Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-β-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127–TRMT112, hereafter termed ‘lysine methyltransferase 9’ (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor–dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
RNA-seq and ChIP-seq data have been deposited in the GEO under accession code GSE117536. The crystallographic data have been deposited in the Protein Data Bank under the accession codes PDB 6H1D and PDB 6H1E. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008965. PRM data have been deposited to PASSEL (peptide Atlas platform) with the dataset identifier PASS01154. Source data for Figs. 2c,f,h, 3c–e, 4b,f,i–k, 5a–g, and 6a,d,e,h are available online. All other data are available upon reasonable request.
Strahl, B. D. & Allis, C. D. The language of covalent histone modifications. Nature 403, 41–45 (2000).
Arrowsmith, C. H., Bountra, C., Fish, P. V., Lee, K. & Schapira, M. Epigenetic protein families: a new frontier for drug discovery. Nat. Rev. Drug Discov. 11, 384–400 (2012).
Dillon, S. C., Zhang, X., Trievel, R. C. & Cheng, X. The SET-domain protein superfamily: protein lysine methyltransferases. Genome Biol. 6, 227 (2005).
van Leeuwen, F., Gafken, P. R. & Gottschling, D. E. Dot1p modulates silencing in yeast by methylation of the nucleosome core. Cell 109, 745–756 (2002).
Petrossian, T. C. & Clarke, S. G. Uncovering the human methyltransferasome. Mol. Cell Proteomics 10, M110 000976 (2011).
Le Guen, L., Santos, R. & Camadro, J. M. Functional analysis of the hemK gene product involvement in protoporphyrinogen oxidase activity in yeast. FEMS Microbiol. Lett. 173, 175–182 (1999).
Cheng, X. Structure and function of DNA methyltransferases. Ann. Rev. Biophys. Biomol. Struct. 24, 293–318 (1995).
Yang, Z. et al. Structural characterization and comparative phylogenetic analysis of Escherichia coli HemK, a protein (N5)-glutamine methyltransferase. J. Mol. Biol. 340, 695–706 (2004).
Ratel, D. et al. Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase. FEBS Lett. 580, 3179–3184 (2006).
Xiao, C. L. et al. N(6)-methyladenine DNA modification in the human genome. Mol. Cell 71, 306–318.e7 (2018).
Schiffers, S. et al. Quantitative LC-MS provides no evidence for m(6) dA or m(4) dC in the genome of mouse embryonic stem cells and tissues. Angew Chem. Int. Ed. Engl. 56, 11268–11271 (2017).
Liu, P. et al. Deficiency in a glutamine-specific methyltransferase for release factor causes mouse embryonic lethality. Mol. Cell Biol. 30, 4245–4253 (2010).
Heurgue-Hamard, V. et al. The zinc finger protein Ynr046w is plurifunctional and a component of the eRF1 methyltransferase in yeast. J. Biol. Chem. 281, 36140–36148 (2006).
Figaro, S., Scrima, N., Buckingham, R. H. & Heurgue-Hamard, V. HemK2 protein, encoded on human chromosome 21, methylates translation termination factor eRF1. FEBS Lett. 582, 2352–2356 (2008).
Zorbas, C. et al. The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis. Mol. Biol. Cell 26, 2080–2095 (2015).
Cai, X. C., Kapilashrami, K. & Luo, M. Synthesis and assays of inhibitors of methyltransferases. Methods Enzymol. 574, 245–308 (2016).
Schubert, H. L., Blumenthal, R. M. & Cheng, X. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28, 329–335 (2003).
Liger, D. et al. Mechanism of activation of methyltransferases involved in translation by the Trm112 ‘hub’ protein. Nucleic Acids Res. 39, 6249–6259 (2011).
Schubert, H. L., Phillips, J. D. & Hill, C. P. Structures along the catalytic pathway of PrmC/HemK, an N5-glutamine AdoMet-dependent methyltransferase. Biochemistry 42, 5592–5599 (2003).
Graille, M. et al. Molecular basis for bacterial class I release factor methylation by PrmC. Mol. Cell 20, 917–927 (2005).
Nadal, R. & Bellmunt, J. The evolving role of enzalutamide on the treatment of prostate cancer. Future Oncol. 12, 607–616 (2016).
Leroy, G. et al. A quantitative atlas of histone modification signatures from human cancer cells. Epigenetics Chromatin 6, 20 (2013).
Feller, C., Forne, I., Imhof, A. & Becker, P. B. Global and specific responses of the histone acetylome to systematic perturbation. Mol. Cell 57, 559–571 (2015).
Ong, S. E., Mittler, G. & Mann, M. Identifying and quantifying in vivo methylation sites by heavy methyl SILAC. Nat. Methods 1, 119–126 (2004).
Liu, X., Kraus, W. L. & Bai, X. Ready, pause, go: regulation of RNA polymerase II pausing and release by cellular signaling pathways. Trends Biochem. Sci. 40, 516–525 (2015).
Kusevic, D., Kudithipudi, S. & Jeltsch, A. Substrate specificity of the HEMK2 protein glutamine methyltransferase and identification of novel substrates. J. Biol. Chem. 291, 6124–6133 (2016).
Hoefer, J. et al. Critical role of androgen receptor level in prostate cancer cell resistance to new generation antiandrogen enzalutamide. Oncotarget 7, 59781–59794 (2016).
Metzger, E. et al. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437, 436–439 (2005).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Thorvaldsdottir, H., Robinson, J. T. & Mesirov, J. P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).
Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589 (2010).
Orlando, D. A. et al. Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep. 9, 1163–1170 (2014).
Metzger, E. et al. Assembly of methylated KDM1A and CHD1 drives androgen receptor-dependent transcription and translocation. Nat. Struct. Mol. Biol. 23, 132–139 (2016).
Robinson, M. D. & Smyth, G. K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008).
Gross, A., Geresh, S. & Whitesides, G. M. Enzymatic synthesis of S-adenosyl-L-methionine from L-methionine and ATP. Appl. Biochem. Biotechnol. 8, 415–422 (1983).
Schmid-Burgk, J. L. et al. OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines. Genome Res. 24, 1719–1723 (2014).
Schmidt, T., Schmid-Burgk, J. L. & Hornung, V. Synthesis of an arrayed sgRNA library targeting the human genome. Sci. Rep. 5, 14987 (2015).
Alabert, C. et al. Two distinct modes for propagation of histone PTMs across the cell cycle. Genes Dev. 29, 585–590 (2015).
Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).
Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, (213–221 (2010).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
We thank Z. Culig (Department of Urology, Medical University of Innsbruck, Innsbruck, Austria) and R. Schneider (Institute of Functional Epigenetics, Helmholtz Zentrum München, Ludwig Maximilians Universität, Munich) for providing reagents. We are obliged to A. Rieder for providing excellent technical assistance. We are grateful to Swiss Light Source (SLS) beam line scientists for the technical support. This work was supported by grants of the European Research Council (ERC AdGrant 322844) and the 15-1 to R. SFB 992, 850, 746, and Schu688/15-1 to R.S.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a, Extracted Ion Chromatograms (XICs) of targeted LC–MS/MS runs of un- or monomethylated H4 aa 4-17 peptides. Histone H4 was incubated in the absence or presence of C21orf127–TRMT112 without or with SAM, heavy SAM (hSAM) or SAM–hSAM. The experiment was repeated three times independently with similar results. b-f, Characterization of the anti-H4K12me1-specific rabbit polyclonal antibody. b, C21orf127–TRMT112 was incubated with wild-type or mutant histone H4 and SAM as indicated. Western blot was decorated with anti-H4K12me1 antibody and proteins were stained by Ponceau red. c-f, Specificity of the anti-H4K12me1 antibody was verified using serial dilutions of peptides carrying various histone modifications as indicated. Dot blots were decorated with anti-H4K12me1 antibody and peptides were stained by Ponceau red. The experiments b-f were repeated two times independently with similar results.
Supplementary Figure 2 C21orf127–TRMT112 heterodimerization is obligatory for SAM binding and H4K12 methylation.
Supplementary Fig. 2 a, Ribbon representation of C21orf127 (grey) TRMT112 (cyan) in complex with H4 aa 11-15 K12me1 and SAH. The H4 aa 11-15K12me1 peptide (yellow) and SAH (magenta) are represented as stick models. b, c, Methylation assays. b, C21orf127–TRMT112 or C21orf127(D103)–TRMT112 were incubated with histone H4 and SAM. c, C21orf127–TRMT112, C21orf127(N122A)–TRMT112, C21orf127, or TRMT112 were incubated with histone H4 and SAM. Proteins were stained by Ponceau red. Histone H4 methylation was revealed by autoradiography. d, C21orf127–TRMT112 and C21orf127(N122A)–TRMT112 heterodimers were analysed by gel filtration chromatography. Both heterodimers show a similar elution profile. Heterodimers were separated by SDS-PAGE and visualised by Coomassie blue staining. e, Representation of the H4 substrate-binding pocket of C21orf127 with H4 aa 11-15K12me1 peptide. The substrate pocket of KMT9α is shown as grey surface and histone H4 aa 11-15 is depicted as yellow sticks. The distance (1.8Å) between the mutated G14A and the main chain of E24 is shown as dashed red line, indicating a possible steric clash between the side chain of H4A14 and the main chain of E24. f, Representation of the amino acid sequences flanking the lysine residues present in histones H3 and H4. g-j, Representative ITC experiments displaying titration of histone H4 aa 1-21 (g-i), H4 aa8-21 (g), H4 aa1-7 (g), H4 aa1-16 (h) and ETF1 aa141-274 (j) to C21orf127–TRMT112 in presence or absence of SAH as indicated. k, Crystal structure of PrmC-Erf1 superimposed with the structure of H4 aa 11-15 peptide-bound C21orf127–TRMT112. C21orf127 and H4 peptide are shown in grey and yellow. PrmC and eRF1 are shown in green and cyan. The conserved ‘NPPY’ motif of C21orf127 and PrmC are shown as grey and green sticks, respectively. H4K12me1 and Q235 of eRF1 are shown as yellow and cyan sticks. The hydrogen bond is showed as yellow dash line. The experiments b-d, g-j were repeated three times independently with similar results.
a, KMT9 protein and H4K12me1 levels in cancer cell lines. The levels of KMT9α, KMT9β, and H4K12me1 present in SK-N-SH glioblastoma, LNCaP and PC-3M prostate tumor, and HT-29 colon carcinoma cells were analysed by Western blotting using the indicated antibodies (left panel). Upon extraction of histone H4, the relative abundance of H4 aa 4-17 peptides monomethylated at K12 in the different cell lines was calculated using parallel reaction monitoring (PRM) quantifications based on three specific fragment ions (y6, y7, y9) and represented as bar graph. n = 4 biologically independent samples. Data represent means + s.d. b-e, Analysis of H4K12 methylation by KMT9 in HEK 293 cells. b, Lysates of proficient control and KMT9α−/− HEK 293 cells were separated by SDS-PAGE and analysed by Coomassie blue staining or Western blotting using the indicated antibodies The experiments were repeated three times independently with similar results. Subsequently, histones H4 were isolated, propionylated, and digested with trypsin. c, The resulting H4 aa 4-17 peptides were selected for targeted MS/MS analysis. XICs of the indicated peptide isoforms are shown. Spectra represent the average of 8 MS/MS scans of the PRM analysis of H4 peptides at the elution time of the monomethylated H4 aa 4-17 peptide at around 65 min RT. Red arrows indicate the retention times of the methylated H4 aa 4-17 peptides. No signal corresponding to methylated H4 aa 4-17 was detected in HEK 293 KMT9α−/− cells. NL: normalized level. AU: arbitrary units. d, MS/MS analysis of H4 aa 4-17 peptides isolated from KMT9-proficient control HEK 293 cells detected incorporation of a methyl group for fragments y6, y7, and y9 (y6+_Me, y7+_Me, y9+_Me) demonstrating H4K12 monomethylation. m/z= mass-to-charge ratio; z= charge state calculation based on the observed isotope distribution. The experiments were repeated four times independently with similar results. e, The relative abundance of H4 aa 4-17 peptides monomethylated at K12 in proficient control and HEK 293 KMT9α−/− cells was calculated by averaging the selected reaction monitoring (SRM) quantifications for all three transitions. n = 4 biologically independent samples. Data represent means + s.d.; *** p < 0.001 by two-tailed Student’s test.
a, b, Pie charts showing genomic distribution of KMT9α (a) and KMT9β (b) peaks in PC-3M cells. c, f, Venn diagrams showing number and overlap of KMT9α (c) and KMT9β (f) peaks in PC-3M cells treated with siCtrl, siKMT9α, or siKMT9β as indicated. d, g, Meta-analyses of sequencing read density based on KMT9α ChIP-seq around KMT9α peaks (d) and KMT9β ChIP-seq around KMT9β peaks (g) in PC-3M cells treated with siCtrl, siKMT9α, or siKMT9β as indicated. e, h, Heat maps of ChIP-seq signals for KMT9α (e) and KMT9β (h) ± 2 kb around KMT9α (e) or KMT9β (h) peak center in PC-3M treated with siCtrl, siKMT9α, or siKMT9β as indicated. i, Number of genes differentially expressed upon RNAi-mediated knockdown of KMT9α in MCF10A cells (top panel) and RNA-seq track showing that KMT9α is not expressed (bottom panel). j, Pie chart showing genomic distribution of H4K12me1 peaks in PC-3M cells. k, Average KMT9α, KMT9β, and H4K12me1 ChIP-seq read density profiles in PC-3M cells. l, Venn diagram showing number and overlap of H4K12me1 peaks in PC-3M cells treated with siCtrl or siKMT9α. m, Meta-analysis of sequencing read density based on H4K12me1 ChIP-seq around H4K12me1 peaks in PC-3M cells treated with siCtrl or siKMT9α. n, Heat maps of ChIP-seq signals for H4K12me1 ± 2 kb around the H4K12me1 peak centers in PC-3M treated with siCtrl or siKMT9α. o, ChIP-seq tracks showing KMT9α, KMT9β, and H4K12me1 at representative genes in PC-3M treated with siCtrl or siKMT9α. The experiment was repeated two times with similar results. p, q, Average Pol II (p), and Pol II CTD S5ph (q) ChIP-seq read density profiles around the transcription start site (TSS) in PC-3M cells treated with siCtrl and siKMT9α for non-differentially expressed genes (left panel) and genes that are non- or low expressed (right panel) upon KMT9α knockdown.
a, Figure exemplifying the gating strategy used to assess by flow cytometry and propidium iodide (PI) the cell cycle phase distribution in PC-3M cells treated with siCtrl and siKMT9α. b, Figure exemplifying the gating strategy used to identify by flow cytometry (using AnnexinV and PI staining) apoptotic cells in PC-3M treated with siCtrl and siKMT9α. c-f, Proliferation of C4-2B (c), LNCaP-abl (d), LNCaP-abl EnzaR (e), and DuCaP EnzaR (f) cells transfected with siCtrl or siKMT9α as indicated. Western blot analyses were performed with the indicated antibodies to verify knockdown of KMT9α and decreased H4K12me1 levels (c-f). n = 4 biologically independent samples. Data represent means ± s.d.; ***p < 0.001 by two-tailed Student’s test. g, Proliferation of MCF10A cells that do not express KMT9α is not affected by treatment with siKMT9α. To verify absence of KMT9α, Western blot analyses were performed using the indicated antibodies. n = 4 biologically independent samples. Data represent means ± s.d. h, The proliferation rate of HEK 293 KMT9α−/− cells is similar to that of proficient HEK 293 indicating that KMT9α depletion does not alter cell proliferation. To verify KMT9α depletion, Western blot analyses were performed using the indicated antibodies. n = 3 biologically independent samples. Data represent means ± s.d. i, Knockdown of KMT9α does not affect proliferation of non-cancerous C2C12 myoblast cells. n = 4 biologically independent samples. Data represent means ± s.d. j-l, Knockdown of KMT9α does not affect proliferation of HepG2 hepatocarcinoma (j), HCT116−/− colon carcinoma (k), and NGP glioblastoma (l) cells. To verify KMT9α knockdown, Western blot analyses were performed with the indicated antibodies. Data represent means ± s.d. n = 4 biologically independent samples. The experiments c-l were repeated three times independently with similar results. m, n, ETF1 is not methylated in PC-3M (m) and LNCaP (n) cells. ETF1 was immunoprecipitated from lysates of PC-3M (m) and LNCaP (n) cells treated with siCtrl or siKMT9α and analysed by Western blot with the indicated antibodies. o, Methylation assay. ETF1 incubated with SAM in the presence or absence of KMT9α–KMT9β, was analysed by Ponceau red staining and Western blotting with the anti-ETF1me antibody. The experiments m-o were repeated three times independently with similar results. p, Proliferation of LNCaP cells transfected with siCtrl or siKMT9α and infected with lentivirus driving expression of either LacZ or ETF1. KMT9α and ETF1 levels were analysed by Western blotting using the indicated antibodies. Data represent means ± s.d.; **p < 0.01, ***p < 0.001 by two-tailed Student’s test; n = 3 biologically independent samples. The experiments were repeated two times independently with similar results.
a-d, Characterization of LNCaP xenograft tumors. a, LNCaP cells were infected with lentiviruses driving expression of either miRNA Ctrl or a miRNA against KMT9α. Growth of LNCaP xenograft tumors was measured over time. b, Western blot analysis using the indicated antibodies verified depletion of KMT9α in LNCaP cells. The experiments were repeated three times independently with similar results. c, Representation of LNCaP xenograft tumors isolated from individual animals at day 50. d, Tumor weight of LNCaP xenografts at day 50. n = 10 mice. Data represent means ± s.e.m (a) and mean + s.e.m (d), **p < 0.01, ***p < 0.001 by two-tailed Student’s test.
About this article
Cite this article
Metzger, E., Wang, S., Urban, S. et al. KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells. Nat Struct Mol Biol 26, 361–371 (2019). https://doi.org/10.1038/s41594-019-0219-9
International Journal of Oncology (2020)
Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA
Nucleic Acids Research (2020)
Depletion of histone methyltransferase KMT9 inhibits lung cancer cell proliferation by inducing non-apoptotic cell death
Cancer Cell International (2020)
Journal of Molecular Biology (2020)
An engineered variant of SETD3 methyltransferase alters target specificity from histidine to lysine methylation
Journal of Biological Chemistry (2020)