Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

Abstract

Whole-genome analysis approaches are identifying recurrent cancer-associated somatic alterations in noncoding DNA regions. We combined somatic copy number analysis of 12 tumor types with tissue-specific epigenetic profiling to identify significant regions of focal amplification harboring super-enhancers. Copy number gains of noncoding regions harboring super-enhancers near KLF5, USP12, PARD6B and MYC are associated with overexpression of these cancer-related genes. We show that two distinct focal amplifications of super-enhancers 3′ to MYC in lung adenocarcinoma (MYC-LASE) and endometrial carcinoma (MYC-ECSE) are physically associated with the MYC promoter and correlate with MYC overexpression. CRISPR/Cas9-mediated repression or deletion of a constituent enhancer within the MYC-LASE region led to significant reductions in the expression of MYC and its target genes and to the impairment of anchorage-independent and clonogenic growth, consistent with an oncogenic function. Our results suggest that genomic amplification of super-enhancers represents a common mechanism to activate cancer driver genes in multiple cancer types.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Pan-cancer analysis identifying focally amplified super-enhancers.
Figure 2: Lineage-specific focal amplification of super-enhancers adjacent to the MYC gene.
Figure 3: The activity of MYC-LASE is predominantly driven by the e3 constituent enhancer.
Figure 4: Identification of transcription factors required for the activity of the e3 enhancer.
Figure 5: KRAB-dCas9–mediated repression of the e3 enhancer identifies MYC as a direct target.
Figure 6: CRISPR/Cas9-mediated deletion of the e3 enhancer impairs the oncogenic effect of e3 enhancer amplification.

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Stratton, M.R., Campbell, P.J. & Futreal, P.A. The cancer genome. Nature 458, 719–724 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Beroukhim, R. et al. The landscape of somatic copy-number alteration across human cancers. Nature 463, 899–905 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Bignell, G.R. et al. Signatures of mutation and selection in the cancer genome. Nature 463, 893–898 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Stephens, P.J. et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 462, 1005–1010 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Weir, B.A. et al. Characterizing the cancer genome in lung adenocarcinoma. Nature 450, 893–898 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550 (2014).

  7. Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers. Nature 489, 519 525 (2012).

  8. Xue, W. et al. A cluster of cooperating tumor-suppressor gene candidates in chromosomal deletions. Proc. Natl. Acad. Sci. USA 109, 8212–8217 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ong, C.-T. & Corces, V.G. Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat. Rev. Genet. 12, 283–293 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Graf, T. & Enver, T. Forcing cells to change lineages. Nature 462, 587–594 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Xie, W. & Ren, B. Developmental biology. Enhancing pluripotency and lineage specification. Science 341, 245–247 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Bulger, M. & Groudine, M. Functional and mechanistic diversity of distal transcription enhancers. Cell 144, 327–339 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. 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 

  14. Visel, A. et al. ChIP-seq accurately predicts tissue-specific activity of enhancers. Nature 457, 854–858 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Heintzman, N.D. et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat. Genet. 39, 311–318 (2007).

    Article  CAS  PubMed  Google Scholar 

  16. 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 

  17. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ernst, J. et al. Mapping and analysis of chromatin state dynamics in nine human cell types. Nature 473, 43–49 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. 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 

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

  21. Roadmap Epigenomics Consortium. Integrative analysis of 111 reference human epigenomes. Nature 518, 317–330 (2015).

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

    CAS  PubMed  Google Scholar 

  23. Pott, S. & Lieb, J.D. What are super-enhancers? Nat. Genet. 47, 8–12 (2015).

    Article  CAS  PubMed  Google Scholar 

  24. Lovén, J. et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 153, 320–334 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Shi, J. et al. Role of SWI/SNF in acute leukemia maintenance and enhancer-mediated Myc regulation. Genes Dev. 27, 2648–2662 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Northcott, P.A. et al. Enhancer hijacking activates GFI1 family oncogenes in medulloblastoma. Nature 511, 428–434 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Mansour, M.R. et al. Oncogene regulation. 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 

  28. Gröschel, S. et al. A single oncogenic enhancer rearrangement causes concomitant EVI1 and GATA2 deregulation in leukemia. Cell 157, 369–381 (2014).

    Article  PubMed  Google Scholar 

  29. Herranz, D. et al. A NOTCH1-driven MYC enhancer promotes T cell development, transformation and acute lymphoblastic leukemia. Nat. Med. 20, 1130–1137 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mermel, C.H. et al. GISTIC2.0 facilitates sensitive and confident localization of the targets of focal somatic copy-number alteration in human cancers. Genome Biol. 12, R41 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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 

  33. Burska, U.L. et al. Deubiquitinating enzyme Usp12 is a novel co-activator of the androgen receptor. J. Biol. Chem. 288, 32641–32650 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Qiu, R.-G., Abo, A. & Steven Martin, G. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCζ signaling and cell transformation. Curr. Biol. 10, 697–707 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Imielinski, M. et al. Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing. Cell 150, 1107–1120 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kagey, M.H. et al. Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430–435 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Deng, W. et al. Controlling long-range genomic interactions at a native locus by targeted tethering of a looping factor. Cell 149, 1233–1244 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Bailey, S.D. et al. ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters. Nat. Commun. 2, 6186 (2015).

    Article  PubMed  Google Scholar 

  40. Wang, J. et al. Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors. Genome Res. 22, 1798–1812 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Gilbert, L.A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kearns, N.A. et al. Functional annotation of native enhancers with a Cas9–histone demethylase fusion. Nat. Methods 12, 401–403 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schlosser, I. et al. Dissection of transcriptional programmes in response to serum and c-Myc in a human B-cell line. Oncogene 24, 520–524 (2005).

    Article  CAS  PubMed  Google Scholar 

  44. Coller, H.A. et al. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc. Natl. Acad. Sci. USA 97, 3260–3265 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zeller, K.I., Jegga, A.G., Aronow, B.J., O'Donnell, K.A. & Dang, C.V. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets. Genome Biol. 4, R69 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Schuhmacher, M. et al. The transcriptional program of a human B cell line in response to Myc. Nucleic Acids Res. 29, 397–406 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Battey, J. et al. The human c-myc oncogene: structural consequences of translocation into the IgH locus in Burkitt lymphoma. Cell 34, 779–787 (1983).

    Article  CAS  PubMed  Google Scholar 

  49. Francis, J.M. et al. EGFR variant heterogeneity in glioblastoma resolved through single-nucleus sequencing. Cancer Discov. 4, 956–971 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Cowper-Sal lari, R. et al. Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression. Nat. Genet. 44, 1191–1198 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Zhang, X., Cowper-Sal lari, R., Bailey, S.D., Moore, J.H. & Lupien, M. Integrative functional genomics identifies an enhancer looping to the SOX9 gene disrupted by the 17q24.3 prostate cancer risk locus. Genome Res. 22, 1437–1446 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    PubMed  PubMed Central  Google Scholar 

  54. Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank G. Ha and other members of the Meyerson laboratory for discussions. We acknowledge support from US Department of Defense grant W81XWH-12-1-0269 (M.M.), National Cancer Institute grant 1R35CA197568 (M.M.) and the American Cancer Society Research Professorship (M.M.). X.Z. is supported by the Lung Cancer Research Foundation and the American Association for Cancer Research–John and Elizabeth Leonard Family Foundation Basic Cancer Research Fellowship. P.S.C. is supported by a fellowship from the International Association for the Study of Lung Cancer and National Cancer Institute grant 1F32CA180662.

Author information

Authors and Affiliations

Authors

Contributions

X.Z., P.S.C., J.M.F. and M.M. designed the research and wrote the manuscript with input from the other authors. X.Z., P.S.C., J.M.F. and H.W. conducted the biological assays, and X.Z., P.S.C., J.M.F., M.I. and A.D.C. conducted the computational analysis.

Corresponding author

Correspondence to Matthew Meyerson.

Ethics declarations

Competing interests

M.M. and A.D.C. have received a commercial research grant from Bayer.

Integrated supplementary information

Supplementary Figure 1 The expression level of genes in tumors with somatic focal amplification of each super-enhancer versus tumors without this focal amplification.

Genes that are expressed and closest to each amplification were analyzed. Box plot: middle bar, median; lower and upper box limits, 25th and 75th percentiles, respectively; whiskers, min and max values. The P value is derived from a t test: **P ≤ 0.01, ***P ≤ 0.001.

Supplementary Figure 2 GISTIC peak identified on chromosome 13q from esophageal carcinomas.

The H3K27ac ChIP-seq profile from esophageal cells is presented. Super-enhancers are also indicated. Expression level of KLF5 in tumors with and without amplified KLF5-ESSE (KLF5 esophageal carcinoma super-enhancer). The P value is derived from a t test.

Supplementary Figure 3 A focally amplified region (MYC-LASE) in lung adenocarcinomas is part of a super-enhancer.

(a) Copy number amplification of the noncoding region ~450 kb 3′ to MYC in lung adenocarcinoma primary tumors from TCGA (n = 11; sample IDs are listed in Supplementary Table 3) and (b) lung adenocarcinoma cell lines (NCI-H2009, HCC78, HOP92 and NCI-H358). Red lines represent relative focal amplification regions identified from each sample. The focal amplification peak called by GISTIC is highlighted. (c) Whole-genome sequencing rearrangement analysis of two lung adenocarcinoma tumors (sample IDs in Supplementary Table 3) identify tandem duplications, indicated by the red curves, as detected by the JaBbA structural rearrangement algorithm (Imielinski et al., in preparation). ChIP-seq profile of H3K27ac and super-enhancer (SE) regions in (d) A549, NCI-H358, NCI-H2009 and (e) HCC95 and NCI-H2171 cells. Thin bars above the ChIP-seq signal represent super-enhancers that are called by the ROSE pipeline. LUAD, lung adenocarcinomas; SqCC, squamous cell lung carcinomas; Small cell, small cell lung cancer.

Supplementary Figure 4 DNase I signal and p300 binding profile of lung adenocarcinoma A549 cells and endometrial carcinoma Ishikawa cells in the focal amplification regions MYC-LASE and MYC-ECSE.

Supplementary Figure 5 Cancer type–specific enhancer activity of the super-enhancer region.

(a) ChIP-seq profile of H3K27ac at the MYC locus. No enrichment of H3K27ac is detectable at the MYC-LASE region for HEK293 cells. (b) Luciferase reporter assay (n = 3) of the five constituent enhancers, e1–e5, in A549 lung adenocarcinoma cells and HEK293 cells. The P value is derived from a t test: **P ≤ 0.01, ***P ≤ 0.001.

Supplementary Figure 6 siRNA silencing of NFE2L2 and CEBPB in A549 cells.

The P value is derived from a t test (n = 3): **P ≤ 0.01, ***P ≤ 0.001.

Supplementary Figure 7 RNA-seq results in NCI-H2009 cells with and without KRAB-dCas9–mediated e3 enhancer repression.

Blue dots represent genes that are significantly differentially expressed (>25%) after e3 enhancer repression. Negative-control (NC) includes sg-Empty and sg-Control, while KRAB includes sg-e3KRAB 1 and sg-e3KRAB 2. RPKM mean indicates the mean expression levels (RPKM) normalized to the negative-control samples.

Supplementary Figure 8 KRAB-dCas9 fusion–mediated repression of the e3 enhancer in NCI-H2009 cells.

KRAB-dCas9 fusion–mediated repression of the e3 enhancer in NCI-H2009 cells leads to a significant reduction in cellular transformation efficiency as measured by anchorage-independent growth assay (a) and cellular proliferation rate as measured by clonogenic growth assay (b). Representative images are shown.

Supplementary Figure 9 CRISPR/Cas9-mediated deletion of the e3 enhancer in NCI-H2009 cells.

CRISPR/Cas9-mediated deletion of the e3 enhancer in NCI-H2009 cells leads to a significant reduction in cellular transformation efficiency as measured by anchorage-independent growth assay (a) and cellular proliferation rate as measured by clonogenic growth assay (b). Representative images are shown.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9. (PDF 1004 kb)

Supplementary Table 1: Pan-cancer copy number alteration analysis.

The number of noncoding focal amplification peaks identified and H3K27ac ChIP-seq data availability are highlighted in each tumor type. (XLSX 69 kb)

Supplementary Table 2: Accession numbers for public data sets used in the study.

A list of accession numbers for ENCODE data, Roadmap project data and other public data sets that were used in the study. (XLSX 44 kb)

Supplementary Table 3: Primers used in the study.

A list of PCR primers and CRISPR sgRNA sequences that were used in the study. (XLSX 13 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, X., Choi, P., Francis, J. et al. Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers. Nat Genet 48, 176–182 (2016). https://doi.org/10.1038/ng.3470

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ng.3470

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer