HULC, a Histone H2B Ubiquitinating Complex, Modulates Heterochromatin Independent of Histone Methylation in Fission Yeast

Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures. Heterochromatin in fission yeast is targeted dynamically by opposing chromatin-modifying activities capable of alleviating or promoting transcriptional gene silencing. In this study, we report the biochemical and genetic characterization of a ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6), which has been shown to negatively affect stability of heterochromatic structures. We show that Rhp6 is a component of the multisubunit protein complex (termed HULC) that also contains two RING finger proteins Rfp1 and Rfp2, sharing homology with budding yeast Bre1 protein and a unique serine-rich protein Shf1. HULC is required for ubiquitination of histone H2B at lysine 119 (H2B-K119), and it localizes to heterochromatic sequences. Moreover, our analyses suggest that Rhp6-induced changes in heterochromatic silencing are mediated predominantly through H2B ubiquitination (ubH2B), and they correlate with increased RNA polymerase II levels at repeat elements embedded within heterochromatin domains. Interestingly, heterochromatic derepression caused by Rhp6 occurs independently of the involvement of HULC subunits and ubH2B in methylation of histone H3 at lysine 4 (H3K4me). These analyses implicate ubH2B in modulation of heterochromatin, which has important implications for dynamics and many functions associated with heterochromatic structures. Histone post-translational modifications play an important role in modulating chromatin structure that underlies various chromosomal processes in eukaryotic cells (1Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7709) Google Scholar, 2Berger S.L. Curr. Opin. Genet. Dev. 2002; 12: 142-148Crossref PubMed Scopus (991) Google Scholar). These modifications, including acetylation, phosphorylation, methylation, and ubiquitination of histones, have been shown to regulate transcription, chromosome condensation, and DNA repair (1Jenuwein T. Allis C.D. 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Nielsen O. Murray J.M. Thon G. Eukaryot. Cell. 2002; 1: 613-625Crossref PubMed Scopus (12) Google Scholar, 57Choi E.S. Kim H.S. Jang Y.K. Hong S.H. Park S.D. Mol. Cell. Biol. 2002; 22: 8366-8374Crossref PubMed Scopus (10) Google Scholar). Whereas loss of Rhp6 enhances silencing at heterochromatic loci, overexpression of Rhp6 disrupts silencing that correlates with increased levels of euchromatin-specific modifications such as H3K4me (56Nielsen I.S. Nielsen O. Murray J.M. Thon G. Eukaryot. Cell. 2002; 1: 613-625Crossref PubMed Scopus (12) Google Scholar, 57Choi E.S. Kim H.S. Jang Y.K. Hong S.H. Park S.D. Mol. Cell. Biol. 2002; 22: 8366-8374Crossref PubMed Scopus (10) Google Scholar). Despite these advances, the mechanism by which Rhp6 antagonizes the repressive effects of heterochromatin is not known. In this study we utilize biochemical and genetic approaches to characterize the mechanism of Rhp6-mediated heterochromatin derepression. We show that Rhp6 is a component of multisubunit protein complex, referred to as HULC (histone H2B ubiquitin ligase complex), which is required for ubiquitination of histone H2B lysine 119 in S. pombe. Interestingly, HULC localizes to heterochromatic loci and modulates stability of heterochromatin structures via ubiquitination of H2B but independently of histone H3 lysine 4 methylation. Our results further suggest that HULC facilitates pol II occupancy at heterochromatic repeat elements in a ubH2B-dependent manner. Strains, Plasmids, and Media—Standard conditions were used for growing yeast strains, sporulation, and tetrad analysis. Strains expressing epitope-tagged proteins (H2B-FLAG, Rfp1-FLAG, Shf1-FLAG, and Rhp6-FLAG) and deletion strains (Δrhp6, Δrfp1, Δrfp2, and Δshf1) were generated using a PCR-based module method. To generate the H2B mutant, we transformed wild type strain with a DNA fragment consisting of mutated htb1 fused to a KanR cassette. The mutation was confirmed by DNA sequencing. Rhp6 expression vector was constructed by inserting rhp6+ open reading frame into the pRep3 plasmid, as described previously (57Choi E.S. Kim H.S. Jang Y.K. Hong S.H. Park S.D. Mol. Cell. Biol. 2002; 22: 8366-8374Crossref PubMed Scopus (10) Google Scholar). Expression of ura4+ reporter inserted at the heterochromatic location was assayed by performing serial dilution plating assay on nonselective (N/S), uracil-deficient (–ura), and counterselective FOA (5-FOA) thiamine-free minimal medium. Immunoaffinity Purification—Whole cell extracts from FLAG epitope tagged and untagged strains were prepared as described (58Jia S. Kobayashi R. Grewal S.I. Nat. Cell Biol. 2005; 7: 1007-1013Crossref PubMed Scopus (170) Google Scholar). Twelve liters of exponentially growing cells (A ∼ 0.75–1.0) were harvested, washed with 2× HC buffer (300 mm HEPES-KOH, pH 7.6, 100 mm KCl, 40% glycerol, 2 mm dithiothreitol, 2 mm EDTA, protease inhibitors), and frozen in liquid nitrogen. Frozen cells were blended with dry ice using a household blender and extracted with 40 ml of 1× HC buffer containing 300 mm KCl for 30 min. The lysates were cleared by centrifugation at 100,000 × g for 2 h. The supernatant was pre-cleared with IgG-Sepharose (Roche Applied Science), combined with 300 μl of anti-FLAG M2-agarose for 2 h, and washed five times with 1× HC buffer supplemented with 300 mm KCl, and 10 times with 1× HC buffer containing 100 mm NaCl. Proteins were eluted with 150 mg/ml 3xFLAG peptide (Sigma). The eluted proteins were resolved on 4–20% SDS-PAGE, visualized by silver staining, and analyzed by mass spectrometry as described previously (58Jia S. Kobayashi R. Grewal S.I. Nat. Cell Biol. 2005; 7: 1007-1013Crossref PubMed Scopus (170) Google Scholar). Chromatin Immunoprecipitation (ChIP)—ChIP was performed as described previously (59Nakayama J. Klar A.J. Grewal S.I. Cell. 2000; 101: 307-317Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) using antibodies against mono-, di-, or trimethylated H3K9 (Upstate Biotechnology), Swi6 (59Nakayama J. Klar A.J. Grewal S.I. Cell. 2000; 101: 307-317Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), FLAG (M2; Sigma), and the largest subunit of pol II (8WG16; Covance). Histone Immunoprecipitation and Western Analysis—Exponentially growing cells (5 × 105) were harvested, washed with NIB buffer (0.25 m sucrose, 15 mm Pipes, pH 6.8, 60 mm KCl, 15 mm NaCl, 5 mm MgCl2, 1 mm CaCl2, 0.8% Triton X-100), and resuspended in 0.5 ml of NIB buffer supplemented with 2 mm ZnSO4, 10 ng/ml trichostatin A, and protease inhibitors. Cells were homogenized using glass bead beater and collected by centrifugation (11,000 × g). Cell pellets were extracted twice with 0.4 m H2SO4. Histones were collected by precipitation with trichloroacetic acid, dissolved in 2.5 m urea, and resolved on 17% SDS-PAGE before subjecting to Western analysis with antibodies against H3K4me2 (Upstate), FLAG (M2, Sigma), or ubiquitin (P4D1, Santa Cruz Biotechnology). For immunoprecipitation, histones dissolved in 2.5 m urea were diluted with IP buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Nonidet P-40, and 0.5 mg/ml bovine serum albumin) to adjust urea to a final concentration of 0.5 m and combined with 30 μl of anti-FLAG M2-agarose for 2 h, washed three times with IP buffer, and eluted with 150 mg/ml 3xFLAG peptide (Sigma). In Vitro Ubiquitination Assay—Ubiquitination was performed at 30 °C for 4 h in 25 μl of mixture containing 20 mm HEPES-KOH, pH 7.6, 5% glycerol, 30 mm KCl, 30 mm NaCl, 5 mm MgCl2, 6 mm ATP, 2.5 μg of ubiquitin (Sigma), 500 ng of histone H2B (Upstate), 500 ng of E1 ubiquitin-activating enzyme (Calbiochem), and 100 ng of immunoprecipitated HULC. Reactions were terminated by addition of 5× SDS sample buffer, and proteins were resolved on 17% SDS-PAGE and subjected to Western blot analysis with anti-ubiquitin antibodies (Santa Cruz Biotechnology). Purification of Rhp6 and Identification of Its Interaction Partners—To gain insight into the mechanism of Rhp6-mediated modulation of heterochromatin, we used immunoaffinity to purify Rhp6 from a strain expressing Rhp6 tagged at its carboxyl terminus with three copies of FLAG epitope (Rhp6-FLAG). The fusion protein was expressed under the control of its native promoter, and its functionality was confirmed by comparing cell morphology and growth rate with Δrhp6 cells. Silver staining analysis of the Rhp6-purified fraction revealed several polypeptides that were specifically present in immunoaffinity-purified fraction of Rhp6-FLAG but not in the control fraction (Fig. 1A). Mass spectrometry analyses of excised bands identified an ∼22-kDa polypeptide to be Rhp6-FLAG, consistent with its predicted molecular weight (Fig. 1, A and B). Peptides from the major ∼80-kDa band identified two proteins encoded by SPCC970.10c and SPCC1919.15. Both of these proteins contain the C3HC4-type RING finger domain implicated in catalysis of ubiquitin transfer by E3 ubiquitin ligases (60Lorick K.L. Jensen J.P. Fang S. Ong A.M. Hatakeyama S. Weissman A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11364-11369Crossref PubMed Scopus (947) Google Scholar) and share similarity with the budding yeast Bre1 (43%) and with each other (42%) (Fig. 1C). We named these proteins Rfp1 and Rfp2 (RING finger protein 1 and 2). Another protein co-purifying with Rhp6-FLAG as an ∼25-kDa band was identified as a product of SPAC22F8.12c. This protein does not have any apparent homologs, and its only identifiable feature is a serine-rich region. We will refer to this protein as Shf1 (small histone ubiquitination factor). To verify that Shf1, Rfp1, Rfp2, and Rhp6 are indeed associated together in a complex, we constructed a strain that produced functional Shf1 fused at its carboxyl terminus with three copies of the FLAG epitope (Shf1-FLAG). Affinity purification followed by mass spectrometry identified Rhp6, Rfp1, and Rfp2 as Shf1-assocciated factors (Fig. 1, A and B). These analyses suggest that Rhp6, Rfp1, Rfp, and Shf1 are associated together in a complex, which we have named HULC. Consistent with these factors forming a complex, deletion of rfp1, rfp2, or shf1 causes phenotypes known to be associated with rhp6 mutant cells, including an elongated cell shape (Fig. 1D) (54Reynolds P. Koken M.H. Hoeijmakers J.H. Prakash S. Prakash L. EMBO J. 1990; 9: 1423-1430Crossref PubMed Scopus (72) Google Scholar). HULC Mediates Ubiquitination of Histone H2B—In S. cerevisiae, Rad6-Bre1 has been implicated in mono-ubiquitination of H2B (26Hwang W.W. Venkatasubrahmanyam S. Ianculescu A.G. Tong A. Boone C. Madhani H.D. Mol. Cell. 2003; 11: 261-266Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 27Wood A. Krogan N.J. Dover J. Schneider J. Heidt J. Boateng M.A. Dean K. Golshani A. Zhang Y. Greenblatt J.F. Johnston M. Shilatifard A. Mol. Cell. 2003; 11: 267-274Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). We therefore sought to explore whether HULC components play a role in H2B ubiquitination. For this purpose, we constructed strains expressing histone H2B fused at its carboxyl terminus with triple FLAG epitope (htb1-FLAG). Western analysis of immunoaffinity-purified H2B-FLAG isolated under denaturing conditions either from wild type or Δrhp6 cells was used to detect ubH2B. We detected a slower migrating H2B-FLAG band in a wild type sample that was missing in the Δrhp6 sample (Fig. 2A, upper panel). The slower migrating H2B band represented ubH2B as it was also detected in Western analysis with anti-ubiquitin antibody (Fig. 2A, lower panel). The intensity of the slower migrating band was enhanced slightly in histones isolated from cells overexpressing Rhp6, further indicating that this band corresponds to ubH2B-FLAG (supplemental Fig. 1). We next analyzed the involvement of Rhp6-associated proteins in H2B ubiquitination. Western analysis of H2B-FLAG isolated from mutants deficient for individual subunits of HULC revealed that slower migrating band representing ubH2B was missing in cells lacking either Bre1 homologs (i.e. Rfp1 or Rfp2) or Shf1. These analyses suggest that components of HULC are required for ubiquitination of H2B. To confirm that HULC is indeed a ubiquitin ligase, we performed an in vitro ubiquitination assay. Purified HULC was incubated with recombinant H2B in the presence of recombinant E1-activating enzyme and ubiquitin, and ubiquitin-tagged proteins were detected by Western blot analysis with anti-ubiquitin antibody. An ∼20-kDa ubiquitin conjugate corresponding to ubH2B was detected specifically in reactions containing purified HULC (Fig. 2C). This band was eliminated when HULC or recombinant H2B was omitted from the reactions. Based on this analysis, we conclude that HULC is a ubiquitinating complex that most likely directly modifies H2B in vivo. To address whether HULC specifically modifies H2B at a lysine residue that corresponds to the known Rad6-Bre1 target (i.e. H2B-K123) in S. cerevisiae, we developed strains expressing mutant H2B carrying lysine 119 to arginine (htb1-K119R) substitution. Mutant H2B expressed under the control of native promoter was the sole source of histone H2B. Like cells carrying deletions of genes encoding HULC subunits, cells carrying htb1-K119R mutant allele showed an elongated cell shape (Fig. 1D). More importantly, Western analysis showed that the band corresponding to ubH2B was undetectable in htb1-K119R mutant cells (Fig. 2B). We conclude from these analyses that the Lys-119 residue is a key target of HULC-mediated H2B ubiquitination. Rhp6-mediated Modulation of Heterochromatin Requires HULC Components—Previous studies have suggested a role for Rhp6 in antagonizing heterochromatic gene silencing (56Nielsen I.S. Nielsen O. Murray J.M. Thon G. Eukaryot. Cell. 2002; 1: 613-625Crossref PubMed Scopus (12) Google Scholar, 57Choi E.S. Kim H.S. Jang Y.K. Hong S.H. Park S.D. Mol. Cell. Biol. 2002; 22: 8366-8374Crossref PubMed Scopus (10) Google Scholar). However, the molecular mechanism by which Rhp6 counteracts heterochromatic silencing has remained unclear. We sought to explore whether components of HULC affect heterochromatic silencing. The ura4+ reporter gene inserted in the outer repeat region of centromere 1 (otr1::ura4+) is subject to heterochromatin-mediated transcriptional silencing (61Allshire R.C. Nimmo E.R. Ekwall K. Javerzat J.P. Cranston G. Genes Dev. 1995; 9: 218-233Crossref PubMed Scopus (385) Google Scholar). However, silencing of the reporter gene is not completely stable. As a result, wild type cells carrying otr1::ura4+ can grow on both medium lacking uracil and counter-selective medium containing FOA, which selects for growth cells in which ura4+ reporter is silenced (Fig. 2D). We assayed the effects of deletion of genes encoding HULC subunits on otr1::ura4+ expression by performing serial dilution plating assay. Consistent with previous reports (56Nielsen I.S. Nielsen O. Murray J.M. Thon G. Eukaryot. Cell. 2002; 1: 613-625Crossref PubMed Scopus (12) Google Scholar, 57Choi E.S. Kim H.S. Jang Y.K. Hong S.H. Park S.D. Mol. Cell. Biol. 2002; 22: 8366-8374Crossref PubMed Scopus (10) Google Scholar), Δrhp6 resulted in enhanced silencing of the otr1::ura4+, as shown by reduced growth on medium lacking uracil (Fig. 2D). Loss of other HULC components such as Rfp1, Rfp2, or Shf1 also resulted in a similar increase in silencing of the ura4+ reporter (Fig. 2D). These results indicate that Rhp6 counteracts heterochromatic silencing as a component of the HULC. Overexpression of Rhp6 abrogates silencing of the otr1::ura4+ reporter, resulting in the loss of cell viability on medium supplemented with FOA (Fig. 3A). Defects in heterochromatic silencing observed in cells overexpressing Rhp