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Assembly of methylated KDM1A and CHD1 drives androgen receptor–dependent transcription and translocation

Nature Structural & Molecular Biology volume 23, pages 132–139 (2016)Cite this article

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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.

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Figure 1: EHMT2 methylates KDM1A at K114 in vitro.
Figure 2: EHMT2 methylates KDM1A at K114 in vitro.
Figure 3: CHD1 interacts with KDM1A K114me2.
Figure 4: KDM1A K114me2 and CHD1 co-occupy AR-binding sites.
Figure 5: KDM1A K114me2 regulates AR-dependent gene expression by controlling chromatin occupancy of CHD1 and AR.
Figure 6: KDM1A K114me2 controls TMPRSS2-ERG gene fusion.

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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

  1. Klinik für Urologie und Zentrale Klinische Forschung, Klinikum der Universität Freiburg, Freiburg, Germany

    Eric Metzger, Dominica Willmann, Joel McMillan, Philipp Metzger, Kerstin Petroll, Sylvia Urban, Anne-Kathrin Schott, Adrien Eberlin, Katrin M Schüle & Roland Schüle

  2. Diamond Light Source, Didcot, UK

    Joel McMillan & Ralf Flaig

  3. Adolf-Butenandt Institut, University of Munich, München, Germany

    Ignasi Forne & Axel Imhof

  4. Institut für Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Stefan Gerhardt, Daniel Wohlwend & Oliver Einsle

  5. Center for Biological Systems Analysis, Freiburg, Germany

    Kerstin Petroll & Jörn Dengjel

  6. Pathology Network of the University Hospital of Luebeck and Leibniz Research Center Borstel, Luebeck and Borstel, Germany

    Anne von Maessenhausen & Sven Perner

  7. Department of Carcinogenesis, University of Texas, Smithville, Texas, USA

    Alexsandra Espejo & Mark T Bedford

  8. Department of New Therapeutic Concept Discovery, Boehringer Ingelheim, Vienna, Austria

    Michael Schleicher

  9. Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany

    Manfred Jung

  10. Deutsches Konsortium für Translationale Krebsforschung, Freiburg, Germany

    Manfred Jung & Roland Schüle

  11. Centre of Biological Signalling Studies, Albert-Ludwigs-University Freiburg, Freiburg, Germany

    Oliver Einsle & Roland Schüle

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  1. Eric Metzger
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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.

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Correspondence to Roland Schüle.

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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.

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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

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