View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector 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 anti- bodies to various methyl marks. ‘‘1x’’ corre- sponds to approximately 500 ng of r. JARID1d. Where indicated, 3m is trimethyl, 2m is di- methyl, 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 cat- alytic 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 re- combinant 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 mu- tants. 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 (À) 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, fol- lowed by western blotting using antibodies to anti-dimethyl or anti-trimethyl H3K4 anti- bodies.