Abstract
Prostate cancer evolution is driven by a combination of epigenetic and genetic alterations such as coordinated chromosomal rearrangements, termed chromoplexy. TMPRSS2-ERG gene fusions found in human prostate tumors are a hallmark of chromoplexy. TMPRSS2-ERG fusions have been linked to androgen signaling and depend on androgen receptor (AR)-coupled gene transcription. Here, we show that dimethylation of KDM1A at K114 (to form K114me2) by the histone methyltransferase EHMT2 is a key event controlling androgen-dependent gene transcription and TMPRSS2-ERG fusion. We identified CHD1 as a KDM1A K114me2 reader and characterized the KDM1A K114me2–CHD1 recognition mode by solving the cocrystal structure. Genome-wide analyses revealed chromatin colocalization of KDM1A K114me2, CHD1 and AR in prostate tumor cells. Together, our data link the assembly of methylated KDM1A and CHD1 with AR-dependent transcription and genomic translocations, thereby providing mechanistic insight into the formation of TMPRSS2-ERG gene fusions during prostate-tumor evolution.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Siegel, R., Naishadham, D. & Jemal, A. Cancer statistics, 2013. CA Cancer J. Clin. 63, 11–30 (2013).
Taylor, B.S. et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22 (2010).
Rubin, M.A., Maher, C.A. & Chinnaiyan, A.M. Common gene rearrangements in prostate cancer. J. Clin. Oncol. 29, 3659–3668 (2011).
Barbieri, C.E. et al. Exome sequencing identifies recurrent SPOP, FOXA1 and MED12 mutations in prostate cancer. Nat. Genet. 44, 685–689 (2012).
Baca, S.C. et al. Punctuated evolution of prostate cancer genomes. Cell 153, 666–677 (2013).
Tomlins, S.A. et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310, 644–648 (2005).
Burkhardt, L. et al. CHD1 is a 5q21 tumor suppressor required for ERG rearrangement in prostate cancer. Cancer Res. 73, 2795–2805 (2013).
Metzger, E. et al. LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437, 436–439 (2005).
Kahl, P. et al. Androgen receptor coactivators lysine-specific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res. 66, 11341–11347 (2006).
Cai, C. et al. Androgen receptor gene expression in prostate cancer is directly suppressed by the androgen receptor through recruitment of lysine-specific demethylase 1. Cancer Cell 20, 457–471 (2011).
Shi, Y. et al. Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119, 941–953 (2004).
Ko, S. et al. Lysine methylation and functional modulation of androgen receptor by Set9 methyltransferase. Mol. Endocrinol. 25, 433–444 (2011).
Xu, K. et al. EZH2 oncogenic activity in castration-resistant prostate cancer cells is Polycomb-independent. Science 338, 1465–1469 (2012).
Vedadi, M. et al. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat. Chem. Biol. 7, 566–574 (2011).
Kubicek, S. et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol. Cell 25, 473–481 (2007).
Kim, J. et al. Tudor, MBT and chromo domains gauge the degree of lysine methylation. EMBO Rep. 7, 397–403 (2006).
Flanagan, J.F. et al. Double chromodomains cooperate to recognize the methylated histone H3 tail. Nature 438, 1181–1185 (2005).
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).
Tan, P.Y. et al. Integration of regulatory networks by NKX3-1 promotes androgen-dependent prostate cancer survival. Mol. Cell. Biol. 32, 399–414 (2012).
Wang, Q., Carroll, J.S. & Brown, M. Spatial and temporal recruitment of androgen receptor and its coactivators involves chromosomal looping and polymerase tracking. Mol. Cell 19, 631–642 (2005).
Haffner, M.C. et al. Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nat. Genet. 42, 668–675 (2010).
Metzger, E. et al. Phosphorylation of histone H3T6 by PKCβI controls demethylation at histone H3K4. Nature 464, 792–796 (2010).
Lee, D.Y., Northrop, J.P., Kuo, M.H. & Stallcup, M.R. Histone H3 lysine 9 methyltransferase G9a is a transcriptional coactivator for nuclear receptors. J. Biol. Chem. 281, 8476–8485 (2006).
Huang, S. et al. Recurrent deletion of CHD1 in prostate cancer with relevance to cell invasiveness. Oncogene 31, 4164–4170 (2012).
Duteil, D. et al. LSD1 promotes oxidative metabolism of white adipose tissue. Nat. Commun. 5, 4093 (2014).
Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).
Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).
Willmann, D. et al. Impairment of prostate cancer cell growth by a selective and reversible lysine-specific demethylase 1 inhibitor. Int. J. Cancer 131, 2704–2709 (2012).
Keller, S. et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Chem. 84, 5066–5073 (2012).
Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).
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).
Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).
Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).
Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Robinson, M.D. & Smyth, G.K. Small-sample estimation of negative binomial dispersion, with applications to SAGE data. Biostatistics 9, 321–332 (2008).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).
Dengjel, J. et al. Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens. Mol. Cell Proteomics 11, M111.014035 (2012).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).
Olsen, J.V. et al. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap. Mol. Cell. Proteomics 4, 2010–2021 (2005).
Hagège, H. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat. Protoc. 2, 1722–1733 (2007).
Perner, S. et al. TMPRSS2:ERG fusion-associated deletions provide insight into the heterogeneity of prostate cancer. Cancer Res. 66, 8337–8341 (2006).
Acknowledgements
We thank Y. Shinkai (Institute for Virus research, Kyoto University, Japan) for providing reagents. We are obliged to D. Hassan, J. Kappel, A. Rieder, B. Diedrich and O. Schilling for providing excellent technical assistance. We are obliged to T. Günther, H. Greschik and J.M. Müller for helpful discussions. The authors would like to thank the Swiss Light Source and Diamond Light Source for beam time and their staff for assistance with data collection. J.M. is supported by a Diamond studentship grant. This work was supported by grants of the European Research Council (ERC AdGrant 322844 to R.S.) and the Deutsche Forschungsgemeinschaft (SFB 992, 850, 746, and Schu688/12-1 to R.S.).
Author information
Authors and Affiliations
Contributions
R.S. and E.M. generated the original hypothesis. E.M., J.M., P.M., I.F., K.P., S.U., S.G., A.v.M., D. Wohlwend, A.-K.S., A. Espejo, A. Eberlin, R.F. and K.M.S. performed experiments. D. Willmann performed bioinformatics analyses. S.P., M.S., M.T.B., J.D., A.I., M.J. and O.E. provided intellectual contributions throughout the project. E.M. and R.S. took primary responsibility for writing the manuscript. All authors edited the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 PRMT1, SETD7 and EZH2 do not methylate KDM1A.
(a,b,c) Autoradiographs and Coomassie blue stainings. Samples are recombinantly expressed and purified GST-KDM1A, GST-KDM1A K114A, or core histones incubated with MBP-PRMT1 (a), His-SETD7 (b), or His-EZH2-His-SUZ12-His-(Embryonic Ectoderm Development) EED complex (c) in presence of SAM[3H]. (a-c) Results are representatives of 2 independent experiments.
Supplementary Figure 2 EHMT2 methylates KDM1A at K114 in vivo.
(a-d) Characterization of the rabbit polyclonal anti-KDM1A K114me2 specific antibody. (a,b) Anti-KDM1A K114me2 Western blots and Ponceau S stainings. Samples are peptides. (c,d) Anti-KDM1A K114me2 (c,d), anti-KDM1A and anti-tubulin (d) Western blots. Samples are nuclear extract from LNCaP cells (c), or extracts from LNCaP cells treated with siRNA control (Ctrl) or siRNA against KDM1A (d). (e) Anti-KDM1A K114me2, anti-KDM1A and anti-EHMT2 Western blots. Samples are recombinantly expressed and purified GST-KDM1A or GST-KDM1A K114A proteins that were incubated in the presence or absence of purified GST-EHMT2 aa786-1210 with or without SAM. (f, g) Anti-Flag, anti-KDM1A K114me2 and anti-KDM1A Western blots. Samples are extracts from 293T cells transfected with Flag-KDM1A or Flag-KDM1A K114A in the presence or absence of Flag-EHMT2 (f, g) and cultured with or without the EHMT2 inhibitor UNC0638 (g). Extracts were immunoprecipitated with anti-Flag antibody. (h) Coomassie blue stainings and anti- EHMT2 and anti-KDM1A K114me2 Western blots. Samples are expressed purified GST-KDM1A or GST-KDM1A K114A expressed alone or in combination with GST-EHMT2. (i) Demethylase activity assay. Samples are H3K4me2 peptides that were incubated with GST-KDM1A or GST-KDM1A K114A expressed alone or in combination with GST-EHMT2. (j) Anti-GST, anti-Strep and anti KDM1A Western blots. Samples are GST-PHF21A expressed either alone or in combination with Strep-RCOR1, His-KDM1A, or His-KDM1A aa171-852 and purified over Glutathione SepharoseTM 4B beads. (k) Anti-KDM1A Western blot. Samples are extracts from LNCaP cells that were immunoprecipitated with anti-KDM1A K114me2 antibody. Numbers represent the quantified KDM1A levels detected with the anti-KDM1A antibody. FT: flow-through; IP: immunoprecipitation. Results are representatives of 2 (a-d,g-k) and 4 (e,f) independent experiments.
Supplementary Figure 3 CHD1 interacts with KDM1A K114me2.
(a) Protein-domain microarray. Samples are GST fusion proteins listed in (b) that were arrayed onto nitrocellulose. M contains GST alone. (c) The microarrays were probed with either anti-GST antibody and visualized with a FITC-conjugated secondary antibody or Cy3-labelled KDM1A and KDM1A K114me2 peptides. (d) Coomassie blue staining and table. Samples are extracts of LNCaP cells incubated with column bound KDM1A peptides that were eluted and analyzed as indicated. The table depicts the proteins enriched with the methylated KDM1A peptide. (e) Representative ITC experiment displaying titration of H3 and H3K4me3 peptides to CHD1. (f) Two-dimensional error surface projections of CHD1 (A) and H3K4me3 (B) ITC fit. (g) View of the intermolecular interactions between the KDM1A peptide and CHD1 residues. (h) Superimposition of KDM1A and H3 in complex with CHD1. The surface that could be exploited for the design of inhibitors specifically interfering with binding of KDM1A K114me2 but not H3K4me3 is shown in red. (i,j) Representative ITC experiments displaying titration of KDM1A R113A-K114me2 mutant peptide to CHD1 (i) and KDM1A K114me2 to CHD1 D425A (j). (k) Anti-CHD1, anti-EHMT2, anti-KDM1A and anti-KDM1A K114A Western blots. Samples are extracts from LNCaP cells cultured with or without DHT and Bix-01294 that were immunoprecipitated with anti-EHMT2 or rIgG. Results are representatives of 1 (c,d), 2 (k, i,j) and 3 (e) independent experiments.
Supplementary Figure 4 KDM1A K114me2 and CHD1 co-occupy AR-binding sites.
(a,b) Venn diagrams showing number and overlap of KDM1A K114me2 (a) and CHD1 (b) peaks in LNCaP cells with or without RNAi mediated knockdown (KD) of KDM1A (a) or CHD1 (b). (c) Venn diagram illustrating number and intersection of KDM1A K114me2 and CHD1 peaks that are refractory to RNAi-mediated knockdown of KDM1A and CHD1. (d) Venn diagram depicting number and intersection of AR peaks with RNAi-refractory KDM1A K114me2 and CHD1 peaks. (e) Venn diagram showing number and intersection of the KDM1A K114me2-CHD1-AR co-locations with SUZ12 in LNCaP cells cultured in the presence of DHT. (f) Venn diagram showing number of KDM1A K114me2 locations remaining in the KDM1A K114me2-CHD1-AR intersection (Fig. 3c, 2941 locations) upon RNAi of EHMT2 in LNCaP cells. (g) Venn diagram showing the intersection and number of genes where AR and the RNAi-refractory KDM1A K114me2 and CHD1 peaks co-localize with genes that are differentially regulated in LNCaP cells upon treatment with DHT. (h,i) Box-and-Whisker plots of KDM1A K114me2 (h) and CHD1 (i) ChIP-seq tags around AR peaks in LNCaP cells treated without (-DHT) and with DHT (+DHT). (j) Average KDM1A K114me2 and CHD1 ChIP-seq read density profiles of the 861 genes, which are co-occupied by KDM1A K114me2, CHD1 and AR and differentially expressed upon treatment with DHT. (a-j) Results of genome-wide analyzes are representatives of 2 independent experiments. *** p<0.0001; ** p<0.001 by two-tailed Student’s test.
Supplementary Figure 5 KDM1A K114me2 regulates AR-dependent gene expression by controlling chromatin occupancy of CHD1 and AR.
(a-f) ChIP analyzes performed with anti-KDM1A, anti-KDM1A K114A, anti-EHMT2, anti-CHD1, anti-AR antibodies and rIgG. Samples originate from LNCaP cells that were cultivated in the presence or absence of DHT, treated with or without Bix-01294 (a-c) or transfected with siRNA (d-f) as indicated. The precipitated chromatin was quantified by qPCR analysis using primers flanking AREs in the enhancer regions of the KLK3 ( a,b,e,f) or TMPRSS2 (d) genes and an unrelated control region (c) as indicated. (g-i) Expression analyzes of androgen-regulated genes. Samples originate from LNCaP (g,i) or LAPC4 (h) cells treated with Bix-01294 (g,h) or RNAi against EHMT2, KDM1A, or CHD1 (i). (j) Expression analyzes. Samples originate from LNCaP cells cultured in the absence or presence of DHT and treated with RNAi against KDM1A or CHD1. (d,f,i) Anti-tubulin (d,f,i), anti-KDM1A (d), anti-EHMT2 (f) and anti-CHD1 (i) Western blots. Samples are lysates of LNCaP cells transfected with an unrelated siRNA control (Ctrl) or a siRNA against EHMT2 (f), CHD1 (i), or KDM1A (d). Error bars, s.d. (a-f) Technical replicates of one representative experiment out of three (n=3). (g-j) Biological replicates (n=3 cell cultures). *** p<0.0001; ** p<0.001; * p<0.005 by two-tailed Student’s test.
Supplementary Figure 6 KDM1A K114me2 controls TMPRSS2-ERG fusion.
(a) Schematic representation of DHT-induced intrachromosomal interaction occurring at the TMPRSS2 locus between the enhancer region and a region where DNA double strand breaks (breakpoint) occur during TMPRSS2-ERG fusion. Androgen-dependent chromosomal loop formation is detected in 3C experiments using the indicated primers. AREs at the enhancer and breakpoint regions are labeled yellow and light blue, respectively. Positions of the NspI sites relevant for performing 3C experiment are indicated. (b) 3C analysis. Samples are DNA from LNCaP cells cultured in absence and presence of DHT that were analyzed for the DHT-dependent spatial chromatin interaction of the TMPRSS2 enhancer region and the breakpoint region during TMPRSS2-ERG fusion. The enhancer-breakpoint 3C PCR product was verified by sequencing. (c) Controls for 3C analysis. (d,e) Anti-Flag (d), anti KDM1A (d) anti-CHD1(e) and anti-tubulin (d,e) Western blots. Samples are lysates of LNCaP cells expressing RNAi-resistant (rr) Flag NLS KDM1A-rr, or Flag NLS KDM1A-rr K114A (d) CHD1-rr or CHD1-rr D425A (e) and treated with siRNA. (f) Model representing how assembly of methylated KDM1A and CHD1 drives AR-dependent transcription. (g) Model representing how assembly of methylated KDM1A and CHD1 drives AR-dependent loop formation and translocation at the TMPRSS2 gene. (b-e) Results are representatives of 3 independent experiments.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 and Supplementary Table 1 (PDF 1984 kb)
Supplementary Data Set 1
Uncropped gel pictures for all gels in the article (PDF 301 kb)
Source data
Rights and permissions
About this article
Cite this article
Metzger, E., Willmann, D., McMillan, J. et al. Assembly of methylated KDM1A and CHD1 drives androgen receptor–dependent transcription and translocation. Nat Struct Mol Biol 23, 132–139 (2016). https://doi.org/10.1038/nsmb.3153
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nsmb.3153
This article is cited by
-
Sterile inflammation via TRPM8 RNA-dependent TLR3-NF-kB/IRF3 activation promotes antitumor immunity in prostate cancer
The EMBO Journal (2024)
-
Emerging evidence that the mammalian sperm epigenome serves as a template for embryo development
Nature Communications (2023)
-
Exploring aromatic cage flexibility of the histone methyllysine reader protein Spindlin1 and its impact on binding mode prediction: an in silico study
Journal of Computer-Aided Molecular Design (2021)
-
Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer
Nature Genetics (2020)
-
LSD1: more than demethylation of histone lysine residues
Experimental & Molecular Medicine (2020)