- Open Access
Inferring regulatory element landscapes and transcription factor networks from cancer methylomes
© Yao et al. 2015
- Received: 18 March 2015
- Accepted: 7 May 2015
- Published: 21 May 2015
Recent studies indicate that DNA methylation can be used to identify transcriptional enhancers, but no systematic approach has been developed for genome-wide identification and analysis of enhancers based on DNA methylation. We describe ELMER (Enhancer Linking by Methylation/Expression Relationships), an R-based tool that uses DNA methylation to identify enhancers and correlates enhancer state with expression of nearby genes to identify transcriptional targets. Transcription factor motif analysis of enhancers is coupled with expression analysis of transcription factors to infer upstream regulators. Using ELMER, we investigated more than 2,000 tumor samples from The Cancer Genome Atlas. We identified networks regulated by known cancer drivers such as GATA3 and FOXA1 (breast cancer), SOX17 and FOXA2 (endometrial cancer), and NFE2L2, SOX2, and TP63 (squamous cell lung cancer). We also identified novel networks with prognostic associations, including RUNX1 in kidney cancer. We propose ELMER as a powerful new paradigm for understanding the cis-regulatory interface between cancer-associated transcription factors and their functional target genes.
- Enhancer Region
- Putative Target Gene
- Squamous Cell Lung Cancer
- Transcription Factor Network
- Putative Enhancer
ENCODE and other large-scale efforts have mapped transcription factor binding sites, histone modifications, and chromatin accessibility in a common set of cell lines [1, 2]. Integration of these genome-wide maps has led to the view that distinct epigenetic marks are not independent but rather that chromatin is organized into discrete functional states marked by particular combinations of individual features [3, 4]. Computational methods such as chromHMM  and Segway  have been developed to identify these states from individual histone and accessibility features, and the state most consistently linked to cellular identity is the ‘active enhancer’ state defined by the presence of histone H3 lysine 27 acetylation and low levels of the canonical promoter mark, H3 lysine 4 tri-methylation [5, 7, 8]. Active enhancers are enriched for sequences bound by cell-type specific transcription factors, reinforcing their preeminent role in encoding the cis-regulatory logic of the genome. Projects such as the NIH Roadmap [2, 9] and Blueprint  have also mapped histone modifications and chromatin accessibility in primary human tissues, identifying a large set of enhancers from many different cell types. Others have employed these datasets to identify large numbers of enhancer-promoter pairs in 12 human cell types [11, 12]. However, approaches such as ChIP-seq or DNAse hypersensitivity assays require careful tissue handling (to avoid protein degradation) and relatively large numbers of cells (106 to 107) and thus have not been applied to the identification of enhancers in primary tumor tissues.
Fortunately, enhancers can also be identified using patterns of 5-methylcytosine, an epigenetic mark that is maintained more stably than protein marks, and can be detected genome-wide in as few as 1,000 cells . Historically, DNA methylation research has focused on gene promoter regions (reviewed in ). While early work suggested that DNA methylation could mark enhancer regions of interest , this was not widely appreciated until the first complete and unbiased study of DNA methylation in human cells revealed enhancer regions as being unmethylated in a cell-type specific manner . A later study used the same whole-genome bisulfite sequencing (WGBS) approach to identify all genomic regions containing little or no methylation; these regions overwhelmingly corresponded to enhancers and other distal regulatory elements . Cell-type specific demethylation of enhancers was confirmed by targeted bisulfite sequencing in the ENCODE project . More recently, WGBS data from 30 diverse human cell types showed that enhancers had highly dynamic methylation patterns - roughly 30% of the most cell type-specific regions in the genome overlapped known enhancers (compared to 5% that overlapped gene promoters). The mechanism underlying these correlations is not well understood, but could involve de-methylation of DNA initiated by transcription factor binding (; reviewed in ) and maintained by DNA methyltransferase protection by Histone H3 lysine 4 monomethyl groups .
In cancer tissues, recent studies have shown that cancer-specific enhancers and transcription factor binding sites can be identified from DNA methylation profiles. The first genome-scale analysis of transcription factor binding sites in cancer found that binding by transcription factors such as Sp1, NRF1, and YY1 could protect CpG island gene promoters from cancer-specific hypermethylation . Our WGBS study of a human colon cancer identified all genomic regions that changed from a methylated state in the normal colon to an unmethylated state in the tumor; 90% of these regions overlapped known enhancers, and a highly disproportionate number contained binding sites for the AP-1 transcription factor . A more recent study showed that DNA methylation changes at enhancer elements were significantly better than those at promoters for predicting gene expression changes of target genes in cancer . WGBS was recently used to show that unmethylated regions were enriched for binding sites for subtype-specific transcription factors in pediatric medulloblastoma (LEF1 for the WNT subtype and GLI2 for the SHH subtype ).
Once an enhancer has been identified by DNA methylation, identification of the specific target gene or genes whose expression is modulated by that enhancer can be challenging because the target genes can be thousands to millions of base pairs away from the enhancer. A study using chromatin conformation sequencing (ChIA-PET) to study enhancer/promoter interactions found that the median distance between an enhancer and a promoter was approximately 50 kb, and that at least 40 % of enhancers skip one or more annotated genes to find their target promoter . The ChIA-PET dataset was used in conjunction with DNA methylation and RNA-seq data from breast cancer cases in The Cancer Genome Atlas (TCGA) to identify enhancer/promoter pairs in vivo . Other reports have also shown that methylation of distal regulatory sites is closely related to gene expression levels across the genome . Here, we present a statistical framework for identification of cancer-specific enhancers and paired gene promoters, and use it to investigate approximately 3,000 cases from 11 tumors types in the TCGA ‘Pan Cancer’ analysis set . Our R software package, ELMER, uses only methylation and expression data, and does not require any chromatin conformation or ChIP-seq data. Furthermore, by identifying transcription factor binding motifs present within enhancers, and incorporating expression patterns of upstream transcription factors, ELMER is able to infer transcription factor networks activated in specific cancer subtypes. This work suggests a general approach for identifying in vivo transcription factor networks and the associated regulatory control sequences altered in cancer.
Identifying cancer-specific DNA methylation changes in distal enhancer regions for 10 cancer types
To identify cancer-specific changes in DNA methylation, we obtained 3,381 DNA methylation datasets for 11 types of primary tumors from the TCGA Pan Cancer analysis set . The cancer types we included in our analyses were leukemia (LAML), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), kidney renal clear cell carcinoma (KIRC), bladder urothelial carcinoma (BLCA), uterine corpus endometrioid carcinoma (UCEC), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), breast cancer (BRCA), colon adenocarcinoma (COAD), and rectal adenocarcinoma (READ). Based on previous TCGA studies , COAD and READ are very similar and are often combined for analyses. Therefore we combined these two cancer types (indicated herein as CRC), resulting in 10 different primary tumor types. The TCGA ID numbers for all samples can be found in Additional file 1.
To identify enhancers that displayed cancer-specific changes in DNA methylation, we applied a t-test to identify enhancer probes that were significantly hypermethylated or hypomethylated within tumor samples of each cancer type, relative to TCGA adjacent normal samples from the same tissue (Fig. 1b; see Methods for details); a list of the identified hypermethylated or hypomethylated enhancer probes for each tumor type can be found in Additional file 3. We identified many more hypomethylated enhancer probes than hypermethylated probes for each of the 10 cancer types (Fig. 1c). Interestingly, most of the probes showing DNA methylation changes were found to have similar changes in DNA methylation in more than one cancer type. However, some probes were uniquely hypermethylated or hypomethylated in only one of the 10 tumor types. We note that it is not possible for us to be certain that the adjacent tissues collected by TCGA correspond to the same cell type from which the cancer arose, and therefore some of these methylation changes may correspond to tissue-specific differences rather than changes arising in the cancer. However, these differentially methylated probes are only candidates, as the next steps of ELMER (described below) use differences across all normal and tumor tissues (of the same cancer type) to determine true regulatory interactions.
Linking methylation-affected enhancers to gene expression
Identification of regulatory TFs in each cancer type
Different members of a TF family have very similar DNA binding domains that can bind very similar or identical motifs. For example, we have previously shown that GATA1 and GATA2 bind to the same regulatory regions  and that members of the E2F family can bind to the same promoters . Thus, identification of a motif does not uniquely identify the TF that binds in vivo to a region containing that motif. However, there is evidence to support the hypothesis that expression levels of a particular TF can correlate with levels of demethylation and subsequent gene expression [18, 42, 43]. To discover which members of a TF family are likely to be responsible for binding in vivo to the hypomethylated enhancer probes identified above and regulating expression of their putative target genes, we analyzed the correlation between the probes containing a particular motif and expression of all known TFs (Fig. 6b, left). We ranked all the TFs by the degree to which their expression inversely correlated with the methylation status of the enhancers containing the motif (Fig. 6b, right), which allowed us to determine the family member most likely to be involved in regulation of the putative target genes in that particular cancer. For example, the GATA motif was enriched in (expression-linked) enhancer probes in BRCA samples (Fig. 6a). There are six members of the GATA family, with different members being linked to different differentiation phenotypes. For example, GATA1–3 have been linked to the specification of different hematopoietic cell fates and GATA4–6 are involved in differentiation of cardiac and lung tissues [44–49]. GATA3 is one of the most highly enriched transcription factors in the mammary epithelium, has been shown to be necessary for mammary cell differentiation, and is specifically required to maintain the luminal cell fate [48, 49]. Studies of human breast cancers have shown that GATA3 is expressed in early stage, well-differentiated tumors but not in advanced invasive cancers. In addition, GATA3 expression is correlated with longer disease-free survival and evidence suggests that it can prevent or reverse the epithelial to mesenchymal transition that is characteristic of cancer metastasis . Not surprisingly, our analysis of the correlation of the methylation of the GATA motif-containing hypomethylated probes identified GATA3 as the most likely member of the GATA family to be responsible for the observed hypomethylation of GATA-containing enhancers in the BRCA samples (Fig. 6b). Not only was GATA3 the second most correlated transcription factor overall, but the extent of correlation made it easily distinguishable from other members (GATA3 had a U test P value less than 10−40, vs. P values greater than 10−5 for all other GATA family members). Furthermore, expression of GATA3 and methylation of GATA-containing enhancer probes were co-linked to breast cancer subtypes. As shown using color-coding in the Fig. 6b scatterplot, Luminal tumors had high expression of GATA3 and low methylation of GATA-containing enhancer probes, while Basal-like subtype tumors showed the converse. Figure 6c shows an example of one of these GATA-containing enhancer probes (cg1396202), along with the target gene (CCND1) predicted by expression to be regulated by this putative enhancer. ENCODE ChIP-seq data in the Luminal-subtype MCF7 cell line confirm that this putative enhancer region is indeed bound by GATA3, confirming the relationship between transcription factor binding and demethylation shown in . This case was among the easier to detect, since breast cancer has two large subtypes (Luminal and Basal-like), which are molecularly quite distinct and are increasingly seen as two different diseases. As with all cancer genomic approaches, rarer subtypes will require larger number of samples to be identified by ELMER. Nevertheless, our results on other more challenging cancer types were also promising, as described below.
In our studies, we have used tumor-specific changes of the DNA methylation status within distal enhancer regions to provide insight into the mechanisms of gene expression, transcription factor networks, and tumor classification. We have shown that this can be a powerful approach for generating hypotheses about master regulators in cancer, and we propose that ELMER analysis be applied along with other hypothesis-generating approaches in high throughput cancer genomics. For the TCGA Pan-Cancer dataset, we provide to the community prioritized lists of putative enhancer-target gene pairs for future validation, and lists of site-specific transcription factors that should be further investigated for their role in the development and progression of specific tumor types.
We found that most of the putative linkages between enhancer probes and local gene expression were cancer type-specific and that within each cancer type, most enhancers correlated with the expression of only one gene. In keeping with previous looping studies, we found that the putative target gene was typically not the nearest gene. In fact, the gene identified was the nearest gene in only approximately 15 % of the hypomethylated enhancer-gene pairs. As in other studies [51, 52], we found that the set of all hypomethylated enhancers was composed of similar proportions of intragenic and intergenic enhancers. We found that as compared to the intergenic enhancers, intragenic enhancers were 75 % more likely to be linked to expression of the nearest TSS (which in 88 % of the cases was the gene in which it resided); see Additional file 12. An intragenic enhancer can loop to regulate the ‘upstream’ promoter of the gene in which it resides but could also act as alternative promoter. Although we have eliminated all known promoters from our set of distal probes, we cannot eliminate the possibility that some of the intragenic enhancers represent as-of-yet unannotated, tumor-specific alternative promoters for the gene in which they reside [53, 54].
Our linking method is based strictly on correlation and therefore cannot absolutely rule out indirect (trans) interactions. For instance, if the same transcription factor or set of factors regulate both enhancer X and enhancer Y, the methylation patterns of X and Y across samples may be so similar that we link enhancer X to a gene that is in fact the direct target of enhancer Y. We have used high-confidence statistical thresholds in order to rule out as many of these indirect interactions as possible. Our search within the nearest 20 genes is unbiased, so the fact that we disproportionally find linkages to the gene nearest the enhancer probe provides strong evidence that we are identifying true direct (cis) interactions. We have provided a robust set of predicted linkages that can serve as a starting point for future experimental validations. Of course, we realize that we are working under a largely untested assumption that anti-correlation between an enhancer and expression level of a nearby gene indicate functional regulation. While this and prior correlative studies [22, 23, 25] provide strong supporting evidence for this, further experimental studies (for example, using CRISPR/Cas9 to delete the enhancers in appropriate tumor cell lines, followed by RNA-seq) will be needed to determine with certainty that the enhancers regulate their putative target genes, and what degree of correlation is required to infer functionality. Similarly, a comparison between our predicted enhancer-target pairs and global analysis of long-range chromatin looping would be of interest. Unfortunately, chromatin conformation assay data are not available for any of the tumor tissue samples and, in fact, very few studies of global chromatin looping have been completed for cancer cell lines. However, we have identified a set of chromatin loops derived from deep-sequenced ChIA-PET data from MCF7 cells . Although MCF7 cells are not representative of all breast cancers (and are cultured cells, not tumor tissues), we did find that 166 of the 2,038 enhancer probes pairs we identified in breast cancer tumors (approximately 8%) were also identified as loops in the MCF7 ChIA-PET data. This was an almost four-fold enrichment over randomized enhancer probe-gene pairs (see Additional file 13 for an enrichment analysis, along with a complete list of BRCA enhancer-gene pairs falling within loops in MCF7 cells). We note that the various assays used to study looping are not yet optimized and do not always identify the same sets of loops ; in addition, some loops may not be related to transcriptional regulation. Thus, enhancer-gene pairs identified by expression assays are not necessarily concordant with the sets of promoter-enhancer loops identified by chromatin confirmation assays. Future comparisons between indirect (that is, correlative) mapping of enhancer-gene interactions of the type we described here, with direct physical mapping of enhancer-gene interactions, will be important to help to resolve the different mechanisms involved. However, in addition to the genome-wide confirmation by ChIA-PET, we note that at least two of the putative enhancer-gene pairs from our analysis have been studied in functional models confirming our results. The putative CCND1 enhancer we identified in breast tumors (Fig. 6c) was shown to directly regulate the CCND1 gene in response to estradiol in breast cancer cells  and a putative MYC enhancer we identified in colon tumors (Additional file 14) was shown to be directly responsible for MYC expression in colon cancer cells , and in vivo in a mouse model of colorectal cancer .
We realize that the relationship between TF binding and DNA methylation can be complex . For example, reduced DNA methylation in an enhancer region in a tumor cell relative to a normal cell could allow a TF to bind and regulate a target gene in a tumor-specific manner without changes in the expression level of that TF in the tumor. However, it is likely that increased levels of a TF in a tumor can result in higher binding at a partially methylated enhancer, directly leading to loss of DNA methylation . Based on this second mechanism, we have attempted to identify TFs that regulate the target genes of enhancers that are hypomethylated in tumors. First, we identified a list of site-specific TF binding motifs that are enriched within the enhancers linked to putative target genes. Then, by examining the expression patterns of each of the TF family members expected to bind to these motifs, we have predicted the TF that regulates specific sets of genes in the different cancer types (Fig. 8). For example, in bladder cancer (BLCA) we have provided a list of 65, 208, and 65 genes that may be regulated by POU3F1, FOXA1, or CEBPA, respectively, by binding to a specific hypomethylated enhancer. In all, utilizing enhancer methylation patterns, expression of putative target genes, motif enrichment, and expression of TF family members that bind to the motif, we have derived a list of 4,280 enhancer-TF-putative target gene linkages.
Some of the cancer type-specific TF networks we show in Fig. 8 are already known to have a functional role in the same tumor type, such as PU.1 in AML  and TCF7L2 in colorectal cancer [28, 60–63]. Two of the four TFs we identified in squamous cell lung cancer (LUSC), TP63 and SOX2, are oncogenes that are overexpressed in LUSC through genomic amplification [64, 65]. Recently, SOX2 and TP63 were shown to interact functionally and co-localize to a large number of genomic binding sites in squamous cell lung cancer . In a number of cases, incorporating TF expression data allowed us to resolve between different members of the same family that would be indistinguishable by binding motif alone. For instance, FOXA1 clearly appears to be responsible for hypomethylation of FOX-containing enhancers in breast (BRCA) and bladder (BLCA) cancers, while FOXA2 appears to be responsible in endometrial (UCEC). Other TF networks we identified, such as RUNX1/2 and its association with poor outcome in kidney cancer, have never been reported and will form the basis for future studies.
The method we describe herein is based on detecting methylation and expression differences between samples of the same tumor type, and is therefore aimed at identifying changes that co-occur within particular subsets of cases. For instance, we found that GATA-containing enhancer hypomethylation occurred primarily in the subset of breast cancer cases belonging to the Luminal subtype, which also had high expression of the GATA3 gene (Fig. 6b, c). While GATA3 is a well-studied case, our method can be applied to identify, understand, and find biomarkers for novel molecular subtypes. Understanding the genome-wide transcriptional consequences of molecular subtypes will be particularly relevant for those that are defined by genetic mutation of transcriptional regulators; indeed, transcription factors make up the largest functional class within the list of 127 cancer genes with so-called ‘driver’ mutations identified by TCGA . A number of the altered transcription factor networks we identified using ELMER (Fig. 8) were also present within the 30 or so transcription factors included in this TCGA driver gene list. These TFs included FOXA1, FOXA2, GATA3, NFE2L2, and SOX17. Intriguingly, ELMER often identified a particular TF in the same cancer type or types where it is most frequently mutated. For instance, FOXA1 is most frequently mutated in Breast and Bladder cancer, and ELMER identified it in these specific cancers. Likewise, FOXA2 and SOX17 are primarily mutated in endometrial cancers, and ELMER identified network alterations specifically in this cancer type (UCEC). NFE2L2 is most frequently mutated in lung squamous cell carcinoma (LUSC), the same cancer type where ELMER detected NFE2L2 alterations. It will take additional work to understand the relationship between genetic mutations of TFs and epigenetic/transcriptomic changes in each of these different examples, but the identification of important cancer driver genes underscores the power of studying enhancers, which sit at the cis-regulatory interface between transcription factors, epigenetic modifiers, and downstream effector genes.
We also note that in some cases, transcription factors that are not expected to bind to the specific motif being analyzed were identified as being highly correlated with the degree of enhancer hypomethylation. In all, we identified 186 TFs frequently correlated with multiple motifs that do not correspond to the known motif for that TF family (Additional file 15). These correlations could be due to indirect effects caused by TF networks. For example, transcription factors regulated by GATA3 may show a similar correlation of expression with the hypomethylated probes in BRCA as does GATA3 itself. Another possible cause is suggested by the case of AP-1. Our results indicate that hypomethylation of AP-1-containing enhancers is a common feature of many or most cancer types (including nine of our 10 cancer types, see Fig. 6a); this confirms our earlier whole-genome observations in colorectal cancer . While the AP1 motif is classically described as a binding sequence for FOS/JUN dimers, it is found to be enriched in many ChIP-seq datasets, including those using antibodies that recognize factors other than FOS or JUN family members . Phosphorylation of JUN can lead to histone acetylation at AP-1 motif-containing enhancers by inhibiting their association with the Mbd3 component of the NuRD complex . This could in turn allow binding of other positive transcriptional regulators, activation of downstream genes, and a proliferative expression program. Because JUN activity is regulated post-transcriptionally, it is logical that our method (which is based on expression) would miss JUN itself, and instead identify the positive regulators binding these regions (which are often cell-type specific). For instance, the most strongly associated TF with the AP-1 motif in kidney cancer is RUNX1, while in breast cancer it is FOXA1, suggesting that many of the AP-1 motif-containing sites may require AP-1 dependent de-repression along with positive RUNX1/FOXA activation.
Also included in the list of 186 ‘commonly correlated’ TFs are around 50 zinc finger domain-containing TFs (known as ZNFs). Although ZNFs are the most abundant class of human site-specific TFs, comprising around half of all site-specific TFs [70–72], few of them have been well studied. One of the commonly correlated factors was ZNF703, which correlated with 16 different motifs in the BRCA samples. Interestingly, high expression of ZNF703 has been shown to correlate with poor prognosis in patients with luminal B breast cancer . We suggest that our analyses can point to a role for other ZNFs in tumorigenesis. In fact, 11 of the identified ZNFs showed associations with survival of the cancer in which they were identified (Additional file 16). For example, ZNF273 was correlated with four motifs in CRC and ZNF683 was correlated with nine motifs in KIRC; neither of these TFs has ever been associated with cancer. However, there is a strong correlation between high expression of ZNF273 and ZNF683 with poor survival rates in colorectal and kidney cancers, respectively. Most of the time, the 186 ‘commonly correlated’ TFs showed cancer type-specific correlations. However, one factor (GRHL2) was identified in the top 1 % of all correlations for 31 different motifs spread among five of the 10 different cancer types studied. GRHL2 has been shown to directly bind and activate the hTERT promoter and has been suggested to be involved in telomerase activation during cellular immortalization . Perhaps GRHL2 plays an important role in tumor development in many cancer types.
The results we describe here use motif analysis primarily to help identify the transcription factors responsible for enhancer hypomethylation. However, the most important output of this work may actually be the identification of enhancers in which mutations in individual transcription factor binding sites can be responsible for cancer risk or cancer progression. A number of studies have shown that population risk alleles for cancer reside preferentially in enhancer regions [31, 75–79] and a recent paper demonstrated that these could be identified in breast cancer by combining DNA methylation and chromatin conformation capture data to identify putative enhancers . Somatic enhancer mutations are predicted to affect cancer progression, although these have not yet been identified due to the overwhelming use of exon sequencing as a means to identify new cancer mutations. The recent availability of whole-genome sequencing of tumors has started to allow the identification of non-coding mutations, which have been shown to affect transcription factor binding sites [80–82]. Methods like ELMER, which can identify in vivo enhancer regions in tumors, will be essential for analyzing non-coding cancer mutations arising from WGS studies.
Although our study is not comprehensive due to the nature of the DNA methylation platform used by TCGA (which only contains coverage of 15 % of known enhancers) and because enhancers have not yet been mapped in all normal and tumor cell types, our analyses have allowed us to identify a number of cancer type-specific transcriptional regulators, along with the cis-regulatory sequences mediating effects on target genes. Large-scale identification of such cis-regulatory regions will be critical for understanding the effects of non-coding genetic polymorphisms on cancer risk and non-coding somatic mutations on cancer progression [28, 59, 60]. Complete tumor methylation profiles using whole-genome bisulfite sequencing [21, 23, 83] are rapidly becoming available, and these will dramatically increase the power of the ELMER approach to reconstruct complete transcription factor network and identify important cis-regulatory regions.
Availability of source code and R package
All source code is available as an R package, ELMER, downloadable from the main Bioconductor repository  or from our GitHub repository . Vignettes illustrating the use of the functions are available as part of the BioConductor package, along with an example replicating the results described in this paper using the ELMER function TCGA.pipe. A user manual and tutorial can be downloaded from the GitHub repository here: , and a full manual can be downloaded here: .
DNA methylation and RNA-seq datasets
TCGA level 3 DNA methylation data based on the Illumina Infinium HumanMethylation450 BeadArray platform was downloaded from . Only the samples whitelisted by TCGA for Pan-Cancer Analysis Working Group were used in the study. The whitelist can be downloaded from Sage Bionetworks Synapse  with identifier syn1571603. The version numbers and final sample IDs for each cancer type are listed in Additional file 1. The DNA methylation level at each CpG is referred to as a beta (β) value, calculated as (M/(M+U)), where M represents the methylated allele intensity and U the unmethylated allele intensity, which are normalized using the TCGA standard pipeline. Beta values are in the range of 0 to 1, reflecting the fraction of methylated alleles at each CpG in the each tumor; beta values close to 0 indicating low levels of DNA methylation and beta values close to 1 indicating high levels of DNA methylation. Since there are no available normal tissues for acute myeloid leukemia (LAML) and glioblastoma multiforme (GBM) in TCGA, we also downloaded Infinium HM450K DNA methylation data from publicly available sources as normal tissue controls for these two cancer types. A set of 58 sorted glial cell samples from GEO (accession number GSE41826) was used as normal reference samples for glioblastoma. A set of 11 sorted blood samples from GEO (accession number GSE49618) was used for normal reference samples for leukemia. These data were generated at the USC Epigenome Center and were processed through the same data analysis pipeline that was used to create the TCGA Level 3 data files (all TCGA data were also generated by the USC Epigenome Center). The sample IDs are also listed in Additional file 1.
TCGA Level 3 RNA-seq data were downloaded from . The version number of each package downloaded is listed in Additional file 1. TCGA uses gene-level expression values, meaning any alternative isoforms are included in a single normalized RSEM expression value. TCGA data production and analysis pipelines are described elsewhere, but a brief description follows: all data were generated on the Illumina HiSeq platform, with the exception of UCEC, which was generated on the Illumina GAII platform. Within each cancer type, data were mapped with MapSplice and quantitated with RSEM (RNA-seq by Expectation Maximization). RSEM outputs expression values that are normalized across samples, so that the third quartile for each sample equals 1,000. Entrez gene IDs were used for mapping to genomic locations using GenomicRanges . The final RNA-seq sample IDs used in our analyses are listed in Additional file 1.
Selecting enhancer probes
Probes overlapping SNPs are removed as part of the standard TCGA Level 3 pipeline. Probes located less than 2 kb from an annotated transcription start site in GENCODE v.15 were filtered out to remove promoter regions from our analysis. ENCODE/REMC chromHMM data were downloaded from  and any HM450 probes falling within the genomic regions annotated as EnhG1, EnhG2, EnhA1, or EnhA2 were selected. FANTOM5 data were downloaded from  and any HM450 probes falling within regions annotated as eRNA were selected. This resulted in 102,518 enhancer probes, which are listed in Additional file 2. This functionality is implemented in the get.feature.probe function of the ELMER BioConductor package.
Identifying enhancer probes with cancer-specific DNA methylation changes
Each of the 10 cancer types was processed independently to identify cancer-specific DNA methylation changes. For each enhancer probe, we first ranked tumor samples and normal samples (within the cancer type) by their DNA methylation beta values. To identify hypomethylated probes, we compared the lower normal quintile (20 % of normal samples with the lowest methylation) to the lower tumor quintile (20 % of tumor samples with the lowest methylation), using an unpaired one-tailed t-test. Only the lower quintiles were used because we did not expect all cases to be from a single molecular subtype, and we sought to identify methylation changes within cases from the same molecular subtype. Twenty percent (that is, a quintile) was picked as a cutoff to include high enough sample numbers to yield t-test P values that could overcome multiple hypothesis correction, yet low enough to be able to capture changes in individual molecular subtypes occurring in 20 % or more of the cases. This number can be set arbitrarily as an input to the get.diff.meth function in the ELMER package, and should be tuned based on sample sizes in individual studies. The one tailed t-test was used to rule out the null hypothesis: μtumor ≥ μnormal, where μtumor is the mean methylation within the lowest tumor quintile and μnormal is the mean within the lowest normal quintile. Raw P values were adjusted for multiple hypothesis testing using the Benjamini-Hochberg method, and probes were selected when they had adjusted P value less than 0.01. For additional stringency, probes were only selected if the methylation difference |Δ|= |μnormal - μtumor | was greater than 0.3. This technique is illustrated in Fig. 1b, and carried out in the get.diff.meth function of the ELMER package. The same method was used to identify hypermethylated probes, except we used upper tumor quintile and upper normal quintile, and chose the opposite tail in the t-test. The full set of hypermethylated and hypomethylated probes we identified are provided in Additional file 3, and can be replicated using the TCGA.pipe vignette in the ELMER package.
Linking enhancer probes with methylation changes to target genes with expression changes
MCF7 ChIA-PET linkage pairs were taken from a previous publication . The random pairs were generated by randomly selecting the same number of probes from the set of distal enhancer probes, and pairing each with one or more of the 20 adjacent genes; the number of links made for each random probe was identical to the corresponding ‘true’ probe. Thus, the random linkage set has both the same number of probes and the same number of linked genes as the true set. One hundred such random datasets were generated to arrive at a 95 % CI (+/-1.96* SD).
Gene Ontology (GO) enrichment analysis
Genes associated with hypo- or hypermethylated enhancer probes in more than one cancer type were selected for GO analysis. GO analyses were performed using the R package ‘topGO’ . The classic Fisher test was used to generate enrichment P values. To select the GO terms that pass a significance cutoff, P values were adjusted using the Benjamini-Hochberg method; only those GO terms with a P value <0.01 and a fold change >1.5 are shown in Fig. 5.
Associating TF expression with TF binding motif methylation
For each motif considered to be enriched within a particular probe set, we compared the average DNA methylation at all distal enhancer probes within +/− 100bp of a motif occurrence, to the expression of 1,777 human TFs ( and with further refinements, see Additional file 17). A statistical test was performed for each motif-TF pair, as follows. The samples (all tumors and normal within a particular cancer type) were divided into two groups: the M group, which consisted of the 20 % of samples with the highest average methylation at all motif-adjacent probes, and the U group, which consisted of the 20 % of samples with the lowest methylation. The 20th percentile cutoff is a parameter to the get.TFs function of the ELMER package, and was set to allow for identification of molecular subtypes present in 20 % of cases. For each candidate motif-TF pair, the Mann-Whitney U test was used to test the null hypothesis that overall gene expression in group M was greater or equal than that in group U. This non-parametric test was used in order to minimize the effects of expression outliers, which can occur across a very wide dynamic range. For each motif tested, this resulted in a raw P value (P r ) for each of the 1,777 TFs. All TFs were ranked by the -log10(P r ), and those falling within the top 5 % of this ranking were considered candidate upstream regulators. The best upstream TFs for each of these cases was automatically extracted as high-value candidates, and presented in Fig. 8. These high-value candidates are also shown in detail in Additional files 9 and 10.
A Kaplan-Meier survival analysis was used to estimate the association of the TF expression with the survival of patients. For each selected TF and cancer type combination, tumor samples with the highest (top 30 %) and lowest (bottom 30 %) transcription factor expression were analyzed using a Log Rank test. Overall survival was calculated from the date of initial diagnosis of cancer to disease-specific death (patients whose vital status is termed dead) and months to last follow-up (for patients who are alive).
The TCGA samples can be downloaded at https://tcga-data.nci.nih.gov/tcgafiles/ftp_auth/distro_ftpusers/anonymous/tumor/. The whitelist from Pan-Can group is available on Synapse (https://www.synapse.org/) as syn1571603. The enhancer genomic coordinates can be downloaded at http://egg2.wustl.edu/roadmap/data/byFileType/chromhmmSegmentations/ChmmModels/coreMarks/jointModel/final/) and http://enhancer.binf.ku.dk/Welcome.html.
We thank the ENCODE Project, the Roadmap Epigenome Mapping Consortium, and the FANTOM Consortium for the use of the genomic locations of enhancers [2, 9, 31, 32]. The results published here are largely based upon data generated by the TCGA Research Network (http://cancergenome.nih.gov/), and our use of these data is in accordance with the guidelines at: http://cancergenome.nih.gov/publications/publicationguidelines. We thank the TCGA Consoritum members for use of these datasets. We thank Simon Coetzee and Toshinori Hinoue for suggestions and help with the ELMER BioConductor package. BPB and LY were supported in part by NCI grant 1U01CA184826, and BPB in part by 5R01HG006705. PJF and LY were supported in part by National Cancer Institute (NCI) grants 1U01ES017154, U54HG006996, and P30CA014089. HS and PWL were supported in part by the TCGA Consortium through NCI grant 1U24CA143882. High-performance computing support was provided by the USC High Performance Computing Center (HPCC).
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