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Science 19 October 2007:
Vol. 318. no. 5849, pp. 444 - 447
DOI: 10.1126/science.1145801
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Reports
JMJD6 Is a Histone Arginine Demethylase
Bingsheng Chang,
Yue Chen,
Yingming Zhao and
Richard K. Bruick*
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390–9038, USA.
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Fig. 1. JMJD6 is a putative Fe(II)- and 2-oxoglutarate–dependent dioxygenase. (A) JMJD6 demethylates H3R2me2 and H4R3me2. Bulk histones were incubated in the presence (+) or absence (–) of purified recombinant JMJD6. Antibodies (Abcam) specific for the indicated histone methylation sites were used to detect loss of these modifications by Western blot analysis. Recognition of the indicated methylated sites was confirmed with blocking peptides (fig. S2A). Total amounts of histone H3 and H4 are shown as loading controls. Similar results were obtained with antibodies from another manufacturer (fig. S2B). (B) The  -H4R3me1 and -H4R3me2 antibodies specifically recognize synthetic peptide substrates, where R3 contains one (1) or two (2) methyl groups, respectively. (C) JMJD6 demethylates both mono- and dimethylarginine residues. Bulk histones or histone H4 peptides were synthesized with symmetric dimethylarginine (p2meR3H4) or monomethylarginine (p1meR3H4) and incubated in the absence (–) or presence of wt JMJD6 protein or a catalytically inactive (mut) JMJD6 (H187A; D189A; H273A). Demethylation was assessed by Western blot analysis.
[View Larger Version of this Image (66K GIF file)]
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Fig. 2. JMJD6 is a Fe(II)- and 2-oxoglutarate–dependent dioxygenase. (A) Formation of the H4R3me1 product from symmetric (SDMA) or asymmetric (ADMA) dimethylarginine containing p2meR3H4 substrates. Peptide substrates derived from histone H4 containing two or zero methyl groups on R3 were incubated in the absence (–) or presence (+) of wt JMJD6 protein or a catalytically dead (mut) JMJD6 (H187A; D189A; H273A). The -H4R3me1 antibody was used to detect formation of this product by Western blot analysis. (B) Formation of the demethylated H4R3me1 product from the p2meR3H4 substrate by JMJD6 requires Fe(II), ascorbate, and 2-oxoglutarate (2-OG). (C) JMJD6-mediated arginine demethylation generates formaldehyde. Recombinant histone H3 was methylated on R2, R17, and R26 by CARM1 (28), and recombinant histone H4 was methylated on R3 by PRMT1 (28) with [3H]-S-adenosyl methionine. As assayed by the modified Nash method (29), 3H-methyl groups were liberated as formaldehyde by wt JMJD6, but not by buffer alone or mut JMJD6. 3H-formaldehyde is not produced by JMJD6 from histone H3 methylated on K36 by Set2 (D), which otherwise serves as a substrate for the JmjC-containing lysine demethylase Yer051w (4). Assays were performed in triplicate with bars indicating standard error.
[View Larger Version of this Image (27K GIF file)]
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Fig. 3. Analysis of demethylation products by mass spectrometry. (A) JMJD6 demethylates p2meR3H4 to the monomethylated product (1me). The p2meR3H4 peptide substrate [expected protonated molecular mass (M + 1) = 3129.6 daltons] was incubated with buffer alone or JMJD6 (wt or mut). Products were immunoprecipitated with the -H4R3me1 antibody before analysis. A p1meR3H4 peptide synthesized with a stable isotope of leucine (containing six 13C and one 15N atoms) was added as an internal standard (S) before immunoprecipitation. The peak with m/z 3112.85 in the top panel is a deaminated impurity that was removed during immunoprecipitation. (B) ETD fragmentation of the demethylation products (+5 charged ions) indicates that in addition to demethylation, JMJD6 can promote oxidation of nearby lysine residues [1me + 1O or 1me + 2O in (A)]. The relevant ion fragments are labeled and the corresponding peptide positions are illustrated. Fragments containing a single oxidation modification are denoted by "***" and fragments containing two oxidation events are denoted by "^^". Shown are the partial (for full spectra, see fig. S4) MS/MS spectrum of the monomethyl product (top panel), the monomethyl product oxidized on K8 (middle panel), and the monomethyl product with oxidation of both K5 and K8 (bottom panel).
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Fig. 4. JMJD6 promotes histone arginine demethylation in cultured HeLa cells. Transient overexpression of V5-tagged wild-type (wt), but not an inactive variant (mut) of JMJD6, reduces global amounts of H3R2me2 (A) and H4R3me2 (B), but not H3R17me2 (C), H3R26me2 (D), or H3K4me3 (E). DAPI (4',6-diamidino-2-phenylindole) staining marks the location of nuclei in the field, and arrows indicate transfected cells.
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