Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer

Heyn et al. Genome Biology (2016) 17:11 DOI 10.1186/s13059-016-0879-2 RESEARCH Open Access Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer Holger Heyn1*†, Enrique Vidal1†, Humberto J. Ferreira1, Miguel Vizoso1, Sergi Sayols1, Antonio Gomez1, Sebastian Moran1, Raquel Boque-Sastre1, Sonia Guil1, Anna Martinez-Cardus1, Charles Y. Lin2,3,4, Romina Royo5, Jose V. Sanchez-Mut1, Ramon Martinez6, Marta Gut7, David Torrents5,8, Modesto Orozco5,9,10, Ivo Gut7, Richard A. Young2,11 and Manel Esteller1,8,12* Abstract Background: One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. Results: Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans-acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. Conclusions: We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes. Keywords: Super-enhancer, DNA methylation, Epigenomics, Cancer Background The naked DNA sequence alone cannot explain the different cellular functions or phenotypes of cells and organisms with identical genetic sequences, such as the presence of different tissues within the same individual [1], monozygotic twins [2], and cloned animals [3]. This is even more pertinent when we try to explain the * Correspondence: ; † Equal contributors 1 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), 08908 L’Hospitalet de Llobregat, Barcelona, Catalonia, Spain Full list of author information is available at the end of the article pathophysiology of the most common human diseases with their multifactorial causes. The existence of different chemical marks, such as DNA methylation and posttranslational modifications of histones, that regulate gene activity in the epigenetic layers has taken center stage in biology and medicine [4]. However, many studies have taken a biased approach in examining the regulatory sequences nearest to the transcriptional start sites of the studied genes and, with rare exceptions [5–7], other potentially important regions have been neglected in attempts to address the role of epigenomics in tissue identity and disease. In this context, the existence of super-enhancers [8] or locus control regions [9, 10], © 2016 Heyn et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Heyn et al. Genome Biology (2016) 17:11 Page 2 of 16 samples in The Cancer Genome Atlas (TCGA) projects (Table 2) [16–18]. Aligning uniquely mapping bisulfite sequencing reads (mean ~480 million reads per sample) of the original 13 samples undergoing whole genome single-nucleotide resolution analysis resulted in a median genomic coverage of 11.1× (14.1× CpG coverage) per sample. Consistent with previous reported results, apart from bimodal DNA methylation levels at promoter sites, the genomes presented high methylation levels, which were globally reduced in cancer samples (Table S1 and Figure S1 in Additional file 1) [5, 6]. To estimate the relationship between super-enhancers and DNA methylation levels, we determined DNA methylation profiles for enhancer regions within their respective tissue types. From the super-enhancers previously described in our normal tissue types through the histone modification H3K27ac (identified as a superior and sufficient mark for the identification of super-enhancers [11]), we could examine 99.3 % (5128 of 5163; >50 % CpGs covered; Table S1 in Additional file 1) using our WGBS data. We found significant enrichment of unmethylated DNA sequences within the super-enhancers compared with the flanking genomic regions (Fisher’s exact test, odds ratio (OR) 5.6, p < 0.001), supporting the relevance of the features in the here interrogated context. In particular, the edges of the enhancers were CpG-unmethylated, clearly marking the boundaries of the regulatory regions (Fig. 1a, b), a phenomenon that was consistent throughout the analyzed tissue types (Figure S2 in Additional file 1) and that could not be observed in traditional enhancers (Figure S3a, b in Additional file 1) [11]. Moreover, super-enhancers were significantly more hypomethylated large clusters of transcriptional enhancers that drive expression of genes that define cell identity, has been described. Most importantly, disease-associated variation is especially enriched in the super-enhancers of the corresponding cell types [11], and new super-enhancers for oncogenes and other transforming genes have been identified in cancer cells [12–15]. Herein, we present human DNA methylomes at single-nucleotide resolution of normal and cancer cells to identify epigenetic shifts in super-enhancers associated with these diseases. Results and discussion We performed whole genome bisulfite sequencing (WGBS) to obtain unique DNA methylation data sets for five normal tissues and eight associated cancer samples (Table 1). Normal samples (n = 5) included brain, blood (CD19+), breast, lung and colon specimens. In order to enable the analysis of DNA methylation variance from different perspectives, we produced references data sets for cancer samples that involved (...truncated)