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. 2007 Nov 20;104(47):18439-44.
doi: 10.1073/pnas.0707292104. Epub 2007 Nov 14.

Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases

Sunhwa Hong  1 , Young-Wook Cho, Li-Rong Yu, Hong Yu, Timothy D Veenstra, Kai Ge
Affiliations

Identification of JmjC domain-containing UTX and JMJD3 as histone H3 lysine 27 demethylases

Sunhwa Hong et al. Proc Natl Acad Sci U S A. .

Abstract

Covalent modifications of histones, such as acetylation and methylation, play important roles in the regulation of gene expression. Histone lysine methylation has been implicated in both gene activation and repression, depending on the specific lysine (K) residue that becomes methylated and the state of methylation (mono-, di-, or trimethylation). Methylation on K4, K9, and K36 of histone H3 has been shown to be reversible and can be removed by site-specific demethylases. However, the enzymes that antagonize methylation on K27 of histone H3 (H3K27), an epigenetic mark important for embryonic stem cell maintenance, Polycomb-mediated gene silencing, and X chromosome inactivation have been elusive. Here we show the JmjC domain-containing protein UTX (ubiquitously transcribed tetratricopeptide repeat, X chromosome), as well as the related JMJD3 (jumonji domain containing 3), specifically removes methyl marks on H3K27 in vitro. Further, the demethylase activity of UTX requires a catalytically active JmjC domain. Finally, overexpression of UTX and JMJD3 leads to reduced di- and trimethylation on H3K27 in cells, suggesting that UTX and JMJD3 may function as H3K27 demethylases in vivo. The identification of UTX and JMJD3 as H3K27-specific demethylases provides direct evidence to indicate that similar to methylation on K4, K9, and K36 of histone H3, methylation on H3K27 is also reversible and can be dynamically regulated by site-specific histone methyltransferases and demethylases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
UTX specifically demethylates histone H3K27 in vitro. (A) Schematic representation of human UTX protein with six TPR (tetratricopeptide repeat) domains and one JmjC domain. (B) Coomassie blue staining of purified UTX protein. 293T cells were transfected with plasmids expressing FLAG- and myc-tagged UTX or vector only (Vec). Whole-cell extracts were prepared with 1% Nonidet P-40-containing buffer and were incubated with anti-FLAG M2–agarose. After extensive washing, bound proteins were eluted with FLAG peptide, resolved on SDS/PAGE 4–15% followed by Coomassie blue staining. IP, immunoprecipitation. M, protein marker. (C) Eluate corresponding to ≈1 μg of purified UTX was incubated with 2.5 μg of calf core histone in histone demethylase assay. Assay products were analyzed by Western blotting, with antibodies indicated on the right. Where indicated, me1 is monomethyl, me2 is dimethyl, and me3 is trimethyl. (D) Increasing amounts of purified UTX were subjected to histone demethylase assay. (E–G) Mass spectrometry analysis of UTX activity toward H3K27me3 (E), H3K27me2 (F), and H3K4me3 (G) peptides. Three micrograms of UTX was incubated with 1 μg of peptide in histone demethylase assay followed by MALDI-TOF mass spectrometry analysis. The masses of the H3K27me3, H3K27me2, and H3K4me3 peptides were 2,958.7, 2,944.7, and 1,189.7 Da, respectively.
Fig. 2.
Fig. 2.
The H3K27 demethylase activity of UTX requires a catalytically active JmjC domain. (A) The TPR domains are required for the optimal demethylase activity of UTX on H3K27me1 but are dispensable for UTX activity on H3K27me2/3. (Upper) Schematic representation of full-length and 401-1401 aa fragment of human UTX protein. The location of the conserved Fe(II)-binding H1146 within JmjC catalytic domain is indicated. (Lower) Plasmids expressing FLAG-tagged UTX, either full-length or 401-1401 aa fragment, were transfected into 293T cells followed by protein isolation and histone demethylase assay on calf core histones as described in Fig. 1. IP, immunoprecipitation. (B) Mutation of the Fe(II)-binding H1146 in the JmjC domain abrogates the H3K27 demethylase activity of UTX. Plasmids expressing FLAG-tagged wild-type or H1146A mutant UTX were transfected into 293T cells followed by protein isolation and histone demethylase assay as described above.
Fig. 3.
Fig. 3.
Overexpression of wild-type UTX but not the H1146A mutant results in reduced H3K27 methylation in cells. COS-7 cells transfected with myc-tagged wild-type or H1146A mutant UTX were stained with anti-myc and anti-H3K27me3 (A), anti-H3K27me2 (B), or anti-H3K27me1 antibody (C). The green corresponds to anti-myc antibody staining; the red corresponds to the staining by specific methylation antibody; and the blue corresponds to DAPI staining of nuclei. Cells with UTX overexpression (indicated by arrows) were scored for the signal intensity of H3K27me3, H3K27me2, and H3K27me1 under the microscope, and the quantitation results are shown in Table 1. The experiments were repeated at least three times, and a representative result is shown.
Fig. 4.
Fig. 4.
JMJD3 is a H3K27-specific demethylase. (A) Schematic representation of protein sequence alignment of UTX, UTY, and JMJD3, with the degree of sequence similarity to UTX shown. The protein sequence identities are shown in parentheses. The JmjC domain containing 1174–1636 aa of JMJD3 shares 84% sequence similarity with the JmjC domain containing 931-1394 aa of UTX. (B) JMJD3 specifically demethylates H3K27me2 and H3K27me3 in vitro. Plasmid expressing FLAG-tagged JMJD3 was transfected into 293T cells followed by protein isolation and histone demethylase assay on calf core histones as in Fig. 1. (C) Purified UTY protein has no H3K27 demethylase activity in vitro. Plasmid expressing FLAG-tagged UTY or UTX was transfected into 293T cells followed by protein isolation and histone demeythylase assay as in Fig. 1. Approximately 120 ng of purified UTX and UTY proteins were used in demethylase assay on calf core histones. (D) Overexpression of JMJD3 results in reduced H3K27me2/3 in cells. COS-7 cells transfected with myc-tagged JMJD3 were stained with anti-myc (green) and anti-H3K27me3 (Upper, red) or anti-H3K27me2 (Lower, red) antibody as described in Fig. 3. The arrows point to cells with JMJD3 overexpression.
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