TMPRSS2–ERG fusion co-opts master transcription factors and activates NOTCH signaling in primary prostate cancer

Abstract

TMPRSS2–ERG (T2E) structural rearrangements typify 50% of prostate tumors and result in overexpression of the ERG transcription factor. Using chromatin, genomic and expression data, we show distinct cis-regulatory landscapes between T2E-positive and non-T2E primary prostate tumors, which include clusters of regulatory elements (COREs). This difference is mediated by ERG co-option of HOXB13 and FOXA1, implementing a T2E-specific transcriptional profile. We also report a T2E-specific CORE on the structurally rearranged ERG locus arising from spreading of the TMPRSS2 locus pre-existing CORE, assisting in its overexpression. Finally, we show that the T2E-specific cis-regulatory landscape underlies a vulnerability against the NOTCH pathway. Indeed, NOTCH pathway inhibition antagonizes the growth and invasion of T2E-positive prostate cancer cells. Taken together, our work shows that overexpressed ERG co-opts master transcription factors to deploy a unique cis-regulatory landscape, inducing a druggable dependency on NOTCH signaling in T2E-positive prostate tumors.

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Figure 1: H3K27ac ChIP-seq defines active CREs proximal to functionally relevant genes in primary prostate tissue samples.
Figure 2: T2E prostate cancer has a distinct chromatin and gene expression landscape.
Figure 3: ERG remodels binding of master transcription factors to the chromatin.
Figure 4: COREs are proximal to key prostate cancer genes and are remodeled in T2E-positive prostate cancer.
Figure 5: The TMPRSS2 CORE spreads into the ERG locus and contributes to ERG expression.
Figure 6: ERG activates CREs surrounding NOTCH pathway genes.

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References

  1. 1

    Siegel, R.L., Miller, K.D. & Jemal, A. Cancer statistics, 2016. CA Cancer J. Clin. 66, 7–30 (2016).

    Article  Google Scholar 

  2. 2

    Tomlins, S.A. et al. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science 310, 644–648 (2005).

    Article  CAS  Google Scholar 

  3. 3

    Cancer Genome Atlas Research Network. The molecular taxonomy of primary prostate cancer. Cell 163, 1011–1025 (2015).

  4. 4

    Lin, B. et al. Prostate-localized and androgen-regulated expression of the membrane-bound serine protease TMPRSS2. Cancer Res. 59, 4180–4184 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Clinckemalie, L. et al. Androgen regulation of the TMPRSS2 gene and the effect of a SNP in an androgen response element. Mol. Endocrinol. 27, 2028–2040 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Perner, S. et al. TMPRSS2-ERG fusion prostate cancer: an early molecular event associated with invasion. Am. J. Surg. Pathol. 31, 882–888 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  7. 7

    Cerveira, N. et al. TMPRSS2-ERG gene fusion causing ERG overexpression precedes chromosome copy number changes in prostate carcinomas and paired HGPIN lesions. Neoplasia 8, 826–832 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 8

    Carver, B.S. et al. Aberrant ERG expression cooperates with loss of PTEN to promote cancer progression in the prostate. Nat. Genet. 41, 619–624 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

    King, J.C. et al. Cooperativity of TMPRSS2-ERG with PI3-kinase pathway activation in prostate oncogenesis. Nat. Genet. 41, 524–526 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Mounir, Z. et al. TMPRSS2:ERG blocks neuroendocrine and luminal cell differentiation to maintain prostate cancer proliferation. Oncogene 34, 3815–3825 (2015).

    Article  CAS  Google Scholar 

  11. 11

    Tomlins, S.A. et al. Role of the TMPRSS2-ERG gene fusion in prostate cancer. Neoplasia 10, 177–188 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Baltzinger, M., Mager-Heckel, A.M. & Remy, P. Xl erg: expression pattern and overexpression during development plead for a role in endothelial cell differentiation. Dev. Dyn. 216, 420–433 (1999).

    Article  CAS  Google Scholar 

  13. 13

    Scott, E.W., Simon, M.C., Anastasi, J. & Singh, H. Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages. Science 265, 1573–1577 (1994).

    Article  CAS  Google Scholar 

  14. 14

    Yang, H. et al. ETS family transcriptional regulators drive chromatin dynamics and malignancy in squamous cell carcinomas. eLife 4, e10870 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  15. 15

    Nikolova-Krstevski, V. et al. ERG is required for the differentiation of embryonic stem cells along the endothelial lineage. BMC Dev. Biol. 9, 72 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Loughran, S.J. et al. The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Nat. Immunol. 9, 810–819 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Meadows, S.M., Salanga, M.C. & Krieg, P.A. Kruppel-like factor 2 cooperates with the ETS family protein ERG to activate Flk1 expression during vascular development. Development 136, 1115–1125 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Wilson, N.K. et al. Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. Cell Stem Cell 7, 532–544 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Chen, Y. et al. ETS factors reprogram the androgen receptor cistrome and prime prostate tumorigenesis in response to PTEN loss. Nat. Med. 19, 1023–1029 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Yu, J. et al. An integrated network of androgen receptor, Polycomb, and TMPRSS2-ERG gene fusions in prostate cancer progression. Cancer Cell 17, 443–454 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Cai, C. et al. ERG induces androgen receptor–mediated regulation of SOX9 in prostate cancer. J. Clin. Invest. 123, 1109–1122 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 22

    Pomerantz, M.M. et al. The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis. Nat. Genet. 47, 1346–1351 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Moorman, C. et al. Hotspots of transcription factor colocalization in the genome of Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 103, 12027–12032 (2006).

    Article  CAS  Google Scholar 

  24. 24

    Gaulton, K.J. et al. A map of open chromatin in human pancreatic islets. Nat. Genet. 42, 255–259 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Song, L. et al. Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res. 21, 1757–1767 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Whyte, W.A. et al. Master transcription factors and Mediator establish super-enhancers at key cell identity genes. Cell 153, 307–319 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Parker, S.C.J. et al. Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants. Proc. Natl. Acad. Sci. USA 110, 17921–17926 (2013).

    Article  Google Scholar 

  28. 28

    Akhtar-Zaidi, B. et al. Epigenomic enhancer profiling defines a signature of colon cancer. Science 336, 736–739 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. 29

    Aran, D. & Hellman, A. DNA methylation of transcriptional enhancers and cancer predisposition. Cell 154, 11–13 (2013).

    Article  CAS  Google Scholar 

  30. 30

    Aran, D., Sabato, S. & Hellman, A. DNA methylation of distal regulatory sites characterizes dysregulation of cancer genes. Genome Biol. 14, R21 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Jia, L. et al. Functional enhancers at the gene-poor 8q24 cancer-linked locus. PLoS Genet. 5, e1000597 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Magnani, L. et al. Genome-wide reprogramming of the chromatin landscape underlies endocrine therapy resistance in breast cancer. Proc. Natl. Acad. Sci. USA 110, E1490–E1499 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  33. 33

    Stergachis, A.B. et al. Developmental fate and cellular maturity encoded in human regulatory DNA landscapes. Cell 154, 888–903 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Heintzman, N.D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Fraser, M. et al. Genomic hallmarks of localized, non-indolent prostate cancer. Nature 541, 359–364 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Taylor, G.C.A., Eskeland, R., Hekimoglu-Balkan, B., Pradeepa, M.M. & Bickmore, W.A. H4K16 acetylation marks active genes and enhancers of embryonic stem cells, but does not alter chromatin compaction. Genome Res. 23, 2053–2065 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Thurman, R.E. et al. The accessible chromatin landscape of the human genome. Nature 489, 75–82 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Koch, C.M. et al. The landscape of histone modifications across 1% of the human genome in five human cell lines. Genome Res. 17, 691–707 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Taylor, B.S. et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. 40

    Lupien, M. et al. FoxA1 translates epigenetic signatures into enhancer-driven lineage-specific transcription. Cell 132, 958–970 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Sérandour, A.A. et al. Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers. Genome Res. 21, 555–565 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Creyghton, M.P. et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. USA 107, 21931–21936 (2010).

    CAS  Google Scholar 

  44. 44

    Lin, C.Y. et al. Active medulloblastoma enhancers reveal subgroup-specific cellular origins. Nature 530, 57–62 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Gao, N. et al. The role of hepatocyte nuclear factor-3α (Forkhead Box A1) and androgen receptor in transcriptional regulation of prostatic genes. Mol. Endocrinol. 17, 1484–1507 (2003).

    Article  CAS  Google Scholar 

  46. 46

    Lupien, M. & Brown, M. Cistromics of hormone-dependent cancer. Endocr. Relat. Cancer 16, 381–389 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Economides, K.D. & Capecchi, M.R. Hoxb13 is required for normal differentiation and secretory function of the ventral prostate. Development 130, 2061–2069 (2003).

    Article  CAS  Google Scholar 

  48. 48

    Chang, H. et al. Synergistic action of master transcription factors controls epithelial-to-mesenchymal transition. Nucleic Acids Res. 44, 2514–2527 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  49. 49

    Hnisz, D. et al. Super-enhancers in the control of cell identity and disease. Cell 155, 934–947 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Zhang, X. et al. Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers. Nat. Genet. 48, 176–182 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Ooi, W.F. et al. Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity. Nat. Commun. 7, 12983 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Gao, N. et al. Forkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation. Development 132, 3431–3443 (2005).

    Article  CAS  Google Scholar 

  53. 53

    Bhatia-Gaur, R. et al. Roles for Nkx3.1 in prostate development and cancer. Genes Dev. 13, 966–977 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Chen, R. et al. Cav1.3 channel α1D protein is overexpressed and modulates androgen receptor transactivation in prostate cancers. Urol. Oncol. 32, 524–536 (2014).

    Article  CAS  Google Scholar 

  55. 55

    Jin, Y. et al. STAMP2 increases oxidative stress and is critical for prostate cancer. EMBO Mol. Med. 7, 315–331 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Weier, C. et al. Nucleotide resolution analysis of TMPRSS2 and ERG rearrangements in prostate cancer. J. Pathol. 230, 174–183 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Hay, D. et al. Genetic dissection of the α-globin super-enhancer in vivo. Nat. Genet. 48, 895–903 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. 58

    Hollenhorst, P.C., Jones, D.A. & Graves, B.J. Expression profiles frame the promoter specificity dilemma of the ETS family of transcription factors. Nucleic Acids Res. 32, 5693–5702 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    McLean, C.Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Cowley, G.S. et al. Parallel genome-scale loss of function screens in 216 cancer cell lines for the identification of context-specific genetic dependencies. Sci. Data 1, 140035 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Klezovitch, O. et al. A causal role for ERG in neoplastic transformation of prostate epithelium. Proc. Natl. Acad. Sci. USA 105, 2105–2110 (2008).

    Article  Google Scholar 

  62. 62

    Wang, Q. et al. A hierarchical network of transcription factors governs androgen receptor–dependent prostate cancer growth. Mol. Cell 27, 380–392 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. 63

    Thangapazham, R. et al. Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer. BMC Cancer 14, 16 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  64. 64

    Mansour, M.R. et al. An oncogenic super-enhancer formed through somatic mutation of a noncoding intergenic element. Science 346, 1373–1377 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. 65

    Affer, M. et al. Promiscuous MYC locus rearrangements hijack enhancers but mostly super-enhancers to dysregulate MYC expression in multiple myeloma. Leukemia 28, 1725–1735 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. 66

    Berger, M.F. et al. The genomic complexity of primary human prostate cancer. Nature 470, 214–220 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. 67

    Ciriello, G. et al. Emerging landscape of oncogenic signatures across human cancers. Nat. Genet. 45, 1127–1133 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. 68

    Rickman, D.S. et al. Oncogene-mediated alterations in chromatin conformation. Proc. Natl. Acad. Sci. USA 109, 9083–9088 (2012).

    Article  Google Scholar 

  69. 69

    Zhang, Y. et al. Down-regulation of Jagged-1 induces cell growth inhibition and S phase arrest in prostate cancer cells. Int. J. Cancer 119, 2071–2077 (2006).

    Article  CAS  Google Scholar 

  70. 70

    Wang, Z. et al. Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF-κB signaling pathways. J. Cell. Biochem. 109, 726–736 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. 71

    Bin Hafeez, B. et al. Targeted knockdown of Notch1 inhibits invasion of human prostate cancer cells concomitant with inhibition of matrix metalloproteinase-9 and urokinase plasminogen activator. Clin. Cancer Res. 15, 452–459 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. 72

    Smyth, G.K. in Bioinformatics and Computational Biology Solutions Using R and Bioconductor (eds. Gentleman, R., Carey, V.J., Huber, W., Irizarry, R.A. & Dudoit, S.) 397–420 (Springer, 2005).

  73. 73

    Wang, D. et al. Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA. Nature 474, 390–394 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. 74

    Takayama, K. et al. RUNX1, an androgen- and EZH2-regulated gene, has differential roles in AR-dependent and -independent prostate cancer. Oncotarget 6, 2263–2276 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  75. 75

    Toropainen, S. et al. SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin. Nucleic Acids Res. 43, 848–861 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. 76

    Asangani, I.A. et al. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 510, 278–282 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. 77

    Huang, Q. et al. A prostate cancer susceptibility allele at 6q22 increases RFX6 expression by modulating HOXB13 chromatin binding. Nat. Genet. 46, 126–135 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. 78

    ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

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Acknowledgements

R. Bristow (Princess Margaret Cancer Centre) provided 22Rv1 and LNCaP cells, and H. He (Princess Margaret Cancer Centre) provided VCaP cells. We acknowledge the Princess Margaret Genomics Centre and the Princess Margaret Bioinformatics group for providing the infrastructure to assist with analysis of the chromatin landscape of primary prostate tumor samples. This work was supported by Prostate Cancer Canada and is funded by the Movember Foundation (RS2014-04 to M.L. and RS2014-01 to P.C.B.), with additional support from the Ontario Institute for Cancer Research funded by the Government of Ontario, the Princess Margaret Cancer Foundation (M.L. and R.G.B.) and the Radiation Medicine Program Academic Enrichment Fund (R.G.B.). K.J.K. is supported by a Canadian Breast Cancer Foundation (CBCF) postdoctoral fellowship recipient. P.C.B. is supported by a Terry Fox Research Institute New Investigator Award and a Canadian Institute of Health Research (CIHR) New Investigator Award. R.G.B. is supported by a Canadian Cancer Society Research Scientist Award. M.L. is supported by an Investigator Award from the Ontario Institute for Cancer Research, a CIHR New Investigator Award and a Movember Rising Star Award from Prostate Cancer Canada. K.J.K. and A.M. are co-first authors and can be interchangeably ordered in reporting documents.

Author information

Affiliations

Authors

Contributions

K.J.K. designed and conducted most of the experiments. A.M. implemented most of the computational and statistical approaches. S.Z. conducted the co-IP assays and assisted in ChIP-reChIP and CRISPR experiments. V.H. and T.N.Y. analyzed the genetic data sets for single nucleotide and copy number variants. Y.-J.S. processed gene expression data sets. T.v.d.K. was responsible for histopathological assessment of the tumor samples. M.F., P.C.B., R.G.B. and M.L. supervised the research. M.L. oversaw the project. Figures were designed and prepared by K.J.K. and A.M. The manuscript was written by K.J.K., A.M. and M.L. with assistance from all authors.

Corresponding author

Correspondence to Mathieu Lupien.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Infiltrating immune cells do not differ between T2E and non-T2E samples and represent a small fraction of total cellularity.

(a) ESTIMATE immune score from mRNA expression array data for non-T2E and T2E samples. (b) Estimated percentage of tumor infiltrating lymphocytes within each tumor sample as assessed by H&E staining.

Supplementary Figure 2 ChIP-seq signal distribution across the genome of primary prostate tissue samples.

(a) Distribution of H3K27ac enriched cis-regulatory elements across promoters, introns, exons, downstream/3′ UTR, and promoter/5′UTR elements in each of the 19 samples profiled by ChIP-seq. Snapshot of H3K27ac levels surrounding (b) the housekeeping ACTB gene and (c) prostate-specific KLK3 gene.

Supplementary Figure 3 Classification of samples into T2E and non-T2E on the basis of ERG expression.

(a) Complete linkage hierarchical clustering of HOXB13, AR, ERG, and FOXA1 mRNA expression values in 17 CPCG tumor samples. (b) Immunohistochemical staining of ERG in 6 T2E and 5 non-T2E CPCG samples.

Supplementary Figure 4 T2E-up and T2E-down elements are proximal to known ERG targets and genomic regions previously not classified as T2E specific.

Snapshots of H3K27ac levels in proximity to (a) LAMC2, (b) LINC00898, (c) POTEB, and (b) HTR7 genes.

Supplementary Figure 5 Promoter and enhancer acetylation changes between T2E and non-T2E primary prostate tumors are significantly predictive of one another and of associated gene expression changes.

Volcano plots of log2 fold change vs -log2 FDR-corrected q-value for statistical significance of differential H3K27ac ChIP-seq signal between T2E and non-T2E tumors at enhancer regions linked to (a) T2E-up promoters or (b) T2E-down promoters. Volcano plots of log fold change vs -log FDR corrected q-value for statistical significance of matched differential mRNA expression between T2E and non-T2E tumors for mRNA associated with genes whose promoters (c) or predicted enhancers (d) show differential H3K27ac ChIP-seq signal between T2E and non-T2E tumors.

Supplementary Figure 6 ERG recruits prostate master transcription factors to T2E-up elements.

(a) Motif enrichment for selected motifs in cis-regulatory elements that were not significantly different between T2E and non-T2E tumors. (FKH: Forkhead; ARE: androgen response element; ETS: E26 transformation specific; HBOX: homebox). (b) Western blot of ERG, FOXA1, and HOXB13 following depletion of ERG with siRNA in VCaP cells. Vinculin was blotted as a loading control. (siCtl: non-targeting siRNA; siERG: ERG targeting siRNA). (c) ChIP-seq signal plots of average HOXB13 tag density in VCaP cells treated with control non-targeting siRNA or ERG targeting siRNA at HOXB13/ERG co-bound T2E-up elements, (d) HOXB13 bound T2E-up elements not ERG bound, (e) HOXB13 bound T2E-down elements, and (f) HOXB13 peaks that are not T2E-up, T2E down, and not bound by ERG. (g) ChIP-seq plots of average FOXA1 tag density in VCaP cells treated with control non-targeting siRNA or ERG targeting siRNA at FOXA1/ERG co-bound T2E-up elements, (h) FOXA1 bound T2E-up elements not ERG bound, (i) FOXA1 bound T2E-down elements, and (j) FOXA1 peaks that are not T2E-up, T2E down, and not bound by ERG. (k). Co-immunoprecipitation of FOXA1, ERG, and HOXB13 transcription factors in VCaP cells. Arrows indicate expected size of protein on blot. (IP: immunoprecipitation; WB: western blot).

Supplementary Figure 7 ERG colocalizes with FOXA1, HOXB13 and AR at T2E-up elements.

Snapshot depicting H3K27ac ChIP-seq signal in primary tumors and VCaP cells as well as FOXA1, HOXB13, AR and ERG binding in VCaP cells at seven loci tested (Fig. 3g) by ChIP-reChIP-qPCR.

Supplementary Figure 8 COREs surround prostate master transcription factors, and CORE deregulation in T2E prostate cancer corresponds to T2E-specific gene expression changes.

Snapshot depicting H3K27ac ChIP-seq signal in T2E and non-T2E primary prostate cancer for (a) FOXA1 and (b) HOXB13. (c) Volcano plot of log2 fold change vs -log2 FDR-corrected q-value for statistical significance of differential expression from MSKCC dataset of all genes that were overlapping a T2E-up or T2E-down CORE.

Supplementary Figure 9 CRISPR-mediated deletions of the TMPRSS2–ERG CORE.

(a) Snapshot depicting H3K27ac ChIP-seq signal in primary tumors, VCaP cells, and HUVEC cells as well as master transcription factor binding in VCaP cells across the control AAVS1 locus in the PPP1R12C gene. (b) PCR blots to verify deletions of EC1, EC2, EC3, and control loci. PCR products running below expected size products represent deletion fragments. (*: non-specific PCR product).

Supplementary Figure 10 Cis-regulatory elements affected by ERG enrich for genes involved in NOTCH and developmental signaling pathways.

−log10 FDR values for top ten GO biological processes terms in (a) VCaP siERG H3K27ac down cis-regulatory elements, (b) primary tissue defined T2E-up cis-regulatory elements, and (c) primary tissue defined T2E-up COREs.

Supplementary Figure 11 Key NOTCH pathway genes associated with T2E-up cis-regulatory elements and COREs are overexpressed in T2E prostate cancer.

Log2 mRNA array expression values for T2E and non-T2E samples within the entire CPCG cohort for (a) HES1, (b) JAG1, and (c) DLL1.

Supplementary Figure 12 BMP and Nodal signaling genes do not show essentiality in a T2E-specific manner.

Average gene level Z-score in the Achilles project dataset from (a) BMP and (b) Nodal signaling pathway genes in 22Rv1and VCaP cells. Distributions were obtained from 1,000 random gene sets.

Supplementary Figure 13 Saturation analysis of called H3K27ac peaks stratified by T2E status suggests we have identified at least 95% of potential peaks.

A non-linear regression model was fit to the number of new peaks obtained sequentially over all samples for 1,000 permutations of sample orders and stratified according to (a) T2E and (b) nonT2E status. Individual points show mean new peaks per sample added and associated standard error of the mean fitted blue line shows fitted model with saturation line (red) marked as well as estimated number of samples needed to identify 95, 97 and 99% of peaks (grey lines).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13 (PDF 1433 kb)

Life Sciences Reproducibility Summary (PDF 190 kb)

Supplementary Tables 1–10

H3K27ac cis-regulatory elements in CPCG0233 (XLSX 19715 kb)

H3K27ac cis-regulatory elements in CPCG0236

H3K27ac cis-regulatory elements in CPCG0242

H3K27ac cis-regulatory elements in CPCG0246

H3K27ac cis-regulatory elements in CPCG0248

H3K27ac cis-regulatory elements in CPCG0258

H3K27ac cis-regulatory elements in CPCG0259

H3K27ac cis-regulatory elements in CPCG0262

H3K27ac cis-regulatory elements in CPCG0265

H3K27ac cis-regulatory elements in CPCG0266

Supplementary Tables 11–19

H3K27ac cis-regulatory elements in CPCG0267 (XLSX 18329 kb)

H3K27ac cis-regulatory elements in CPCG0269

H3K27ac cis-regulatory elements in CPCG0339

H3K27ac cis-regulatory elements in CPCG0340

H3K27ac cis-regulatory elements in CPCG0341

H3K27ac cis-regulatory elements in CPCG0342

H3K27ac cis-regulatory elements in CPCG0348

H3K27ac cis-regulatory elements in CPCG0365

H3K27ac cis-regulatory elements in CPCG0366

Supplementary Table 20

GREAT analysis of cis-regulatory elements found in all 19 tumors (XLSX 465 kb)

Supplementary Table 21

T2E-up cis-regulatory elements (XLSX 160 kb)

Supplementary Table 22

T2E-down cis-regulatory elements (XLSX 203 kb)

Supplementary Tables 23–32

Cis-regulatory elements with increased H3K27ac in T2E samples with PTEN deletion (XLSX 33 kb)

Cis-regulatory elements with decreased H3K27ac in T2E samples with PTEN deletion

Cis-regulatory elements with increased H3K27ac in non-T2E samples with NKX3-1 deletion

Cis-regulatory elements with decreased H3K27ac in non-T2E samples with NKX3-1 deletion

Cis-regulatory elements with increased H3K27ac in T2E samples with RB1 deletion

Cis-regulatory elements with decreased H3K27ac in T2E samples with RB1 deletion

Cis-regulatory elements with increased H3K27ac in T2E samples with CDKN1B deletion

Cis-regulatory elements with decreased H3K27ac in T2E samples with CDKN1B deletion

Cis-regulatory elements with increased H3K27ac in non-T2E samples with CDKN1B deletion

Cis-regulatory elements with decreased H3K27ac in non-T2E samples with CDKN1B deletion

Supplementary Table 33

Catalog of COREs defined across 19 tumors (XLSX 97 kb)

Supplementary Table 34

T2E-up COREs (XLSX 18 kb)

Supplementary Table 35

T2E-down COREs (XLSX 18 kb)

Supplementary Table 36

GREAT analysis from VCaP siERG H3K27ac down cis-regulatory elements (XLSX 1100 kb)

Supplementary Table 37

GREAT analysis from T2E-up cis-regulatory elements (XLSX 467 kb)

Supplementary Table 38

GREAT analysis from T2E-up COREs (XLSX 193 kb)

Supplementary Table 39

Oligos used in this study (XLSX 11 kb)

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Kron, K., Murison, A., Zhou, S. et al. TMPRSS2–ERG fusion co-opts master transcription factors and activates NOTCH signaling in primary prostate cancer. Nat Genet 49, 1336–1345 (2017). https://doi.org/10.1038/ng.3930

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