Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription1,2,3. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species4,5,6. H3K9me3 has long been regarded as a ‘permanent’ epigenetic mark7,8. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1)9, which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C10. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and α-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas9,11,12, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.
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We are grateful to U. Toftegaard and S. Keshtar for technical assistance. We thank A. Bracken, A. H. Lund and C. Storgaard Sørensen for critical reading of the manuscript. We thank members of the Helin laboratory and M. Salek for technical advice and support. This work was supported by grants from the Danish Cancer Society, the Novo Nordisk Foundation, the Danish Medical Research Council, the Danish Natural Science Research Council and the Danish Ministry of Science, Technology and Innovation. Author Contributions P.A.C.C. performed the in vitro binding experiments identifying GASC1 as an H3K9me3 interactor, suggested that GASC1 could be an H3K9me3-specific demethylase and co-wrote the paper. J.C. performed most DNA cloning steps and produced recombinant proteins. P.A.C.C. and J.C. established the in vitro demethylation assays, identified GASC1 as an H3K9me3-specific demethylase and performed various biochemical experiments. K.A. performed the in vivo experiments and immunofluorescence studies. K.H.H. designed the peptides and implemented the in vitro binding assays, which identified H3K9me3 interactors, and performed various biochemical experiments including preparation of samples for mass spectrometry. A.M. and J.R. performed mass spectrometry. T.A. performed in silico modelling studies. K.H. suggested the strategy to identify proteins binding to the modified histone tails, contributed to the planning of experiments and co-wrote the manuscript. All authors discussed the results and commented on the manuscript.
This file contains the Supplementary Methods, additional references and Supplementary Figures 1–13.
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