Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma Abstract Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma. Introduction DNA methylation at the fifth position of cytosine [5-methylcytosine (5mC)] has been one of the most studied epigenetic modifications in mammals to date. 5mC is involved in the regulation of multiple physiologic and pathologic processes, including cancer, and when located at gene promoters, it is usually linked to transcriptional repression. As distinctive features of tumorigenesis, local DNA hypermethylation and global hypomethylation have been attributed to changes in 5mC levels (1, 2). However, the discovery a few years ago, of 5-hydroxymethylcytosine (5hmC), a new epigenetic mark resulting from 5mC oxidation, is reshaping our view of the cancer epigenome (3, 4). This 5mC to 5hmC conversion in mammals is mediated by ten–eleven translocation proteins 1, 2 and 3 (TET1, TET2 and TET3), a family of α-ketoglutarate and Fe(II)-dependent dioxygenases (4, 5). Global levels of 5hmC in the genome fluctuate considerably according to tissue type, and are consistently around 10-fold lower than those of 5mC, though it is interesting that the highest levels of both marks are found in brain (6–11). Several studies have shown that 5hmC is an intermediate of DNA demethylation (7, 12, 13), and that it is also associated with cancer (14–18). In this same vein, loss of 5hmC has been reported across a wide range of human cancers including melanoma, glioma, breast, colon, gastric, kidney, liver, lung, pancreatic and prostate cancers (9, 14, 18–22). Although none of these studies actually demonstrate that changes in 5hmC are directly affecting cancer biology independent of its role in DNA demethylation, other studies identifying 5hmC-specific protein binders suggest that this epigenetic mark might have its own biologic role (23, 24). The fact that there are now methods available that distinguish 5mC and 5hmC positions at single-base resolution within the genome prompted us to reassess the role of DNA methylation status in tumorigenesis from a 5hmC perspective. The method used here allowed us to describe global and genome-wide locus-specific 5mC and 5hmC patterns in brain samples, to identify a specific chromatin signature associated with changes of these epigenetic marks in glioma and, most importantly, to describe a novel non-canonical type of aberrant DNA hypermethylation. Results Global changes of 5mC and 5hmC in cancer To evaluate the role of 5hmC in the changes of DNA methylation observed in glioma, we first analyzed the levels of 5hmC and 5mC at DNA repeats in 11 normal and 50 tumor samples. We used oxidative bisulfite conversion (oxBS) of DNA to discriminate between 5mC and 5hmC (see Materials and Methods) and bisulfite pyrosequencing was used to determine the level of both epigenetic modifications in four different types of repeated DNA: the retrotransposons LINE-1 and AluYb8, and the pericentromeric tandem repeats Sat-alpha and NBL-2 (25). These four DNA regions contain most of the genomic methylation and, consequently, global DNA methylation level is highly dependent on their 5mC content (26). As expected, 5mC levels at repeated DNA in healthy tissue were high but were reduced in tumor samples, a change that was statistically significant in LINE-1 and Sat-alpha (linear model, P < 0.001) (Fig. 1A). In contrast, levels of 5hmC at repeated DNA in healthy tissue were low, while tumoral tissue showed even lower levels of 5hmC in the same DNA regions, although this was only statistically significant in LINE-1 (linear model, P < 0.05) (Fig. 1B). Figure 1. View largeDownload slide 5mC and 5hmC levels at repetitive DNA sequences in glioma. 5mC (A) and 5hmC (B) values of several repetitive regions (AluYb8, LINE-1, NBL-2 and Sat-alpha) measured by pyrosequencing in controls and gliomas. Individual CpG site values for each repeat are displayed, and a linear model including both ‘sample group’ and ‘CpG site’ as covariates was fitted. Significant P-values for any repetitive region are shown. Figure 1. View largeDownload slide 5mC and 5hmC levels at repetitive DNA sequences in glioma. 5mC (A) and 5hmC (B) values of several repetitive regions (AluYb8, LINE-1, NBL-2 and Sat-alpha) measured by pyrosequencing in controls and gliomas. Individual CpG site values for each repeat are displayed, and a linear model including both ‘sample group’ and ‘CpG site’ as covariates was fitted. Significant P-values for any repetitive region are shown. 5mC and 5hmC profiling in brain tissue As changes in 5hmC at repeated DNA were not able to explain the global loss of this epigenetic mark previously observed by mass spectrometry (14,20,27,28), we hypothesized that these changes primarily occur at single copy sequences. To investigate this possibility in more detail, we first used 450K Infinium methylation arrays to determine the level and genomic distribution of 5mC and 5hmC in five healthy brain tissue samples. A preliminary examination of the data revealed that the beta values of the oxidized samples (true 5mC) were lower than their non-oxidized counterparts (5mC + 5hmC) (Wilcoxon rank sum test; P < 0.001; W = 2.34e13) (Fig. 2A). Specifically, 111 633 hydroxymethylated CpG sites (5hmC sites) distributed throughout the whole genome were identified, irrespective of the chromosome analyzed (Fig. 2A;Supplementary Material, Table S1). Figure 2. View largeDownload slide Characterization of DNA 5hmC in normal brain. (A) Bean plots showing differences between average Beta values of 5mC + 5hmC (BS) and true 5mC (OxBS) values in normal brain. The diamond inside the bean indicates the mean value. Hilbert curve showing the amount and genomic distribution of 5hmC in brain. A Level-9 Hilbert curve was used. Each region delimited by black lines represents a chromosome. A point in the image represents a genomic segment of ∼950 bp. A blue point indicates the presence of at least one 5hmC enriched CpG in the given segment. A Hilbert curve including all CpG sites analyzed in the methylation array (450 K) is also shown (B) Associations between 5hmC and CpG density. (C) Distribution of 5hmC CpG sites relative to CpG island status and compared with the array background (450 K). (D) Distribution of 5hmC CpG sites relative to different genomic regions. (E) Heatmaps showing significant enrichment of the 5hmC CpG sites identified in brain, with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Color code indicates the significant enrichment based on log2 OR. (F) Heatmaps showing significant enrichment of 5hmC CpG sites with fifteen ‘chromatin states’ generated by an HMM. Color codes indicate the significant enrichment based on log2 OR. Figure 2. View largeDownload slide Characterization of DNA 5hmC in normal brain. (A) Bean plots showing differences between average Beta values of 5mC + 5hmC (BS) and true 5mC (OxBS) values in normal brain. The diamond inside the bean indicates the mean value. Hilbert curve showing the amount and genomic distribution of 5hmC in brain. A Level-9 Hilbert curve was used. Each region delimited by black lines represents a chromosome. A point in the image represents a genomic segment of ∼950 bp. A blue point indicates the presence of at least one 5hmC enriched CpG in the given segment. A Hilbert curve including all CpG sites analyzed in the methylation array (450 K) is also shown (B) Associations between 5hmC and CpG density. (C) Distribution of 5hmC CpG sites relative to CpG island status and compared with the array background (450 K). (D) Distribution of 5hmC CpG sites relative to different genomic regions. (E) Heatmaps showing significant enrichment of the 5hmC CpG sites identified in brain, with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Color code indicates the significant enrichment based on log2 OR. (F) Heatmaps showing significant enrichment of 5hmC CpG sites with fifteen ‘chromatin states’ generated by an HMM. Color codes indicate the significant enrichment based on log2 OR. To further validate the consistency of our results we compared our data with published data for 5hmC in human normal brain (29) obtained by Tet-Assisted Bisulfite Sequencing (TAB-Seq), an alternative technique that directly measures the 5hmC rather than inferring it, and we found that more than 75% of the 5hmC-enriched CpG sites identified in our study overlapped with 5hmC-enriched CpG sites analyzed by the TAB-Seq (Supplementary Material, Fig. S1). The analysis of the genomic distribution of the 5hmC sites showed that hydroxymethylation is enriched at the low CpG-density regions interrogated by the array (Wilcoxon non-parametric test; P < 0.001, D=−0.5) (Fig. 2B). Consequently, the 5hmC sites were enriched in non-CpG islands (non-CGI) [χ2 test; P < 0.001; odds ratio (OR) = 1.93, and P < 0.001, OR = 3.45, respectively] and infrequent in CGIs (χ2 test; P < 0.001, OR = 0.14, and P < 0.001, OR = 0.13) (Fig. 2C). With respect to genes, 5hmC sites were enriched in introns (χ2 test; P < 0.001, OR = 1.82, and P < 0.001, OR = 1.76, respectively), but were less frequent than expected in gene promoters (χ2 test; P < 0.001, OR = 0.58 and P < 0.001, OR = 0.6) (Fig. 2D). To identify possible chromatin marks associated with 5hmC sites, we compared these CpG sites with previously published data on a range of histone modifications and chromatin modifiers in 10 different cell types (see Materials and Methods) (Fig. 2E). This approach identified statistically significant associations (Fisher’s exact test; P < 0.05) between the 5hmC sites in brain and the active histone marks H3K4me1, H3K36me3 and H4K20me1 (Fig. 2E). A similar framework was used to test for the enrichment of our selected probes over the computer-generated chromatin segmentation states from the ENCODE ChromHMM project (see Materials and Methods). In total, fifteen states were used to segment the genome, and these were then grouped and colored to highlight predicted functional elements. This approach showed that the hmC sites were significantly enriched in states associated with enhancers and transcription (Fisher’s exact test; P < 0.05) (Fig. 2F). These associations were further corroborated by using available chromatin immunoprecipitation sequencing (ChIP-seq) tracks from epigenomes of eight brain cell types obtained from the NIH Roadmap Epigenome consortia (30) (Fisher’s exact test; P < 0.05) (Supplementary Material, Fig. S2). Locus-specific alterations of 5hmC in glioma To identify differentially hydroxymethylated CpG sites (d5hmC) at single copy sequences in cancer, we used 450K methylation arrays to analyze nine primary tumors obtained from patients with glioma (see Materials and Methods). A total of 49 601 CpG sites that were hypo-hydroxymethylated were identified in gliomas, but almost no hyper-hydroxymethylated sites were found (see Materials and Methods) (Fig. 3A;Supplementary Material, Table 2). To validate the results obtained with the methylation arrays with an alternative technique nondependent on the oxBS of DNA, we randomly selected five of the 100 most variable sequences previously identified and analyzed their 5hmC status using a hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit (Epigentek; see Materials and Methods) in five brain samples and five gliomas. The results corroborated the massive loss of 5-hydroxymethylation in glioma in all the candidate sequences (Mann–Whitney test; P < 0.05) (Supplementary Material, Fig. S3). Figure 3. View largeDownload slide Alterations of 5hmC in glioma. (A) Bar plot showing the number of d5hmC sites in glioma. (B) Unsupervised hierarchical clustering and heatmap including CpG sites with 5hmC loss in glioma (3000 random probes). (C) Associations between 5hmC loss in glioma and density of CpGs (upper panel), CpG island status (middle panel), and different genomic regions (lower panel). (D) Heatmaps showing significant enrichment of hypo5hmC CpGs identified in glioma with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. (E) Heatmaps showing significant enrichment of hypo5hmC CpGs in gliomas with 15 ‘chromatin states’ generated by an HMM (right panel). Color codes indicate the significant enrichment based on log2 OR. Figure 3. View largeDownload slide Alterations of 5hmC in glioma. (A) Bar plot showing the number of d5hmC sites in glioma. (B) Unsupervised hierarchical clustering and heatmap including CpG sites with 5hmC loss in glioma (3000 random probes). (C) Associations between 5hmC loss in glioma and density of CpGs (upper panel), CpG island status (middle panel), and different genomic regions (lower panel). (D) Heatmaps showing significant enrichment of hypo5hmC CpGs identified in glioma with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. (E) Heatmaps showing significant enrichment of hypo5hmC CpGs in gliomas with 15 ‘chromatin states’ generated by an HMM (right panel). Color codes indicate the significant enrichment based on log2 OR. Hierarchical clustering using the d5hmC showed the correct classification of normal and tumor samples (Fig. 3B). The analysis of the genomic distribution of the hypo-hydroxymethylated CpG sites in gliomas showed an enrichment at low CpG density regions (Wilcoxon rank sum test, P < 0.001, D = -0.41), and consequently at non-CpG islands (χ2 test, P < 0.001, OR = 2.53) (Fig. 3C). With respect to gene location, hypo-hydroxymethylation was more frequent in introns (χ2 test, P < 0.001, OR = 1.77) (Fig. 3C). To identify possible chromatin signatures associated with DNA hypo-hydroxymethylation in gliomas, we compared our list of hypo-hydroxymethylated CpG sites with previously published data on a range of histone modifications and chromatin modifiers in 10 different cell types (see Materials and Methods) (Fig. 3D). Interestingly, this approach showed an enrichment of hypo-hydroxymethylation at chromatin regions marked with the activating histone post-translational modifications (PTMs) H3K4me1, H3K36me3, H4K20me1 and H3K79me2 (Fisher’s exact test, P < 0.05) (Fig. 3D), but not with the repressive histone modification H3K27me3, which has been previously shown to be associated with aberrant DNA hypermethylation in cancer (31,32) (Fig. 3D). A similar framework was used to test for the enrichment of our selected probes over the computer-generated chromatin segmentation states from the ENCODE ChromHMM project. Using this approach, we found that hypohydroxymethylated CpG sites were significantly associated with transcription regulation and enhancers (Fisher’s exact test; P < 0.05) (Fig. 3E). These associations were further corroborated using available ChIP-seq tracks from the epigenomes of 8 types of brain cell obtained from the NIH Roadmap Epigenome consortia (30) (Fisheŕs exact test; P < 0.05) (Supplementary Material, Fig. S4). DNA hypo-hydroxymethylation identifies a novel type of non-canonical aberrant DNA hyper-methylation in glioma To study the relationship between changes in 5mC and 5hmC in glioma, we first identified aberrantly methylated CpG (d5mC) sites. The comparison of the methylation data between tumoral and control samples (see Materials and Methods) identified 2727 hypo- and 12 050 hyper-methylated CpG sites in gliomas (Supplementary Material, Tables S3 and S4). Next, we compared these d5mC sites with the previously identified hypo-hydroxymethylated CpG sites (Fig. 3A;Supplementary Material, Table S2). This approach showed that 4627 (38.4%) of the CpG sites aberrantly hypermethylated in gliomas also lost 5hmC (Fig. 4A;Supplementary Material, Table S5). Interestingly, those CpG sites were those that showed the highest values of 5hmC in normal tissue (Supplementary Material, Fig. S5) (Wilcoxon rank sum test, P < 0.001). Figure 4. View largeDownload slide Relationships between changes in 5mc and 5hmc in glioma. (A) Euler diagram illustrating overlap of CpGs that lose 5hmC (hypo5hmC) and gain 5mC (hyper 5mC) in glioma. (B) Associations between hypermethylated CpG sites that lose (or not) 5hmC and CpG density (upper panel) and CpG island status (lower panel), compared to the array background (450 K). (C) Unsupervised hierarchical clustering and heatmap including 3000 randomly chosen CpG sites with 5mC changes (hyper- and hypomethylation) in glioma. Hypo- (purple) and non-hypo (orange) 5hmC overlapped CpGs are indicated by colored lines on the annexed track. Average beta methylation values are displayed from 0 (blue) to 1 (yellow). Figure 4. View largeDownload slide Relationships between changes in 5mc and 5hmc in glioma. (A) Euler diagram illustrating overlap of CpGs that lose 5hmC (hypo5hmC) and gain 5mC (hyper 5mC) in glioma. (B) Associations between hypermethylated CpG sites that lose (or not) 5hmC and CpG density (upper panel) and CpG island status (lower panel), compared to the array background (450 K). (C) Unsupervised hierarchical clustering and heatmap including 3000 randomly chosen CpG sites with 5mC changes (hyper- and hypomethylation) in glioma. Hypo- (purple) and non-hypo (orange) 5hmC overlapped CpGs are indicated by colored lines on the annexed track. Average beta methylation values are displayed from 0 (blue) to 1 (yellow). To verify that these hypermethylated CpG sites that also lose 5hmC in gliomas (hyper5mC–hypo5hmC) had not been identified in previous studies, owing to no distinction being made between 5mC and 5hmC, we compared the DNA methylation values of our samples with those of gliomas obtained from the same type of methylation array available in The Cancer Genome Atlas (TCGA) (33). We observed that not separating 5mC and the 5hmC resulted in many false negatives for hypermethylation in gliomas since the gain of 5mC in tumors was masked by the high levels of 5hmC in normal brain (Supplementary Material, Fig. S6A). However, irrespective of the issue of separating or not 5mC and 5hmC, similar results were found when we performed the same comparisons using hypermethylated CpG sites that showed no changes in 5hmC in gliomas, since, in this case, these CpGs showed very low levels of 5hmC in normal brain (Supplementary Material, Fig. S6B). To investigate, at a functional genomic level, the characteristics of these two classes of aberrantly hypermethylated CpG sites in gliomas we first analyzed their genomic distribution in relation to density of CpG sites and we found that the hypermethylated CpG sites that lose 5hmC (hyper5mC–hypo5hmC) were enriched in low density CpG regions (Wilcoxon rank sum test, P < 0.001, D = -0.11) as compared with the hypermethylated CpG sites that showed no changes in 5hmC (hyper5mC) (Wilcoxon rank sum test, P < 0.001, D = -0.23) (Fig. 4B;Supplementary Material, Tables S5 and S6). Furthermore, hyper5mC–hypo5hmC sites were strongly depleted from CGIs (χ2 test, P < 0.001, OR = 0.42) and enriched in CGI shores (χ2 test, P < 0.001, OR = 2.03) (Fig. 4B). Hierarchical clustering using the differentially methylated CpG sites showed that the hyper5mC–hypo5hmC sites were slightly more methylated in control brain samples than the hyper5mC sites, and that they were more uniformly hypermethylated in glioma (Fig. 4C). To further corroborate our results, we took advantage of recently published data on the whole-genome bisulfite sequencing (WGBS), albeit data were from one glioblastoma (GBM) patient only (34). We found that, in addition to a large percentage of CpGs (n: 4051; 88%) showing the same patterns of change as in our methylation arrays, the WGBS analysis identified more than 106 new hyper5mC–hypo5hmC sites, thus confirming that this is a frequent event in glioma (Supplementary Material, Fig. S7). Next, to identify possible chromatin signatures associated with the two classes of aberrantly hypermethylated CpG sites in gliomas, we compared our data with previously published data on a range of histone modifications and chromatin modifiers in 10 different cell types (see Materials and Methods) (Fig. 5A). This approach confirmed the association between hyper5mC and the repressive histone marks H3K9me3 and H3K27me3 (Fisher’s exact test, P < 0.05) (31,32,35). The hyper5mC–hypo5hmC sites showed a completely different chromatin signature, with enrichment in the activating histone PTMs H3K4me1, H3K36me3, H3K79me2 and H4K20me1 (Fisher’s exact test, P < 0.05) (Fig. 5A). Note, when compared with the chromatin signature of the whole set of hypo-hydroxymethylated CpGs in glioma, these CpG sites were particularly enriched at the H3K4me2 histone mark (Fisher's exact test, P < 0.001, OR in [1.19, 1.78] for all cell lines in the Broad Histone project) (Fig. 5B). Figure 5. View largeDownload slide Canonical and non-canonical hypermethylation in glioma. (A) Heatmaps showing significant enrichment of CpG sites in glioma which exclusively gain 5mC (canonical hypermethylation) (upper panel), and both lose 5hmC and gain 5mC (non-canonical hypermethylation) (lower panel), with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Histone PTMs related to activation and repression are distinguished by colors as indicated in the key. (B) Circular representation of two representative chromosomes (12 and 17), indicating genomic location of canonical (orange) and non-canonical (purple) hypermethylation in glioma. Inner tracks display chromatin marks (H3K9me3, H3K27me3 and H3K4me2), generated for NH-A cells. Two examples of genes showing canonical and non-canonical hypermethylation associated with specific chromatin signatures are displayed below. Figure 5. View largeDownload slide Canonical and non-canonical hypermethylation in glioma. (A) Heatmaps showing significant enrichment of CpG sites in glioma which exclusively gain 5mC (canonical hypermethylation) (upper panel), and both lose 5hmC and gain 5mC (non-canonical hypermethylation) (lower panel), with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Histone PTMs related to activation and repression are distinguished by colors as indicated in the key. (B) Circular representation of two representative chromosomes (12 and 17), indicating genomic location of canonical (orange) and non-canonical (purple) hypermethylation in glioma. Inner tracks display chromatin marks (H3K9me3, H3K27me3 and H3K4me2), generated for NH-A cells. Two examples of genes showing canonical and non-canonical hypermethylation associated with specific chromatin signatures are displayed below. These results indicate that the hyper5mC sites behave like the aberrantly hypermethylated canonical CpG sites in cancer (i.e. enriched in CGIs and repressive histone marks), whilst the hyper5mC–hypo5hmC sites represent a novel and functionally different non-canonical type of aberrantly methylated DNA sequence in glioma (Fig. 5A and B;Supplementary Material, Tables S5 and S6). In support of this notion, experiments focused on the computational prediction of functional elements confirmed the enrichment of canonical aberrant hypermethylation in promoters and repressed sequences and revealed a completely different pattern for non-canonical hypermethylation, one which is more closely associated with enhancers and transcriptional regulation (Fisher’s exact test; P < 0.05) (Supplementary Material, Fig. S8). These associations were also corroborated by using available ChIP-seq tracks from epigenomes of eight types of brain cell obtained from the NIH Roadmap Epigenome consortia (30) (Fisheŕs exact test; P < 0.05) (Supplementary Material, Fig. S9). Distinct functional role of canonical and non-canonical aberrant hypermethylation in glioma To identify possible differences between the functional role of canonical and non-canonical aberrant DNA hypermethylation in glioma we first ascribed CpG sites to specific genes and then used HOMER to carry out gene ontology (GO) analyses of each group of genes (see Materials and Methods). Using this approach, we identified 1921 genes displaying canonical hypermethylation, 2042 displaying non-canonical hypermethylation and 938 displaying both types of aberrant hypermethylation (Fig. 6A;Supplementary Material, Tables S7–S9). As expected, GO analyses showed an enrichment of development and differentiation processes in canonical genes (36) (Fig. 6A;Supplementary Material, Table S10). In contrast, non-canonical genes were enriched in cell signaling and protein processing pathways (Fig. 6A;Supplementary Material, Table S11). Figure 6. View largeDownload slide Functional role of canonical and non-canonical hypermethylation in glioma. (A) Euler diagrams showing number of genes associated with canonical hypermethylation, non-canonical hypermethylation or both. On the right are representative GO terms (biological process) of genes associated with canonical (orange) and non-canonical (purple) hypermethylation, ranked by Q-value, and enrichment score (relative risk). (B) Euler diagram showing overlap of canonical and non-canonical hypermethylated genes with down-regulation. (C) Associations of canonical and non-canonical hypermethylation in glioma with different genomic regions. (D) Representative example of one gene (SLC1A4) showing non-canonical hypermethylation in glioma (orange frame). Organization of the gene, locations of CpGs included in the methylation array (black dots), and TSS are shown below. 5mC hypermethylation (blue to yellow) and 5hmC loss (gray to blue) in glioma are shown above. WGBS data (34) including all the CpG sites in the same region are shown on the right. The associated change in gene expression is displayed below. Figure 6. View largeDownload slide Functional role of canonical and non-canonical hypermethylation in glioma. (A) Euler diagrams showing number of genes associated with canonical hypermethylation, non-canonical hypermethylation or both. On the right are representative GO terms (biological process) of genes associated with canonical (orange) and non-canonical (purple) hypermethylation, ranked by Q-value, and enrichment score (relative risk). (B) Euler diagram showing overlap of canonical and non-canonical hypermethylated genes with down-regulation. (C) Associations of canonical and non-canonical hypermethylation in glioma with different genomic regions. (D) Representative example of one gene (SLC1A4) showing non-canonical hypermethylation in glioma (orange frame). Organization of the gene, locations of CpGs included in the methylation array (black dots), and TSS are shown below. 5mC hypermethylation (blue to yellow) and 5hmC loss (gray to blue) in glioma are shown above. WGBS data (34) including all the CpG sites in the same region are shown on the right. The associated change in gene expression is displayed below. To further investigate the functional role of canonical and non-canonical hypermethylation in cancer, we compared our methylation data with previously published gene expression data in the same type of tumor (see Materials and Methods). Results showed that 681 (23.8%) of the canonical and 585 (19.6%) of the non-canonical aberrantly hypermethylated genes were repressed in gliomas (Fig. 6B). Genomic distribution analysis of both types of aberrant hypermethylation confirmed the enrichment of canonical hypermethylation in exons (χ2 test, P < 0.001, OR = 1.79 for general exons, OR = 2.01 for first exons), while non-canonical hypermethylation was more frequent in introns (χ2 test, P < 0.001, OR = 1.7) (Fig. 6C). The genes frequently downregulated in glioma, SLC14A and the SMAD7, represent two bona fide examples of this pattern of non-canonical aberrant hypermethylation (Fig. 6D;Supplementary Material, Fig. S10). Taken as a whole, these results indicate that both types of aberrant hypermethylation have a similar effect on gene expression, but that they affect different types of genes and gene regions. Discussion During recent decades, it has largely been accepted that aberrant genomic DNA methylation is a hallmark of cancer (1,2) and the best-known DNA methylation alterations in tumors were the aberrant hypermethylation of CpG island promoters, and global DNA hypomethylation. In both cases, the alterations were mostly attributed to changes in the overall content and genomic distribution of 5mC (1,2). The vast majority of studies on DNA methylation and cancer have been based on the sodium bisulfite modification of the genomic DNA, a chemical reaction that allows C and 5mC to be distinguished by polymerase chain reaction (37). However, this approach cannot distinguish between 5mC and 5hmC, the latter being a chemical modification of the cytosine first identified in bacteriophages in 1952 (38), and which has recently been found to be quite abundant in specific mammalian tissue (3). 5hmC is synthesized from 5mC by the Tet Enzymes, a family of proteins that can also catalyze the successive conversion of 5hmC to 5-formylcytosine and then to 5-carboxylcytosine, both of which can be transformed to unmodified C (39). Although 5hmC was originally described as simply a demethylation intermediate of C (7,12,13), recent data suggest that this may be an epigenetic mark in its own right (40,41). Thus, as most previous studies did not distinguish between 5mC and 5hmC, and it appears that DNA hydroxymethylation might play a specific role in cancer, in this work we aimed to re-evaluate changes in DNA methylation in cancer, paying special attention to the specific contribution of 5hmC. To identify the DNA regions affected by hydroxymethylation changes in cancer, we first focused on four types of repeated DNA (LINE1, Sat-alpha, NBL2 and AluYb8). Among them, the LINE1 repeat is of particular interest because it contains ∼20% of the genomic 5mC, and it has been proposed to be a surrogate of global DNA methylation (26). Our results confirmed that tumors lose 5mC at repeated DNA (42). However, the level of 5hmC at repeated DNA in healthy samples was very low and no significant differences were observed compared to tumors, which indicates that the global DNA hypo-hydroxymethylation previously observed in cancer (14,20,27,28,43) does not principally occur at repeated DNA. As changes in 5hmC at repeated DNA could not explain the global differences previously observed by mass spectrometry (14,20,27,28), we decided to study the possible contribution of single copy sequences. Genome-wide profiling of 5mC and 5hmC of healthy tissue identified more than 100 000 CpG sites frequently hydroxymethylated in brain, providing evidence that the level of this epigenetic mark is very abundant in this tissue (3,7–11). Moreover, 5hmC was enriched in specific regions, i.e. those with low CpG density and in introns, indicating that 5hmC is not simply a demethylation intermediate (7,12,13). Interestingly, 5hmC co-localized in regions marked with the activating histone PTM H3K4me1. This histone mark has been previously associated with gene enhancers (44,45), which suggests that DNA hydroxymethylation might play a role in gene regulation in trans. Moreover, we have recently found an association between H3K4me1 and DNA hypomethylation during aging in stem and differentiated cells (46), which may represent an interesting link between aging and cancer at these genomic regions. The cell type(s) from which GBMs originate is not well understood at this moment in time, although there is some evidence that they might be neuronal stem cells or a glial precursor (47,48). In our study, normal brain cells were obtained from the frontal cortex, which principally comprises neural and glial cells, thus we cannot rule out some differences found in our analysis possibly reflecting differences between normal cell types. That said, the great number of hypo-hydroxymethylated single CpG sites in glioma could explain the global differences previously observed by mass spectrometry (14,20,27,28) and suggests that, in contrast to 5mC, most DNA hypo-hydroxymethylation in brain tumors occurs at single copy sequences. The behavior of 5hmC led us to next identify two types of CpG sites aberrantly hypermethylated in glioma: (i) aberrantly hypermethylated CpG sites that showed no changes in 5hmC and (ii) hypermethylated CpG sites that lose 5hmC. This negative correlation between changes in 5mC and in 5hmC agrees with that previously found in cancer differentially methylated regions of both liver and lung tumors (49). The former sites display similar chromatin signatures to previously described genes aberrantly hypermethylated in cancer (i.e. enrichment in the repressive histone marks H3K9me3 and H3K27me3) (31,32,35). In contrast, the latter type of aberrantly hypermethylated CpG sites were enriched in the activating histone PTMs H3K4me1, H3K36me3, H3K79me2, H4K20me1 and H3K4me2. As these CpG sites present a genuine chromatin signature which is different to the repressive chromatin signature of the classical genes aberrantly hypermethylated in cancer (31,32,35), we conclude that they represent a novel 5hmC-dependent non-canonical class of aberrant DNA hypermethylation in glioma (Fig. 7). As this gain in 5mC is inversely correlated with loss of 5hmC, it was not possible to identify this significant alteration in previous studies using the classical sodium bisulfite-based technologies, since they are not able to distinguish between the two chemical modifications. Figure 7. View largeDownload slide Schematic representation of genomic regions and related histone marks associated with canonical and non-canonical DNA hypermethylation in glioma. CpG sites that suffered canonical hypermethylation are overrepresented in CpG islands (CGI) and in poised promoters and repressed regions enriched in H3K27me3 and H3K9me3. In contrast, non-canonical hypermethylated CpG sites are enriched in CGI shores, and enhancers and transcribed regions characterized for activating histone marks. Figure 7. View largeDownload slide Schematic representation of genomic regions and related histone marks associated with canonical and non-canonical DNA hypermethylation in glioma. CpG sites that suffered canonical hypermethylation are overrepresented in CpG islands (CGI) and in poised promoters and repressed regions enriched in H3K27me3 and H3K9me3. In contrast, non-canonical hypermethylated CpG sites are enriched in CGI shores, and enhancers and transcribed regions characterized for activating histone marks. Aberrant DNA hypermethylation in cancer was discovered more than 30 years ago, but the underlying molecular mechanisms are still poorly understood. For example, it has been proposed that genes enriched in bivalent histone modifications (H3K4me3 and H3K27me3) and polycomb group proteins during embryo development are prone to become aberrantly hypermethylated in cancer (31,32,35) but the molecular basis of this is unknown. Our data suggest that tumor cells might in fact acquire aberrant DNA methylation through various different pathways. Moreover, in the case of the non-canonical hypermethylation, the previous loss of 5hmC suggests that aberrant hypermethylation at these DNA regions could be due to an attempt by the cell to reverse or repair the loss of 5hmC at functionally sensible loci. This possibility is supported by the fact that the non-canonical aberrant hypermethylation described here seems to play an important role in gene regulation. Intriguingly, 5hmC at gene promoters has also been proposed to protect from aberrant hypermethylation in colorectal cancer (28). Thus, although it seems that 5hmC plays an important role in the regulation of the DNA methylation changes in cancer, more research is needed to fully understand its role. The non-canonical aberrant hypermethylation described here seems to have a similar overall effect on gene expression as classical canonical hypermethylation, although the type of genes and the genomic regions affected are very different. Previous research has shown that the repression of developmental genes affected by canonical aberrant hypermethylation promotes tumorigenesis (36). However, the possible functional role of disruption of cell signaling and protein processing pathways affected by the non-canonical hypermethylation described in this study remains to be elucidated. Future research is thus needed to address this issue, and to determine whether the two types of aberrant DNA hypermethylation have distinct functional roles in cancer. Materials and Methods Normal samples and primary tumors Brain samples analyzed in this study were collected at the Hospital Universitario Central de Asturias (HUCA), the Hospital Virgen de la Salud, Toledo and the Hospital Universitario Puerta de Hierro, Madrid. The samples studied comprised 12 normal brains and 53 GBMs. The study was approved by the Clinical Research Ethics Committee and all the individuals involved provided written informed consent. Pyrosequencing assays 5mC and 5hmC patterns at repetitive sequences (LINE1, AluYb8, Sat-alpha and NBL2) were analyzed by pyrosequencing using previously described primers (25). To calculate 5hmC levels, each sample was analyzed using two methods performed in parallel; an oxBS and a bisulfite-only conversion (BS), in accordance with the TrueMethyl® Array Kit User Guide (CEGX, Version 2) with some modifications. Briefly, DNA samples were cleaned using Agencourt AMPure XP (Beckman Coulter) then oxidated with 1 μl of a KRuO4 (Alpha Aeser) solution (375 mM in 0.3 M NaOH), after which BS was performed using EpiTect bisulfite kit (Qiagen®). After PCR amplification of the region of interest in oxBS and BS samples, pyrosequencing was performed using PyroMark Q24 reagents, and vacuum prep workstation, equipment and software (Qiagen®). To avoid negative methylation values due to the substraction of the oxBS and BS signals, 5mC and 5hmC estimations were calculated by means of a maximum likelihood model using the OxyBS R CRAN package (version 1.5) (50). Briefly, percentages of CpG methylation obtained from the PyroMark Q24 software were used as beta values for the BS or the oxBS treated samples, and signal intensities from the oxBS and BS experiments were obtained from the peak height signals of the corresponding nucleotides measured in the pyrosequencing reaction. Genome-wide DNA methylation analysis with high-density arrays Microarray-based DNA methylation profiling was performed with the HumanMethylation 450 BeadChip (51). Oxidative bisulfite (oxBS) and bisulfite-only (BS) conversion was performed using the TrueMethyl® protocol for 450K analysis (Version 1.1, CEGX) following the manufacturer’s recommended procedures. Processed DNA samples were then hybridized to the BeadChip (Illumina), following the Illumina Infinium HD Methylation Protocol. Genotyping services were provided by the Spanish Centro Nacional de Genotipado (CEGEN-ISCIII) (www.cegen.org). Array data were deposited in ArrayExpress accession numbers E-MTAB-6003. HumanMethylation450 BeadChip data preprocessing Raw IDAT files were processed using the R/Bioconductor package minfi (52) (version 1.14.0), implementing the Subset-quantile Within Array Normalization (SWAN) algorithm (53) to correct for differences in the microarray probe designs. No background correction or control probe normalization was applied. Probes where at least two samples had detection P-values >0.01, and samples where at least 5500 probes had detection P-values >0.01 were filtered out. M-values and beta values were computed as the final step in the preprocessing procedure. In line with a previously published methodology (54), M-values were used for the statistical analyses and beta values for effect size thresholding, visualization and report generation. Batch effect correction In order to detect whether there was any batch effect associated with technical factors, the visualization technique of multidimensional scaling was employed to highlight any strange interaction affecting the different samples. Where necessary, posterior adjustment of the samples was performed by means of the surrogate variable analysis (SVA) method (55) implemented in the R/Bioconductor sva package (version 3.14.0). Computation of hydroxymethylation levels Beta values from oxBS samples were subtracted from their corresponding BS treated pairs, generating an artificial dataset representing the level of 5hmC for each probe and sample as per a previously published methodology (56). One further dataset was created to represent the 5mC levels using beta values from oxBS samples. Detection of differentially methylated probes Differential methylation and hydroxymethylation of an individual probe was determined by a moderated t-test implemented in the R/Bioconductor package limma (57). A linear model, with methylation or hydroxymethylation levels as response and the sample group (normal/tumoral) as the principal covariate of interest, was then fitted to the methylation or hydroxymethylation data. Surrogate Variables generated using SVA were also included in the model definition but excluding those found to be correlated to the phenotype of interest. P-values were corrected for multiple testing using the Benjamini–Hochberg method for controlling false discovery rate (FDR). An FDR threshold of 0.001 was employed to determine differentially methylated and hydroxymethylated probes. Additionally, these probes were filtered according to their effect size, keeping only those probes with methylation or hydroxymethylation changes between-groups that exceeded the median of all differences for the same comparison. The probes without no significant 5hmC signal on control samples were filtered out from the set of hypo-hydroxymethylated probes in glioma. In order to describe the genomic distribution of 5hmC in brain, we used Hilbert curves (58), which are especially suited to the visualization of simple measurements, such as the location of the 5hmC enriched probes, over large scales. These curves allow for compact representation on a genomic scale and have an interesting property by which two points which are near each other in the one-dimensional genomic location space are also closely located in the two-dimensional transformation generated by the curve. The converse may not always be true. Identification of hydroxymethylated probes In order to identify those probes representing the regions where the 5hmC mark is located, a differential hydroxymethylation analysis was performed as described previously (59) using a dataset containing both oxBS and BS versions of the control samples. Probes with significant differences in beta values between the BS and oxBS samples were considered to be enriched for the 5hmC mark. An FDR threshold of 0.001 was employed. No filtering on effect size was applied in this case. 5hmC immunoprecipitation-qPCR assay Immunoprecipitation of 5hmC was carried out using the EpiQuik Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit (Epigentek), according to the manufacturer’s instructions. Input, non-specific IgG- and 5hmC-enriched fractions were obtained from 11 samples corresponding to 5 normal brains, 5 glioma tumors and 1 glioma cell line. All these fractions were amplified by qPCR with oligonucleotides specific for the CpGs detailed in Supplementary Material, Table S12. After confirming there were no significant differences between input DNAs, 5hmC relative enrichment was calculated as a fold change relative to input Ct mean. Histone enrichment analysis In order to analyze the enrichment of histone marks on a subset of probes, we used the information contained in the UCSC Genome Browser Broad Histone tracks from the ENCODE Project (10 different cell types) and the NIH Roadmap Epigenome consortium (8 brain cell types, but less available ChIP-seq data). Histone mark peaks were downloaded for every combination of cell line and antibody. For each track, a 2× 2 contingency table was built to represent the partition of the whole set of possible probes in the microarray with respect to the membership of the subset of interest and the overlap between the probes and the histone peaks. A Fisher’s exact test was used to determine whether there was significant enrichment of the selected histone mark for the subset of interest. P-values were adjusted for multiple comparisons using the Benjamini–Hochberg method for controlling FDR. A significance level of 0.05 was used to determine whether the given combination of histone mark and cell line presented a significant change in proportion. Additionally, the base-2 logarithm of the OR was used as a measure of effect size. Chromatin segment enrichment analysis Data from the BROAD ChromHMM Project were downloaded from the UCSC Genome Browser and the NIH Roadmap Epigenome consortium. Each of the tracks comprising these datasets represents a different segmentation (15 and 18 chromatin states, respectively) generated by a hidden Markov model (HMM) using Chip-Seq signals from the Broad Histone Project as inputs. The segmentations were later curated and labeled according to their functional status (60,61). In order to detect any significant enrichment in the proportion of probes in a given subset of interest belonging to one functional category, an analysis strategy similar to the one employed for the detection of histone enrichment was performed. In this case, a 2× 2 contingency table was built using segments of a given functional status rather than antibodies. A Fisher’s exact test was employed, and significant combinations were detected using an FDR threshold of 0.05 (Benjamini–Hochberg procedure). Again, the base-2 logarithm of the OR was used as a measure of effect size. Genomic region analysis The probes in the microarray were assigned to a genomic region according to their position relative to the transcript information extracted from the R/Bioconductor package TxDb.Hsapiens.UCSC.hg19.knownGene (package version 3.1.2). A probe was said to be in a promoter region if it was located in a region up to 2 kb upstream of the transcription start site (TSS) of any given transcript. Similarly, a set of mutually exclusive regions were defined inside the transcripts, namely 5UTR, 3UTR, First Exon, Exon and Intron. A probe could only belong to one category, hence if the location of a probe overlapped with two or more regions in different transcripts, it was assigned to the region with a higher level of precedence (i.e. in the order stated above, earlier mention indicates higher precedence). If a probe was not assigned to any of these special regions, it was labeled by default as Intergenic. A contingency table was built for each of the subsets, partitioning the whole set of probes according to membership to a given category and the subset of interest. A Pearson’s χ2 test was used to determine whether there was any significant change in proportion between the number of probes marked as belonging to a given region inside and outside the subset of interest. A significance level of 0.05 was employed, and effect size measured by OR. CGI status analysis Similar to the genomic region analysis, probes were labeled according to their relative position to CGIs, the locations of which were obtained from the R/Bioconductor package FDb.InfiniumMethylation.hg19 (package version 2.2.0). The generation procedure of these CGIs is described by (62), i.e. ‘CpG shores’ were defined as the 2 kb regions flanking a CGI. ‘CpG shelves’ were defined as the 2 kb regions either upstream of or downstream from each CpG shore. Probes not belonging to any of the regions thus far mentioned were assigned to the special category ‘non-CGI’ with each probe being assigned to only one of the categories. A 4× 2 contingency table was constructed for each subset of probes in order to study the association between the given subset and the different CGI categories. A χ2 test was used to determine whether any of the categories had a significant association with the given subset. For each of the CGI status levels, a 2× 2 contingency table was defined and another χ2 test used to independently evaluate the association of the given subset with each status level, a significance level of 0.05 being employed for all tests. Effect size was reported as the OR for each of the individual tests. Analysis of CpG density For each of the probes in the HumanMethylation450 microarray, CpG density was measured as the number of CG 2-mers present divided by the number which would be theoretically possible in a 2 kb window with the CpG under study at its center. A Wilcoxon non-parametric test was used to determine if any significant difference existed between the CpG density of each subset of interest and that of the array probes in the background. A significance level of 0.05 was employed for all tests. Effect size was measured using Cliff's delta (D). Gap distance analysis Distance to both the centromere and telomere was measured for each of the probes in the HumanMethylation450 microarray. In order to find significant differences between the probes within the subset of interest and those in the background, a Wilcoxon non-parametric test was used. Once again, a significance level of 0.05 was employed for all tests, and Cliff's delta (D) was used as a measure of effect size. Microarray background correction Although it is sometimes referred to as a genome-wide solution, the HumanMethylation450 BeadChip only covers a fraction of the entire genome. In its 27K predecessor, the probes were mainly located at gene promoter regions, while the newer HumanMethylation450 BeadChip additionally includes probes located inside genes and in intergenic regions (63). The irregular distribution of probes can, however, lead to unwanted biases when studying whether a selected subset of probes is enriched with respect to any functional or clinical mark. For this reason, here a reference to the background distribution of features was included in all statistical tests performed in order to prevent our conclusions from being driven by the irregular distribution of probes. In qualitative tests (CGI status, genomic region or histone mark enrichment), the contingency matrix was built to represent the background distribution of the microarray. In quantitative tests (CpG density, distance to centromeres and telomeres) the corresponding metric was compared between the subset of interest and the remaining probes in the microarray. Thus, any significant result would indicate a departure from the fixed background distribution and ignore any bias inherent in the test. GO analysis and annotation Probe sets were converted into gene sets by using the annotation information from the R/Bioconductor package TxDb.Hsapiens.UCSC.hg19.knownGene (version 3.1.2). A probe was assigned to a gene if the probe was contained within the overlap of all the genomic regions represented by the different transcripts belonging to that gene, or in a 2 kb region upstream of the corresponding TSS. Probes converted this way can be assigned to one or more genes, or to zero (i.e. intergenic probes). After gene conversion, each subset of interest was analyzed using the HOMER software tool (64). The software was configured to use the whole set of genes represented in the HumanMethylation450 architecture as a background. HOMER tested the genes in each subset of interest against 21 different databases, including the GO biological process, molecular function and cellular component ontologies, as well as KEGG and Reactome pathway databases, among many others. Circular visualization and track smoothing In order to plot the CpG and histone peak information on the circular genome-wide and example graphs, smoothing was applied to the data. CpG enrichment information for canonical and non-canonical hypermethylation was generated by partitioning the genome into intervals of 10 kb and assigning to each a score corresponding to the average coverage of the selected CpGs in the interval. WGBS datasets TAB-Seq data, corresponding to an adult brain prefrontal cortex tissue sample (GSM1135082) (29), was used as a validation dataset for the location of 5hmC in controls. Additionally, TrueMethyl (ox-BS) Whole Genome data referenced in (34) (E-MTAB-5171), obtained from a single GBM patient, was used as a validation dataset. Previously processed data in the form of quantified methylation for each CpG measured in both strands of the genome was downloaded and filtered. The resulting dataset comprised only two samples (normal and tumoral), hence a descriptive strategy was used to distinguish the different types of probe according to their methylation status. For both the TAB-Seq and TrueMethyl-seq validation datasets, hydroxymethylated probes were identified as those having a 5hmC measure higher than 0.1. In the case of WGBS, differentially methylated probes were defined as those having an absolute difference in their methylation values between the control and tumor samples which was above a given threshold (0.2 for 5mC and 0.1 for 5hmC). Only methylation measures from CpGs having a total read count higher than 10 were retained. The validation datasets may contain either one or two methylation measures for each CpG in the genome as they measure methylation in both strands. Strand-agnostic CpG regions representing the CpG dinucleotides with at least one measure were defined in order to compute the degree of intersection between the WGBS and methylation arrays results. TCGA expression and methylation dataset In order to analyze changes in gene expression, samples of GBM multiforme were selected from among the data generated by the TCGA Research Network (http://cancergenome.nih.gov). DNA methylation data for GBM were additionally obtained from TCGA for visualization and validation purposes. Expression Level-3 pre-processed data were obtained for 572 GBM samples (10 controls and 562 tumors). The moderated t-test approach in the R/Bioconductor package limma was used to assess the differential expression status of each gene in the TCGA datasets. The normalized expression ratio in the TCGA datasets was used as the response variable, and the sample group (normal/tumoral) as the covariate of interest. No adjustment for possible confounders was performed in this case. An FDR threshold of 0.001 was used to correct for multiple hypotheses. No filtering on effect size was applied in this case. DNA Methylation Level-1 raw data for the Illumina 450k architecture was obtained for 162 GBM samples (33). The raw values were normalized using the SWAN algorithm. No additional filtering was performed on the samples. Data analysis workflow All the necessary steps for upstream and downstream analyses were defined and implemented using the Snakemake tool (65), which helps data scientists to generate a reproducible and inherently parallel processing pipeline. Individual workflow tasks were implemented in R (version 3.2.2) and Python (version 3.4.3). Acknowledgements We thank Ronnie Lendrum for editorial assistance. We also thank the Tumor Bank of the Hospital Virgen de la Salud (BioB-HVS, Toledo, Spain) for providing tumor samples. Conflict of Interest statement: None declared. Funding This work has been financially supported by: the Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892 to M.F.F. and A.F.F.; RTC-2015-3393-1 to A.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, and the Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); the Fundación Científica de la AECC (to R.G.U.); the Fundación Ramón Areces (to M.F.F); FICYT (to E.G.T., M.G.G. and A.C.); and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). Work in P.M. laboratory is supported by the European Research Council (CoG-2014-646903), the Spanish Ministry of Economy-Competitiveness (SAF-SAF2013-43065), the Obra Social La Caixa-Fundaciò Josep Carreras, and the Generalitat de Catalunya. P.M. is an investigator in the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain. References 1 Esteller M. 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Abstract

Abstract Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma. Introduction DNA methylation at the fifth position of cytosine [5-methylcytosine (5mC)] has been one of the most studied epigenetic modifications in mammals to date. 5mC is involved in the regulation of multiple physiologic and pathologic processes, including cancer, and when located at gene promoters, it is usually linked to transcriptional repression. As distinctive features of tumorigenesis, local DNA hypermethylation and global hypomethylation have been attributed to changes in 5mC levels (1, 2). However, the discovery a few years ago, of 5-hydroxymethylcytosine (5hmC), a new epigenetic mark resulting from 5mC oxidation, is reshaping our view of the cancer epigenome (3, 4). This 5mC to 5hmC conversion in mammals is mediated by ten–eleven translocation proteins 1, 2 and 3 (TET1, TET2 and TET3), a family of α-ketoglutarate and Fe(II)-dependent dioxygenases (4, 5). Global levels of 5hmC in the genome fluctuate considerably according to tissue type, and are consistently around 10-fold lower than those of 5mC, though it is interesting that the highest levels of both marks are found in brain (6–11). Several studies have shown that 5hmC is an intermediate of DNA demethylation (7, 12, 13), and that it is also associated with cancer (14–18). In this same vein, loss of 5hmC has been reported across a wide range of human cancers including melanoma, glioma, breast, colon, gastric, kidney, liver, lung, pancreatic and prostate cancers (9, 14, 18–22). Although none of these studies actually demonstrate that changes in 5hmC are directly affecting cancer biology independent of its role in DNA demethylation, other studies identifying 5hmC-specific protein binders suggest that this epigenetic mark might have its own biologic role (23, 24). The fact that there are now methods available that distinguish 5mC and 5hmC positions at single-base resolution within the genome prompted us to reassess the role of DNA methylation status in tumorigenesis from a 5hmC perspective. The method used here allowed us to describe global and genome-wide locus-specific 5mC and 5hmC patterns in brain samples, to identify a specific chromatin signature associated with changes of these epigenetic marks in glioma and, most importantly, to describe a novel non-canonical type of aberrant DNA hypermethylation. Results Global changes of 5mC and 5hmC in cancer To evaluate the role of 5hmC in the changes of DNA methylation observed in glioma, we first analyzed the levels of 5hmC and 5mC at DNA repeats in 11 normal and 50 tumor samples. We used oxidative bisulfite conversion (oxBS) of DNA to discriminate between 5mC and 5hmC (see Materials and Methods) and bisulfite pyrosequencing was used to determine the level of both epigenetic modifications in four different types of repeated DNA: the retrotransposons LINE-1 and AluYb8, and the pericentromeric tandem repeats Sat-alpha and NBL-2 (25). These four DNA regions contain most of the genomic methylation and, consequently, global DNA methylation level is highly dependent on their 5mC content (26). As expected, 5mC levels at repeated DNA in healthy tissue were high but were reduced in tumor samples, a change that was statistically significant in LINE-1 and Sat-alpha (linear model, P < 0.001) (Fig. 1A). In contrast, levels of 5hmC at repeated DNA in healthy tissue were low, while tumoral tissue showed even lower levels of 5hmC in the same DNA regions, although this was only statistically significant in LINE-1 (linear model, P < 0.05) (Fig. 1B). Figure 1. View largeDownload slide 5mC and 5hmC levels at repetitive DNA sequences in glioma. 5mC (A) and 5hmC (B) values of several repetitive regions (AluYb8, LINE-1, NBL-2 and Sat-alpha) measured by pyrosequencing in controls and gliomas. Individual CpG site values for each repeat are displayed, and a linear model including both ‘sample group’ and ‘CpG site’ as covariates was fitted. Significant P-values for any repetitive region are shown. Figure 1. View largeDownload slide 5mC and 5hmC levels at repetitive DNA sequences in glioma. 5mC (A) and 5hmC (B) values of several repetitive regions (AluYb8, LINE-1, NBL-2 and Sat-alpha) measured by pyrosequencing in controls and gliomas. Individual CpG site values for each repeat are displayed, and a linear model including both ‘sample group’ and ‘CpG site’ as covariates was fitted. Significant P-values for any repetitive region are shown. 5mC and 5hmC profiling in brain tissue As changes in 5hmC at repeated DNA were not able to explain the global loss of this epigenetic mark previously observed by mass spectrometry (14,20,27,28), we hypothesized that these changes primarily occur at single copy sequences. To investigate this possibility in more detail, we first used 450K Infinium methylation arrays to determine the level and genomic distribution of 5mC and 5hmC in five healthy brain tissue samples. A preliminary examination of the data revealed that the beta values of the oxidized samples (true 5mC) were lower than their non-oxidized counterparts (5mC + 5hmC) (Wilcoxon rank sum test; P < 0.001; W = 2.34e13) (Fig. 2A). Specifically, 111 633 hydroxymethylated CpG sites (5hmC sites) distributed throughout the whole genome were identified, irrespective of the chromosome analyzed (Fig. 2A;Supplementary Material, Table S1). Figure 2. View largeDownload slide Characterization of DNA 5hmC in normal brain. (A) Bean plots showing differences between average Beta values of 5mC + 5hmC (BS) and true 5mC (OxBS) values in normal brain. The diamond inside the bean indicates the mean value. Hilbert curve showing the amount and genomic distribution of 5hmC in brain. A Level-9 Hilbert curve was used. Each region delimited by black lines represents a chromosome. A point in the image represents a genomic segment of ∼950 bp. A blue point indicates the presence of at least one 5hmC enriched CpG in the given segment. A Hilbert curve including all CpG sites analyzed in the methylation array (450 K) is also shown (B) Associations between 5hmC and CpG density. (C) Distribution of 5hmC CpG sites relative to CpG island status and compared with the array background (450 K). (D) Distribution of 5hmC CpG sites relative to different genomic regions. (E) Heatmaps showing significant enrichment of the 5hmC CpG sites identified in brain, with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Color code indicates the significant enrichment based on log2 OR. (F) Heatmaps showing significant enrichment of 5hmC CpG sites with fifteen ‘chromatin states’ generated by an HMM. Color codes indicate the significant enrichment based on log2 OR. Figure 2. View largeDownload slide Characterization of DNA 5hmC in normal brain. (A) Bean plots showing differences between average Beta values of 5mC + 5hmC (BS) and true 5mC (OxBS) values in normal brain. The diamond inside the bean indicates the mean value. Hilbert curve showing the amount and genomic distribution of 5hmC in brain. A Level-9 Hilbert curve was used. Each region delimited by black lines represents a chromosome. A point in the image represents a genomic segment of ∼950 bp. A blue point indicates the presence of at least one 5hmC enriched CpG in the given segment. A Hilbert curve including all CpG sites analyzed in the methylation array (450 K) is also shown (B) Associations between 5hmC and CpG density. (C) Distribution of 5hmC CpG sites relative to CpG island status and compared with the array background (450 K). (D) Distribution of 5hmC CpG sites relative to different genomic regions. (E) Heatmaps showing significant enrichment of the 5hmC CpG sites identified in brain, with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Color code indicates the significant enrichment based on log2 OR. (F) Heatmaps showing significant enrichment of 5hmC CpG sites with fifteen ‘chromatin states’ generated by an HMM. Color codes indicate the significant enrichment based on log2 OR. To further validate the consistency of our results we compared our data with published data for 5hmC in human normal brain (29) obtained by Tet-Assisted Bisulfite Sequencing (TAB-Seq), an alternative technique that directly measures the 5hmC rather than inferring it, and we found that more than 75% of the 5hmC-enriched CpG sites identified in our study overlapped with 5hmC-enriched CpG sites analyzed by the TAB-Seq (Supplementary Material, Fig. S1). The analysis of the genomic distribution of the 5hmC sites showed that hydroxymethylation is enriched at the low CpG-density regions interrogated by the array (Wilcoxon non-parametric test; P < 0.001, D=−0.5) (Fig. 2B). Consequently, the 5hmC sites were enriched in non-CpG islands (non-CGI) [χ2 test; P < 0.001; odds ratio (OR) = 1.93, and P < 0.001, OR = 3.45, respectively] and infrequent in CGIs (χ2 test; P < 0.001, OR = 0.14, and P < 0.001, OR = 0.13) (Fig. 2C). With respect to genes, 5hmC sites were enriched in introns (χ2 test; P < 0.001, OR = 1.82, and P < 0.001, OR = 1.76, respectively), but were less frequent than expected in gene promoters (χ2 test; P < 0.001, OR = 0.58 and P < 0.001, OR = 0.6) (Fig. 2D). To identify possible chromatin marks associated with 5hmC sites, we compared these CpG sites with previously published data on a range of histone modifications and chromatin modifiers in 10 different cell types (see Materials and Methods) (Fig. 2E). This approach identified statistically significant associations (Fisher’s exact test; P < 0.05) between the 5hmC sites in brain and the active histone marks H3K4me1, H3K36me3 and H4K20me1 (Fig. 2E). A similar framework was used to test for the enrichment of our selected probes over the computer-generated chromatin segmentation states from the ENCODE ChromHMM project (see Materials and Methods). In total, fifteen states were used to segment the genome, and these were then grouped and colored to highlight predicted functional elements. This approach showed that the hmC sites were significantly enriched in states associated with enhancers and transcription (Fisher’s exact test; P < 0.05) (Fig. 2F). These associations were further corroborated by using available chromatin immunoprecipitation sequencing (ChIP-seq) tracks from epigenomes of eight brain cell types obtained from the NIH Roadmap Epigenome consortia (30) (Fisher’s exact test; P < 0.05) (Supplementary Material, Fig. S2). Locus-specific alterations of 5hmC in glioma To identify differentially hydroxymethylated CpG sites (d5hmC) at single copy sequences in cancer, we used 450K methylation arrays to analyze nine primary tumors obtained from patients with glioma (see Materials and Methods). A total of 49 601 CpG sites that were hypo-hydroxymethylated were identified in gliomas, but almost no hyper-hydroxymethylated sites were found (see Materials and Methods) (Fig. 3A;Supplementary Material, Table 2). To validate the results obtained with the methylation arrays with an alternative technique nondependent on the oxBS of DNA, we randomly selected five of the 100 most variable sequences previously identified and analyzed their 5hmC status using a hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit (Epigentek; see Materials and Methods) in five brain samples and five gliomas. The results corroborated the massive loss of 5-hydroxymethylation in glioma in all the candidate sequences (Mann–Whitney test; P < 0.05) (Supplementary Material, Fig. S3). Figure 3. View largeDownload slide Alterations of 5hmC in glioma. (A) Bar plot showing the number of d5hmC sites in glioma. (B) Unsupervised hierarchical clustering and heatmap including CpG sites with 5hmC loss in glioma (3000 random probes). (C) Associations between 5hmC loss in glioma and density of CpGs (upper panel), CpG island status (middle panel), and different genomic regions (lower panel). (D) Heatmaps showing significant enrichment of hypo5hmC CpGs identified in glioma with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. (E) Heatmaps showing significant enrichment of hypo5hmC CpGs in gliomas with 15 ‘chromatin states’ generated by an HMM (right panel). Color codes indicate the significant enrichment based on log2 OR. Figure 3. View largeDownload slide Alterations of 5hmC in glioma. (A) Bar plot showing the number of d5hmC sites in glioma. (B) Unsupervised hierarchical clustering and heatmap including CpG sites with 5hmC loss in glioma (3000 random probes). (C) Associations between 5hmC loss in glioma and density of CpGs (upper panel), CpG island status (middle panel), and different genomic regions (lower panel). (D) Heatmaps showing significant enrichment of hypo5hmC CpGs identified in glioma with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. (E) Heatmaps showing significant enrichment of hypo5hmC CpGs in gliomas with 15 ‘chromatin states’ generated by an HMM (right panel). Color codes indicate the significant enrichment based on log2 OR. Hierarchical clustering using the d5hmC showed the correct classification of normal and tumor samples (Fig. 3B). The analysis of the genomic distribution of the hypo-hydroxymethylated CpG sites in gliomas showed an enrichment at low CpG density regions (Wilcoxon rank sum test, P < 0.001, D = -0.41), and consequently at non-CpG islands (χ2 test, P < 0.001, OR = 2.53) (Fig. 3C). With respect to gene location, hypo-hydroxymethylation was more frequent in introns (χ2 test, P < 0.001, OR = 1.77) (Fig. 3C). To identify possible chromatin signatures associated with DNA hypo-hydroxymethylation in gliomas, we compared our list of hypo-hydroxymethylated CpG sites with previously published data on a range of histone modifications and chromatin modifiers in 10 different cell types (see Materials and Methods) (Fig. 3D). Interestingly, this approach showed an enrichment of hypo-hydroxymethylation at chromatin regions marked with the activating histone post-translational modifications (PTMs) H3K4me1, H3K36me3, H4K20me1 and H3K79me2 (Fisher’s exact test, P < 0.05) (Fig. 3D), but not with the repressive histone modification H3K27me3, which has been previously shown to be associated with aberrant DNA hypermethylation in cancer (31,32) (Fig. 3D). A similar framework was used to test for the enrichment of our selected probes over the computer-generated chromatin segmentation states from the ENCODE ChromHMM project. Using this approach, we found that hypohydroxymethylated CpG sites were significantly associated with transcription regulation and enhancers (Fisher’s exact test; P < 0.05) (Fig. 3E). These associations were further corroborated using available ChIP-seq tracks from the epigenomes of 8 types of brain cell obtained from the NIH Roadmap Epigenome consortia (30) (Fisheŕs exact test; P < 0.05) (Supplementary Material, Fig. S4). DNA hypo-hydroxymethylation identifies a novel type of non-canonical aberrant DNA hyper-methylation in glioma To study the relationship between changes in 5mC and 5hmC in glioma, we first identified aberrantly methylated CpG (d5mC) sites. The comparison of the methylation data between tumoral and control samples (see Materials and Methods) identified 2727 hypo- and 12 050 hyper-methylated CpG sites in gliomas (Supplementary Material, Tables S3 and S4). Next, we compared these d5mC sites with the previously identified hypo-hydroxymethylated CpG sites (Fig. 3A;Supplementary Material, Table S2). This approach showed that 4627 (38.4%) of the CpG sites aberrantly hypermethylated in gliomas also lost 5hmC (Fig. 4A;Supplementary Material, Table S5). Interestingly, those CpG sites were those that showed the highest values of 5hmC in normal tissue (Supplementary Material, Fig. S5) (Wilcoxon rank sum test, P < 0.001). Figure 4. View largeDownload slide Relationships between changes in 5mc and 5hmc in glioma. (A) Euler diagram illustrating overlap of CpGs that lose 5hmC (hypo5hmC) and gain 5mC (hyper 5mC) in glioma. (B) Associations between hypermethylated CpG sites that lose (or not) 5hmC and CpG density (upper panel) and CpG island status (lower panel), compared to the array background (450 K). (C) Unsupervised hierarchical clustering and heatmap including 3000 randomly chosen CpG sites with 5mC changes (hyper- and hypomethylation) in glioma. Hypo- (purple) and non-hypo (orange) 5hmC overlapped CpGs are indicated by colored lines on the annexed track. Average beta methylation values are displayed from 0 (blue) to 1 (yellow). Figure 4. View largeDownload slide Relationships between changes in 5mc and 5hmc in glioma. (A) Euler diagram illustrating overlap of CpGs that lose 5hmC (hypo5hmC) and gain 5mC (hyper 5mC) in glioma. (B) Associations between hypermethylated CpG sites that lose (or not) 5hmC and CpG density (upper panel) and CpG island status (lower panel), compared to the array background (450 K). (C) Unsupervised hierarchical clustering and heatmap including 3000 randomly chosen CpG sites with 5mC changes (hyper- and hypomethylation) in glioma. Hypo- (purple) and non-hypo (orange) 5hmC overlapped CpGs are indicated by colored lines on the annexed track. Average beta methylation values are displayed from 0 (blue) to 1 (yellow). To verify that these hypermethylated CpG sites that also lose 5hmC in gliomas (hyper5mC–hypo5hmC) had not been identified in previous studies, owing to no distinction being made between 5mC and 5hmC, we compared the DNA methylation values of our samples with those of gliomas obtained from the same type of methylation array available in The Cancer Genome Atlas (TCGA) (33). We observed that not separating 5mC and the 5hmC resulted in many false negatives for hypermethylation in gliomas since the gain of 5mC in tumors was masked by the high levels of 5hmC in normal brain (Supplementary Material, Fig. S6A). However, irrespective of the issue of separating or not 5mC and 5hmC, similar results were found when we performed the same comparisons using hypermethylated CpG sites that showed no changes in 5hmC in gliomas, since, in this case, these CpGs showed very low levels of 5hmC in normal brain (Supplementary Material, Fig. S6B). To investigate, at a functional genomic level, the characteristics of these two classes of aberrantly hypermethylated CpG sites in gliomas we first analyzed their genomic distribution in relation to density of CpG sites and we found that the hypermethylated CpG sites that lose 5hmC (hyper5mC–hypo5hmC) were enriched in low density CpG regions (Wilcoxon rank sum test, P < 0.001, D = -0.11) as compared with the hypermethylated CpG sites that showed no changes in 5hmC (hyper5mC) (Wilcoxon rank sum test, P < 0.001, D = -0.23) (Fig. 4B;Supplementary Material, Tables S5 and S6). Furthermore, hyper5mC–hypo5hmC sites were strongly depleted from CGIs (χ2 test, P < 0.001, OR = 0.42) and enriched in CGI shores (χ2 test, P < 0.001, OR = 2.03) (Fig. 4B). Hierarchical clustering using the differentially methylated CpG sites showed that the hyper5mC–hypo5hmC sites were slightly more methylated in control brain samples than the hyper5mC sites, and that they were more uniformly hypermethylated in glioma (Fig. 4C). To further corroborate our results, we took advantage of recently published data on the whole-genome bisulfite sequencing (WGBS), albeit data were from one glioblastoma (GBM) patient only (34). We found that, in addition to a large percentage of CpGs (n: 4051; 88%) showing the same patterns of change as in our methylation arrays, the WGBS analysis identified more than 106 new hyper5mC–hypo5hmC sites, thus confirming that this is a frequent event in glioma (Supplementary Material, Fig. S7). Next, to identify possible chromatin signatures associated with the two classes of aberrantly hypermethylated CpG sites in gliomas, we compared our data with previously published data on a range of histone modifications and chromatin modifiers in 10 different cell types (see Materials and Methods) (Fig. 5A). This approach confirmed the association between hyper5mC and the repressive histone marks H3K9me3 and H3K27me3 (Fisher’s exact test, P < 0.05) (31,32,35). The hyper5mC–hypo5hmC sites showed a completely different chromatin signature, with enrichment in the activating histone PTMs H3K4me1, H3K36me3, H3K79me2 and H4K20me1 (Fisher’s exact test, P < 0.05) (Fig. 5A). Note, when compared with the chromatin signature of the whole set of hypo-hydroxymethylated CpGs in glioma, these CpG sites were particularly enriched at the H3K4me2 histone mark (Fisher's exact test, P < 0.001, OR in [1.19, 1.78] for all cell lines in the Broad Histone project) (Fig. 5B). Figure 5. View largeDownload slide Canonical and non-canonical hypermethylation in glioma. (A) Heatmaps showing significant enrichment of CpG sites in glioma which exclusively gain 5mC (canonical hypermethylation) (upper panel), and both lose 5hmC and gain 5mC (non-canonical hypermethylation) (lower panel), with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Histone PTMs related to activation and repression are distinguished by colors as indicated in the key. (B) Circular representation of two representative chromosomes (12 and 17), indicating genomic location of canonical (orange) and non-canonical (purple) hypermethylation in glioma. Inner tracks display chromatin marks (H3K9me3, H3K27me3 and H3K4me2), generated for NH-A cells. Two examples of genes showing canonical and non-canonical hypermethylation associated with specific chromatin signatures are displayed below. Figure 5. View largeDownload slide Canonical and non-canonical hypermethylation in glioma. (A) Heatmaps showing significant enrichment of CpG sites in glioma which exclusively gain 5mC (canonical hypermethylation) (upper panel), and both lose 5hmC and gain 5mC (non-canonical hypermethylation) (lower panel), with different histone marks contained in the UCSC Browser Broad Histone track from the ENCODE project. Histone PTMs related to activation and repression are distinguished by colors as indicated in the key. (B) Circular representation of two representative chromosomes (12 and 17), indicating genomic location of canonical (orange) and non-canonical (purple) hypermethylation in glioma. Inner tracks display chromatin marks (H3K9me3, H3K27me3 and H3K4me2), generated for NH-A cells. Two examples of genes showing canonical and non-canonical hypermethylation associated with specific chromatin signatures are displayed below. These results indicate that the hyper5mC sites behave like the aberrantly hypermethylated canonical CpG sites in cancer (i.e. enriched in CGIs and repressive histone marks), whilst the hyper5mC–hypo5hmC sites represent a novel and functionally different non-canonical type of aberrantly methylated DNA sequence in glioma (Fig. 5A and B;Supplementary Material, Tables S5 and S6). In support of this notion, experiments focused on the computational prediction of functional elements confirmed the enrichment of canonical aberrant hypermethylation in promoters and repressed sequences and revealed a completely different pattern for non-canonical hypermethylation, one which is more closely associated with enhancers and transcriptional regulation (Fisher’s exact test; P < 0.05) (Supplementary Material, Fig. S8). These associations were also corroborated by using available ChIP-seq tracks from epigenomes of eight types of brain cell obtained from the NIH Roadmap Epigenome consortia (30) (Fisheŕs exact test; P < 0.05) (Supplementary Material, Fig. S9). Distinct functional role of canonical and non-canonical aberrant hypermethylation in glioma To identify possible differences between the functional role of canonical and non-canonical aberrant DNA hypermethylation in glioma we first ascribed CpG sites to specific genes and then used HOMER to carry out gene ontology (GO) analyses of each group of genes (see Materials and Methods). Using this approach, we identified 1921 genes displaying canonical hypermethylation, 2042 displaying non-canonical hypermethylation and 938 displaying both types of aberrant hypermethylation (Fig. 6A;Supplementary Material, Tables S7–S9). As expected, GO analyses showed an enrichment of development and differentiation processes in canonical genes (36) (Fig. 6A;Supplementary Material, Table S10). In contrast, non-canonical genes were enriched in cell signaling and protein processing pathways (Fig. 6A;Supplementary Material, Table S11). Figure 6. View largeDownload slide Functional role of canonical and non-canonical hypermethylation in glioma. (A) Euler diagrams showing number of genes associated with canonical hypermethylation, non-canonical hypermethylation or both. On the right are representative GO terms (biological process) of genes associated with canonical (orange) and non-canonical (purple) hypermethylation, ranked by Q-value, and enrichment score (relative risk). (B) Euler diagram showing overlap of canonical and non-canonical hypermethylated genes with down-regulation. (C) Associations of canonical and non-canonical hypermethylation in glioma with different genomic regions. (D) Representative example of one gene (SLC1A4) showing non-canonical hypermethylation in glioma (orange frame). Organization of the gene, locations of CpGs included in the methylation array (black dots), and TSS are shown below. 5mC hypermethylation (blue to yellow) and 5hmC loss (gray to blue) in glioma are shown above. WGBS data (34) including all the CpG sites in the same region are shown on the right. The associated change in gene expression is displayed below. Figure 6. View largeDownload slide Functional role of canonical and non-canonical hypermethylation in glioma. (A) Euler diagrams showing number of genes associated with canonical hypermethylation, non-canonical hypermethylation or both. On the right are representative GO terms (biological process) of genes associated with canonical (orange) and non-canonical (purple) hypermethylation, ranked by Q-value, and enrichment score (relative risk). (B) Euler diagram showing overlap of canonical and non-canonical hypermethylated genes with down-regulation. (C) Associations of canonical and non-canonical hypermethylation in glioma with different genomic regions. (D) Representative example of one gene (SLC1A4) showing non-canonical hypermethylation in glioma (orange frame). Organization of the gene, locations of CpGs included in the methylation array (black dots), and TSS are shown below. 5mC hypermethylation (blue to yellow) and 5hmC loss (gray to blue) in glioma are shown above. WGBS data (34) including all the CpG sites in the same region are shown on the right. The associated change in gene expression is displayed below. To further investigate the functional role of canonical and non-canonical hypermethylation in cancer, we compared our methylation data with previously published gene expression data in the same type of tumor (see Materials and Methods). Results showed that 681 (23.8%) of the canonical and 585 (19.6%) of the non-canonical aberrantly hypermethylated genes were repressed in gliomas (Fig. 6B). Genomic distribution analysis of both types of aberrant hypermethylation confirmed the enrichment of canonical hypermethylation in exons (χ2 test, P < 0.001, OR = 1.79 for general exons, OR = 2.01 for first exons), while non-canonical hypermethylation was more frequent in introns (χ2 test, P < 0.001, OR = 1.7) (Fig. 6C). The genes frequently downregulated in glioma, SLC14A and the SMAD7, represent two bona fide examples of this pattern of non-canonical aberrant hypermethylation (Fig. 6D;Supplementary Material, Fig. S10). Taken as a whole, these results indicate that both types of aberrant hypermethylation have a similar effect on gene expression, but that they affect different types of genes and gene regions. Discussion During recent decades, it has largely been accepted that aberrant genomic DNA methylation is a hallmark of cancer (1,2) and the best-known DNA methylation alterations in tumors were the aberrant hypermethylation of CpG island promoters, and global DNA hypomethylation. In both cases, the alterations were mostly attributed to changes in the overall content and genomic distribution of 5mC (1,2). The vast majority of studies on DNA methylation and cancer have been based on the sodium bisulfite modification of the genomic DNA, a chemical reaction that allows C and 5mC to be distinguished by polymerase chain reaction (37). However, this approach cannot distinguish between 5mC and 5hmC, the latter being a chemical modification of the cytosine first identified in bacteriophages in 1952 (38), and which has recently been found to be quite abundant in specific mammalian tissue (3). 5hmC is synthesized from 5mC by the Tet Enzymes, a family of proteins that can also catalyze the successive conversion of 5hmC to 5-formylcytosine and then to 5-carboxylcytosine, both of which can be transformed to unmodified C (39). Although 5hmC was originally described as simply a demethylation intermediate of C (7,12,13), recent data suggest that this may be an epigenetic mark in its own right (40,41). Thus, as most previous studies did not distinguish between 5mC and 5hmC, and it appears that DNA hydroxymethylation might play a specific role in cancer, in this work we aimed to re-evaluate changes in DNA methylation in cancer, paying special attention to the specific contribution of 5hmC. To identify the DNA regions affected by hydroxymethylation changes in cancer, we first focused on four types of repeated DNA (LINE1, Sat-alpha, NBL2 and AluYb8). Among them, the LINE1 repeat is of particular interest because it contains ∼20% of the genomic 5mC, and it has been proposed to be a surrogate of global DNA methylation (26). Our results confirmed that tumors lose 5mC at repeated DNA (42). However, the level of 5hmC at repeated DNA in healthy samples was very low and no significant differences were observed compared to tumors, which indicates that the global DNA hypo-hydroxymethylation previously observed in cancer (14,20,27,28,43) does not principally occur at repeated DNA. As changes in 5hmC at repeated DNA could not explain the global differences previously observed by mass spectrometry (14,20,27,28), we decided to study the possible contribution of single copy sequences. Genome-wide profiling of 5mC and 5hmC of healthy tissue identified more than 100 000 CpG sites frequently hydroxymethylated in brain, providing evidence that the level of this epigenetic mark is very abundant in this tissue (3,7–11). Moreover, 5hmC was enriched in specific regions, i.e. those with low CpG density and in introns, indicating that 5hmC is not simply a demethylation intermediate (7,12,13). Interestingly, 5hmC co-localized in regions marked with the activating histone PTM H3K4me1. This histone mark has been previously associated with gene enhancers (44,45), which suggests that DNA hydroxymethylation might play a role in gene regulation in trans. Moreover, we have recently found an association between H3K4me1 and DNA hypomethylation during aging in stem and differentiated cells (46), which may represent an interesting link between aging and cancer at these genomic regions. The cell type(s) from which GBMs originate is not well understood at this moment in time, although there is some evidence that they might be neuronal stem cells or a glial precursor (47,48). In our study, normal brain cells were obtained from the frontal cortex, which principally comprises neural and glial cells, thus we cannot rule out some differences found in our analysis possibly reflecting differences between normal cell types. That said, the great number of hypo-hydroxymethylated single CpG sites in glioma could explain the global differences previously observed by mass spectrometry (14,20,27,28) and suggests that, in contrast to 5mC, most DNA hypo-hydroxymethylation in brain tumors occurs at single copy sequences. The behavior of 5hmC led us to next identify two types of CpG sites aberrantly hypermethylated in glioma: (i) aberrantly hypermethylated CpG sites that showed no changes in 5hmC and (ii) hypermethylated CpG sites that lose 5hmC. This negative correlation between changes in 5mC and in 5hmC agrees with that previously found in cancer differentially methylated regions of both liver and lung tumors (49). The former sites display similar chromatin signatures to previously described genes aberrantly hypermethylated in cancer (i.e. enrichment in the repressive histone marks H3K9me3 and H3K27me3) (31,32,35). In contrast, the latter type of aberrantly hypermethylated CpG sites were enriched in the activating histone PTMs H3K4me1, H3K36me3, H3K79me2, H4K20me1 and H3K4me2. As these CpG sites present a genuine chromatin signature which is different to the repressive chromatin signature of the classical genes aberrantly hypermethylated in cancer (31,32,35), we conclude that they represent a novel 5hmC-dependent non-canonical class of aberrant DNA hypermethylation in glioma (Fig. 7). As this gain in 5mC is inversely correlated with loss of 5hmC, it was not possible to identify this significant alteration in previous studies using the classical sodium bisulfite-based technologies, since they are not able to distinguish between the two chemical modifications. Figure 7. View largeDownload slide Schematic representation of genomic regions and related histone marks associated with canonical and non-canonical DNA hypermethylation in glioma. CpG sites that suffered canonical hypermethylation are overrepresented in CpG islands (CGI) and in poised promoters and repressed regions enriched in H3K27me3 and H3K9me3. In contrast, non-canonical hypermethylated CpG sites are enriched in CGI shores, and enhancers and transcribed regions characterized for activating histone marks. Figure 7. View largeDownload slide Schematic representation of genomic regions and related histone marks associated with canonical and non-canonical DNA hypermethylation in glioma. CpG sites that suffered canonical hypermethylation are overrepresented in CpG islands (CGI) and in poised promoters and repressed regions enriched in H3K27me3 and H3K9me3. In contrast, non-canonical hypermethylated CpG sites are enriched in CGI shores, and enhancers and transcribed regions characterized for activating histone marks. Aberrant DNA hypermethylation in cancer was discovered more than 30 years ago, but the underlying molecular mechanisms are still poorly understood. For example, it has been proposed that genes enriched in bivalent histone modifications (H3K4me3 and H3K27me3) and polycomb group proteins during embryo development are prone to become aberrantly hypermethylated in cancer (31,32,35) but the molecular basis of this is unknown. Our data suggest that tumor cells might in fact acquire aberrant DNA methylation through various different pathways. Moreover, in the case of the non-canonical hypermethylation, the previous loss of 5hmC suggests that aberrant hypermethylation at these DNA regions could be due to an attempt by the cell to reverse or repair the loss of 5hmC at functionally sensible loci. This possibility is supported by the fact that the non-canonical aberrant hypermethylation described here seems to play an important role in gene regulation. Intriguingly, 5hmC at gene promoters has also been proposed to protect from aberrant hypermethylation in colorectal cancer (28). Thus, although it seems that 5hmC plays an important role in the regulation of the DNA methylation changes in cancer, more research is needed to fully understand its role. The non-canonical aberrant hypermethylation described here seems to have a similar overall effect on gene expression as classical canonical hypermethylation, although the type of genes and the genomic regions affected are very different. Previous research has shown that the repression of developmental genes affected by canonical aberrant hypermethylation promotes tumorigenesis (36). However, the possible functional role of disruption of cell signaling and protein processing pathways affected by the non-canonical hypermethylation described in this study remains to be elucidated. Future research is thus needed to address this issue, and to determine whether the two types of aberrant DNA hypermethylation have distinct functional roles in cancer. Materials and Methods Normal samples and primary tumors Brain samples analyzed in this study were collected at the Hospital Universitario Central de Asturias (HUCA), the Hospital Virgen de la Salud, Toledo and the Hospital Universitario Puerta de Hierro, Madrid. The samples studied comprised 12 normal brains and 53 GBMs. The study was approved by the Clinical Research Ethics Committee and all the individuals involved provided written informed consent. Pyrosequencing assays 5mC and 5hmC patterns at repetitive sequences (LINE1, AluYb8, Sat-alpha and NBL2) were analyzed by pyrosequencing using previously described primers (25). To calculate 5hmC levels, each sample was analyzed using two methods performed in parallel; an oxBS and a bisulfite-only conversion (BS), in accordance with the TrueMethyl® Array Kit User Guide (CEGX, Version 2) with some modifications. Briefly, DNA samples were cleaned using Agencourt AMPure XP (Beckman Coulter) then oxidated with 1 μl of a KRuO4 (Alpha Aeser) solution (375 mM in 0.3 M NaOH), after which BS was performed using EpiTect bisulfite kit (Qiagen®). After PCR amplification of the region of interest in oxBS and BS samples, pyrosequencing was performed using PyroMark Q24 reagents, and vacuum prep workstation, equipment and software (Qiagen®). To avoid negative methylation values due to the substraction of the oxBS and BS signals, 5mC and 5hmC estimations were calculated by means of a maximum likelihood model using the OxyBS R CRAN package (version 1.5) (50). Briefly, percentages of CpG methylation obtained from the PyroMark Q24 software were used as beta values for the BS or the oxBS treated samples, and signal intensities from the oxBS and BS experiments were obtained from the peak height signals of the corresponding nucleotides measured in the pyrosequencing reaction. Genome-wide DNA methylation analysis with high-density arrays Microarray-based DNA methylation profiling was performed with the HumanMethylation 450 BeadChip (51). Oxidative bisulfite (oxBS) and bisulfite-only (BS) conversion was performed using the TrueMethyl® protocol for 450K analysis (Version 1.1, CEGX) following the manufacturer’s recommended procedures. Processed DNA samples were then hybridized to the BeadChip (Illumina), following the Illumina Infinium HD Methylation Protocol. Genotyping services were provided by the Spanish Centro Nacional de Genotipado (CEGEN-ISCIII) (www.cegen.org). Array data were deposited in ArrayExpress accession numbers E-MTAB-6003. HumanMethylation450 BeadChip data preprocessing Raw IDAT files were processed using the R/Bioconductor package minfi (52) (version 1.14.0), implementing the Subset-quantile Within Array Normalization (SWAN) algorithm (53) to correct for differences in the microarray probe designs. No background correction or control probe normalization was applied. Probes where at least two samples had detection P-values >0.01, and samples where at least 5500 probes had detection P-values >0.01 were filtered out. M-values and beta values were computed as the final step in the preprocessing procedure. In line with a previously published methodology (54), M-values were used for the statistical analyses and beta values for effect size thresholding, visualization and report generation. Batch effect correction In order to detect whether there was any batch effect associated with technical factors, the visualization technique of multidimensional scaling was employed to highlight any strange interaction affecting the different samples. Where necessary, posterior adjustment of the samples was performed by means of the surrogate variable analysis (SVA) method (55) implemented in the R/Bioconductor sva package (version 3.14.0). Computation of hydroxymethylation levels Beta values from oxBS samples were subtracted from their corresponding BS treated pairs, generating an artificial dataset representing the level of 5hmC for each probe and sample as per a previously published methodology (56). One further dataset was created to represent the 5mC levels using beta values from oxBS samples. Detection of differentially methylated probes Differential methylation and hydroxymethylation of an individual probe was determined by a moderated t-test implemented in the R/Bioconductor package limma (57). A linear model, with methylation or hydroxymethylation levels as response and the sample group (normal/tumoral) as the principal covariate of interest, was then fitted to the methylation or hydroxymethylation data. Surrogate Variables generated using SVA were also included in the model definition but excluding those found to be correlated to the phenotype of interest. P-values were corrected for multiple testing using the Benjamini–Hochberg method for controlling false discovery rate (FDR). An FDR threshold of 0.001 was employed to determine differentially methylated and hydroxymethylated probes. Additionally, these probes were filtered according to their effect size, keeping only those probes with methylation or hydroxymethylation changes between-groups that exceeded the median of all differences for the same comparison. The probes without no significant 5hmC signal on control samples were filtered out from the set of hypo-hydroxymethylated probes in glioma. In order to describe the genomic distribution of 5hmC in brain, we used Hilbert curves (58), which are especially suited to the visualization of simple measurements, such as the location of the 5hmC enriched probes, over large scales. These curves allow for compact representation on a genomic scale and have an interesting property by which two points which are near each other in the one-dimensional genomic location space are also closely located in the two-dimensional transformation generated by the curve. The converse may not always be true. Identification of hydroxymethylated probes In order to identify those probes representing the regions where the 5hmC mark is located, a differential hydroxymethylation analysis was performed as described previously (59) using a dataset containing both oxBS and BS versions of the control samples. Probes with significant differences in beta values between the BS and oxBS samples were considered to be enriched for the 5hmC mark. An FDR threshold of 0.001 was employed. No filtering on effect size was applied in this case. 5hmC immunoprecipitation-qPCR assay Immunoprecipitation of 5hmC was carried out using the EpiQuik Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit (Epigentek), according to the manufacturer’s instructions. Input, non-specific IgG- and 5hmC-enriched fractions were obtained from 11 samples corresponding to 5 normal brains, 5 glioma tumors and 1 glioma cell line. All these fractions were amplified by qPCR with oligonucleotides specific for the CpGs detailed in Supplementary Material, Table S12. After confirming there were no significant differences between input DNAs, 5hmC relative enrichment was calculated as a fold change relative to input Ct mean. Histone enrichment analysis In order to analyze the enrichment of histone marks on a subset of probes, we used the information contained in the UCSC Genome Browser Broad Histone tracks from the ENCODE Project (10 different cell types) and the NIH Roadmap Epigenome consortium (8 brain cell types, but less available ChIP-seq data). Histone mark peaks were downloaded for every combination of cell line and antibody. For each track, a 2× 2 contingency table was built to represent the partition of the whole set of possible probes in the microarray with respect to the membership of the subset of interest and the overlap between the probes and the histone peaks. A Fisher’s exact test was used to determine whether there was significant enrichment of the selected histone mark for the subset of interest. P-values were adjusted for multiple comparisons using the Benjamini–Hochberg method for controlling FDR. A significance level of 0.05 was used to determine whether the given combination of histone mark and cell line presented a significant change in proportion. Additionally, the base-2 logarithm of the OR was used as a measure of effect size. Chromatin segment enrichment analysis Data from the BROAD ChromHMM Project were downloaded from the UCSC Genome Browser and the NIH Roadmap Epigenome consortium. Each of the tracks comprising these datasets represents a different segmentation (15 and 18 chromatin states, respectively) generated by a hidden Markov model (HMM) using Chip-Seq signals from the Broad Histone Project as inputs. The segmentations were later curated and labeled according to their functional status (60,61). In order to detect any significant enrichment in the proportion of probes in a given subset of interest belonging to one functional category, an analysis strategy similar to the one employed for the detection of histone enrichment was performed. In this case, a 2× 2 contingency table was built using segments of a given functional status rather than antibodies. A Fisher’s exact test was employed, and significant combinations were detected using an FDR threshold of 0.05 (Benjamini–Hochberg procedure). Again, the base-2 logarithm of the OR was used as a measure of effect size. Genomic region analysis The probes in the microarray were assigned to a genomic region according to their position relative to the transcript information extracted from the R/Bioconductor package TxDb.Hsapiens.UCSC.hg19.knownGene (package version 3.1.2). A probe was said to be in a promoter region if it was located in a region up to 2 kb upstream of the transcription start site (TSS) of any given transcript. Similarly, a set of mutually exclusive regions were defined inside the transcripts, namely 5UTR, 3UTR, First Exon, Exon and Intron. A probe could only belong to one category, hence if the location of a probe overlapped with two or more regions in different transcripts, it was assigned to the region with a higher level of precedence (i.e. in the order stated above, earlier mention indicates higher precedence). If a probe was not assigned to any of these special regions, it was labeled by default as Intergenic. A contingency table was built for each of the subsets, partitioning the whole set of probes according to membership to a given category and the subset of interest. A Pearson’s χ2 test was used to determine whether there was any significant change in proportion between the number of probes marked as belonging to a given region inside and outside the subset of interest. A significance level of 0.05 was employed, and effect size measured by OR. CGI status analysis Similar to the genomic region analysis, probes were labeled according to their relative position to CGIs, the locations of which were obtained from the R/Bioconductor package FDb.InfiniumMethylation.hg19 (package version 2.2.0). The generation procedure of these CGIs is described by (62), i.e. ‘CpG shores’ were defined as the 2 kb regions flanking a CGI. ‘CpG shelves’ were defined as the 2 kb regions either upstream of or downstream from each CpG shore. Probes not belonging to any of the regions thus far mentioned were assigned to the special category ‘non-CGI’ with each probe being assigned to only one of the categories. A 4× 2 contingency table was constructed for each subset of probes in order to study the association between the given subset and the different CGI categories. A χ2 test was used to determine whether any of the categories had a significant association with the given subset. For each of the CGI status levels, a 2× 2 contingency table was defined and another χ2 test used to independently evaluate the association of the given subset with each status level, a significance level of 0.05 being employed for all tests. Effect size was reported as the OR for each of the individual tests. Analysis of CpG density For each of the probes in the HumanMethylation450 microarray, CpG density was measured as the number of CG 2-mers present divided by the number which would be theoretically possible in a 2 kb window with the CpG under study at its center. A Wilcoxon non-parametric test was used to determine if any significant difference existed between the CpG density of each subset of interest and that of the array probes in the background. A significance level of 0.05 was employed for all tests. Effect size was measured using Cliff's delta (D). Gap distance analysis Distance to both the centromere and telomere was measured for each of the probes in the HumanMethylation450 microarray. In order to find significant differences between the probes within the subset of interest and those in the background, a Wilcoxon non-parametric test was used. Once again, a significance level of 0.05 was employed for all tests, and Cliff's delta (D) was used as a measure of effect size. Microarray background correction Although it is sometimes referred to as a genome-wide solution, the HumanMethylation450 BeadChip only covers a fraction of the entire genome. In its 27K predecessor, the probes were mainly located at gene promoter regions, while the newer HumanMethylation450 BeadChip additionally includes probes located inside genes and in intergenic regions (63). The irregular distribution of probes can, however, lead to unwanted biases when studying whether a selected subset of probes is enriched with respect to any functional or clinical mark. For this reason, here a reference to the background distribution of features was included in all statistical tests performed in order to prevent our conclusions from being driven by the irregular distribution of probes. In qualitative tests (CGI status, genomic region or histone mark enrichment), the contingency matrix was built to represent the background distribution of the microarray. In quantitative tests (CpG density, distance to centromeres and telomeres) the corresponding metric was compared between the subset of interest and the remaining probes in the microarray. Thus, any significant result would indicate a departure from the fixed background distribution and ignore any bias inherent in the test. GO analysis and annotation Probe sets were converted into gene sets by using the annotation information from the R/Bioconductor package TxDb.Hsapiens.UCSC.hg19.knownGene (version 3.1.2). A probe was assigned to a gene if the probe was contained within the overlap of all the genomic regions represented by the different transcripts belonging to that gene, or in a 2 kb region upstream of the corresponding TSS. Probes converted this way can be assigned to one or more genes, or to zero (i.e. intergenic probes). After gene conversion, each subset of interest was analyzed using the HOMER software tool (64). The software was configured to use the whole set of genes represented in the HumanMethylation450 architecture as a background. HOMER tested the genes in each subset of interest against 21 different databases, including the GO biological process, molecular function and cellular component ontologies, as well as KEGG and Reactome pathway databases, among many others. Circular visualization and track smoothing In order to plot the CpG and histone peak information on the circular genome-wide and example graphs, smoothing was applied to the data. CpG enrichment information for canonical and non-canonical hypermethylation was generated by partitioning the genome into intervals of 10 kb and assigning to each a score corresponding to the average coverage of the selected CpGs in the interval. WGBS datasets TAB-Seq data, corresponding to an adult brain prefrontal cortex tissue sample (GSM1135082) (29), was used as a validation dataset for the location of 5hmC in controls. Additionally, TrueMethyl (ox-BS) Whole Genome data referenced in (34) (E-MTAB-5171), obtained from a single GBM patient, was used as a validation dataset. Previously processed data in the form of quantified methylation for each CpG measured in both strands of the genome was downloaded and filtered. The resulting dataset comprised only two samples (normal and tumoral), hence a descriptive strategy was used to distinguish the different types of probe according to their methylation status. For both the TAB-Seq and TrueMethyl-seq validation datasets, hydroxymethylated probes were identified as those having a 5hmC measure higher than 0.1. In the case of WGBS, differentially methylated probes were defined as those having an absolute difference in their methylation values between the control and tumor samples which was above a given threshold (0.2 for 5mC and 0.1 for 5hmC). Only methylation measures from CpGs having a total read count higher than 10 were retained. The validation datasets may contain either one or two methylation measures for each CpG in the genome as they measure methylation in both strands. Strand-agnostic CpG regions representing the CpG dinucleotides with at least one measure were defined in order to compute the degree of intersection between the WGBS and methylation arrays results. TCGA expression and methylation dataset In order to analyze changes in gene expression, samples of GBM multiforme were selected from among the data generated by the TCGA Research Network (http://cancergenome.nih.gov). DNA methylation data for GBM were additionally obtained from TCGA for visualization and validation purposes. Expression Level-3 pre-processed data were obtained for 572 GBM samples (10 controls and 562 tumors). The moderated t-test approach in the R/Bioconductor package limma was used to assess the differential expression status of each gene in the TCGA datasets. The normalized expression ratio in the TCGA datasets was used as the response variable, and the sample group (normal/tumoral) as the covariate of interest. No adjustment for possible confounders was performed in this case. An FDR threshold of 0.001 was used to correct for multiple hypotheses. No filtering on effect size was applied in this case. DNA Methylation Level-1 raw data for the Illumina 450k architecture was obtained for 162 GBM samples (33). The raw values were normalized using the SWAN algorithm. No additional filtering was performed on the samples. Data analysis workflow All the necessary steps for upstream and downstream analyses were defined and implemented using the Snakemake tool (65), which helps data scientists to generate a reproducible and inherently parallel processing pipeline. Individual workflow tasks were implemented in R (version 3.2.2) and Python (version 3.4.3). Acknowledgements We thank Ronnie Lendrum for editorial assistance. We also thank the Tumor Bank of the Hospital Virgen de la Salud (BioB-HVS, Toledo, Spain) for providing tumor samples. Conflict of Interest statement: None declared. Funding This work has been financially supported by: the Plan Nacional de I+D+I 2013–2016/FEDER (PI15/00892 to M.F.F. and A.F.F.; RTC-2015-3393-1 to A.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, and the Plan Nacional de I+D+I 2008–2011/FEDER (CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); the Fundación Científica de la AECC (to R.G.U.); the Fundación Ramón Areces (to M.F.F); FICYT (to E.G.T., M.G.G. and A.C.); and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). Work in P.M. laboratory is supported by the European Research Council (CoG-2014-646903), the Spanish Ministry of Economy-Competitiveness (SAF-SAF2013-43065), the Obra Social La Caixa-Fundaciò Josep Carreras, and the Generalitat de Catalunya. P.M. is an investigator in the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain. References 1 Esteller M. 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Human Molecular GeneticsOxford University Press

Published: Sep 1, 2018

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