Monoubiquitination of Human Histone H2B: The Factors Involved and Their Roles in HOX Gene Regulation

Molecular Cell, Vol. 20, 601–611, November 23, 2005, Copyright ª2005 by Elsevier Inc.

DOI 10.1016/j.molcel.2005.09.025

Monoubiquitination of Human Histone H2B: The Factors Involved and Their Roles in HOX Gene Regulation Bing Zhu,1 Yong Zheng,1 Anh-Dung Pham,1 Subhrangsu S. Mandal,1 Hediye Erdjument-Bromage,2 Paul Tempst,2 and Danny Reinberg1,* 1 Howard Hughes Medical Institute Division of Nucleic Acids Enzymology Department of Biochemistry University of Medicine and Dentistry of New Jersey Robert Wood Johnson Medical School 683 Hoes Lane Piscataway, New Jersey 08854 2 Molecular Biology Program Memorial Sloan-Kettering Cancer Center 1275 York Avenue New York, New York 10021

Summary In yeast, histone H2B monoubiquitination is a cotranscriptional event regulating histone H3 methylation at lysines 4 and 79. However, mammalian H2B monoubiquitination remains poorly understood. We report that in humans, the 600 kDa RNF20/40 complex is the E3 ligase and UbcH6 is the ubiquitin E2-conjugating enzyme for H2B-Lys120 monoubiquitination. RNF20 and RNF40 are both homologs of Bre1, the E3 ligase in the yeast case. UbcH6 physically interacts with RNF20/40 and with the hPAF complex. Formation of a trimeric complex with hPAF stimulates H2B monoubiquitination activity in vitro. Accordingly, UbcH6, RNF20/40, and the hPAF complex are recruited to transcriptionally active genes in vivo. RNF20 overexpression leads to elevated H2B monoubiquitination, subsequently higher levels of methylation at H3 lysines 4 and 79, and stimulation of HOX gene expression. In contrast, RNAi against the RNF20/40 complex or hPAF complex reduces H2B monoubiquitination, lowers methylation levels at H3 lysines 4 and 79, and represses HOX gene expression. Introduction The eukaryotic genome is packaged into chromatin, the natural substrate for transcription. Many dynamic and broad-range changes in chromatin are involved during RNA Polymerase II-mediated transcription (for reviews, see Narlikar et al. [2002], Orphanides and Reinberg [2002], and Hampsey and Reinberg [2003]). Both ATPdependent chromatin remodeling and posttranslational histone modifications are key to regulating these transcription-related chromatin changes (for reviews, see Jenuwein and Allis [2001], Zhang and Reinberg [2001], Fischle et al. [2003], Kurdistani and Grunstein [2003], Sims et al. [2004], and Margueron et al. [2005]). The relatively well-studied mark of histone acetylation is known to be associated with transcription activation, and there are others as well. In recent years, several his*Correspondence: [email protected]

tone marks have been linked to active transcription: histone H2B-K123 (K120 in vertebrates) monoubiquitination and H3-K4, H3-K36, and H3-K79 methylation (for reviews, see Hampsey and Reinberg [2003], Gerber and Shilatifard [2003], Osley [2004], and Margueron et al. [2005]). In yeast, histone H2B-K123 monoubiquitination is mediated by the E2-conjugating enzyme Rad6 and the E3 ligase Bre1 (Robzyk et al., 2000; Hwang et al., 2003; Wood et al., 2003a). Additionally, the PAF complex is also required in vivo (Ng et al., 2003; Wood et al., 2003b). Furthermore, histone H2B-K123 monoubiquitination is a prerequisite for histone H3-K4 and H3-K79 methylation (Sun and Allis, 2002; Wood et al., 2003a). Interestingly, deubiquitination of H2B-K123 is required for histone H3-K36 methylation (Henry et al., 2003). In vertebrates, histone H2B is monoubiquitinated at lysine 120 (Thorne et al., 1987), the equivalent of yeast histone H2B lysine 123. Recently, the p53 binding protein Mdm2 (Momand et al., 1992) was reported to function as the E3 ligase for histone H2B monoubiquitination in mammals (Minsky and Oren, 2004). However, the study did not show conclusively that Mdm2 targets histone H2B-K120. Moreover, histone H2A is apparently a better substrate for Mdm2 (Minsky and Oren, 2004). Therefore, the enzymes responsible for histone H2BK120 monoubiquitination and the functional significance for this modification in mammals are not clear. Here, we report the purification of the human RNF20/ 40 complex that functions as the E3 ligase and UbcH6 as the ubiquitin E2-conjugating enzyme for histone H2BK120 monoubiquitination. UbcH6 specifically interacts with the RNF20/40 complex and with the hPAF complex. The trimeric association of UbcH6 and the hPAF and RNF20/40 complexes is required for efficient histone H2B-K120 monoubiquitination activity in vitro. Moreover, Hox genes are the specific targets regulated by the level of histone H2B-K120 monoubiquitination. Results and Discussion Identification of a Human Histone H2B-K120 Monoubiquitination Activity We devised an assay to score for monoubiquitination activity specific to histone H2B-K120. The assay consisted of recombinant oligonucleosome substrates that were assembled with either wild-type (wt) histones or with histones H2A/B carrying a single substitution, H2B-K120A, or H2A-K119A. A monoubiquitination activity specific for histone H2B-K120 was detected in extracts (data not shown) and was partially purified by conventional chromatography as shown in Figure 1A. The activity is specific for histone H2B-K120 (Figure 1B) and was nucleosome specific (Figure 1C). Because native human histone H2B migrates slightly slower than recombinant histone H2B on SDS-polyacrylamide gels, the ubiquitinated native H2B polypeptide migrated slightly slower than that of the ubiquitinated recombinant protein (Figure 1C). On a Superose 6 gel filtration column, the H2B-K120 monoubiquitination activity eluted as an w600 kDa complex (Figure 1D).

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Figure 1. Partial Purification of a Histone H2B-K120 Monoubiquitination Activity from HeLa Extracts and the Purification of Human RNF20/40 Complex (A) The purification scheme for a partially purified histone H2B-K120 monoubiquitination activity. (B) The monoubiquitination activity is specific for histone H2B-K120. (C) The histone H2B-K120 monoubiquitination activity is nucleosome specific. (D) The histone H2B-K120 monoubiquitination activity is a w600 kDa complex. Fractions were from a 2 ml Smart Superose 6 gel filtration column. The fraction numbers are shown in the top of the panel. (E) Silver stain of the affinity-purified human RNF20/40 complex. Fractions were from a 2 ml Smart Superose 6 gel filtration column. The fraction numbers are shown at the top of the panel. The subunits of the human RNF20/40 complex are indicated at the right side.

Purification of the Human RNF20/40 Complex Further purification of the histone H2B-K120 monoubiquitination activity was unsuccessful, likely due to the separation of E2-conjugating enzyme and/or other required factors from the E3 ligase. However, we realized that this activity differs from that of Mdm2. Mdm2 catalyzes monoubiquitination of purified core histones (Minsky and Oren, 2004), but the activity we observed is specific for nucleosomal substrates. Additionally, the activity does not target histone H2A and other lysines on histone H2B as Mdm2 does (Minsky and Oren, 2004). Finally, Mdm2 protein was not detected in the partially purified activity by Western analysis (data not shown). We therefore sought to identify this activity that is distinct from MDM2 by using an alternative approach. In the human genome, there are two Bre1 homolog RING finger domain-containing proteins, RNF20 and RNF40. RNF20 shares 14.9% sequence identity and 28.4% similarity with the yeast Bre1, whereas RNF40 shares 14% sequence identity and 26% similarity (Figure S1 available in the Supplemental Data with this arti-

cle online). We established a stable cell line expressing FLAG-RNF20. Nuclear extracts derived from the stable cell line were subjected to affinity purification by using M2 anti-FLAG resin under stringent conditions (for details see the Experimental Procedures), followed by Superose 6 gel filtration. Interestingly, FLAG-RNF20 was present in a complex with another protein, and they coeluted with an apparent mass of w600 kDa (Figure 1E), similar to that found for the purified native histone H2B-K120 monoubiquitination activity (Figure 1D). However, the RNF20 containing fractions alone was devoid of activity (data not shown), likely due to the absence of E2-conjugating enzyme (see below). The proteins shown in Figure 1E were excised and subjected to MALDI-TOF mass spectrometry analyses. Interestingly, the polypeptide coeluting with FLAG-RNF20 was identified as RNF40. We designated this complex as the RNF20/40 complex. Given the molecular weight of these two proteins and the apparent mass of the complex on a gel filtration column, we suggest that the RNF20/40 polypeptides likely exist as a tetramer, with two copies of each polypeptide. The finding that two

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RING finger domain-containing proteins with close sequence homology form the RNF20/40 complex bears remarkable similarity to the recently published human histone H2A-K119 monoubiquitination E3 ligase complex, which contains two RING finger proteins with close homology, Ring1 and Ring2, as its subunits (Wang et al., 2004). The RNF20/40 Complex Together with UbcH6 Catalyzes Monoubiquitination of Histone H2B-K120 That the RNF20/40 complex composed of two Bre1 homologs has a similar apparent molecular mass as the purified native H2B-K120 monoubiquitination activity strongly suggested that it might function as the E3 ligase for H2B-K120 monoubiquitination. However, another key player essential for the activity was missing, the ubiquitin E2-conjugating enzyme that apparently separates from the E3 ligase during native and affinity purifications of the RNF20/40 complex. Therefore, we tested eight known E2 enzymes for their ability to function in monoubiquitination of nucleosomal substrates in the presence or absence of the RNF20/40 complex. Only UbcH6 was active in a RNF20/40-dependent manner (Figure 2A). Furthermore, the activity mediated by UbcH6 and the RNF20/40 complex was specific for histone H2B-K120 (Figure 2B) and also specific for nucleosomes (Figure 2C), similar to the case for the purified native activity (Figures 1B and 1C). Taken together, our data strongly suggested that the native activity we partially purified (Figure 1A) is composed of the RNF20/40 complex and UbcH6. It is generally known that ubiquitin E3 ligases interact with specific ubiquitin E2-conjugating enzymes to confer substrate specificity to the reaction. Therefore, we next tested whether the RNF20/40 complex interacts with UbcH6. Indeed, among three different E2-conjugating enzymes tested, only UbcH6 specifically interacted with the RNF20/40 complex in a nickel-agarose pulldown assay (Figure 2D). Importantly, in the reciprocal experiment, antibody against FLAG immunoprecipitated UbcH6 protein only in the presence of purified FLAG-RNF20/40 complex, whereas the control IgG did not (Figure 2E). The above data identified UbcH6 and the RNF20/40 complex as the E2 and E3 enzymes, respectively, in histone H2B-K120 monoubiquitination in vitro. Trimeric Association of UbcH6, hPAF, and RNF20/40 Complexes Is Essential for Efficient Monoubiquitination of Histone H2B-K120 In Vitro Recently, several groups, including ours, reported the purification of the human PAF complex (hPAF), which contains four subunits, Ctr9, Leo1, Paf1, and Cdc73, that are homologs of those contained in the yeast PAF complex (Rozenblatt-Rosen et al., 2005; Yart et al., 2005; Zhu et al., 2005) as well as a higher eukaryotic specific subunit, Ski8 (Zhu et al., 2005). The yeast PAF complex is genetically required for histone H2B-K123 monoubiquitination, a step requisite for subsequent methylation of histone H3 at lysines 4 and 79 (Krogan et al., 2003; Ng et al., 2003; Wood et al., 2003b). The hPAF complex is also required for histone H3 monoand trimethylation at lysine 4 and dimethylation at lysine 79 (Zhu et al., 2005). However, the functional role of the

Figure 2. UbcH6 Is the E2-Conjugating Enzyme, and RNF20/40 Complex Is the E3 Ligase for Histone H2B-K120 Monoubiquitination (A) UbcH6 and RNF20/40 specifically monoubiquitinate the nucleosome substrate. (B) The activity mediated by UbcH6 and RNF20/40 complex is specific for histone H2B-K120. (C) The activity mediated by UbcH6 and RNF20/40 complex is specific for nucleosome substrate. (D) His-tagged UbcH6 specifically pulls down the purified RNF20/ 40 complex in a nickel-agarose pull-down assay. (E) Antibody against FLAG immunoprecipitates UbcH6 in the presence of purified FLAG-RNF20/40 complex.

human (and yeast) PAF complex in histone H2B monoubiquitination is unknown. Because hPAF is a w600 kDa complex (Zhu et al., 2005), similar to that of the purified native histone H2B-K120 monoubiquitination activity (Figure 1D), we next tested if the hPAF complex participates in this activity. First, we performed Western analysis for all the hPAF subunits and detected their presence in the partially purified H2B-K120 monoubiquitination activity (Figure 3A). We then performed immunodepletion experiments by using antibody against the hPAF subunit Leo1 (Figure 3B). Interestingly, after hPAF depletion, the H2B-K120 monoubiquitination activity was reduced to 26% and the lost activity was rescued upon the addition of purified hPAF complex to the reaction (Figure 3C). Of note, the purified hPAF complex alone did not have histone H2B-K120 monoubiquitination activity (data not shown and Figure 3G). A nonspecific ubiquitination activity was also observed in this fraction (Figures 1B, 1C, and 3C), but importantly, this nonspecific activity was not affected by antibodies against hPaf1 or by the addition of the purified hPAF complex (Figure 3C). This result strongly suggested that the hPAF complex is directly involved in stimulating histone H2B monoubiquitination. The yeast PAF complex interacts

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Figure 3. Trimeric Association of the PAF Complex, UbcH6, and RNF20/40 Complex Stimulates Histone H2B-K120 Monoubiquitination In Vitro (A) All the subunits of the PAF complex are present in the native partially purified histone H2B-K120 monoubiquitination activity assayed in Figures 1B and 1C. (B) Westerns indicating the immunodepletion of the PAF complex from the native partially purified histone H2B-K120 monoubiquitination activity. (C) Depletion of PAF complex from the native partially purified histone H2B-K120 monoubiquitination activity reduces the activity that was rescued upon supplementation with purified PAF complex. (D) His-UbcH6 specifically pulls down the purified PAF complex in a nickel-agarose pulldown assay. (E) Antibody against Paf1 immunoprecipitates UbcH6 in the presence of purified PAF complex. (F) RNF20/40 complex and PAF complex interact only in the presence of UbcH6. (G) Trimeric association of PAF complex, UbcH6, and RNF20/40 complex stimulates histone H2B-K120 monoubiquitination activity.

with the E2 enzyme Rad6 (Xiao et al., 2005), so it was likely that the hPAF complex contributes to histone H2B-K120 monoubiquitination through its interaction with E2 or E3 enzymes. To examine if the hPAF complex and UbcH6 directly interact, nickel-agarose pull-down experiments were performed. UbcH6, but not the other two E2 enzymes tested, specifically pulled down the purified hPAF complex (Figure 3D). Antibodies against hPaf1, but not control IgG, immunoprecipitated UbcH6, dependent upon the presence of the hPAF complex (Figure 3E). On the other hand, direct interaction between the hPAF and RNF20/40 complexes was not observed (data not shown and Figure 3F). Yet in the presence of UbcH6, antibodies against hPaf1 immunoprecipitated the RNF20/40 complex together with the hPAF complex, indicating that a trimeric association of UbcH6, hPAF, and RNF20/40 complexes exists. Most importantly, the hPAF complex stimulated the ubiquitination activity detected in the presence of UbcH6 and the RNF20/40 complex (Figure 3G). Taken together, our data indicate that hPAF directly interacts with UbcH6 and forms a trimeric association with UbcH6 and RNF20/40, and this is essential for efficient H2B-K120 monoubiquitination in vitro.

UbcH6, RNF20/40, and hPAF Complexes Colocalize with RNA Polymerase II at Transcriptionally Active Genes In Vivo In yeast, Rad6 is associated with transcriptionally active genes (Wood et al., 2003a; Kao et al., 2004; Xiao et al., 2005); in both yeast and mammals, the PAF complex is associated with transcriptionally active genes (Pokholok et al., 2002; Zhu et al., 2005). To examine whether the physical interactions amongst UbcH6, hPAF, and RNF20/40 complexes described above represent a functional interaction in vivo, we investigated if UbcH6 and the RNF20/40 complex are present at transcriptionally active genes by using chromatin immunoprecipitation (ChIP) experiments. Because antibodies against RNF20 and RNF40 are not available, we took advantage of the stable cell line expressing FLAG-RNF20 as the material source. First, we examined a constitutively expressed gene, RPB1, that encodes the largest subunit of RNA Polymerase II. As expected, RNA Polymerase II (Rpb4) and the hPAF complex (hPaf1) localized at both promoter and coding regions of the RPB1 gene, whereas TFIIB localized only at the promoter region (Figure 4A). UbcH6 and the RNF20/40 complex localized to both the promoter and coding regions. The signals appeared

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Figure 4. Human RNF20/40 Complex, UbcH6, and the PAF Complex Are Present at Transcriptionally Active Genes (A) ChIP experiments showing RNF20/40 complex, UbcH6, and PAF complex are present at the constitutively expressed RPB1 gene. (B) ChIP experiment showing the recruitment of RNF20/40 complex, UbcH6, and PAF complex together with the RNA Polymerase II transcription machinery to the MAGE-A1 gene upon its induction.

weaker at the polyadenylation site and were undetectable at the downstream nontranscribed region (Figure 4A). To further analyze the presence of UbcH6 and the RNF20/40 complex at transcriptionally active genes, we chose an inducible gene, MAGE-A1. MAGE genes (melanoma antigen genes) are heavily methylated, heterochromatic, and transcriptionally inactive in most human tissues and cell lines (De Smet et al., 1996). The DNA demethylating reagent 5-aza-deoxycytidine (5aza-dC) induces the expression of MAGE genes in various cells (De Smet et al., 1996). In HEK293 cells used in this study, the MAGE-A1 gene is also methylated and silent. However, expression of the MAGE-A1 gene is restored after 48 hr of treatment with 5-aza-dC along with the recruitment of the transcription machinery (Zhu et al., 2005). ChIP experiments were performed with or without 5-aza-dC treatment by using antibodies against MeCP2, TFIIB, RNA Polymerase II (Rpb4), the hPAF complex (hPaf1), UbcH6, and the RNF20/40 complex (FLAG).

In untreated cells, although MeCP2 was detected, components of the transcription machinery were not present at the inert MAGE-A1 gene (Figure 4B). In contrast, after 5-aza-dC treatment, the presence of MeCP2 at the MAGE-A1 gene was drastically reduced, whereas components of the transcription machinery were now detectable. Similar to the results obtained with the constitutive RPB1 gene, UbcH6 and the RNF20/40 complex were detected at both promoter and coding regions, but not the downstream nontranscribed region (Figure 4B). The Levels of Histone H2B Monoubiquitination and Resultant Levels of Histone H3-K4 Mono- and Trimethylation and H3-K79 Dimethylation Are Dependent on RNF20/40 and PAF Complexes In Vivo To further understand the functional importance of the RNF20/40 complex in vivo, we investigated the levels of various histone modifications in cells overexpressing RNF20. Total histones were affinity purified from wt control cells and cells overexpressing RNF20. Figure 5A is

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Figure 5. Overexpression of RNF20 Results in Elevated Histone H2B Monoubiquitination In Vivo (A) Coomassie blue staining of hydroxyapatite-purified core histones. (B) Histone H2B monoubiquitination levels are elevated upon overexpression of RNF20. (C) Overexpression of RNF20 also leads to elevated levels of histone H3-K4 mono- and trimethylation and H3-K79 dimethylation. (D) Quantification of (B); error bars represent the standard error of the mean. (E) Knockdown of RNF20 and RNF40. RNAi targeting RNF20 and/or RNF40 verified by RT-PCR. (F) The level of u-H2B, but not of u-H2A, is reduced upon RNAi against the RNF20/40 complex.

a Coomassie blue-stained SDS/PAGE gel showing the purity of the core histones. We then subjected the purified histone samples to Western analysis by using various antibodies. Figure 5B shows that in cells overexpressing RNF20, the levels of monoubiquitinated histone H2B were elevated, as scored by reactivity with antibodies against histone H2B and ubiquitin. The cellular levels of total histone H3 and different methylation marks on histone H3 were also examined. The levels of histone H3 monomethyl-K4, trimethyl-K4, and dimethyl-K79 are significantly higher in cells overexpressing RNF20, whereas other H3-methylation marks such as dimethyl-K4, trimethyl-K9, trimethyl-K27, dimethyl-K36, and trimethyl-K36 remained unchanged (Figures 5C and 5D). We next examined the repercussions to H2B monoubiquitination and to resultant H3 methylation marks when cells are made deficient in RNF20/40 complex by using RNAi (Figure 5E). Because the endogenous level of monoubiquitinated histone H2B was barely detectable

in our earlier assay (Figure 5B), we improved the sensitivity by purifying the histone H2A/H2B dimer from cells (Figure 5F, panel 1) and loading increased amounts for Western analysis. With antibody against histone H2B, we were able to detect the endogenous u-H2B and its decrease upon RNAi against the RNF20/40 complex (Figure 5F, panel 2). We detected both u-H2B and u-H2A by using antibody against ubiquitin (Figure 5F, panel 3). Only the level of the slower-migrating u-H2B decreases upon RNAi against the RNF20/40 complex (Figure 5F, panels 2, 3, and 4). Of note, we also detected decreased u-H2B levels upon RNAi against the PAF complex by using the same assay system (Figure S2). RNAi against the RNF20/40 complex consistently gave rise to lower levels of monomethyl-H3-K4, trimethyl-H3-K4, and dimethyl-H3-K79 (Figure S3A). Given the association of hPAF and RNF20/40 through UbcH6 and the role of hPAF in stimulating H2B monoubiquitination shown above, these data are in accordance with our previous published findings that RNAi against the

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Figure 6. Perturbing the Level of Histone H2B Monoubiquitination Results in Misregulation of the Hox Genes (A) Overexpression of RNF20 leads to elevated Hox gene expression. (B) Quantification for selected genes tested in (A). (C) RNAi targeting hSki8 or hCtr9 reduces Hox gene expression. (D) Quantification of selected genes tested in (C).

human PAF subunits reduced the cellular levels of monomethyl-K4, trimethyl-K4, and dimethyl-K79 (Zhu et al., 2005). Taken together, these data support that, like the case in yeast, the hPAF complex is required for histone H2B monoubiquitination, which is in turn essential for histone H3-K4 and H3-K79 methylation. However, we did notice an important difference while examining the status of the different methylation marks in the case of histone H3-K4 methylation. Monomethylation and trimethylation appeared to be downstream of the histone H2B monoubiquitination pathway, whereas dimethylation was not. This is likely due to the existence of multiple histone H3-K4 methyltransferases in humans (for a review, see Margueron et al. [2005]) and the distinct roles for the disparate H3-K4 methylation status. Trimethylated H3-K4 is well documented to be associated with active gene transcription (Santos-Rosa et al., 2002; Schneider et al., 2004); however, little is known about H3-K4 monomethylation. Notably, knockdown of WDR5 protein, a component of several histone H3-K4 methylation complexes (Wysocka et al., 2003; Yokoyama et al., 2004; Hughes et al., 2004), leads to reduced histone H3-K4 monoand trimethylation, but not dimethylation (Wysocka et al., 2005). The reports on WDR5 (Wysocka et al., 2005), our previously published data on the hPAF complex (Zhu et al., 2005), and our current results on the RNF20/40 complex all show the same effect on the methylation status of histone H3-K4. Thus, these data suggest that histone H3-K4 monomethylation may also be a mark associated with active transcription.

Levels of Histone H2B-K120 Monoubiquitination Specifically Regulate Hox Gene Expression Although histone H2B-K120 monoubiquitination is essential for subsequent H3-K4 and H3-K79 methylation and these modifications are marks for active gene transcription, none of the factors required for the histone H2B-K120 monoubiquitination pathway such as Rad6, Bre1, and PAF subunits are essential for yeast survival, suggesting that this pathway is dispensable for general transcription. However, deletion of the PAF complex (or its subunits) in yeast and plants did affect the expression of certain specific genes (Porter et al., 2002; Oh et al., 2004; He et al., 2004). Therefore, it was important to identify target genes for the histone H2B-K120 monoubiquitination pathway. Human Hox gene expression is regulated by MLL1 (Yu et al., 1995), a histone methyltransferase with specificity for histone H3-K4 (Milne et al., 2002; Dou et al., 2005). Interestingly, a recent report demonstrated that Hox gene regulation entails increased levels of histone H3-K79 methylation, mediated by Dot1 (Okada et al., 2005). Because both histone H3-K4 methylation and H3-K79 methylation are downstream events of histone H2BK120 monoubiquitination in mammals as shown above, we next examined Hox gene expression as a function of histone H2B monoubiquitination levels. To perturb the levels of histone H2B monoubiquitination and thus the levels of downstream methylation marks, we took two different approaches. We examined conditions in which RNF20 was overexpressed as shown above and conditions in which the hPAF subunits or RNF 20/40

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gion we surveyed, with the exception being the region corresponding to the promoter of the HoxA11 gene. We also tested two repressive marks. Histone H3-K9 trimethylation was not detected along the region, whereas histone H3-K27 trimethylation levels were reduced upon RNF20 overexpression. These ChIP data, along with the observations that RNF20 overexpression leads to elevated global histone H3-K4 mono- and trimethylation and histone H3-K79 dimethylation (Figures 5C and 5D) and elevated Hox gene expression (Figures 6A and 6B), substantiate histone H2B-K120 monoubiquitination as a step upstream of histone H3-K4 and H3K79 methylation and Hox gene expression.

Figure 7. Overexpression of RNF20 Leads to Elevated Levels of Histone H3-K4 Trimethylation and H3-K79 Dimethylation at the Hox Genes

were depleted by using RNAi (Zhu et al. [2005] and this study, respectively). Overexpression of RNF20 triggered robust overexpression of many Hox genes tested, whereas the expression of actin, GAPDH, UbcH6, and the hPAF subunits remained unchanged (Figures 6A and 6B). Interestingly, RNF40 expression was also upregulated, suggesting a potential coregulatory mechanism between the two subunits of the RNF20/40 complex (Figures 6A and 6B). In contrast, RNAi against the hPAF complex or RNF20/40 complex specifically downregulated the same set of Hox genes (Figures 6C and 6D and Figure S3B), demonstrating that the levels of histone H2B-K120 monoubiquitination regulate Hox gene expression. Overexpression of RNF20 Elevates Levels of Histone H3-K4 and H3-K79 Methylation at Hox Genes We next compared the levels of histone methylation marks at the Hox genes in wt cells versus cells overexpressing RNF20 by using ChIP experiments. The region spanning the HoxA11 and HoxA10 genes was chosen because both of these genes are upregulated robustly after RNF20 overexpression (Figures 6A and 6B). As shown in Figure 7, histone H3-K4 trimethylation was consistently higher in cells overexpressing RNF20 throughout the entire region surveyed, whereas histone H3-K4 dimethylation levels were unchanged relative to the wt case. Interestingly, little histone H3-K4 monomethylation was observed along the entire region, and the only signal we did detect was at the region in between HoxA11 and HoxA10 genes; the signal was higher in cells overexpressing RNF20 as well. Histone H3-K79 dimethylation levels were also elevated throughout the re-

Concluding Remarks In this report, we present the isolation, biochemical characterization, and functional analysis of a human factor that catalyzes monoubiquitination of nucleosomal H2B-K120. The factor is composed of RNF20/40 and the E2-conjugating enzyme UbcH6. Although these three polypeptides represent the core of the complex, optimal activity requires the association of the core complex with the hPAF complex, an interaction mediated through UbcH6. Most importantly, we demonstrated that the RNF20/40-UbcH6-hPAF complex is required for transcription of the Hox genes in the context of the following cascade in mammals: hPAF / histone H2B ubiquitination / histone H3-K4 and H3-K79 methylation / Hox gene expression. PAF / Histone H2B Monoubiquitination / Histone H3-K4 Methylation Cascade in Transcription The hPAF and RNF20/40 complexes localized to transcriptionally active genes that were chosen randomly (RPB1 and MAGE-A1), suggesting a global role for transcription. Yet in contrast with the Hox genes, the expression level of these genes remained unchanged upon perturbing factors that catalyze H2B-K120 monoubiquitination (Figure 6 and Zhu et al. [2005]). The Hox genes appear to be a set of specific target genes under the control of histone H2B-K120 monoubiquitination. Hox gene expression is specifically regulated by MLL1 (Yu et al., 1995), an enzyme capable of mediating histone H3-K4 di- and trimethylation (Milne et al., 2002, Dou et al., 2005). MLL1 and H3-K4 trimethylation exhibit a global presence at transcriptionally active genes (Guenther et al., 2005; Santos-Rosa et al., 2002; Schneider et al., 2004), yet MLL1 specifically regulates the Hox loci (Yu et al., 1995). This discrepancy may be explained by the finding that in contrast to the other transcriptionally active loci that are MLL1 independent, MLL1 and the histone H3-K4 trimethylation mark occupy an extensive area of the transcriptionally active region of the Hox genes (Guenther et al., 2005). Although the functional significance of this is presently unclear, it may pertain to the dependence of Hox gene regulation on H2BK120 monoubiquitination, an event upstream of histone H3-K4 trimethylation. In the case of the PAF complex, both human and yeast PAF also exhibit global promoter occupancy on transcriptionally active genes, and yet, PAF is not required in yeast for expression of most genes (Porter et al., 2002) and appears not to be required for global gene expression in mammals (Figure 6 and Zhu et al. [2005]). In fact, that hPAF is required for Hox

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gene expression is the first such case identified. This finding is in accordance with the participation of hPAF in H2B monoubiquitination, an event upstream of MLL1 activity. Two E3 Ligases in One Complex, Another Level of Complexity for the ‘‘Histone Code’’ Hypothesis? Two copies each of the E3 ligases RNF20 and RNF40 are present in the same complex catalyzing histone H2BK120 monoubiquitination. Remarkably, the complex that catalyzes histone H2A-K119 monoubiquitination also contains two E3 ligases, Ring1 and Ring2 (Wang et al., 2004). Why are E3 ligases for histones present as heteromultimers? As every nucleosome consists of two copies of the core histones, it is possible that each E3 dimer targets one H2B tail per nucleosome or that each E3 ligase targets one H2B tail in two adjacent nucleosomes. Yet, why is the complex composed of nonidentical E3 ligases, i.e., RNF20 and RNF40? One possibility is that the nonidentical regions are important for the interaction between RNF20 and RNF40. An intriguing possibility is that the regions of nonhomology may be functionally relevant to the specific H2B tail targeted for monoubiquitination. The activity of the two E3 ligases could well be regulated differently and potentially trigger different outcomes with regard to downstream methylation events (tri- versus dimethylation of H3-K4, for example). During transcription, one histone H2A/H2B dimer is removed and then placed back by the chromatin transcription elongation factor FACT, which associates with nucleosomes in a transcription-dependent manner (Kireeva et al., 2002; Belotserkovskaya et al., 2003; Schwabish and Struhl, 2004; Kaplan et al., 2003). This may be an event subject to asymmetric H2B targeting whereby the remaining or the newly placed H2A/H2B dimer may be monoubiquitinated. Although the above speculations remain to be elucidated, the presence of two copies of each of two nonidentical E3 ligases is intriguing and may add one more level of complexity to the ‘‘histone code’’ theory. Functional Significance of the PAF / Histone H2B Ubiquitination / Histone H3-K4 and H3-K79 Methylation / Hox Genes Cascade The identification of the Hox gene family as a set of target genes for the hPAF and RNF20/40 complexes has several important implications. One of the subunits of the human PAF complex, Cdc73/parafibromin (RozenblattRosen et al., 2005; Yart et al., 2005; Zhu et al., 2005), is encoded by HRPT2, a tumor suppressor gene found frequently mutated in hyperparathyroidism-jaw tumor syndrome (HPT-JT) and sporadic parathyroid tumors (Carpten et al., 2002; Howell et al., 2003; Shattuck et al., 2003). HPT-JT is an autosomal-dominant, multiple neoplasia syndrome characterized by parathyroid tumors (Jackson et al., 1990). In the original identification of mutations within the HRPT2 gene, only about half of the kindreds exhibited germ-line inactivating mutations (Carpten et al., 2002). It was thought that many of the remaining mutations might exist in the promoter and/or other regulatory sequences that had not been screened. Recent studies revealed the identity of the hPAF complex subunits that included cdc73 and suggested that some of the unde-

tected mutations may reside in genes encoding these other hPAF subunits (Rozenblatt-Rosen et al., 2005; Yart et al., 2005; Zhu et al., 2005). In our present study, we identified downstream factors of hPAF complex that in conjunction with the hPAF subunits may be fruitful additions to mutational screening for HPT-JT. The Hox genes are required for proper development, and as our studies demonstrate, the hPAF complex is required for Hox gene expression. These correlative observations may have important consequences to the mechanism underlying human HPT-JT, that is, defects in the transcriptional regulation of some Hox genes. This possibility can be tested through analyses of Hox gene expression profiles in patient samples. Finally, the gene encoding MLL1 protein exhibits translocations in some leukemias (Ziemin-van der Poel et al., 1991; Ford et al., 1993). Our discovery that histone H3-K4 trimethylation is an event downstream of the hPAF complex (Zhu et al., 2005) and of histone H2BK120 monoubiquitination, along with the identification of factors involved in the upstream events, provides tenable and alternative lines of investigation into mechanisms underlying MLL1-related leukemias. Experimental Procedures Antibodies Antibodies against hPaf1, Leo1, and Ski8 were generated in earlier studies (Zhu et al., 2005). Antibody against hCtr9 was kindly provided by Dr. Desiderio from the University of Pennsylvania. Antibodies against hCdc73 were kindly provided by the Program for Genomic Applications and the Center for Biomedical Inventions, University of Texas Southwestern Medical Center at Dallas. The M2 anti-FLAG antibody was purchased from Sigma. Antibodies against UbcH6 and ubiquitin were purchased from Boston Biochem. Antibodies against Rpb4, TFIIB, trimethylated H3-K4, trimethylated H3-K9, and trimethylated H3-K27 were generated in our laboratory. Antibodies against histones H2B, H3, and H2A were purchased from Cell Signaling. Antibodies against monomethylated H3-K4 were purchased from Abcam. Antibodies against dimethylated H3-K4, dimethylated H3-K36, trimethylated H3-K36, and dimethylated H3-K79 were purchased from Upstate. E2 Enzymes All the E2 enzymes tested in this study were purchased from Boston Biochem. Partial Purification of a Histone H2B-K120 Monoubiquitination Activity Approximately 15 grams of proteins derived from HeLa cell nuclear extract were fractionated by chromatography onto a 1.5 liter phosphocellulose column (Sigma) equilibrated with buffer C (20 mM Tris-HCl [pH 7.9], 1 mM EDTA, 10% glycerol, 1 mM DTT, and 0.2 mM PMSF) containing 0.1 M KCl. Proteins were step eluted with buffer C containing 0.1 M KCl, 0.3 M KCl, 0.5 M KCl, and 1 M KCl, respectively. The 0.3 M KCl fraction was dialyzed against buffer C containing 0.1 M KCl and loaded onto a 300 ml DEAE-celulose column (Whatman). After extensive washing with buffer C containing 0.1 M KCl, the proteins were eluted with buffer C containing 0.35 M KCl. The eluate was dialyzed against buffer C containing 0.1 M KCl and loaded onto a 100 ml Heparin agarose column. After extensive washing with buffer C containing 0.1 M KCl, the proteins were eluted with buffer C containing 0.5 M KCl. The eluate was dialyzed against buffer C containing 0.1 M KCl and loaded onto a 8 ml Mono Q column, and proteins were eluted with a 15 column volume linear gradient from 0.1 to 0.7 M KCl in buffer C. The histone H2B-K120 monoubiquitination activity eluted between 0.25 and 0.3 M KCl. For determining the native mass of the histone H2B-K120 monoubiquitination activity, active fractions derived from the Mono Q column were concentrated to 50 ml and loaded onto a Smart Superose 6 gel filtration column.

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The histone H2B-K120 monoubiquitination activity eluted as an w600 kDa complex. Affinity Purification of the RNF20/40 Complex M2 anti-FLAG agarose (Sigma) was equilibrated with the same buffer used in nuclear extract preparation (20 mM Tris-HCl [pH 7.9], 1.5 mM MgCl2, 0.42 M NaCl, and 0.2 mM PMSF) and then incubated overnight at 4ºC with nuclear extract derived from the stable cells expressing RNF20. The resin was washed with excess amount of buffer C containing 0.5 M KCl plus 0.1% NP40, and proteins remaining bound were eluted with buffer C containing 0.5 M KCl plus 0.1 mg/ml FLAG peptide. Protein Identification Gel-resolved proteins were digested with trypsin and fractionated, and resulting peptide pools were analyzed by matrix-assisted laser-desorption/ionization reflectron time-of-flight (MALDI-reTOF) MS as described (Erdjument-Bromage et al., 1998; Winkler et al., 2002). Mass spectrometric sequencing of selected peptides was done by MALDI-TOF/TOF (MS/MS) analysis on the same prepared samples. Any identification thus obtained was verified by comparing the computer-generated fragment ion series of the predicted tryptic peptide with the experimental MS/MS data. Histone Monoubiquitination Assays Histone monoubiquitination assays were performed in a 30 ml reaction buffer containing 20 mM Hepes-KOH (pH 8.0), 12.5 mM MgCl2, 0.1 mM EDTA, and 0.1 mM ATP supplemented with 50 ng of 32P labeled ubiquitin, 100 ng of E1 ubiquitin-activating enzyme, and 1 mg of recombinant oligo-nucleosomes. Reactions were incubated at 37ºC for 1 hr. ChIP ChIP was performed according to the protocol described by Upstate. For each assay, cells were derived from approximately one confluent 15 cm plate. RNAi Knockdown of PAF Complex and RNF20/40 Complex siRNAs targeting Ski8, Ctr9, RNF20, and RNF40 were purchased from Dharmacon. siRNA was delivered by using RNAiFect from Qiagen. Supplemental Data Supplemental Data include three figures and are available with this article online at http://www.molecule.org/cgi/content/full/20/4/ 601/DC1/. Acknowledgments We are grateful to Dr. Stephen Desiderio from the Department of Molecular Biology and Genetics, John Hopkins University School of Medicine for kindly providing antibody against hCtr9/p150TSP. We thank the Program in Genomics Application antibody core center for biomedical inventions, and the University of Texas Southwestern Medical Center at Dallas for providing antibody against hCdc73. We are grateful to Dr. L.D. Vales for valuable comments on the manuscript. This study was supported by a grant from the National Institutes of Health (GM37120) and by the Howard Hughes Medical Institute (to D.R.) and by NCI Cancer Center Support Grant P30 CA08748 to P.T. Received: July 19, 2005 Revised: September 22, 2005 Accepted: September 30, 2005 Published: November 22, 2005 References Belotserkovskaya, R., Oh, S., Bondarenko, V.A., Orphanides, G., Studitsky, V.M., and Reinberg, D. (2003). FACT facilitates transcription-dependent nucleosome alteration. Science 301, 1090–1093. Carpten, J.D., Robbins, C.M., Villablanca, A., Forsberg, L., Presciuttini, S., Bailey-Wilson, J., Simonds, W.F., Gillanders, E.M., Kennedy, A.M., Chen, J.D., et al. (2002). HRPT2, encoding parafibromin, is

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