Physical and Functional Association of a Trimethyl H3K4 Demethylase and Ring6a/MBLR, a Polycomb-Like Protein

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Physical and Functional Association of a Trimethyl H3K4 Demethylase and Ring6a/MBLR, a Polycomb-like Protein

Min Gyu Lee,1 Jessica Norman,1 Ali Shilatifard,2 and Ramin Shiekhattar1,3,* 1 The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA 2 Department of Biochemistry and Molecular Biology, Saint Louis University Health Sciences Center, St. Louis, MI 63104, USA 3 Present address: Center de Regulacio Genomica, Dr. Aiguader, 88, 08003 Barcelona, Spain. *Correspondence: [email protected] DOI 10.1016/j.cell.2007.02.004

SUMMARY studied example of this mode of regulation was histone acetylation (Berger, 2002). This modification is enacted Histone methylation is a posttranslational mod- by enzymes called histone acetyltransferses and can be ification regulating chromatin structure and reversed by histone deacetylases. In addition to acetyla- gene regulation. BHC110/LSD1 was the first tion, histones are known to be phosphorylated, ubiquity- histone demethylase described to reverse di- lated, SUMOylated, and methylated (Vaquero et al., 2003). methyl histone H3 lysine 4 (H3K4). Here we Histone methylation has emerged as an important plat- show that JARID1d, a JmjC-domain-containing form for gene regulation. Specific lysines in histones H3 or H4 can be mono-, di- or trimethylated, adding another protein, specifically demethylates trimethyl layer of complexity to this mode of regulation (Strahl H3K4. Detailed mapping analysis revealed that et al., 1999). Moreover, these modifications may either besides the JmjC domain, the BRIGHT and signal activation (H3K4, H3K36, and H3K79), or the silenc- zinc-finger-like C5HC2 domains are required ing (H3K9, H3K27, and H4K20) of gene expression (Martin for maximum catalytic activity. Importantly, iso- and Zhang, 2005; Sims et al., 2003). These effects on tran- lation of native JARID1d complexes from human scriptional regulation have biological consequences rang- cells revealed the association of the demethy- ing from cell differentiation to X inactivation (Kouzarides, lase with a polycomb-like protein Ring6a/ 2002; Margueron et al., 2005; Martin and Zhang, 2005). MBLR. Ring6a/MBLR not only directly interacts Methylation of histone H3K4 results in an array of func- with JARID1d but also regulates its enzymatic tional outcomes. While trimethyl H3K4 is associated with activity. We show that JARID1d and Ring6a an increased rate of transcription and is generally located near the promoter of genes (Bernstein et al., 2002), occupy human Engrailed 2 gene and regulate dimethyl H3K4 on active genes is often localized in the its expression and H3K4 methylation levels. coding region. This methylation is performed by several Depletion of JARID1d enhanced recruitment of SET-domain-containing enzymes including the MLL/ the chromatin remodeling complex, NURF, and COMPASS complexes, SET1, SET7/9, and SMYD3 the basal transcription machinery near the tran- (Briggs et al., 2001; Bryk et al., 2002; Dou et al., 2005; scriptional start site, revealing a role for JAR- Goo et al., 2003; Hamamoto et al., 2004; Krogan et al., ID1d in regulation of transcriptional initiation 2002; Miller et al., 2001; Nakamura et al., 2002; Wang through H3K4 demethylation. et al., 2001; Wysocka et al., 2003; Yokoyama et al., 2004). Historically, histone methylation was considered a per- manent modification. However, in the past few years sev- INTRODUCTION eral groups have provided evidence for the existence of histone demethylases (Cloos et al., 2006; Cuthbert et al., Eukaryotic DNA is packaged into chromatin whose funda- 2004; Fodor et al., 2006; Klose et al., 2006; Lee et al., mental unit is the nucleosome consisting of 146 base pairs 2005; Shi et al., 2004; Tsukada et al., 2006; Wang of DNA wrapped around a histone octamer (Kornberg and et al., 2004b; Whetstine et al., 2006; Yamane et al., Lorch, 1999). The nucleosome is a dynamic structure 2006). BHC110/LSD1, the first known lysine demethylase, whose physical interactions with other chromatin-associ- demethylates di- and monomethyl H3K4 (Lee et al., 2005; ated proteins play a central role in gene regulation (Jenu- Shi et al., 2004, 2005). The most recently discovered fam- wein and Allis, 2001; Strahl and Allis, 2000). Covalent ily of histone demethylases consists of the JmjC-domain- modification of histones has been shown to result in either containing proteins, which are capable of demethylating activation or silencing of gene expression. The first well- di- and trimethyl H3K9 and H3K36 (Cloos et al., 2006;

Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. 877 Figure 1. JARID1d Demethylates Tri- and Dimethyl H3K4 (A) Schematic representation of the JARID1d family members. Overall sequence identities are indicated between JARID1d and other proteins including human family members (JARID1a-1c) and the D. melanogaster JARID1d homolog Lid. (B) Analysis of recombinant JARID1d (r. JARID1d) isolated from Sf9 insect cells by Col- loidal blue staining. Asterisk indicates a non- speciﬁc polypeptide. (C) Demethylation assay using recombinant JARID1d. Histones were used as substrate, and reaction mixtures were analyzed by SDS- PAGE, followed by western blotting using antibodies to various methyl marks. ‘‘1x’’ corresponds to approximately 500 ng of r. JARID1d. Where indicated, 3m is trimethyl, 2m is dimethyl, and 1m is monomethyl.

Fodor et al., 2006; Klose et al., 2006; Tsukada et al., 2006; (Gildea et al., 2000)(Figure 1A). Recombinant Flag- Whetstine et al., 2006; Yamane et al., 2006). JARID1d was subjected to affinity purification, and the To identify enzymes capable of demethylating trimethyl affinity eluate was used to assess JARID1d activity in de- H3K4, we characterized JARID1d, JmjC-domain-contain- methylation assays. Analysis of increasing concentrations ing protein capable of demethylating di- and trimethylated of JARID1d revealed its specific demethylation activity H3K4. JARID1d interacts with the polycomb protein toward tri- and dimethyl histone H3K4 without affecting Ring6a/MBLR. We demonstrate that this association is di- methylation on H3K9, H3K27, H3K36, H3K79, and H4K20 rect and leads to enhanced demethylation activity. Addi- (Figure 1C). Monomethylated H3K4 was not affected tionally, we show that JARID1d is recruited to the Engrailed using similar concentrations of JARD1d that diminished 2 gene to regulate its transcriptional output by demethylat- di- and trimethylated species (Figures 1CandS1). JARID1d ing trimethyl H3K4. Importantly, our results suggest a role also displayed enzymatic activity toward di- and trimethy- for JARID1d in regulation of transcription through modula- lated peptides corresponding to the H3 tail (Figure S1). tion of H3K4 methylation at transcriptional start sites. To ascertain that the catalytic activity was endowed in JARID1d, we generated a recombinant enzyme contain- RESULTS ing point mutations (H477A, E479A) in the described catalytic JmjC domain of JARID1d (Figure 2A). This mutation JARID1d Displays Trimethyl H3K4 abrogated the enzymatic activity of JARID1d, substantiat- Demethylation Activity ing its role as an H3K4 demethylase (Figure 2B). Further- In search of enzymes capable of catalyzing the demethy- more, consistent with the previously described mode of lation of trimethylated histone H3 lysine 4, we generated enzymatic action for the JmjC class of enzymes, the recombinant human JARID1d, a member of the JmjC fam- demethylation activity by JARID1d required addition ily of histone demethylases using a baculovirus expres- of Fe(II) and ascorbate to the demethylation reaction sion system (Figures 1A and 1B). In addition to a JmjC (Tsukada et al., 2006)(Figure 2C). However, we did not domain, the JARID family members also contain JmjN, find a requirement for exogenously added a-ketoglutarate BRIGHT/ARID (A/T-rich interaction domain), plant homeo- (Figure 2C). We performed additional experiments sub- domain (PHD), and zinc finger-like domains (Figure 1A). jecting the recombinant enzyme to increasing salt washes Drosophila posses a single gene termed little imaginal in order to assess its requirement for a-ketoglutarate. discs (lid) with close homology with JARID proteins As shown in Figure S2, even following a 1 M KCl wash,

878 Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. Figure 2. BRIGHT and Zinc Finger-like

C5HC2 Domains Contribute to JARID1d’s Catalytic Activity (A) Analysis of recombinant JARID1d (HE/AA) isolated from Sf9 insect cells by Colloidal blue staining. Histidine (aa 477) and glutamate (aa 479) residues in JARID1d were mutated to alanine. (B) Comparison of demethylase activities of recombinant JARID1d and its mutant JARID1d (HE/AA). ‘‘1x’’ corresponds to approximately 500 ng of recombinant proteins. (C) Effect of cofactors on JARID1d activities. (D) Analysis of recombinant deletion mutants

(DJmjN, DBRIGHT, DPHDa, DC5HC2,or

DPHDb) isolated from Sf9 or Sf21 insect cells by Colloidal blue staining. (E) Comparison of demethylation activities of recombinant JARID1d and its deletion mutants. Approximately 1000 ng of recombinant proteins were used. (F) Quantiﬁcation of trimethyl H3K4 levels shown in (E). The trimethyl H3K4 levels in the absence of JARID1d () were set as 100%. Data are presented as the mean ± SEM. His- tones were used as substrate, and reaction mixtures were analyzed by SDS-PAGE, followed by western blotting using antibodies to anti-dimethyl or anti-trimethyl H3K4 antibodies. Asterisks indicate nonspeciﬁc poly- peptides.

JARID1d did not require additional a-ketoglutarate for en- Analysis of similar amounts of proteins in the histone de- zymatic activity. Although absence of requirement for ex- methylation assays revealed a significant contribution by ogenously added a-ketoglutarate for the enzymatic activ- the BRIGHT and the C5HC2 domains of JARID1d to the ity of JARID1d may reflect a difference for the mechanism demethylation activity (Figures 2E and 2F). We find that of demethylation by JARID1d, we cannot rule out the pos- the JmjN and the PHD domains are dispensable for deme- sibility that the cofactor is tightly associated with the en- thylation activity. While previous analysis have revealed zyme and cannot be removed by increasing salt washes. a role for the zinc finger domain in catalytic activity of Future detailed structural and functional analysis of the the JmjC class of enzymes (Yamane et al., 2006), the con- enzyme will be required to address the precise catalytic tribution of the BRIGHT domain to enzymatic activity is mechanism. unique to the JARID1d family of proteins. Indeed, it is

the presence of the combination of BRIGHT, C5HC2, and BRIGHT/ARID (AT-Rich Interaction Domain) the JmjC domains that deﬁnes the JARID1 proteins as a and the Zinc Finger-like C5HC2 Domains putative H3K4 di- and trimethyl family of demethylases. Contribute to the Enzymatic Activity We next asked whether recombinant JARID1d or the We next asked whether the other domains present in the JARID1d complex mediate the demethylation of nucleo- JARID1d protein are required for enzymatic activity. We somal substrates. While both the recombinant JARID1d produced recombinant proteins in insect cells following and the JARID1d complex (see below) readily demethy- systematic deletion of JmjN, BRIGHT, PHDa, C5HC2, late histones H3K4, the nucleosomal substrates were re- and the PHDb domains of JARID1d (Figures 2D and 1A). fractory to the action of the enzyme (Figures S3A and

Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. 879 Figure 3. JARID1d Associates with a Polycomb-like Protein Ring6a/MBLR (A) Affinity purification and analysis of JARID1d-associated proteins. Asterisks indicate either breakdowns or nonspecific poly- peptides. (B) Schematic representation of the Ring6a/ MBLR and other polycomb proteins (Bmi1 and Mel18). Sequence identities are indicated between Ring6a and other proteins for RING and C-terminal domains. (C) Demethylation assay using JARID1d complex (approximately 200 ng of JARID1d in the complex). Histones were used as substrate, and reaction mixtures were analyzed by SDS- PAGE, followed by western blotting using antibodies to tri- or dimethyl H3K4 marks. (D) Analysis of recombinant Ring6a isolated from Sf21 insect cells by Colloidal blue staining. (E) Coimmunoprecipitation assay between recombinant proteins JARID1d and Ring6a. Re- combinant JARID1d and Ring6a were mixed, immunoprecipitated with anti-JARID1d antibodies, and then washed extensively. The eluates were analyzed by SDS-PAGE followed by western blotting using anti-FLAG antibodies. Both recombinant proteins were FLAG tagged.

S3B). It is likely that JARID1d may require additional regu- Ring6a/MBLR Directly Interacts with JARID1d latory components such as transcriptional repressors or and Regulates Its Enzymatic Activity corepressors for mediating nucleosomal demethylation. We next asked whether Ring6a directly interacts with Previously, we showed that the SANT domains of the JARID1d. We generated recombinant Flag-Ring6a using CoREST protein were required to confer nucleosomal de- the baculovirus expression system and tested its associ- methylation to BHC110 (Lee et al., 2005). It will be interest- ation with recombinant JARID1d produced in insect cells ing if JARID1d also requires a SANT-domain-containing (Figure 3D). We consistently detect a doublet correspond- protein to mediate such nucleosomal demethylation. ing to Ring6a consistent with the contention that the protein may be posttransationally modified in insect cells Association of Ring6a/MBLR with JARID1d (Figure 3D) (Akasaka et al., 2002). Our analysis of the To address whether JARID1d is a component of a multi- two recombinant proteins revealed the specific physical protein complex, we developed stable-cell lines express- association of JARID1d and Ring6a (Figure 3E). To extend ing Flag-JARID1d. Analysis of Flag-JARID1d affinity elu- these results we examined the possible functional con- ate by silver staining revealed the presence of multiple sequence of this association for JARID1d demethylase specific bands in the 50 kDa molecular weight range (Fig- activity. Titration of increasing concentrations of Ring6a ure 3A). Mass-spectrometric analysis of these polypep- moderately enhanced the demethylation activity of tide revealed that while the majority constitute the break- JARID1d (Figures 4A and 4B). The increased activity of down products of JARID1d, an approximately 55 kDa JARID1d by Ring6a was specific as addition of similar band was identified as Ring6a/MBLR (Akasaka et al., concentrations of other proteins (BSA or AOF1) to 2002). Ring6a/MBLR is a protein with close homology to JARID1d had no effect on its demethylase activity (Fig- Bmi1 and Mel18 proteins that are shown to be compo- ure 4C). Taken together, these results indicate that Ring6a nents of the polycomb complex PRC1 (Shao et al., 1999) can directly associate with JARID1d, and this association (Figure 3B). Analysis of JARID1d complex in demethyla- leads to modulation of JARID1d demethylation activity. tion assays confirmed its specific activity toward tri- and To determine whether Ring6a interacts with a specific dimethylated H3K4 (Figure 3C). domain of JARID1d, we analyzed deletion mutants of

880 Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. Figure 4. Ring6a Enhances JARID1d’s Enzymatic Activity, and Its C Terminus Is Responsible for the Association with JARID1d (A) Effect of Ring6a on JARID1d activity. The amounts of recombinant proteins used were as follows: approximately 500 ng of JARID1d and 250–500 ng of Ring6a. (B) Quantiﬁcation of trimethyl H3K4 levels shown in panel (A). Trimethyl H3K4 levels in the absence of JARID1d () were set as 100%. Data are presented as the mean ± SEM. (C) Effect of other proteins on JARID1d activity. Approximately 500 ng of JARID1d, Ring6a, BSA, and AOF1 were used. Histones were used as substrate, and reaction mixtures were analyzed by SDS-PAGE followed by western blotting using antibodies to trimethyl H3K4. (D) Schematic representation of Ring6a and its deletion mutants (DRING, Ring6a-N, and Ring6a-C). (E) Analysis of recombinant Ring6a and its deletion mutants by Colloidal blue staining. Recombinant DRING was isolated from Sf21 insect cells using a baculovirus system while the other proteins (Ring6a-N and Ring6a-C) were from bacteria. The closed circles indicate breakdowns. (F) Coimmunoprecipitation assay between recombinant JARID1d and Ring6a mutants. Recombinant JARID1d and Ring6a mutants were mixed, immunoprecipitated with anti- JARID1d antibodies, and then washed extensively. The eluates were analyzed by SDS-PAGE followed by western blotting using anti-FLAG antibodies. All recombinant proteins were FLAG tagged.

JARID1d protein for their association with Ring6a (Fig- regulated genes (Figure S4A). Although we observed ure S3C). These experiments revealed that the deletion 2- to 3-fold enrichment of JARID1d at Synapsin and of a single domain of JARID1d does not affect the associ- Engrailed 1 genes, Engrailed 2 gene displayed a 5-fold ation of the demethylase and Ring6a (Figure S3C), sug- enrichment of JARID1d (Figure 5A). Engrailed 2 also gesting that the interaction interface encompasses more displayed a 4-fold enrichment for Ring6a occupancy (Fig- than a single domain. However, analysis of the Ring6a do- ure 5C). Therefore, we examined Engrailed 2 in more detail main that mediates the association with JARID1d indi- following the depletion of JARID1d and Ring6a using small cated that only the C-terminal domain of Ring6a is respon- interfering RNAs (Figures 5B and 5D). Interestingly, sible for this interaction (Figures 4D–4F). Interestingly, JARID1d depletion leads to a concurrent loss of Ring6a sequence alignments of Ring6a, Bmi1, and Mel18 re- at the Engrailed 2 gene consistent with their association vealed that beside the Ring ﬁnger, the C-terminal domain in a multisubunit complex (Figure S4B). Decreased levels of Ring6a, represent the highest degree of sequence iden- of JARID1d and Ring6a resulted in enhanced transcription tities between the three proteins (Figure 3B). These results of Engrailed 2 as measured by quantitative reverse tran- suggest a conserved function for the C-terminal domain scriptase-polymerase chain reaction (qRT-PCR) (Fig- of this class of Ring ﬁnger proteins for interaction with ure 5E). Concomitant with this increased transcription, JARID1d family of histone demethylases. we detected increased levels of di- and trimethylated H3K4 at the Engrailed 2 gene (Figure 5F). These results JARID1d and Ring6a Occupy Engrailed 2 Gene are consistent with an in vivo role for JARID1d as a and Regulate Its Expression H3K4 demethylase involved in transcriptional regulation To assess the demethylation properties of JARID1d in of Engrailed 2 gene. Furthermore, it extends the role for vivo, we used chromatin immunoprecipitation (ChIP) to Ring6a protein in modulating the demethylation of H3K4 examine its occupancy on a group of developmentally in vivo.

Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. 881 Figure 5. In Vivo Knockdown of JARID1d and Ring6a Increases Transcriptional and Methylated H3K4 Levels of a Target Gene (A and C) Enrichment of JARID1d (A) and Ring6a (C) on the Engrailed2 gene was analyzed by quantitative chromatin immunoprecipitation (qChIP) assay using H1299 cells. (B and D) The effect of knockdown of JARID1d (B) and Ring6a (D). Stable-cell lines expressing FLAG-JARID1d or FLAG-Ring6a were transfected with siRNA against JARID1d or Ring6a, respectively, and harvested. Whole-cell extracts were immunoprecipitated with anti- FLAG resin. The eluates were analyzed by SDS-PAGE followed by western blotting using anti-FLAG antibodies. (E) Analysis of Engrailed2 mRNA levels by qRT- PCR after treatment of H1299 cells with siRNA against luciferase, JARID1d, or Ring6a. (F) Analysis of promoter occupancy of di- or trimethyl H3K4 on Engrailed2 promoter by quantitative ChIP assay after treatment of H1299 cells with siRNA against luciferase, JARID1d, or Ring6a. ChIP was performed in around +300 (d region in Figure 6). Data are presented as the mean ± SEM. *(<0.05) and **(<0.01) indicate statistically signiﬁcant increases.

JARID1d Regulates Transcription through JARID1d Regulates the Recruitment of Chromatin Modulation of Trimethyl H3K4 Levels at Remodeling Complex, NURF, and the Basal the 50 End of the Engrailed 2 Gene Transcription Machinery We used ChIP to map the occupancy of JARID1d on the Previously, we characterized the human chromatin re- Engrailed 2 promoter and its coding region (Figure 6A). modeling complex, hNURF, composed of SNF2L, BPTF, This analysis revealed the predominant presence of and RbAP48/46, and showed its recruitment to the pro- JARID1d at the 50 end of the Engrailed 2 gene (Figure 6B). moter of the Engrailed 2 gene to regulate its transcriptional We also detect a smaller but significant JARID1d binding activity (Barak et al., 2003). More recent experiments iden- site at about 1 kb upstream of the transcriptional start tified the large subunit of hNURF, the BPTF protein, capa- site (Figure 6B). Presence of JARID1d in this region may ble of specific association with trimethylated H3K4 marks represent the recruitment of the demethylase through (Li et al., 2006; Wysocka et al., 2006). Therefore, we as- specific DNA binding proteins involved in transcriptional sessed whether recruitment of the NURF complex to the repression. Importantly, depletion of JARID1d resulted in Engrailed 2 promoter is regulated by JARID1d. Interest- enhanced H3K4 methylation at the 50 end of the Engrailed ingly, depletion of JARID1d resulted in a 3-fold enhance- 2 gene coincident with sites of enrichment for JARID1d ment in the occupancy of BPTF close to the 50 end of (Figure 6C). There is also about a 2-fold increase in the tri- the Engrailed 2 gene (Figures 7A and 7B). These results methylated H3K4 levels at the JARID1d upstream binding are consistent with the enhanced recruitment of NURF site, which is masked in Figure 6C due to a 100-fold through increased trimethylated H3K4 that is the conse- enrichment of trimethylation near the transcription quence of the depletion of histone demethylase JARID1d. start site. Taken together, our results suggest a role for We next asked whether increased recruitment of NURF JARID1d in regulation of transcription through demethyla- at the 50 end of Engrailed 2 gene following JARID1d depletion of tri-and dimethyl H3K4 that predominates in the tion may coincide with enhanced occupancy of the pro- region of the transcriptional start site. moter by the basal transcription machinery. Analysis of

882 Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. Figure 6. Localization of JARID1d and Trimethyl H3K4 on the Engrailed 2 Gene (A) Schematic representation of the promoter and 50 end of the Engrailed 2 gene. The transcriptional start site is putative and based on an initiator sequence (CTAATTC) and mRNA sequence of the Engrailed 2 gene (GenBank accession number NM_001427). (B) JARID1d occupancy at different regions of the Engrailed 2 gene was analyzed by quantitative ChIP assay using H1299 cells. (C) Trimethyl H3K4 levels at different regions of the Engrailed 2 gene were analyzed by quantitative ChIP assay after treatment of H1299 cells with siRNA against luciferase or JARID1d. Data are presented as the mean ± SEM. *(<0.05) and **(<0.01) indicate statistically signiﬁcant increases.

the Engrailed 2 promoter upstream of the transcriptional ated H3K4 (Lee et al., 2006a, 2005; Shi et al., 2004); how- start site revealed the presence of basal transcription ever, this enzyme was unable to demethylate H3K4 trime- machinery consisting of TBP, TFIIB, TFIIH, and RNAPII thylated species. Therefore, this is the first demonstration (Figure 7A). Importantly, depletion of JARID1d led to a of enzymatic activity toward trimethylated H3K4. Second, 2-fold enhancement of TBP, TFIIB, TFIIH, and RNAPII it shows that besides the JmjC catalytic domain, there is binding at the promoter consistent with increased tran- a role for the BRIGHT and zinc finger-like C5HC2 domains scriptional activity of Engrailed 2 (Figures 7A and 5E). of JARID1d toward the demethylation activity. Third, Moreover, such enhanced recruitment of TFIIH, RNAPII, through biochemical isolation of native JARID1d-contain- and BPTF could also be seen at the 50 end of the Engrailed ing complexes, we reveal an association between 2 gene (Figure 7B). Taken together, our results suggest JARID1d and the polycomb-like protein Ring6a/MBLR. a scenario in which demethylation of trimethyl H3K4 close Fourth, through analysis of recombinant Ring6a/MBLR to transcription start sites by JARID1d results in de- and JARID1d, we demonstrate a direct physical and func- creased occupancy of the chromatin remodeling com- tional association between the two proteins. Fifth, in vivo plex, NURF, and the basal transcription machinery leading analysis using ChIP demonstrated the regulation of to transcriptional repression (Figure 7C). Engrailed 2 transcription as well as H3K4 methylation levels by JARID1d and Ring6a. Finally, it provides evi- DISCUSSION dence for JARID1d’s role in regulation of Engrailed 2 transcription through modulating the recruitment of NURF and The key findings of this work are the following. First, we the basal transcription machinery close to the transcrip- identify a family of JmjC-domain-containing proteins tional start site. with the ability to specifically demethylate trimethylated Enzymes containing the amine-oxidase domain were H3K4. Previously, we and others have shown that the first to be identified as histone demethylase capable BHC110/LSD1 displays enzymatic activity toward methyl- of catalyzing di- and monomethylated H3K4 (Lee et al.,

Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. 883 Figure 7. In Vivo Knockdown of JARID1d Results in Increased Levels of Transcrip- tional Initiation Machinery and BPTF (A and B) Occupancy of various transcription factors, RNA polymerase II (RNAPII), and a 3mH3K4 binding protein BPTF at region c (around 200) (A) and region d (around +300) (B) of the Engrailed 2 gene was analyzed by quantitative ChIP assay after treatment of H1299 cells with siRNA against luciferase or JARID1d. Regions c and d are indicated in Fig- ure 6. Data are presented as the mean ± SEM. *(<0.05) indicates statistically signiﬁcant increases. (C) Schematic representation depicting inhibi- tory effect of histone demethylation by JARID1d on transcriptional initiation and BPTF-trimethyl H3K4 association. Demethyla- tion of trimethyl H3K4 by JARID1d complex results in decreased levels of transcription initiation machinery and BPTF/ trimethyl H3K4 interaction. The length of DNA and sizes of proteins are not drawn to scale, and only some components of transcription initiation complex are shown for simplicity.

2005; Shi et al., 2004). However, due to the intrinsic limita- Ring6a/MBLR is a polycomb-like Ring-finger-containing tions in their catalytic mechanism, this family of histone protein that was previously characterized to be a compo- demethylases was deemed unable to remove trimethy- nent of an E2F6-containing complex (Akasaka et al., 2002; lated H3K4. The discovery of a second class of demethy- Ogawa et al., 2002). These complexes also contained lating enzymes containing the JmjC domain paved the RING1 and RING2 that were recently shown to be capable way for uncovering enzymes with activities toward tri- of ubiquitinating lysine 119 of histone H2A (Wang et al., methyl marks (Cloos et al., 2006; Fodor et al., 2006; Klose 2004a). In our mass-spectrometric analysis of JARID1d et al., 2006; Yamane et al., 2006). We employed a candi- affinity eluate, we did not obtain any peptide sequences date approach to identify JmjC-domain-containing pro- corresponding to Ring1 and Ring2. Moreover, we could teins with the ability to demethylate trimethylated not detect Ring1 or Ring2 using western blot analysis of H3K4. This resulted in the identification of the JARID1 JARID1d-affinity eluate (data not shown). This leads us family of JmjC-domain-containing proteins as histone de- to conclude that the JARID1d/Ring6a complex constitutes methylases able to remove tri- and dimethyl H3K4. Inter- a distinct complex from that of E2F6-containing com- estingly, we show that while the JmjC domain is the cata- plexes. Our functional reconstitution experiments re- lytic heart of the enzyme, two other domains (BRIGHT and vealed that not only does Ring6a directly associate with

C5HC2 domains) play a regulatory role in the enzymatic JARID1d, but it also moderately regulates its demethyla- reaction. Future structural analysis of this family of JmjC- tion activity. Since these assays were performed in the ab- domain proteins should shed light on the mechanism sence of ubiquitin and E1 and E2 enzymes, the facilitation by which these domains contribute to the enzymatic of JARID1d demethylation activity by Ring6a is not due to reaction. its ubiquitination activity but may rather result from a con- Puriﬁcation of JARID1d from human cells resulted in the formational change induced in JARID1d upon Ring6a discovery of Ring6a/MBLR as an associated protein. association.

884 Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. Recent studies have pointed to trimethylated H3K4 as were from Santa Cruz. Anti-dimethyl H3K27 was a generous gift a specific chromatin modification that is targeted by sev- from Dr. Thomas Jenuwein. Anti-trimethyl H3K4, anti-dimethyl eral chromatin-associated proteins (Huang et al., 2006; H3K36, and anti-dimethyl H4K20 were from Dr. Danny Reinberg. Anti-JARID1d and anti-Ring6a antibodies were raised in rabbit (Open Kim et al., 2006; Li et al., 2006; Pena et al., 2006; Shi Biosystems). Stable-cell lines expressing FLAG-JARID1d or FLAG- et al., 2006; Wysocka et al., 2005, 2006). Trimethylation Ring6a were generated using HEK (human embryonic kidney) 293 cells of H3K4 has often been associated with the transcriptional and H1299 cells by cotransfecting with FLAG-JARID1d or FLAG- start site of active genes (Shilatifard, 2006). Therefore, an Ring6a expression plasmid, respectively, and a selectable marker for enzymatic activity reversing such modifications is ex- puromycin resistance. pected to display transcriptional-repressor activity. Con- Affinity Purification of JARID1d-Containing Complex sistent with this contention, we find that depletion of and Recombinant Proteins JARID1d not only enhances di- and trimethylation To purify the JARID1d-containing complex, nuclear extracts (150–200 of H3K4 but also leads to increased transcription of mg) isolated from a JARID1d stable-cell line were incubated with anti- Engrailed 2, a target of JARID1d. We further show that FLAG M2 affinity resin (Sigma). Affinity chromatography using anti- such JARID1d depletions result in enhanced recruitment FLAG resin was performed as previously described (Gregory et al., of chromatin remodeling complex, NURF, and the basal 2006; Lee et al., 2006b). JARID1d-associated proteins were identified transcription machinery providing a mechanistic frame- by liquid chromatography-tandem mass spectroscopy. Baculoviral- recombinant proteins (FLAG-(His) -JARID1d, FLAG-(His) -JARID1d work for the action of JARID1d. These results also indicate 6 6 (HE/AA), -DJmjN, -DBRIGHT, -DPHDa,-DJmjC, -DC5HC2,-DPHDb, that trimethylation of H3K4 at the 50 end of genes may be FLAG-(His)6-Ring6a, FLAG-(His)6-DRING), and bacterial recombinant an important regulator of preinitiation complex assembly proteins (FLAG-Ring6a-N and FLAG-Ring6a-C) were purified by and/or stability. methods previously described (Dong et al., 2003). The amounts of Human cells contain four members of JARID1 family JARID1d in complexes and recombinant proteins were deduced by (see Figure 1A). Therefore, it is conceivable that different comparing band intensities on silver- and Colloidal blue-stained gel with known amounts of bovine serum albumin (BSA), respectively. JARID1 family members are recruited to different pro- moters to influence H3K4 methylation levels and conse- Demethylation Assay quently promote transcriptional repression. Interestingly, The histone-demethylase assay was performed as previously de- mutations in JARID1c are associated with X-linked mental scribed (Lee et al., 2006a, 2005; Lee et al., 2006c; Tsukada et al., retardation (Jensen et al., 2005). Moreover, the single Dro- 2006). In brief, bulk histones (4 mg, Sigma) or methylated H3K4 pep- sophila homolog, lid, is an essential gene, resulting in gen- tides (0.1 mg, Upstate) were incubated with the indicated amounts of eral larval lethality with a limited number of flies surviving recombinant proteins or JARID1d complex in a histone demethylase (HDM) assay buffer (50 mM HEPES [pH 8.0], 100 mM [NH4]2[SO4]2, metamorphosis that die soon after they eclose (Gildea 1mMa-ketoglutarate, 2 mM ascorbate, 5% glycerol, and 0.2 mM et al., 2000). Taken together, our results extend the role PMSF) in a final volume of 10 ml for 5–7 hr at 37C. The reaction mixture of histone demethylases in chromatin modification and was analyzed by SDS-PAGE, followed by western blotting as previ- gene regulation by identifying the JARID1 family of ously described (Lee et al., 2006b). Antibodies against various methyl JmjC-domain-containing proteins as tri- and dimethyl marks were used to detect methylation levels. H3K4 demethylases. Coimmunoprecipitation Assay To test in vitro interaction between JARID1d and either Ring6a or its EXPERIMENTAL PROCEDURES mutants, recombinant JARID1d (1 mg) and either Ring6a or its mutants (0.5–2 mg) were mixed in BC250 buffer (20 mM Tris-HCl [pH 7.9], Plasmids, Antibodies, and Cell Lines 250 mM KCl, 10% glycerol, 0.2 mM EDTA, 1 mM dithiothreitol, and A mammalian expression vector encoding JARID1d (also known as 0.2 mM PMSF) containing 0.1% Nonidet P40, incubated for 1 hr at KIAA0234, GenBank accession number D87072) was generated using 4C, and then immunoprecipitated with anti-JARID1d antibodies, fol- pFLAG-CMV2 (Sigma) vector. Baculoviral-expression vectors encod- lowed by adding rProtein A bead. The beads were washed extensively ing FLAG-(His)6-JARID1d, and FLAG-(His)6-Ring6a were constructed with BC250 buffer containing 0.1% NP40 and eluted by boiling in 1x using pFastBacHTa plasmid (Invitrogen). FLAG-(His)6-JARID1d (HE/ SDS-PAGE sample buffer (62.5 mM Tris [pH 6.8], 5% b-mercaptoetha- AA) and deletion mutants (DJmjN (amino acids [aa] 13–54), DBRIGHT nol, 2% SDS, 10% glycerol, 0.01% Bromophenol blue). The eluates

(aa 80–158), DPHDa (aa 259–305), DJmjC (aa 401–567), DC5HC2 (aa were analyzed by SDS-PAGE followed by western blotting using

640–693), and DPHDb (aa 1117–1178) were derived from FLAG- anti-FLAG antibodies.

(His)6-JARID1d. Amino acid positions of JARID1d (GenBank accession number BAA13241) were renumbered from methionine. FLAG-(His)6- RNA Interference, qRT-PCR, and ChIP

DRING (aa 136–174) was derived from FLAG-(His)6-Ring6a (GenBank For RNA interference, siRNAs were transfected according to the man- accession number NM_001011663). FLAG-Ring6a-N (aa 1–135) and ufacturer’s instructions (Dharmacon) using Lipofectamine 2000 (Invi- FLAG-Ring6a-C (aa 177–350) were cloned into the bacterial expres- trogen) (Dong et al., 2003) and were purchased from Dharmacon’s sion plasmid pET28a. Anti-dimethyl H3K4 (07436), anti-dimethyl siGENOME collection. Small interfering RNA against luciferase was H3K4 (07030), anti-trimethyl H3K4 (07473), anti-dimethyl H3K9 used as a control. The siRNA sequences were as follows: JARID1d (07521), anti-trimethyl H3K9 (07442), anti-trimethyl H3K27 (05851), siRNA in sense strand, 50-ACAAGGAGAUGUUCAUUAUUU-30; Ring6a and anti-dimethyl H3K79 (07366) antibodies were purchased from siRNA in sense strand, 50-GAGGAGGAGCGCCUGAUUAUU-30; lucif- Upstate/Millipore. The anti-trimethyl H3K36 (ab9050), anti-trimethyl erase siRNA (control) in sense strand, 50-AACGUACGCGGAAUA H3K79 (ab2621), anti-trimethyl H4K20 (ab9053), and anti-H3 anti- CUUCGA-30. Total RNA was prepared using the Qiagen RNeasy kit bodies (ab1791) and anti-TBP (ab28175) were from Abcam Ltd. Anti- and reverse-transcribed using Invitrogen’s First Strand Synthesis kit, FLAG antibody (F3165) was from Sigma. Anti-RNA polymerase II (sc- followed by double digestion with RNase H and RNase A. Quantitative 899), anti-TFIIB (sc-225), and anti-CDK7 (a subunit of TFIIH, sc-856x) PCR was carried out using the Opticon2 (MJ Research) with

Cell 128, 877–887, March 9, 2007 ª2007 Elsevier Inc. 885 Finnzymes DyNAmo HS SYBR Green qPCR kit. Each sample was an- Bannister, A.J., et al. (2004). Histone deimination antagonizes arginine alyzed for mRNA levels of both GAPDH and Engrailed 2 (forward, 50- methylation. Cell 118, 545–553. 0 0 GGT CTA CTG TAC GCG CT -3 ; reverse, 5 - TGG TCT GGA AC Dong, Y., Hakimi, M.A., Chen, X., Kumaraswamy, E., Cooch, N.S., 0 TCG GC -3 ) and quantified using Opticon software. Engrailed 2 Godwin, A.K., and Shiekhattar, R. (2003). Regulation of BRCC, a holo- mRNA levels were normalized by GAPDH levels. The relative mRNA enzyme complex containing BRCA1 and BRCA2, by a signalosome- level represents the fold increase over the control. ChIP assay was car- like subunit and its role in DNA repair. Mol. Cell 12, 1087–1099. ried out as previously described (Hakimi et al., 2002). Promoter-occu- Dou, Y., Milne, T.A., Tackett, A.J., Smith, E.R., Fukuda, A., Wysocka, pancy levels were measured by Finnzymes DyNAmo HS SYBR Green J., Allis, C.D., Chait, B.T., Hess, J.L., and Roeder, R.G. (2005). Physical qPCR kit using the Opticon2 (MJ Research), normalized by input, and association and coordinate function of the H3 K4 methyltransferase are expressed as the fold increase over the control. PCR was per- MLL1 and the H4 K16 acetyltransferase MOF. Cell 121, 873–885. formed using primers spanning the promoter and 50 end of Engrailed 2 gene. The primer sequences are available upon request. Data are Fodor, B.D., Kubicek, S., Yonezawa, M., O’Sullivan, R.J., Sengupta, presented as the mean ± SEM. R., Perez-Burgos, L., Opravil, S., Mechtler, K., Schotta, G., and Jenu- wein, T. (2006). Jmjd2b antagonizes H3K9 trimethylation at pericentric heterochromatin in mammalian cells. Genes Dev. 20, 1557–1562. Supplemental Data Supplemental Data include four figures and can be found with this Gildea, J.J., Lopez, R., and Shearn, A. (2000). A screen for new article online at http://www.cell.com/cgi/content/full/128/5/877/DC1/. trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156, 645–663. ACKNOWLEDGMENTS Goo, Y.H., Sohn, Y.C., Kim, D.H., Kim, S.W., Kang, M.J., Jung, D.J., Kwak, E., Barlev, N.A., Berger, S.L., Chow, V.T., et al. (2003). Activat- We thank Drs. D. Reinberg, T. Jenuwein, and H. Koga for their anti- ing signal cointegrator 2 belongs to a novel steady-state complex that bodies; Dr. D. Bochar for recombinant AOF1; and Kazusa DNA contains a subset of trithorax group proteins. Mol. Cell. Biol. 23, 140– Research Institute Foundation for KIAA0234 cDNA. We extend our 149. gratitude to Drs. D. Baillat, C. Wynder, S. McMahon, and X. Zhang for helpful advice and technical assistance; to Henry Hoff for protein Gregory, R.I., Chendrimada, T.P., and Shiekhattar, R. (2006). expression; and to Matt Hart, Tom Beer, Kaye Speicher, and Dr. D. MicroRNA biogenesis: isolation and characterization of the micropro- Speicher for help with mass spectrometry. R.S. was supported by cessor complex. Methods Mol. Biol. 342, 33–47. a grant from NIH RO1CA090758. Hakimi, M.A., Bochar, D.A., Chenoweth, J., Lane, W.S., Mandel, G., and Shiekhattar, R. (2002). A core-BRAF35 complex containing his- Received: September 14, 2006 tone deacetylase mediates repression of neuronal-specific genes. Revised: November 29, 2006 Proc. Natl. Acad. Sci. USA 99, 7420–7425. Accepted: February 2, 2007 Hamamoto, R., Furukawa, Y., Morita, M., Iimura, Y., Silva, F.P., Li, M., Published online: February 22, 2007 Yagyu, R., and Nakamura, Y. (2004). SMYD3 encodes a histone methyltransferase involved in the proliferation of cancer cells. Nat. Cell Biol. 6, 731–740. 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