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Originally published In Press as doi:10.1074/jbc.M700292200 on March 15, 2007

J. Biol. Chem., Vol. 282, Issue 19, 14065-14072, May 11, 2007
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HULC, a Histone H2B Ubiquitinating Complex, Modulates Heterochromatin Independent of Histone Methylation in Fission Yeast*Formula

Martin Zofall and Shiv I. S. Grewal1

From the Laboratory of Biochemistry and Molecular Biology, NCI, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, January 10, 2007 , and in revised form, March 9, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Histone post-translational modifications play an important role in modulating chromatin structure that underlies various chromosomal processes in eukaryotic cells (1, 2). These modifications, including acetylation, phosphorylation, methylation, and ubiquitination of histones, have been shown to regulate transcription, chromosome condensation, and DNA repair (1, 3–5). Specific histone modifications can modify chromatin structure either directly by influencing histone-DNA and/or histone-histone interactions, as suggested in the case of histone acetylation (6), or mediate recruitment of other factors capable of modifying chromatin (7, 8). Factors containing interaction motifs for binding to histones methylated at specific lysine residues have been identified and shown to mediate targeting of chromatin-modifying activities implicated in either activating or repressing transcription (9–12). For example, histone H3 methylated at lysine 9 (H3K9me) recruits conserved heterochromatin protein HP1 via its chromodomain to mediate assembly of condensed heterochromatic structures (13–15). In contrast, methylation of H3 lysine 4 (H3K4me), found at transcriptionally poised euchromatic regions, provides binding sites for PHD, chromodomain, or tudor domain-containing proteins to recruit gene activation machinery (16–20).

In addition to well studied functions of histone acetylation and methylation in modulating chromatin, histone ubiquitination has emerged as an important modification for gene regulation. In particular, mono-ubiquitination of H2B (ubH2B)2 has been shown to play a crucial role in the maintenance of transcriptionally poised chromatin environment (3, 21, 22). Ubiquitin is transferred to H2B by the sequential action of three enzymatic activities as follows: ubiquitin activating ATPase E1, conjugating E2, and ligating E3 enzymes (23, 24). In Saccharomyces cerevisiae the multifunctional Rad6 E2 ubiquitin-conjugating enzyme associates with the RING finger-containing Bre1 E3 ligase to specifically direct mono-ubiquitination of H2B (25–27). In addition to histone H2B, Rad6 targets several other factors involved in a variety of nuclear processes. For example, Rad6 associates with the Rad18 ubiquitin ligase to control post-replication DNA repair (28, 29), and in complex with the Ubr1 1igase, it mediates protein degradation (30).

It has been shown that Rad6-mediated mono-ubiquitination of H2B at lysine 123 is necessary for methylation of lysine 4 and 79 of histone H3 (31–33), thus providing a mechanism for linking ubH2B to control of gene expression. A direct involvement of histone ubiquitination in gene expression is also suggested by a recent study showing that ubH2B facilitates function of the FACT complex (34), which has been implicated in elongation of RNA polymerase II (pol II) transcription through chromatin templates (35). Consistent with the involvement of ubH2B in gene expression, the removal of this modification by deubiquitinating enzymes is essential for preservation of silenced chromatin structures such as at telomeres and the mating-type loci in S. cerevisiae (36). Moreover, maintenance of low levels of H2B ubiquitination is important for recruitment of silent information regulator proteins involved in assembly of silenced heterochromatin-like structures (36).

Heterochromatin assembly pathway in fission yeast Schizosaccharomyces pombe share similarities to mechanisms in higher eukaryotes. The assembly of heterochromatic structures involves histone deacetylation that requires multiple histone deacetylases, including Clr3, Clr6, and Sir2 (37–41), and methylation of H3K9 by the Su(var)3-9 homolog Clr4 (13). H3K9me provides a binding site for recruiting chromodomain proteins, including Chp1, Chp2, and Swi6 (a homolog of mammalian HP1) to heterochromatic loci such as centromeres, telomeres, and the silent mating-type locus (13, 14, 42–44). Chromodomain proteins in turn mediate recruitment of a variety of factors involved in different aspects of heterochromatin assembly and functions (45). For example, Swi6 mediates recruitment/spreading of not only the transcriptional repressor complex SHREC (46, 47), but it also recruits an anti-silencing factor Epe1 that facilitates pol II occupancy and transcription of dg and dh centromeric elements (48). The transcripts originating from repeat elements are processed by RNAi machinery into small RNAs, which are believed to provide specificity for RNAi-mediated targeting of heterochromatin (49–53). Although significant progress has been made in defining the requirements for heterochromatic transcriptional silencing, the factors involved in transcription of heterochromatic repeats are poorly defined.

Previous studies have shown that the S. pombe Rhp6 protein, which shares structural and functional similarities to the budding yeast Rad6 ubiquitin-conjugating enzyme (54, 55), is a negative regulator of heterochromatic transcriptional gene silencing (56, 57). 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 (56, 57). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 ({Delta}rhp6, {Delta}rfp1, {Delta}rfp2, and {Delta}shf1) 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 (57). 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 (58). Twelve liters of exponentially growing cells (A ~ 0.75–1.0) were harvested, washed with 2x 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 1x HC buffer containing 300 mM KCl for 30 min. The lysates were cleared by centrifugation at 100,000 x 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 1x HC buffer supplemented with 300 mM KCl, and 10 times with 1x 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 (58).

Chromatin Immunoprecipitation (ChIP)—ChIP was performed as described previously (59) using antibodies against mono-, di-, or trimethylated H3K9 (Upstate%20Biotechnology">Upstate Biotechnology), Swi6 (59), FLAG (M2; Sigma), and the largest subunit of pol II (8WG16; Covance).

Histone Immunoprecipitation and Western Analysis—Exponentially growing cells (5 x 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 x 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 5x SDS sample buffer, and proteins were resolved on 17% SDS-PAGE and subjected to Western blot analysis with anti-ubiquitin antibodies (Santa Cruz Biotechnology).


Figure 1
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  		FIGURE 1.
Rhp6 is a component of a multisubunit protein complex. A, immunoaffinity purifications. Extract prepared from cells expressing untagged or FLAG-tagged proteins was used to perform immunoaffinity purification. Purified fractions were resolved on 4–20% SDS-PAGE and silver-stained. The bands were excised from gel and subjected to mass spectrometry. The identities of proteins are indicated to the right of the gel. B, number of peptides matching individual subunits of Rhp6-containing protein complex are indicated. C, two Rhp6-associated proteins contain an E3 ubiquitin-ligase signature C3HC4 type RING finger domain. D, defects in Rhp6 associated factors and in the H2B ubiquitination site (htb1K119R) cause an elongated cell shape, similar to the phenotype displayed by {Delta}rhp6 cells. wt, wild type.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 {Delta}rhp6 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 (60) 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) (54).

HULC Mediates Ubiquitination of Histone H2B—In S. cerevisiae, Rad6-Bre1 has been implicated in mono-ubiquitination of H2B (26, 27). 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 {Delta}rhp6 cells was used to detect ubH2B. We detected a slower migrating H2B-FLAG band in a wild type sample that was missing in the {Delta}rhp6 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.


Figure 2
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  		FIGURE 2.
Rhp6-associated proteins Rfp1, Rfp2, and Shf1 are required for H2B ubiquitination. A, detection of ubiquitinated H2B. Acid-extracted histone H2B-FLAG was immunoprecipitated (IP) from the indicated strains and analyzed by Western analysis with anti-FLAG M2 (Sigma) and anti-ubiquitin antibody (P4D1, Santa Cruz Biotechnology). A slower migrating band corresponds to mono-ubiquitinated H2B. wt, wild type. B, Rhp6-associated proteins Rfp1, Rfp2, and Shf1 are required for H2B ubiquitination. Histones purified form the indicated strains were analyzed by Western analysis with antibody against FLAG. C, HULC complex catalyzes histone H2B ubiquitination in vitro. Purified HULC was incubated with ubiquitin and recombinant H2B in the presence of E1-activating enzyme at 30 °C. The reaction was resolved by SDS-PAGE and subjected to Western blot analysis to detect ubiquitinated proteins. Asterisks denote presumed ubiquitin conjugates of HULC subunits. These bands are present only in the lanes corresponding to reactions containing HULC and ubiquitin. D, deletions of genes encoding HULC subunits are associated with enhanced silencing of otr1::ura4+ reporter. A serial dilution plating assay was performed to monitor ura4+ expression. NS, nonselective medium.

 
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 (56, 57). 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 (61). 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 (56, 57), {Delta}rhp6 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 Rhp6 correlate with distinctive changes in heterochromatin-specific histone modification patterns. Interestingly, levels of trimethylated H3K9 (H3K9me3) were significantly reduced, although the levels of monomethylated H3K9 (H3K9me1) were increased at the dg repeat element and otr1::ura4+ (Fig. 3B). Furthermore, dimethylated H3K9 (H3K9me2) levels were slightly elevated. We also found that levels of Swi6 at centromeres were drastically reduced in cells overexpressing Rhp6 (Fig. 3B). Reduced H3K9me3 and Swi6 levels at heterochromatic loci could not be accounted for by a reduction in nucleosome density associated with pol II transcription, as we observed only a small change in nucleosome occupancy at the pericentric regions when compared with constitutively transcribed act1 control (supplemental Fig. 2). We next tested whether HULC subunits are required for Rhp6-mediated changes in heterochromatin. In contrast to the wild type cells, overexpression of Rhp6 in {Delta}rfp1{Delta}rfp2 or {Delta}shf1 mutant cells had no significant effect on silencing and Swi6 localization at the otr1::ura4+ reporter gene (Fig. 4, A and B). These analyses further support the suggestion that HULC plays an important role in Rhp6-mediated modulation of heterochromatin. However, it remains possible that Rhp6 cooperates with additional E3 ligases capable of modifying heterochromatin. To this end, we note that a slight decrease in silencing is observed when Rhp6 is expressed in {Delta}rfp1{Delta}rfp2 and {Delta}shf1 mutant background cells.

HULC Subunits Are Localized at Heterochromatic Repeat Elements—Rad6 and Bre1 are recruited to promoters and coding regions of genes in S. cerevisiae to ubiquitinate H2B that has an important role in pol II transcription of targeted loci (27, 34, 62–64). Because pol II transcribes heterochromatic repeats in S. pombe, we investigated whether components of HULC localize to the heterochromatic repeat elements. We used strains expressing FLAG-tagged Shf1, Rfp2, or Rhp6 fusion protein expressed under the control of their native promoters to perform ChIP assays. Our analyses showed that all three proteins localize to the centromeric dg/dh repeats, albeit at levels lower than observed with euchromatic genes (Fig. 4C; data not shown). These results support the idea that HULC directly antagonizes heterochromatin-mediated transcriptional silencing.


Figure 3
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  		FIGURE 3.
Rhp6-induced loss of heterochromatic silencing is linked to differential changes in mono-, di-, and trimethylation of H3K9 and delocalization of Swi6. A, Rhp6 overexpression disrupts centromeric silencing. Cells carrying plasmid containing rhp6+ under the control of thiamine-repressible nmt1 promoter (nmt1-rhp6+) or control vector Rep3 were grown in thiamine-free media to induce Rhp6 expression. Serial dilution plating assays in the presence or absence of 5-FOA were performed to assay otr1::ura4+ expression. B, overexpression of Rhp6 affects H3K9me and Swi6 localization at centromeric repeats. ChIP analysis was used to measure levels of Swi6, H3K9me1, H3K9me2, or H3K9me3 at centromeric dg repeats or otr1::ura4+. DNA isolated from ChIP and WCE fractions was analyzed by multiplex PCR to measure enrichment of otr1::ura4+ and a DNA fragment corresponding to dg centromeric repeat (dg223). Intensities of bands in ChIP and WCE lanes were used to calculate relative enrichment values shown below each lane. NS, nonselective medium.

 
ubH2B Counteracts Heterochromatic Transcriptional Silencing—The results presented above prompted us to investigate whether ubiquitination of H2B is critical for HULC-mediated modulation of heterochromatin structures. Two different experiments were performed. First, we analyzed the effect of H2B-K119R mutation on silencing of an otr1::ura4+ reporter. Interestingly, the H2B-K119R mutation significantly enhanced silencing of the otr1::ura4+ (Fig. 5A), in a manner similar to mutations in genes encoding HULC subunits (Fig. 2D). Moreover, an H2B-K119R mutation reduced pol II localization at the dg repeat element to an extent comparable with {Delta}rhp6 cells (supplemental Fig. 3). Second, we investigated whether H2B-K119 is required for changes in heterochromatin induced by overexpression of Rhp6. Although overexpression of Rhp6 abolished otr1::ura4+ silencing in wild type background cells, we observed only a minor change in otr1::ura4+ expression in the H2B-K119R mutant background cells (Fig. 5B). Furthermore, H2B-K119R mutation suppressed Rhp6-induced loss of Swi6 and H3K9me3, as well as accompanying increase in H3K9me1 (Fig. 5C). Taken together, these results indicate that ubiquitination of H2B-K119 by HULC plays an important role in modifying heterochromatin structures.

Heterochromatin formation in general correlates with assembly of condensed structures that are refractory to transcriptional machinery such as pol II (65). Based on results presented above, H2B ubiquitination might alter accessibility of heterochromatic sequences to transcriptional machinery and other factors. Indeed, ChIP analyses showed increased pol II occupancy and a slight reduction in histone density at pericentromeric regions (supplemental Fig. 2 and Fig. 5D), suggesting the existence of a more accessible chromatin structure in cells overexpressing Rhp6. An increase in pol II levels requires H2B ubiquitination as we did not detect a change in pol II occupancy in cells expressing mutant H2B (K119R) (Fig. 5D). Therefore, HULC-mediated ubiquitination of H2B-K119 might be functionally important for regulating chromatin dynamics at heterochromatic regions, in addition to the previously described role for ubH2B in pol II transcription at euchromatic loci (34, 62–64).

ubH2B-mediated Transcriptional Derepression by HULC Occurs Independently of H3K4me—In S. cerevisiae, ubiquitination of H2B-K123 by Rad6-Bre1 is required for methylation of H3K4 and H3K79 (26, 27, 31–33). The requirement for ubH2B in H3K4 methylation is conserved in S. pombe, as {Delta}rhp6 causes severe reduction in the levels of H3K4me, which is catalyzed by a single SET domain protein, Set1 (66, 67) (Fig. 6A). In addition to {Delta}rhp6, defects in H3K4 methylation were also detected in {Delta}rfp1, {Delta}rfp2, {Delta}shf1, and htb1K119R mutant cells (Fig. 6A). Because Rhp6-induced heterochromatic derepression in S. pombe was shown to correlate with an increase in H3K4me levels (57), we sought to determine whether ubH2B-mediated modulation of heterochromatin by HULC requires methylation of H3K4. Our analysis showed that loss of H3K4me in {Delta}set1 cells has no consequences for Rhp6-induced heterochromatic derepression at the pericentromeric region. As shown in Fig. 6B, overexpression of Rhp6 resulted in a comparable decrease in otr1::ura4+ silencing in wild type and {Delta}set1 cells. Thus, although components of HULC and ubH2B are required for H3K4 methylation, H3K4me is not functionally important for Rhp6-induced modulation of heterochromatin.


Figure 4
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  		FIGURE 4.
HULC components localize to centromeric heterochromatin and are required for Rhp6-mediated changes in heterochromatin. A, serial dilution assay was performed to examine defects in centromeric (otr1::ura4+) silencing caused by Rhp6 overexpression in the indicated strain backgrounds. Because the results presented in Fig. 1 showed identical phenotypes for single or double rfp1 and rfp2 mutants, only the rfp1rfp2 double mutant was used in this experiment. B, HULC components are required for Rhp6-induced loss of Swi6 at centromeres. ChIP analysis was used to measure Swi6 levels at centromeric dg repeats. C, components of HULC localize to the centromeric heterochromatin. ChIP analysis was performed using strains expressing Rhp6-FLAG, Shf1-FLAG, and Rfp2-FLAG. DNA isolated from ChIP and WCE fractions was analyzed by multiplex PCR with primers that amplify portion of dg centromeric repeat (dg223) or the mitochondrial tRNA locus (mtRNA). wt, wild type; NS, nonselective medium.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Several reports have implicated the ubiquitin-conjugating enzyme Rhp6 (a homolog of budding yeast Rad6) in modifications of heterochromatin structures in S. pombe (56, 57, 68). However, there had been no clear demonstration of the molecular mechanism by which Rhp6-based ubiquitination activity counteracts heterochromatic repression. In this study, we report the biochemical characterization of Rhp6, and we investigate the mode of action for this enzyme in modifying heterochromatin. We show that Rhp6 forms a complex with two RING finger proteins, Rfp1 and Rfp2, and a novel serine-rich protein Shf1. The components of the Rhp6-containing complex HULC are required for mono-ubiquitination of histone H2B at lysine 119, and purified complex ubiquitinates histone H2B in vitro. We provide evidence that HULC subunits localize to heterochromatic regions and that ubiquitination of H2B-K119 by HULC is likely functionally important for Rhp6-mediated heterochromatin remodeling, resulting in increased pol II occupancy at centromeric repeats. Our analysis also suggests that HULC-mediated changes in heterochromatin do not rely on ubH2B/HULC-dependent methylation of histone H3 at lysine 4, a modification known to be associated with active chromatin domains (66, 69, 70).

RING finger proteins Rfp1 and Rfp2 share structural similarities with other E3 ubiquitin ligase proteins such as S. cerevisiae Bre1, which also directs Rad6 to H2B (26, 27). Evidence suggests that Rad6-Bre1-dependent H2B ubiquitination defines active chromatin regions and facilitates pol II transcription of genes by promoting elongation of transcriptional complexes (34, 62–64). However, in contrast to our results showing that HULC subunits are localized at heterochromatic regions, both Rad6 and ubH2B have been shown to be absent from transcriptionally silent heterochromatin regions in S. cerevisiae (36, 63). This difference is most likely because of the fact that in S. pombe heterochromatic repeat sequences are transcribed by pol II, to generate transcripts for production of small RNAs, believed to be essential for RNAi-mediated targeting of heterochromatin (50, 71, 72). An interesting possibility is that ubiquitination of H2B by HULC, along with other activities such as FACT (34, 35), contributes to pol II transcription of centromeric repeat elements by antagonizing the repressive effects of heterochromatin. Indeed, although H2B-K119R and {Delta}rhp6 mutants show a decrease in pol II levels at heterochromatic repeats (supplemental Fig. 3), Rhp6 overexpression is associated with a significant increase in pol II localization to heterochromatic sequences in a manner dependent on ubH2B (Fig. 5). The increased occupancy of transcriptional machinery at heterochromatic loci could in general destabilize heterochromatin, resulting in defects in H3K9 methylation and Swi6 localization (Fig. 3B). However, another possibility is that ubH2B directly interferes with the assembly of heterochromatin complexes. Nonetheless, we note that changes in heterochromatin modification patterns caused by Rhp6 overexpression are reminiscent of patterns observed in cells carrying deletion of Clr3 histone deacetylase (a component of SHREC), which has been shown to limit pol II accessibility to heterochromatic repeat elements (46, 47). In both cases, loss of heterochromatic silencing correlates with a dramatic reduction in H3K9me3 levels that is accompanied by an increase in H3K9me1 levels and defects in Swi6 localization (Fig. 3B) (47).


Figure 5
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  		FIGURE 5.
Rhp6-induced disruption of heterochromatin and pol II localization requires H2B-K119. A, mutation of conserved H2B ubiquitination site (H2B-K119R) enhanced centromeric silencing. Serial dilution plating assay was performed to monitor the expression of otr1::ura4+in wild type (wt), {Delta}rhp6, or htb1 K119R strain backgrounds. B, ubH2B is required for Rhp6-induced disruption of heterochromatic silencing. Serial dilution assays using cells carrying either a plasmid expressing rhp6+ gene under the control of the nmt1 promoter (nmt1-rhp6+) or a control empty plasmid (Rep3) were used to compare otr1::ura4+ expression in the indicated strain backgrounds. C, H2b-K119R mutation suppresses Rhp6-induced changes in heterochromatin. ChIP analysis was used to measure H3K9me1, H3K9me3, and Swi6 levels at otr1::ura4+ in wild type and htb1K119R cells harboring empty vector Rep3 or rhp6+ overexpression vector nmt1-rhp6+. D, H2B ubiquitination facilitates pol II localization to heterochromatin. ChIP analysis was used to measure pol II at otr1::ura4+ and dg centromeric repeat (dg660) in wild type and htb1K119R cells harboring empty vector Rep3 or rhp6+ overexpression vector. NS, nonselective medium.

 


Figure 6
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  		FIGURE 6.
H2B ubiquitination antagonizes heterochromatic silencing independently of H3K4 methylation. A, methylation of histone H3 at lysine 4 requires ubH2B and HULC subunits. Histones were purified from the indicated strains and analyzed by Western analysis with H3K4me2 antibody (upper panel). Coomassie Brilliant Blue (CBB) staining is shown as loading control (lower panel). B, histone H3 lysine 4 methylation is not required for Rhp6-induced heterochromatin derepression. Centromeric (otr1::ura4+) silencing was monitored by serial dilution assay using wild type and {Delta}set1 cells carrying empty vector Rep3 or overexpressing Rhp6 under the control of inducible nmt1promoter (nmt1-rhp6+).

 
Apart from Rhp6, other factors capable of disrupting heterochromatic silencing have been identified (73). One of these factors is a jumonji domain C-containing protein Epe1, which is preferentially enriched at heterochromatic loci and facilitates pol II accessibility to repeat elements coated with heterochromatin (48, 73). Whether Epe1 and HULC/ubH2B cooperate to destabilize heterochromatic structures is not known. However, it should be noted that Rhp6- or Epe1-mediated derepression of heterochromatic sequences is correlated with distinct patterns of histone modifications. Although the levels of H3K9me3 and Swi6 at centromeres are significantly decreased upon Rhp6 overexpression (this study and see Ref. 57), no such change in these heterochromatin markers is observed in cells overexpressing Epe1 (48).

Finally, we emphasize that HULC might also target additional factors other than H2B. Indeed, it has been shown that Rad6-mediated ubiquitination of Uhp1 protein is important for proper maintenance of heterochromatin at silent mating-type loci (74). Future analysis of the mechanism by which HULC-mediated ubiquitination of H2B and other factors modulate gene silencing is likely to provide important insights into the assembly and maintenance of heterochromatic structures.


   FOOTNOTES
 
* This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3. Back

1 To whom correspondence should be addressed. Tel.: 301-594-6389; Fax: 301-435-3697; E-mail: grewals{at}mail.nih.gov.

2 The abbreviations used are: ubH2B, ubiquitinated histone H2B; Shf, small histone ubiquitination factor; Clr, cryptic loci regulator; pol II, DNA-dependent RNA polymerase II; RNAi, RNA interference; FOA, 5-fluoroorotic acid; WCE, whole cell extract; ChIP, chromatin immunoprecipitation; FACT, facilitates chromatin transcription; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; Pipes, 1,4-piperazinediethanesulfonic acid. Back


   ACKNOWLEDGMENTS
 
We thank R. Kobayashi (MD Anderson Cancer Center) for help with mass spectrometry analyses and M. Lichten (NCI, National Institutes of Health) for comments on the manuscript.



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