and to A.W.; a doctoral fellowship from the University of Pennsylvania BGS to K.L.; the American Cancer Society, NIH, and a Mallinckrodt Foundation Award to A.S.; a doctoral fellowship from the Canadian Institutes of Health Research (CIHR) to N.J.K; the CIHR, the Ontario Genomics Institute grants, and the National Cancer Institute of Canada with funds from the Canadian Cancer Society to J.F.G.

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Received: July 16, 2004 Revised: October 21, 2004 Accepted: January 5, 2005 Published: February 17, 2005

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