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Jmjd2b Antagonizes H3K9 Trimethylation at Pericentric Heterochromatin in Mammalian Cells

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Jmjd2b Antagonizes H3K9 Trimethylation at Pericentric Heterochromatin in Mammalian Cells Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION 2004) and resist reprogramming in early mammalian em- Jmjd2b antagonizes H3K9 bryos (Santos et al. 2003). trimethylation at pericentric Until recently, no enzymatic mechanism had been de- scribed that would directly remove histone lysine meth- heterochromatin in ylation, although amine oxidation or radical attack by mammalian cells hydroxylation could destabilize the amino–methyl bond (Kubicek and Jenuwein 2004; Bannister and Kouzarides Barna D. Fodor,1 Stefan Kubicek,1 2005; Trewick et al. 2005). Indeed, a lysine-specific de- methylase 1 (LSD1) has been shown to demethylate Masato Yonezawa, Roderick J. O’Sullivan, H3K4me2 (an active mark) (Shi et al. 2004) and, when Roopsha Sengupta, Laura Perez-Burgos, associated with a different protein complex, also appears Susanne Opravil, Karl Mechtler, Gunnar Schotta, to convert H3K9me2 (a repressive mark) (Metzger et al. and Thomas Jenuwein2 2005). Based on the reaction mechanism, LSD1 cannot act on a lysine trimethyl state, and more potent mecha- Research Institute of Molecular Pathology (IMP), The Vienna nisms, involving attack by oxygen radicals via hydroxy- Biocenter, A-1030 Vienna, Austria lases/dioxygenases, have been postulated (Clissold and Ponting 2001; Trewick et al. 2005). This could be in- Histone lysine trimethyl states represent some of the ferred from the activities of the Escherichia coli AlkB most robust epigenetic modifications in eukaryotic chro- enzyme to counteract alkylating damage of DNA or from matin. Using a candidate approach, we identified the other protein hydroxylases, such as the jmjC (jumonji) subgroup of murine Jmjd2 proteins to antagonize domain containing protein Factor Inhibiting Hypoxia H3K9me3 at pericentric heterochromatin. H3K27me3 (FIH) (Lando et al. 2002). In Schizosaccharomyces and H4K20me3 marks are not impaired in inducible pombe, there is one silencing modifier, Epe1, which also Jmjd2b-GFP cell lines, but Jmjd2b also reduces H3K36 carries a jmjC domain, and this factor antagonizes re- pressive H3K9me2 at centromeric regions (Ayoub et al. methylation. Since recombinant Jmjd2b appears as a 2003). In addition, there are several jmjC proteins that very poor enzyme, we applied metabolic labeling with have been shown to be important transcriptional/chro- heavy methyl groups to demonstrate Jmjd2b-mediated matin regulators (Takeuchi et al. 1995; Gildea et al. removal of chromosomal H3K9me3 as an active process 2000; Ahmed et al. 2004; Jin et al. 2004; Katoh and that occurs well before replication of chromatin. These Katoh 2004; Gray et al. 2005; Zhang et al. 2005). Exten- data reveal that certain members of the jmjC class of sive biochemical analyses have recently disclosed the hydroxylases can work in a pathway that actively an- jmjC protein Fbxl11 as the first bona fide histone lysine tagonizes a histone lysine trimethyl state. demethylase of the class of hydroxylases, and this en- zyme was named JHDM1 (Tsukada et al. 2006). Surpris- Supplemental material is available at http://www.genesdev.org. ingly, JHDM1 is selective to remove H3K36me2, but Received November 30, 2005; revised version accepted April seems again unable to attack a trimethyl state, which 26, 2006. may be a consequence of limited substrate recognition or restricted potential of the catalytic pocket to accommo- date a histone lysine trimethyl state (Tsukada et al. 2006). Histone lysine methylation is a central epigenetic modi- fication with both activating and repressive roles in eu- Results and Discussion karyotic chromatin (Kouzarides 2002; Fischle et al. 2003; Lachner et al. 2004; Reinberg et al. 2004). There are five Histone lysine demethylation and the family lysine residues in the histone N termini that are promi- of murine jmjC domain proteins nently methylated, with H3K4 and H3K36 methylation To address the potential of mammalian jmjC proteins as primarily transducing activating functions, whereas putative demethylases for a histone lysine trimethyl H3K9, H3K27, and H4K20 methylation is mainly asso- state, we first compared the amino acid sequences of the ciated with repressed chromatin. Histone lysine methyl- conserved jmjC domain of known chromatin modifiers ation can be presented as mono-, di-, or trimethyl states, (e.g., Epe1) (Ayoub et al. 2003) or of the protein hydroxy- where each distinct methyl state confers different bio- lase FIH (Lando et al. 2002) with those of murine jmjC logical read-outs (Santos-Rosa et al. 2002; Lachner et al. factors, as had been done previously (Trewick et al. 2004). Histone lysine trimethyl states, particularly for 2005). This sequence alignment resulted in the discrimi- the repressive functions, appear relatively robust, since nation of ∼30 jmjC-containing mouse proteins, 14 of they are stably propagated during several cell divisions which can be categorized into three classes comprising (Lachner et al. 2004; Reinberg et al. 2004; Schotta et al. an Epe1-JHDM1 subgroup, a Jarid subgroup, and a Jmjd2 subgroup (Supplementary Fig. S1). While the jmjC do- main is the sole signature motif for the Epe1-JHDM1 sub- [Keywords: H3K9me3; heterochromatin; Jmjd2b hydroxylase; demethyl- group (Fbx110, Fbx111, Phf2, Phf8, and A630082K20RIK), ation] the Jarid subgroup (Rbbp2, Plu1, Smcx, and Smcy) has 1These authors contributed equally to this work. additional domains, such as BRIGHT/ARID, Zn-fingers, 2 Corresponding author. and a jmjN domain of no proposed function (Clissold and E-MAIL [email protected]; FAX 43-1-798-7153. Article published online ahead of print. Article and publication date are Ponting 2001). The Jarid subgroup also contains a Dro- online at http://www.genesdev.org/cgi/doi/10.1101/gad.388206. sophila gene, termed lid (Gildea et al. 2000), that classi- GENES & DEVELOPMENT 20:1557–1562 © 2006 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/06; www.genesdev.org 1557 Downloaded from genesdev.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Fodor et al. fies as an E(var) gene in position effect variegation (PEV) fragment displays enrichment at pericentric regions (G. Reuter and H. Baisch, pers. comm.). This genetic (arrows in Fig. 1B) that is not observed for the 1–424 characterization would be consistent with the wild-type (H189A) mutant nor for the full-length protein, which Lid protein being capable of counteracting a repressive rather broadly localize in the nucleus. Moreover, peri- function in heterochromatin or enhancing activating centric accumulation for the 1–424 Jmjd2b-GFP variant properties of euchromatin, as is the case for Epe1 in S. is lost in Suv39h dn iMEFs (Fig. 1B, bottom panel). pombe (Ayoub et al. 2003). The Jmjd2 subgroup (Jmjd2a, These data demonstrate that both the jmjN and jmjC Jmjd2b, Jmjd2c, Jmjd2d, and XP916094) has jmjN and domains are essential for the observed reduction of peri- jmjC domains, and three members also contain addi- centric H3K9me3, whereas the PHD Zn-fingers and Tu- tional PHD Zn-fingers and two Tudor domains that dor domains are dispensable, although they may contrib- would indicate a possible chromatin association (Katoh ute to overall Jmjd2b activity, for example, via chroma- and Katoh 2004; Gray et al. 2005; Kim et al. 2006). tin affinity and/or protein complex formation with associated factors. Jmjd2b abrogates pericentric H3K9me3 Jmjd2b reduces H3K9me3 and H3K36me2 GFP fusion constructs of murine jmjC proteins were transiently expressed in female iMEFs, followed by indi- in bulk histones rect immunofluorescence (IF) with highly selective From the above in vivo analyses, we reasoned to have H3K9, H3K27, and H4K20 methyl-lysine histone anti- identified the major components (enzyme and substrate) bodies. For each of these repressive marks, we analyzed for the direct demonstration of biochemical activity of the described distributions for mono-, di-, and trimeth- Jmjd2b. However, all our efforts with recombinant full- ylation profiles (Lachner et al. 2004) and also included length Jmjd2b or with the 1–424 Jmjd2b variant, either stainings for H3K4 methylation. As shown in Figure 1A, purified from bacteria or insect cells and even when iso- overexpression of Jmjd2b results in loss of H3K9me3 at lated as a complex from mammalian cells, have been the characteristic pericentric foci in GFP-positive cells unsuccessful in in vitro demethylation assays with (top panel). There was no apparent difference for the dis- H3K9me3 peptides, calf thymus histones, and in vitro persed H3K9me2 distribution; however, a significant in- methylated nucleosomes. These negative results suggest crease for H3K9me1 was observed. These data suggest that recombinant Jmjd2b has only very poor enzymatic conversion from H3K9me3 to H3K9me1 by overexpres- activity or that Jmjd2b-mediated removal of H3K9me3 sion of Jmjd2b-GFP. Also accumulation of pericentric may not be a simple two-component system or requires HP1␣ was lost, but there was no detectable shift for native chromatin substrates. H3K9 acetylation toward pericentric heterochromatin We therefore focused again on in vivo approaches by (Fig. 1A, bottom panels). In parallel experiments, we ob- generating stable mouse cell lines (NIH3T3 Tet-off) that served no gross changes in the characteristic staining inducibly express GFP-tagged full-length Jmjd2b (clone patterns for H3K27me1 (pericentric), H3K27me3 (Xi), or IB21), the 1–424 truncation variant (clone T2J3), and the for H4K20me1 (Xi) and
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