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. Author manuscript; available in PMC: 2012 Feb 18.
Published in final edited form as: Mol Cell. 2011 Feb 18;41(4):384–397. doi: 10.1016/j.molcel.2011.01.024

WAC, a functional partner of RNF20/40, regulates histone H2B ubiquitination and gene transcription

Feng Zhang 1, Xiaochun Yu 1,*
PMCID: PMC3062166  NIHMSID: NIHMS270279  PMID: 21329877

Summary

Histone H2B ubiquitination plays an important role in regulating chromatin organization during gene transcription. It has been shown that RNF20/40 regulates H2B ubiquitination. Here, using protein affinity purification, we have identified WAC as a functional partner of RNF20/40. Depletion of WAC abolishes H2B ubiquitination. WAC interacts with RNF20/40 through its C-terminal coiled-coil region and promotes RNF20/40’s E3 ligase activity for H2B ubiquitination. The N-terminal WW domain of WAC recognizes RNA polymerase II. During gene transcription, WAC targets RNF20/40 to associate with RNA polymerase II complex for H2B ubiquitination at active transcription sites, which regulates transcription. Moreover, WAC-dependent transcription is important for cell cycle checkpoint activation in response to genotoxic stress. Taken together, our results demonstrate an important regulator for transcription-coupled histone H2B ubiquitination.

Introduction

Genomic DNA is packed into chromatin to wrap nucleosomal histones in the eukaryotic cell nucleus. N-terminal and C-terminal histone tails, which extend away from the core of nucleosome, are available for covalent modifications, such as acetylation, methylation, phosphorylation, sumoylation, ADP-ribosylation and ubiquitination (Berger, 2007; Kouzarides, 2007; Weake and Workman, 2008). Covalent modifications of histones and transcription factors are closely associated with gene transcription, controlled by the RNA polymerase II (Pol II) complex (Couture and Trievel, 2006; Egloff and Murphy, 2008; Shilatifard, 2006; Suganuma and Workman, 2008). One important histone modification that regulates transcription is the monoubiquitination of histone H2B (ubH2B).

Histone H2B is ubiquitinated at the C-terminal tail in most organisms. In S. cerevisiae, monoubiquitination of H2B at Lys123 requires yRad6 and yBre1, which function as E2 ubiquitin conjugase and E3 ubiquitin ligase, respectively (Hwang et al., 2003; Kao et al., 2004; Robzyk et al., 2000; Wood et al., 2003a). In S. pombe, a similar complex containing the yRad6 ortholog Rhp6 and the yBre1 orthologs Brl1/Rfp1 and Brl2/Rfp2 are required for H2B ubiquitination at Lys119 (Tanny et al., 2007; Zofall and Grewal, 2007). In human cells, hRAD6A and hRAD6B, two homologs of yRad6, act as E2 conjugases to catalyze H2B ubiquitination at Lys120 (Kim et al., 2009; Koken et al., 1991; Roest et al., 1996; Wood et al., 2003a); two homologs of yBre1, RNF20 and RNF40, function as E3 ligases to mediate hRAD6A and hRAD6B for H2B ubiquitination (Kim et al., 2009; Kim et al., 2005; Zhu et al., 2005b). Interestingly, although both RNF20 and RNF40 contain a C-terminal RING domain, the RING domains of RNF20 and RNF40 do not interact with hRAD6A and hRAD6B. Instead, the coiled-coil motifs in RNF20 and RNF40 directly bind to their E2 partners (Kim et al., 2009).

Histone H2B is one of the most abundant ubiquitinated proteins in the cell nucleus. It is estimated that 1 – 5 % of H2B is monoubiquitinated (West and Bonner, 1980). Like other histone modifications, accumulated evidence suggests that ubH2B is involved in gene transcription (Henry et al., 2003; Kao et al., 2004; Sun and Allis, 2002; Weake and Workman, 2008; Xiao et al., 2005; Zhang, 2003). Genome wide analysis shows that ubH2B is associated with the transcribed region of highly expressed, but not lowly expressed genes (Minsky et al., 2008). Elevated ubH2B caused by RNF20 overexpression increases transcription of a set of HOX genes (Zhu et al., 2005b). Loss of ubH2B by depleting RNF20 suppresses the expression of p53 targeting genes, such as p21 (Kim et al., 2005; Minsky et al., 2008; Shema et al., 2008). Transcriptional regulation activity of ubH2B is dependent on the Pol II complex (Ng et al., 2003; Pirngruber et al., 2009; Xiao et al., 2005). Instead of modulating transcription initiation, ubH2B associates with the PAF and FACT complexes to regulate transcription elongation (Kim et al., 2009; Pavri et al., 2006). It has also been shown that ubH2B functionally interacts with Spt16, a histone chaperone and a subunit of the FACT complex, for proper chromatin setting during Pol II-dependent elongation (Fleming et al., 2008). Consistent with these observations, ubH2B is often enriched downstream of promoter region (Kim et al., 2009; Minsky et al., 2008).

Although the functional significance of ubH2B in transcription has been addressed, the molecular mechanism underlying transcription-coupled H2B ubiquitination is not fully understood. In this study, using protein affinity purification, we identified WAC (WW domain containing adaptor with coiled-coil) as a functional partner of RNF20/40. WAC regulates H2B ubiquitination through physical interaction with RNF20 and RNF40. During transcription, WAC targets RNF20/40 to associate with the Pol II complex to control H2B ubiquitination and transcription. Collectively, our data demonstrate that WAC is an important player in RNF20/40-dependent H2B ubiquitination and Pol II-dependent transcription.

Results

WAC is a binding partner of RNF20/40

RNF20/40 mediates H2B ubiquitination, which is important for gene transcription (Kim et al., 2009; Kim et al., 2005; Pavri et al., 2006; Zhu et al., 2005b). To explore the molecular mechanisms underlying this event, we have searched for functional partner(s) of RNF20 by affinity purification. The N-terminus of RNF20 was linked with SFB triple tags. Cell lysates of 293T cell stably expressing SFB-RNF20 were subjected to two rounds of affinity purification. As shown in Figure 1A, RNF20 associated with RNF40. Interestingly, besides RNF40, RNF20 also interacted with another protein migrating around 80 kDa. Mass spectrometry analysis revealed that this protein was WAC (Figure 1A). To validate our initial purification results, we examined RNF40 and WAC-associated protein(s) using a similar purification approach. Again, the predominant binding partner of RNF20/40 was WAC (Figure 1A). To further confirm the interactions between WAC and RNF20/40, we generated two anti-WAC antibodies using N-terminus and C-terminus of WAC as antigens, respectively. Both antibodies specifically recognized a band around 80 kDa. Moreover, siWAC treatment diminished the expression of this protein, indicating that both antibodies recognize endogenous WAC (Figure S1A).RNF20 and RNF40 co-immunoprecipitated (co-IPed) with WAC from 293T cell lysates, suggesting that indeed WAC is a binding partner of RNF20/40 (Figure 1B).

Figure 1. WAC associates with RNF20/40.

Figure 1

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(A) Silver staining of affinity-purified RNF20/40 complex. Cell lysates of 293T cell stably expressing SFB-RNF20, SFB-WAC or SFB-RNF40 were subjected to affinity purification. Eluted protein were visualized by silver staining. Arrows indicate proteins corresponding to RNF20, RNF40, and WAC. Peptide coverage was shown in the table. (B) WAC interacts with RNF20/40. 293T cell lysates were analyzed by IP and western blotting with the antibodies indicated. (C–D) 293T cell extracts (C) and purified RNF20/40/WAC complex from 293T cells stably expressing SFB-RNF20 (D) were analyzed by size-exclusion chromatography on a Superose 6 gel filtration column. Proteins eluted from the indicated fractions were blotted with the indicated antibodies. (E) WAC directly binds RNF20/40. SF9 cells were co-infected with baculoviruses encoding SFB-RNF40 and SBP-RNF20 together with GST-WAC or GST-hPAF1. Protein complex was purified by glutathione agarose beads. The purified proteins were visualized by Coomassie blue staining and blotted with antibodies against RNF20 and RNF40.

To investigate whether RNF20/40 and WAC form a stable complex in vivo, we analyzed these proteins in 293T cell nuclear extracts using size-exclusion chromatography. As shown in Figure 1C, WAC and RNF20/40 existed in similar cell lysate fractions corresponding to a molecular mass of approximately 600 kDa. Next, we purified RNF20 from 293T cells stably expressing SFB-RNF20 and examined RNF20 complex by size-exclusion chromatography. Again, WAC co-eluted with RNF20 and RNF40, suggesting that RNF20, RNF40 and WAC formed a stable complex with a molecular weight around 600 kDa (Figure 1D). Moreover, SF9 insect cells were co-infected with baculoviruses encoding SBP-RNF20, SFB-RNF40 and GST-WAC. When GST-WAC was purified from SF9 cell lysates by glutathione agarose beads, approximately equal amount of RNF20 and RNF40, which were recognized by anti-RNF20 and anti-RNF40 antibodies in western blotting to confirm the specificity, were associated with recombinant WAC, indicating that the complex contains stoichiometric amounts of RNF20, RNF40 and WAC (Figure 1E). To provide more direct evidence for the formation of the RNF20/40/WAC complex, we analyzed this recombinant protein complex by size-exclusion chromatography. To avoid GST dimer formation, baculoviruses encoding untagged WAC were generated and used for this analysis. The recombinant RNF20 and RNF40 were purified by streptavidin beads from extracts of SF9 cells coinfected with baculoviruses expressing WAC, SFB-RNF40 and SBP-RNF20. As shown in Figure S1B, WAC was associated with RNF20 and RNF40 in SF9 cell lysates. The molecular weight of this complex was around 600 kDa (Figure S1B). Considering the molecular weight of each subunit in this complex, we estimate that RNF20, RNF40 and WAC form a heterohexamer with 2:2:2 stoichiometry. Taken together, these results demonstrate that RNF20, RNF40 and WAC are constitutively associated and form a stable complex both in vitro and in vivo.

In addition to WAC, previous reports indicate that hPAF1 also interacts with RNF20/40 (Kim and Roeder, 2009). To examine whether PAF1 is in the same complex with RNF20, RNF40 and WAC, we analyzed hPAF1 by size-exclusion chromatography. hPAF1 existed in two different fractions (Figure 1C). The molecular mass of the first fraction is about 2 MDa, indicating that hPAF1 is in a huge complex, which might stably associate with other transcription machinery. However, RNF20, RNF40 and WAC are not detectable in this fraction (Figure 1C). The molecular mass of the second fraction is 600 – 700 kDa. Although hPAF1 co-migrated with RNF20, RNF40 and WAC in this fraction, the molecular weight of the hPAF complex in which hPAF1 stably associates with other subunits is also about 600 – 700 kDa (Kim et al., 2010; Zhu et al., 2005a). To examine whether hPAF1 stably associates with RNF20/40, we examined the RNF20 complex by size-exclusion chromatography. However, we could only detect WAC but not hPAF1 by analyzing the RNF20 complex (Figure 1D). This is in agreement with our protein affinity purification results, in which we did not find any hPAF complex peptide from RNF20, RNF40 or WAC purifications (Figure 1A). To examine whether hPAF1 could directly interact with RNF20 and RNF40, we generated baculoviruses encoding GST-hPAF1. SF9 insect cells were co-infected with baculoviruses expressing SFB-RNF20, SBP-RNF40 and GST-hPAF1. When we purified GST-hPAF1 from cell lysates, in contrast to purified GST-WAC, neither RNF20 nor RNF40 was detected (Figure 1E). To confirm this result, we performed in vitro GST pull down assay by incubating GST-hPAF1 or GST-WAC with recombinant RNF20/40. Only WAC, but not hPAF1, could interact with RNF20/40 (Figure S1C). Taken together, these results suggest that hPAF1 does not directly interact with RNF20/40 or WAC.

The coiled-coil domain of WAC associates with the coiled-coil domain of RNF20 and RNF40

Analysis of the primary sequence of WAC showed that WAC contains a WW domain at the N-terminus and a coiled-coil region at the C-terminus (Xu and Arnaout, 2002). Both domains are evolutionarily conserved from Drosophila to Homo sapiens, indicating that the WW domain and the coiled-coil region of WAC may be important for its function (Figure S2A).

To map the interaction regions in RNF20/40 and WAC, we generated a series of internal deletion mutants of WAC, RNF20 and RNF40. As shown in Figure 2A, the WD7 mutant of WAC, but not wild type WAC or other WAC mutants, abolished the interactions between WAC and RNF20/40. Since this mutant lacks the C-terminal coiled-coil region, it is likely that the coiled-coil motif of WAC recognizes RNF20/40. On the other hand, the D4 mutant of RNF20 and D7 mutant of RNF40 disrupted the interactions with WAC (Figure 2B and 2C). Interestingly, both D4 mutant of RNF20 and D7 mutant of RNF40 lack a highly conserved coiled-coil motif (Figure S2B). This coiled-coil motif also exists in Drosophila Bre1, an ortholog of RNF20/40, suggesting that the coiled-coil motifs in WAC and RNF20/40 may interact with each other to form a stable oligomeric complex. Of note, this coiled-coil motif in RNF20/40 is also involved in the interaction with hRAD6A/B (Kim et al., 2009).

Figure 2. Mapping the interaction regions of WAC and RNF20/40.

Figure 2

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(A) SFB-tagged wild type WAC and its deletion mutants were expressed in 293T cells. Cell lysates were IPed and blotted with the indicated antibody. The expression level of exogenous WAC was blotted with anti-FLAG antibody from whole cell lysates (WCL). (B–C) A coiled-coil region of RNF20 (B) or RNF40 (C) interacts with WAC. HA-tagged wild type RNF20 or RNF40 and their internal deletion mutants were expressed in 293T cells. The interaction between RNF20/40 and WAC were IPed and blotted with the indicated antibodies.

WAC regulates H2B ubiquitination

It has been reported that RNF20/40 regulates H2B ubiquitination through the E2 conjugase hRAD6 (Kim et al., 2009). Like RNF20/40, WAC co-IPed with hRAD6 (Figure 3A). Similar to the interaction with RNF20/40, WD7 mutant of WAC failed to interact with hRAD6, indicating that the coiled-coil motif of WAC is also important for the association with hRAD6 (Figure 3A). Usually, the interactions between E2 conjugases and E3 ligases are transient in vivo. Thus, we only detected a small amount of hRAD6 associated with WAC or RNF20/40. In addition, most hRAD6 exists as the monomeric form in the cell lysates (Figure 1C), which might explain the reason why we have difficulty in detecting hRAD6 in our protein affinity purification analyses (Figure 1A). To examine the role of WAC in histone H2B ubiquitination, we used siRNA to deplete WAC in HCT116 cells. Interestingly, depletion of WAC significantly reduced the level of RNF20, suggesting that WAC stabilizes RNF20/40 (Figure 3B left panel, Figure S3A). To investigate whether WAC affects the interaction between RNF20/40 and hRAD6, we normalized the level of RNF20 in the presence or absence of WAC. In the RNF20 and hRAD6 co-IP assay, depletion of WAC moderately affected the interaction between RNF20 and RAD6 (Figure 3B right panel, Figure S3A). Moreover, depletion of RNF20 apparently abrogated the association between WAC and hRAD6; depletion of hRAD6 also slightly affected the interaction between RNF20 and WAC (Figure 3C, and D, Figure S3B, and C). Thus, these results indicate that although WAC is not essential for the interaction between RNF20/40 and hRAD6, it is core subunit of RNF20/40 complex and facilitates the association between RNF20/40 and hRAD6 in vivo.

Figure 3. WAC regulates H2B ubiquitination.

Figure 3

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(A) Upper panel: 293T cell lysates were analyzed by IP and western blotting with the indicated antibodies. Lower panel: 293T cell expressing wild type or mutant WAC were analyzed by IP and western blotting with the antibodies indicated. The expression level of WAC and its mutants were examined by anti-FLAG antibody. (B–D) HCT116 cells were treated with indicated siRNA. Cell lysates were IPed and blotted with the indicated antibody. The level of WAC, RNF20 and hRAD6 was blotted from whole cell lysates. (B Right panel): The input level of RNF20 was normalized, the interaction between RNF20 and hRAD6 was examined. (E) HCT116 cells were treated with the indicated siRNA. Histone marks were examined with the indicated antibodies. Blots with anti-actin and anti-H3 were used as protein loading controls. (F) Coomassie blue staining of purified protein. (G) WAC facilitates RNF20/40 and hRAD6-mediated H2B ubiquitination in vitro. In vitro chromatin ubiquitination assays contained nucleosomal histones, hE1, hRAD6A, RNF20/40, HA-tagged ubiquitin, and GST-WAC (0, 50, 0, 10, 25, 50 ng from lane 1 to 6). Nucleosomal histones were examined by western blotting with anti-ubH2B antibody (upper panel) and then anti-H2B antibody (lower panel).

Previous studies in yeast showed that the yPAF complex and yBre1 mediate the loading of yRad6 to the transcribed genes and the elongating form of Pol II (Wood et al., 2003b; Xiao et al., 2005). To investigate whether the hPAF complex is directly involved in the interaction among the ubH2B machinery, we examined the association of WAC with RNF20/40 and with hRAD6 in the presence or absence of hPAF1. As shown in Figure S3D, although depletion of hPAF1 remarkably reduced the level of ubH2B, it altered neither the interaction between WAC and RNF20 nor that between WAC and hRAD6. These results suggest that the hPAF complex affects ubH2B most likely through regulating the transcription machinery but not directly through the interaction with ubH2B machinery.

It has been shown that knockdown of RNF20/40 or hRAD6 abolishes H2B ubiquitination (Kim et al., 2009). Similar to knockdown of RNF20 or RNF40, depletion of WAC led to significant down-regulation of H2B ubiquitination (Figure 3E). In addition, RNF20/40-dependent H2B ubiquitination modulates the methylation status of H3K4 and K79 through the COMPASS complex and Dot1 (Lee et al., 2007; McGinty et al., 2008; Ng et al., 2002; Sun and Allis, 2002; Wood et al., 2005). Similarly, loss of WAC resulted in down-regulation of H3K4 trimethylation and H3K79 trimethylation (Figure 3E), suggesting that WAC is a core subunit in the RNF20/40 complex.

Since RNF20/40 is the E3 ligase for ubH2B, and the data presented above indicate that RNF20/40 and WAC form a stable complex, we carried out an in vitro chromatin ubiquitination assay to examine whether WAC is important for the E3 ligase activity of RNF20/40 and H2B ubiquitination. Without RNF20/40, WAC alone could not catalyze H2B ubiquitination (Figure 3G, lane2). Also, in the absence of WAC, RNF20/40 had very limited activity to promote H2B ubiquitination in vitro (Figure 3G, lane 3). However, in the presence of WAC, nucleosomal H2B was significantly ubiquitinated by RNF20/40 (Figure 3G, lane4–6), suggesting that WAC facilitates RNF20/40 and hRAD6-mediated H2B ubiquitination in vitro. Taken together, these results suggested that WAC is an important subunit in the RNF20/40 E3 ligase complex for H2B-Lys120 monoubiquitination.

WAC is recruited to the p21 gene locus upon transcription activation

Accumulated evidence suggests that H2B ubiquitination is often enriched at active gene transcription sites and regulates transcription elongation (Minsky et al., 2008; Xiao et al., 2005). To examine whether the physical interactions among WAC, RNF20/40 and hRAD6 described above represent a functional interaction in vivo, we investigated if WAC, like RNF20/40, was also recruited to a transcriptionally active gene by using chromatin immunoprecipitation (ChIP) assay in an established system (Kim and Roeder, 2009; Minsky et al., 2008). Doxorubicin is known to generate DNA double strand breaks, and thus activates p53-induced p21 transcription. Seven regions in the p21 locus were probed with the corresponding primers (Figure 4A). Without doxorubicin treatment, WAC was broadly distributed at the p21 locus with relatively high level at transcription starting site and the following regions (Figure 4A). Upon doxorubicin treatment, the level of WAC occupancy at the p21 gene was increased similar to what was reported on RNF20 (Kim et al., 2009). In agreement with previous observations (Kim et al., 2009; Minsky et al., 2008), ubH2B was enriched downstream of transcription starting site and was increased in response to doxorubicin treatment (Figure 4A). These results indicate that WAC is likely to function together with RNF20/40 and regulate transcription-coupled H2B ubiquitination. Interestingly, depletion of WAC significantly reduced RNF20 occupancy and H2B ubiquitination level at the p21 locus (Figure 4B, Figure S4A), suggesting that WAC mediates RNF20-dependent H2B ubiquitination during transcription. In contrast, depletion of RNF20 only slightly affected WAC at the p21 locus although ubH2B was obviously decreased (Figure 4B, Figure S4A), suggesting that RNF20 is dispensable for the recruitment of WAC to the p21 locus. Similar to the role of RNF20/40 and hRAD6 in transcription, loss of WAC also disrupted p53-dependent p21 mRNA transcription. It is known that in response to genotoxic stress, p53 induces not only p21 transcription but also other genes transcription, such as Gadd45 and MDM2. Like p21 mRNA, the mRNA levels of Gadd45 and MDM2 were down-regulated in the absence of WAC (Figure 4C). To examine whether WAC plays a role in general transcription regulated by ubH2B other than genotoxic stress-induced p53-dependent gene transcription, we also analyzed TNFα-induced IκBα transcription (Figure S4B, C). Following TNFα treatment, both WAC and ubH2B were enriched at IκBα gene coding region, which coincided with the up-regulation of IκBα transcription. Depletion of WAC in the cell not only significantly decreased ubH2B at IκBα gene coding region but also attenuated TNFα-induced IκBα transcription. Taken together, these results suggest that WAC is an important regulator for gene transcription.

Figure 4. WAC is recruited to the p21 gene locus upon transcription activation.

Figure 4

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(A) DNA damage induces WAC to the p21 gene locus. HCT116 cells were treated with or without 0.5 µM doxorubicin for 12 hours. ChIP analyses on the p21 locus were performed using the indicated antibodies. An irrelevant IgG was used for negative control shown as the dotted lines. Primer pairs of p21 used for quantitative PCR are indicated in schematic diagram. (B) WAC regulates RNF20 and ubH2B enrichment at the p21 locus during p53-dependent transcription. HCT116 cells were transfected with the indicated siRNA followed by doxorubicin treatment. ChIP analyses on the p21 locus were performed using the indicated antibodies. Error bars: SD of triplicate experiments. (C) Total RNA was extracted from HCT116 cells transfected with the indicated siRNA followed by doxorubicin treatment. Quantitative PCR analyses of p53-dependent transcription are shown. Error bars: SD of triplicate experiments.

We also examined the role of the hPAF complex in ubH2B enrichment at actively transcribed gene loci. In agreement with previous report (Kim et al., 2009), the hPAF complex is important for the recruitment of RNF20/40/WAC complex to actively transcribed gene loci and ubH2B enrichment (Figure S4D–F). Although the hPAF complex does not directly interact with the RNF20/40/WAC complex, it is possible that the hPAF complex regulates transcription machinery, such as the status of Pol II (Mueller et al., 2004), which indirectly controls the RNF20/40/WAC complex and ubH2B.

The WW domain of WAC associates with RNA polymerase II

The results from ChIP assay indicate that WAC mediates RNF20/40-dependent H2B ubiquitination at the p21 locus, which in turn regulates p21 transcription. To investigate the molecular mechanism, we searched for other possible WAC partners. Besides the C-terminal coiled-coil region, WAC has an evolutionarily conserved WW domain. Many nuclear WW domain containing proteins that participate in transcription directly interact with Pol II transcriptional machinery (Sudol et al., 2001). Therefore, to explore the possibility that the WW domain of WAC is also involved in mediating the interactions between WAC and Pol II, we performed GST-fusion protein pull down assays. Wild type WAC, but not the WW domain deletion mutant (ΔWW), could pull down Pol II (Figure 5A, Figure S5A), suggesting that the WW domain of WAC could interact with Pol II. Since most nuclear WW domains recognize phosphorylated Pol II C-terminal domain (CTD) (Sudol et al., 2001), we generated recombinant GST-CTD (Figure S5B) and incubated it with CDK9 and Cyclin T1. In vitro, the CDK9/Cyclin T1 complex could phosphorylate GST-CTD on both Ser2 and Ser5 in the tandem heptapeptide repeats (Figure 5B). Compared with non-phosphorylated Pol II CTD, phosphorylated Pol II CTD could efficiently pull down wild type WAC but not the ΔWW mutant (Figure 5B), suggesting that like other WW domain containing proteins, WAC recognizes phosphorylated Pol II CTD through the WW domain. Since both Ser2 and Ser5 in the tandem heptapeptide repeats of CTD are heavily phosphorylated during transcription, we next examined which phosphorylation sites in CTD are recognized by the WW domain of WAC. Using a peptide pull down assay, we observed that phospho-Ser2 peptide could pull down WAC (Figure 5C). Moreover, phospho-Ser2 peptide only associated with wild type WAC but not ΔWW mutant (Figure 5C). To investigate the direct interaction between WAC and phosphorylated Ser2 in Pol II CTD, GST-WAC was incubated with phospho- or non-phospho-peptides. Again, phospho-Ser2 peptide could interact with WAC in vitro (Figure 5C). Although WAC also weakly interacted with phospho-Ser5 peptide, we found that Ser5 phosphorylated Pol II was distributed at promoter region of the p21 locus; whereas Ser2 phosphorylated Pol II mainly localized downstream of the promoter region, which is very similar to that of WAC (Figure 5D). The observation that different phosphorylated forms of Pol II have different localization is also supported by previous reports from other groups, who proposed different functions of Pol II during gene transcription (Gomes et al., 2006; Komarnitsky et al., 2000). The interaction between WAC and Ser2-phosphorylated Pol II in vivo is further confirmed by co-IP and reciprocal co-IP assays (Figure 5E). Collectively, these results indicate that phospho-Ser2 in tandem heptapeptide repeats is the primary binding site to interact with the WW domain of WAC.

Figure 5. WAC associates with RNA polymerase II through the WW domain.

Figure 5

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(A) GST-WAC or GST- ΔWW were used to pull down endogenous Pol II from WCL of 293T cells. (B) GST-CTD was incubated with or without CDK9/Cyclin T1 (left panel). The non- phosphorylated GST-CTD or phosphorylated GST-CTD was immobilized onto glutathione beads and used to pull down cell lysates from 293T cell expressing SFB-WAC and ΔWW respectively (right panel). (C) Left panel: biotinylated non-phospho–CTD peptide, pSer5 or pSer2 CTD peptide was immobilized onto streptavidin beads and used to pull down endogenous WAC from WCL of 293T cells. Empty beads were used as a negative control. Middle panel: pSer2 CTD peptide was used to pull down cell lysates from 293T cell expressing FLAG-WAC or ΔWW mutant. Right panel: non-phospho–CTD peptide, pSer5 or pSer2 CTD peptide was immobilized onto streptavidin beads and incubated with recombinant GST-WAC, and then peptide pull down assay was performed. (D) Localization of phospho-Pol II at the p21 locus. HCT116 cells were treated with or without 0.5 µM doxorubicin for 12 hours. ChIP analyses on the p21 locus were performed using indicated antibodies. An irrelevant IgG was used for negative control presented as the dotted lines. Error bars: SD of triplicate experiments. (E) WAC associates with phosphorylated Pol II in vivo. 293T cell lysates were analyzed by IP and western blotting with indicated antibodies. (F) HCT116 cells were treated with the indicated siRNAs. Cell lysates were examined by IP and western.

To examine whether the interaction between WAC and Pol II bridges the association between Pol II and RNF20/40, we used siRNA to deplete WAC (Figure 5F, Figure S5). In the absence of WAC, RNF20 failed to associate with Pol II. In contrast, knockdown of RNF20 did not affect the interaction between WAC and Pol II (Figure 5F, Figure S5C). These results suggest that WAC is the key adaptor to mediate the interaction between RNF20/40/WAC complex and Pol II.

WAC is recruited by RNA polymerase II to gene transcription sites

The interaction between WAC and Ser2 phosphorylated Pol II indicates that WAC is a functional partner of Pol II during transcription. In the absence of WAC, Ser2 phosphorylated Pol II was still recruited to the p21 locus, suggesting that WAC is dispensable for Pol II localization (Figure 6A). In contrast, when cells were treated with Pol II inhibitor α-amanitin, which induces Ser2 phosphorylated Pol II degradation (Nguyen et al., 1996), the expression level of WAC and RNF20 was not altered, but ubH2B was reduced (Figure 6B). It has been reported that CDK9 controls Pol II Ser2 phosphorylation and directs H2B ubiquitination (Pirngruber et al., 2009). Two CDK9 inhibitors, 5,6-Dichloro-1-β-D-ribofuranosylbenzimidazol (DRB) and flavopiridol, could suppress Pol II Ser2 phosphorylation and down-regulate ubH2B (Figure S6C). These results indicated that Ser2 phosphorylated Pol II is required for global ubH2B. Furthermore, when Ser2 phosphorylated Pol II was suppressed following α-amanitin or DRB treatment, the recruitment of RNF20 and WAC was significantly attenuated (Figure 6C and Figure S6C). Correspondingly, transcription-coupled H2B ubiquitination was also impaired at the p21 locus (Figure 6C, Figure S6A and Figure S6C). To further confirm that Ser2 phosphorylated Pol II regulates WAC-mediated H2B ubiquitination, we stably expressed SFB-tagged ΔWW mutant of WAC in cells. The expression level of the ΔWW mutant is similar to that of endogenous WAC (Figure 6D). The ΔWW mutant is resistant to the WAC siRNA treatment because siRNA targeting region was absent. Moreover, this mutant could interact with and stabilize RNF20/40 when depleting endogenous WAC (Figure 6D). However, since Ser2 phosphorylated Pol II does not interact with the ΔWW mutant, the ΔWW mutant of WAC failed to recruit RNF20/40 to the p21 locus and ubiquitinate histone H2B (Figure 6E and Figure S6B). Taken together, these results suggest that Pol II is an upstream partner of WAC and recruits WAC to transcribed gene sites for H2B ubiquitination.

Figure 6. RNA polymerase II is important for WAC enrichment at the p21 locus.

Figure 6

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(A) WAC is dispensable for pSer2-Pol II occupancy at the p21 locus. HCT116 cells were transfected with control or WAC siRNA. ChIP analyses on the p21 locus were performed using anti-Ser2P-Pol II antibody. (B) Pol II regulates H2B ubiquitination. HCT116 cells were treated with or without 10 µg/ml α-amanitin for 12 hours. Total cells were subjected to western blotting with indicated antibodies. (C) Pol II is important for WAC occupancy at the p21 locus. Cells were treated the same as in (B). ChIP analyses on the p21 locus were performed using indicated antibodies. (D) HCT116 cells and HCT116 cells stably expressing SFB-ΔWW mutant were treated with control or WAC siRNA, respectively. Cell lysates were analyzed by western blotting with indicated antibodies to examine the protein levels of SFB-ΔWW mutant, endogenous WAC and RNF20. IP and western blotting were performed to investigate the interactions between RNF20 and WAC or between RNF20 and SFB-ΔWW mutant. (E) Cells were treated the same as in (D). ChIP analyses were performed by using indicated antibodies at the p21 locus. An irrelevant IgG was used as the negative control shown as the dotted lines (A, C and E). Error bars: SD of triplicate experiments.

WAC regulates cell cycle checkpoint activation in response to DNA damage

Genotoxic stress activates p53-dependent p21 transcription and arrests the cell cycle at G1/S transition This cell cycle checkpoint allows cells to have enough time to repair DNA lesions before restart of DNA replication, and thus avoid duplicating damaged DNA (Abbas and Dutta, 2009). Since WAC regulates p21 transcription through modulating ubH2B enrichment at the p21 locus, we wonder whether WAC controls G1/S checkpoint in response to DNA damage. As doxorubicin treatment constitutively induces DNA double strand breaks and cells may adapt to the hazardous environment and bypass the checkpoint, therefore we used ionizing radiation (IR) treatment to examine cell cycle checkpoint. As shown in Figure 7A, following IR treatment, transcription of p21 gene was induced in normal cells. However, knockdown of WAC and RNF20 significantly suppressed the transcription of p21, suggesting that the transcription of p21 was controlled by WAC and RNF20 (Figure 7A). Since p21 is a major CDK2 inhibitor during G1/S transition and intra-S phase progression, IR-induced p21 expression arrests cells at G1/S transition and intra-S phase. Thus, DNA replication in normal cells, measured by BrdU incorporation, was abrogated in response to DNA damage (Figure 7B). However, although IR-induced p21 expression was abrogated in WAC or RNF20-depleted cells, DNA replication still occurred regardless of DNA damage in WAC or RNF20-depleted cells, indicating that G1/S checkpoint is impaired in the absence of WAC or RNF20 (Figure 7B). Collectively, these results suggest that WAC regulates p53-dependent gene transcription, which controls G1/S phase checkpoint activation in response to genotoxic stress.

Figure 7. WAC is important for G1/S checkpoint activation in response to DNA damage.

Figure 7

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(A) HCT116 cells were transfected with the indicated siRNA followed by 15 Gy IR treatments. mRNA level of p21 was examined by qPCR 12 hours after IR. Error bars: SD of triplicate experiments. (B) As in (A) except that 12 hours after IR, cells were pulse labeled with 20 µM BrdU for 30 minutes and stained with FITC-conjutated anti-BrdU and PI. Incorporation of BrdU and total DNA content were determined by FACS. Cells at G1/S transition and early S phase with positive BrdU staining were calculated. (C) A model of WAC mediates transcription-coupled H2B ubiquitination. WAC recognizes RNA polymerase II C-terminal domain and recruits RNF20/40 and RAD6 to gene transcription sites for H2B ubiquitination.

Discussion

Histone H2B ubiquitination is one of the important histone modifications that regulate gene transcription. In our study, we have identified WAC as a key regulator that links Pol II and RNF20/40 during transcription. These results shed light on the mechanism of transcription-coupled H2B ubiquitination.

WAC has an N-terminal WW domain that recognizes Pol II CTD, and a C-terminal coiled-coil motif that interacts with RNF20/40 and hRAD6. Thus, WAC is likely to be the major linker between Pol II transcriptional machinery and RNF20/40. Besides WAC, it has been shown that the PAF complex also mediates H2B ubiquitination and RNF20/40 localization at gene transcription sites (Kim et al., 2009; Ng et al., 2003; Wood et al., 2003b; Xiao et al., 2005; Zhu et al., 2005b). However, previous studies did not show evidence that the hPAF complex directly interacts with RNF20/40. Particularly, Zhu et al. reported that direct interaction between the hPAF and the RNF20/40 complex was not observed (Zhu et al., 2005b). Consistent with these reports, we did not find that the hPAF complex strongly associated with RNF20/40. It is not clear yet whether WAC exists in yeast, therefore, it is possible that the yPAF complex may substitute partial function of WAC and bridge yBre1 with RNA Pol II machinery in yeast (Kim and Roeder, 2009). The PAF complex is an important functional partner of RNA Pol II during transcription elongation (Chaudhary et al., 2007; Kim et al., 2010). In human cells, it is likely that the hPAF complex indirectly regulates RNF20/40/WAC-mediated H2B ubiquitination through RNA Pol II during transcription elongation. Interestingly, the yPAF complex has been shown to regulate Ser2 phosphorylation of RNA Pol II at some loci (Mueller et al., 2004). It is possible that the hPAF complex regulates H2B ubiquitination at least in part by modulating RNA Pol II Ser2 phosphorylation. The detailed mechanism about the hPAF complex in H2B ubiquitination needs to be further elucidated.

In agreement with previous reports, ubH2B is mainly enriched downstream of transcription starting sites (Kim et al., 2009; Minsky et al., 2008), suggesting a role of ubH2B for transcription elongation instead of transcription initiation (Fleming et al., 2008; Kim et al., 2009; Pavri et al., 2006; Xiao et al., 2005). However, ubH2B and RNF20/40/WAC occupancy at transcribed gene is not 100 % overlapped (Kim et al., 2009). From previous ChIP analyses, Rad6 and RNF20/40 localize close to the transcription starting site (Kim et al., 2009; Wood et al., 2003b), whereas ubH2B is enriched at the downstream gene coding region (Kim et al., 2009; Minsky et al., 2008). It is possible that chromatin forms a highly ordered structure, such as a loop, and the RNF20/40/WAC complex ubiquitinates H2B at the adjacent region and facilitates on-going transcription elongation (Figure 7C). Alternatively, deubiquitination process could also contribute to this phenomenon. It has been shown that H2B ubiquitination is dynamically regulated during transcription. Both H2B ubiquitination and deubiquitination could be equally important for transcription activation (Daniel et al., 2004; Henry et al., 2003; Song and Ahn, 2009; Weake and Workman, 2008). In addition, the occupancy of RNF20/40/WAC and phospho-Ser2 form of Pol II is not 100 % overlapped, either (Figure 4A or Figure 5D). Since the WW domain of WAC also has weak affinity for phospho-Ser5 of Pol II (Figure 5C), it is possible that Phospho-Ser5 of Pol II might play a limited role in recruiting RNF20/40/WAC to transcribed genes.

WAC is an evolutionarily conserved protein, especially in the WW domain and the coiled-coil region, which are functionally important. The WW domain of WAC is very similar to group IV WW domains that recognize Pol II CTD (Peng et al., 2007). The coiled-coil motif of WAC interacts with the coiled-coil motif of RNF20/40, which could be the molecular basis for an oligomer formation that provides docking sites for RAD6A/B. From gene homologous analysis, the interaction between WAC and RNF20 (Bre1) might exist from Drosophila to Homo sapiens. However, the ortholog of WAC in yeast is still not clear. It has been shown that Lge1 in S. cerevisiae and Shf1 in S. pombe associate with Bre1/Rad6 complex (Song and Ahn, 2009; Zofall and Grewal, 2007). Although they both lack the WW domain, Lge1 and Shf1 have a C-terminal coiled-coil region that may mimic the function of WAC C-terminus to facilitate the interaction between yBre1 and RAD6. And other partners of yBre1, such as the yPAF complex, may facilitate the interaction between the Bre1 complex and the Pol II complex (Kim and Roeder, 2009). More detailed analysis of the yBre1 complex in future will elucidate the molecular mechanism by which H2B is ubiquitinated during transcription elongation in yeast.

In response to genotoxic stress, p53 induces p21 transcription to arrest cell cycle at G1/S boundary and intra-S phase (Abbas and Dutta, 2009; Brugarolas et al., 1995). This DNA damage-induced checkpoint facilitates DNA damage repair and prevents the duplication of DNA lesions. Since WAC is required for H2B ubiquitination that regulates p53-dependent p21 transcription, WAC plays an important role in cell cycle checkpoint activation following DNA damage. In summary, we have demonstrated the functional significance and molecular mechanism of the RNF20/40/WAC complex at least in DNA damage response. Namely, this complex regulates p53 targeting genes transcription through modulating H2B ubiquitination under genotoxic stress.

Experimental procedures

Plasmids, antibodies, RNAi, RT-qPCR

Full details of plasmids, antibodies, RNAi and RT-qPCR are listed in the Supplemental information.

Protein purification and size-exclusion chromatography

Purification of SFB triple tagged protein (S, FLAG and SBP tags) was described previously (Zhang et al., 2009). To search for binding partners of RNF20, RNF40 and WAC, 1 liter culture of 293T cells stably expressing SFB-RNF20, SFB-RNF40 and SFB-WAC were harvested and washed with PBS. Cells were lysed with 30 ml ice-cold NETN buffer (0.5 % NP-40, 50 mM Tris-HCl pH 8.0, 2 mM EDTA and 150 mM NaCl). The soluble fraction was incubated with 0.5 ml streptavidin-conjugated agarose beads. The beads were washed with NETN buffer for three times. Associated proteins were eluted with 2 mM biotin in PBS and further incubated with 50 µl S beads (Novagen). The bound proteins were eluted with SDS sample, and analyzed with 10% SDS-PAGE and mass spectrometry. Cells expressing empty vector were used as purification controls. Baculoviruses and recombinant protein purification from insect cells and size-exclusion chromatography are listed in the Supplemental information.

In Vitro Chromatin assembly and ubiquitylation assay

In Vitro Chromatin assembly was performed according to manufacturer’s instructions (Active Motif). Chromatin generated from 0.2 µg input DNA was mixed with 50 ng hE1, 100 ng hRAD6A, 300 ng RNF20/40, 1 µg HA-ubiquitin, and the indicated amount of WAC in 50 mM Tris-Cl (pH 7.9), 5 mM MgCl2, 2 mM NaF, 0.4 mM DTT, and 4 mM ATP in a final volume of 20 µl, and then incubated for 1 hr at 37°C. The resulting products were resolved by SDS-PAGE and subjected to western blotting.

Chromatin immunoprecipitation (ChIP) Assay

ChIP assay was performed according to the protocol described by Upstate. Primers for ChIP quantitative PCR are summarized in Table S1A. The mean value was calculated by three independent experiments.

Peptide pull down assays

Peptide binding assays were conducted as described with minor modifications (Licatalosi et al., 2002). Biotinylated CTD peptides (YSPTSPS)4, (YpSPTSPS)4, (YSPTpSPS)4 were purchased from Bio Basic. Biotinylated peptides (0.15 nmol of each) were immobilized on 50 µl streptavidin beads (Pierce). Following washed with buffer D (20 mM Hepes, pH 7.9, 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 100 mM NaCl, 0.1% Nonidet P-40, and 1 mM β-glycerophosphate) for three times, 1 µg purified GST –WAC in buffer D was incubated with CTD peptide-bound beads for 1 hour at 4°C. After that, the beads were washed four times, and bound protein were separated by SDS-PAGE and analyzed by western blotting.

Full details of all other experimental procedures are listed in Supplemental information.

Supplementary Material

01

Acknowledgement

We thank Drs. Eric Fearon and Yang Liu for sharing equipments. We thank Drs. Jay Hess and Yali Dou for reagents. This work was supported by American Cancer Society (RSG-08-125-01-CCG to X.Y.), National Institute of Health (CA132755 and CA130899 to X.Y.), the University of Michigan Cancer Center and GI Peptide Research Center. X.Y. is a recipient of the Era of Hope Scholar Award from the Department of Defense.

Footnotes

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