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Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles

Nature Medicine volume 22, pages 685–691 (2016)Cite this article

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Abstract

Extensive cross-linking introduced during routine tissue fixation of clinical pathology specimens severely hampers chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) analysis from archived tissue samples. This limits the ability to study the epigenomes of valuable, clinically annotated tissue resources. Here we describe fixed-tissue chromatin immunoprecipitation sequencing (FiT-seq), a method that enables reliable extraction of soluble chromatin from formalin-fixed paraffin-embedded (FFPE) tissue samples for accurate detection of histone marks. We demonstrate that FiT-seq data from FFPE specimens are concordant with ChIP-seq data from fresh-frozen samples of the same tumors. By using multiple histone marks, we generate chromatin-state maps and identify cis-regulatory elements in clinical samples from various tumor types that can readily allow us to distinguish between cancers by the tissue of origin. Tumor-specific enhancers and superenhancers that are elucidated by FiT-seq analysis correlate with known oncogenic drivers in different tissues and can assist in the understanding of how chromatin states affect gene regulation.

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Figure 1: Development of FiT-seq, and its effect on strength and resolution of histone mark signals.
Figure 2: Differences in H3K4me2 marking in CRC tissue and matched normal colon mucosa.
Figure 3: Additional histone marks mapped by FiT-seq.
Figure 4: Tumor-specific enhancers and H3K4me2-defined superenhancers identified in multiple cancer types.

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Acknowledgements

This work was supported by the DFCI–Novartis Drug Discovery Program (R.A.S.), the US NIH grant P50CA127003 (R.A.S.), the Susan F. Smith Center for Women's Cancers (M. Brown), grants from the Susan G. Komen and the Breast Cancer Research foundations (M. Brown), the Medical Oncology Translational Grant Program from the Dana-Farber Cancer Institute (C.J.S.), a grant from Instituto de Salud Carlos III the Spanish Economy and Competitiveness Ministry (grant PI13-01818; P.C.), the ConSEPOC–Comunidad de Madrid (grant S2010/BMD-2542; P.C.), and fellowships from the Asociación Española Contra el Cáncer (Programa de Formación Avanzada en Oncología 2010; P.C.) and the Fundación Caja Madrid (P.C.). We thank E. Díaz and M. de Miguel for technical assistance.

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Authors and Affiliations

  1. Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Paloma Cejas, Lewyn Li, Melissa Duarte, Prakash Rao, X Shirley Liu, Myles Brown, Ramesh A Shivdasani & Henry W Long

  2. Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, USA

    Paloma Cejas, Lewyn Li, Nicholas K O'Neill, Melissa Duarte, Prakash Rao, Joaquim Bellmunt, Stephanie Mullane, Christopher J Sweeney, Myles Brown, Ramesh A Shivdasani & Henry W Long

  3. Department of Medical Oncology, Hospital La Paz Institute for Health Research (IdiPAZ), Madrid, Spain

    Paloma Cejas, Marta Mendiola & Jaime Feliu

  4. Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

    Michaela Bowden & Chensheng W Zhou

  5. Department of Pathology, Hospital La Paz Institute for Health Research (IdiPAZ), Madrid, Spain

    Marta Mendiola & Emilio Burgos

  6. Precision Oncology Laboratory, Infanta Sofia University Hospital, Madrid, Spain

    Juan Moreno-Rubio

  7. Department of Surgery, Jimenez Diaz Foundation, Madrid, Spain

    Héctor Guadalajara & Damián García-Olmo

  8. Clinical Research Unit, Jimenez Diaz Foundation, Madrid, Spain

    Víctor Moreno

  9. Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA

    Michelle Hirsch & Andrea Richardson

  10. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts, USA

    X Shirley Liu

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Contributions

P.C., R.A.S., M. Brown and H.W.L. conceived and designed the study; L.L., P.C., H.W.L. and N.K.O'N. performed computational and statistical analyses; M.D., P.R., M. Bowden and C.W.Z. acquired data; E.B. and M.M. reviewed CRC pathology; J.F., J.M.-R., H.G., V.M., S.M., J.B., D.G.-O., C.J.S., M.H. and A.R. supervised collection of tumor samples; X.S.L., M. Brown, H.W.L. and R.A.S. provided overall supervision; and P.C., R.A.S. and H.W.L. drafted the manuscript, with input from all of the authors.

Corresponding authors

Correspondence to Ramesh A Shivdasani or Henry W Long.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 3763 kb)

Supplementary Table 1

Sample characteristics, study design, and ChIP-seq library statistics. (XLSX 28 kb)

Supplementary Table 2

Comparison of PAT-ChIP-seq and FiT-seq performance on FFPE samples. (XLSX 25 kb)

Supplementary Table 3

Genes differentially H3K4me2 marked at promoters between normal and tumoral colorectal samples. (XLSX 1866 kb)

Supplementary Table 4

Gene Set Enrichment Analysis of the differentially H3K4me2 marked promoters between normal and tumoral colorectal samples for both the FF and FFPE cohorts. (XLSX 1682 kb)

Supplementary Table 5

Motif analysis at the differentially marked enhancers between normal and tumoral colorectal samples. (XLSX 55 kb)

Supplementary Table 6

Locations of the most discriminant enhancers across-tumortypes as determined by ANOVA. (XLSX 340 kb)

Supplementary Table 7

Motif analysis at the most discriminant enhancers acrosstumor-types. (XLSX 97 kb)

Supplementary Table 8

List of super-enhancers called for the series of tumors included in the cross tumor study in Figure 4. (XLSX 9396 kb)

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Cejas, P., Li, L., O'Neill, N. et al. Chromatin immunoprecipitation from fixed clinical tissues reveals tumor-specific enhancer profiles. Nat Med 22, 685–691 (2016). https://doi.org/10.1038/nm.4085

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