FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues

Abstract

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.

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Fig. 1: Schematic overview of the FiTAc-seq H3K27ac profiling protocol in FFPE samples.
Fig. 2: Optimization of the FiTAc-seq protocol and application for enhancer and SE annotation.
Fig. 3: FiTAc-seq performance on clinical FFPE specimens in multiple tumor types.

Data availability

Figures 2 and 3, and Extended Data Figs. 1, 3 and 4 all have associated raw data. The ChIP-seq data have been uploaded to the Gene Expression Omnibus (GSE140808). In Extended Data Fig. 3, we compare our data to previously published data available on the Gene Expression Omnibus (GSE128202).

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Acknowledgements

A.F.-T. has been supported by Associació per a la recerca oncològica (APRO) for the present study. P.C. acknowledges funding from the Ministry of Economy and Competitiveness, Instituto de Salud Carlos III (Institute of Health Carlos III)—PI18-01604. E.M.V.A acknowledges funding from US NIH grant R37CA222574. M.B. acknowledges funding from NIH grant 2PO1CA163227. We thank R. Vadhi, M. A. Berkeley and Z. Herbert from the Molecular Biology Core Facility (MBCF) at the Dana-Farber Cancer Institute for preparing and sequencing Illumina libraries. We acknowledge X. Qiu, K. Lim and S. Syamala for valuable technical support.

Author information

Affiliations

Authors

Contributions

A.F.-T., P.C. and H.W.L. designed the study. A.F.-T. developed the protocol and performed the experiments. N.K., Y.X., L.T. and A.F.-T. performed the computational and statistical analyses. A.L., F.S.H., D.V., L.S.Y., A.A.H., E.M.V.A., C.J.S., E.G. and J.B. supplied the clinical tissue and histological analysis. A.F.-T., P.C. and H.W.L. wrote the paper. A.F.-T., M.B., P.C. and H.W.L. supervised the study and revised the paper.

Corresponding authors

Correspondence to Paloma Cejas or Henry W. Long.

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Competing interests

The authors declare no competing interests.

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Related links

Key references using this protocol

Cejas, P. et al. Nat. Med. 22, 685–691 (2016): https://doi.org/10.1038/nm.4085

Cejas, P. et al. Nat. Med. 25, 1260–1265 (2019): https://doi.org/10.1038/s41591-019-0493-4

Extended data

Extended Data Fig. 1 FiT-seq performance on FFPE mouse liver tissue.

(a) Bar plot of total and 10-fold enriched peaks called from H3K4Me2 and H3K27Ac data generated by the optimized original FiT-seq protocol. (b) Average H3K4Me2 and H3K27Ac signal distribution for normal mouse liver tissue obtained by the optimized original FiT-Seq protocol centered at H3K4Me2 called peaks. (c) IGV traces at GAPDH locus. Y axis indicates the number of reads per million per base pair (rbm). Appropriate institutional regulatory board permission was obtained for animal experiments.

Extended Data Fig. 2 FiTAc-seq performance on clinical FFPE specimens.

Unsupervised hierarchical clustering of the sample-sample correlation of the H3K27Ac signal at the union of all enhancers annotated for the five tumor types (total enhancers = 117397): bladder cancers (BlCa), breast cancer brain metastasis (BrCaBM), melanomas (MEL), pancreatic neuroendocrine tumors (PNETs) and seminomas (SEM). Each row and column represents a different sample. Scale represents Pearson’s correlation.

Extended Data Fig. 3 Comparison of FiTAc-seq and ChromEX-PE performance for H3K27Ac on FFPE mouse liver tissue.

ChromEX-PE15 publicly available data was analyzed at the same sequencing depth and with the exact same analysis parameters as the FiTAc-seq data. (a) Average distribution of H3K27Ac signal at enhancers (TSS excluded) in normal mouse liver FFPE tissue obtained from FiTAc-seq and ChromEX-PE protocols in duplicate as indicated in the legend. The union of enhancer peaks from all the replicates was used to center the H3K27Ac signal to perform the aggregation plot. (b) Integrative genomics viewer (IGV) tracks at the Hnf4a locus comparing replicates of the FiTAc-seq (tracks 2 and 3) and ChromEX-PE (tracks 5 and 6) datasets. FF data from ChromEX-PE publication (track 4) and from our mouse model system (track 1) are used as reference. Y axis represents the number of reads per million per base pair (rbm). Appropriate institutional regulatory board permission was obtained for animal experiments.

Extended Data Fig. 4 Representative traces of chromatin fragmentation.

(a) Fragment size distribution achieved with 5 min sonication (tissue homogenization at 65 °C, two replicates) and with 40 min sonication (tissue homogenization at 50 °C, 65 °C or 80 °C) for mouse liver tissue sample. (b) Fragment size distribution for several clinical FFPE archived samples.

Extended Data Fig. 5 Peak calling performance on FiTAc-seq signal.

(a) Bar plot of total, 10-fold and 20-fold enriched peaks for two replicates of mouse FFPE normal liver tissue (FiTAc-seq 65 °C_5min) called with and without input. (b) Corresponding IGV tracks at representative Hnf4a locus with annotation of peaks (black boxes below signal). Y axis indicates the number of reads per million per base pair (rbm). Appropriate institutional regulatory board permission was obtained for animal experiments.

Extended Data Fig. 6 H3K27Ac signal conservation at peaks.

(a) Plots representing evolutionary conservation across mammalian genomes for FF and FFPE 65 °C_5min and 65 °C_40min mouse liver samples. Appropriate institutional regulatory board permission was obtained for animal experiments. (b) Plots representing evolutionary conservation across mammalian genomes for all FiTAc-seq FFPE clinical samples. Y axis represents PhastCons score, X axis represents distance from center in kb.

Supplementary information

Reporting Summary

Supplementary Table 1

Sequencing and peak calling statistics results from normal mouse liver FFPE FiTAc-seq and FF counterpart.

Supplementary Table 2

List of SE annotated by ROSE algorithm for H3K27Ac signal from FF and FiTAc-seq 65°C_5min FFPE mouse liver samples.

Supplementary Table 3

RP scores from marge-potential analysis on H3K27Ac signal for FF and FiTAc-seq 65°C_5min FFPE mouse liver samples.

Supplementary Table 4

Sequencing and peak calling statistics of FiTAc-seq in clinical tissue samples, including (a) mean values in each tissue-type and (b) specific results for each of the samples.

Supplementary Table 5

List of SE identified by ROSE algorithm for FiTAc-seq FFPE clinical tumor samples, including bladder cancers (BlCa), breast cancer brain metastasis (BrCaBM), melanoma (MEL), pancreatic neuroendocrine tumors (PNETs) and seminomas (SEM).

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Font-Tello, A., Kesten, N., Xie, Y. et al. FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues. Nat Protoc (2020). https://doi.org/10.1038/s41596-020-0340-6

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