BACKGROUND
dna methylation is one of the main epigenetic mechanisms, alongside histone modifications, that plays a significant role in gene silencing  <cit> , tissue differentiation  <cit> , cellular development  <cit> , x-chromosome inactivation  <cit> , or genetic imprinting  <cit> .
aberrant hyper-methylation of high-density cpg regions known as cpg islands   <cit>  and genome-wide hypo-methylation  <cit>  have often been associated with cancer and there has been an increasing effort to understand the specific epigenetic modifications that contribute to carcinogenesis .
in addition to promoter cgis themselves, their surrounding area called shores  and shores  have also a cancer- and tissue-specific methylation  <cit> , while even larger cancer-specific methylation variations were reported in so called open sea regions, far from cgis  <cit> .
in this study, we focus on methylation in promoter cgis and surrounding regions only, in order to investigate its association in cis with gene expression.
the possibility to quantify dna methylation genome-wide on normal and cancer tissues, with microarray or sequencing technologies, has triggered a lot of data-driven research to clarify the role of methylation in gene regulation and cancer.
several studies have highlighted a correlation between differentially methylated regions near promoter regions and gene expression changes .
however, it has also been reported that aberrant over-methylation occurs mostly in normally down-regulated genes, questioning the role of methylation as a causal mechanism for gene repression .
more recently, timp et al.
have proposed a model where epigenetic aberrations contribute to carcinogenesis by dysregulating the functions of specific genes that regulate the epigenome itself  <cit> .
reddington et al.
speculate that epigenetic reprogramming might lead to an altered polycomb binding landscape which could impact genome regulation  <cit> .
to gain further insight into the role of dna methylation in cancer, we perform a large-scale meta-analysis of methylation profiles of normal and cancerous samples from multiple tissues from the cancer genome atlas .
for each cgi and surrounding area, we focus on  its average methylation profile and  the association between variations of its methylation profile and variations in the expression of the target gene.
comparing these parameters between normal and cancerous samples of different tissues suggests that the interplay between promoter methylation and gene expression, and how they are modified in cancer, is not simple.
on the one hand, while each promoter cgi tends to be either hypo- or hypermethylated in all normal samples, we observe hypermethylation of a common subset of cgis in several cancer samples of different tissues , supporting the existence of a cpg island methylator phenotype  with prognostic value, as introduced by toyota et al.
<cit> .
however, we did not find evidence that the genes associated with hypermethylated promoter cgis were less expressed in the cancer samples, as most of the genes concerned are already lowly expressed in normal tissues, as already observed by  <cit> .
on the other hand, looking more precisely at associations between promoter methylation level and gene expression within a set of samples, we observe for each tissue and each normal or cancerous sample set a subset of genes, different from the genes hypermethylated in the cimp phenotype, for which this association is important and strongest outside of the cgis, namely in the n-shores and n-shelves.
this subset of genes varies across tissues but also whether we consider healthy or cancerous samples.
however, transcription factors are over-represented in all subsets.
this suggests that epigenetic reprogramming might contribute to carcinogenesis in part by modifying gene expression susceptibility to changes in dna methylation.
RESULTS
classification of genes based on their cgi methylation profiles in normal and cancerous tissues
we first assess how promoter methylation profiles differ between genes, when for each gene we consider the average methylation profile across normal or cancerous samples.
for that purpose, we collected high-density methylation datasets from the cancer genome atlas  data portal providing more than 485k cpg methylation levels for  <dig> normal and cancerous samples from three tissues of origin: breast, colon and lung .
for each cgi, we combine the probes in the cgi and in the shore and shelves of the cgi, defined as the regions up to 4kb outside of the cgi  <cit> , in a unique cgi, shores and shelves  methylation profile.
we restrict our analysis to the  <dig> cgi + ss where at least  <dig> cpg probes are measured by the technology in order to have high enough coverage to measure the methylation variation within each cgi + ss.
for each of the three tissue of origin, and each normal or cancerous set of tissues, we compute the average methylation profile of each cgi + ss by averaging the methylation values of each cpg across the samples.
hence we compute 3×2= <dig> average profile for each cgi + ss, with we refer to below as cgi+ss signatures.
to assess the diversity of cgi + ss signatures across genes, we perform an unsupervised classification of all signatures for each of the  <dig> types of samples, using ward hierarchical clustering.
since different cgi + ss may contain a different number of gpg probes, we use a specific distance based on dynamic time warping to compare signatures of different lengths.
figure 1a shows the cgi + ss clustering obtained for signatures measured on normal samples from breast samples.
similar figures were obtained for lung  and colon samples .
we observe two clusters, which are largely conserved across the  <dig> tissues of origin .
to clarify the types of signatures captured by each cluster, we represent on a standardized cgi + ss x-axis the  <dig> medoid cgi + ss signatures for each cluster and each tissue .
we clearly observe that the large cluster  <dig>  which contains about  <dig> % of all cgi + ss, corresponds to hypo-methylated islands with hemi-methylated cgi shores and hyper-methylated cgi shelves, while the smaller cluster  <dig> contains about  <dig> % of cgi + ss which are fully hyper-methylated.
a closer look at cluster  <dig> shows that, in some cases, the variation of methylation between islands and shores is unclear, in the sense that some shores are fully hypo-methylated.
as cgis, shores and shelves regions are delimited based on somehow arbitrary criteria, a systematic analysis of these signatures could lead to a refinement of currently accepted boundaries.
fig.
<dig> cgi + ss patterns in breast tissues.
hierarchical clustering of cgi + ss dna methylation patterns for breast normal tissues  and breast cancerous tissues  using dtw as a distance metric and a “ward” linkage.
the colorbar represents the clusters association
fig.
<dig> characteristic profiles for each clusters.
visualization of the cgi + ss dna methylation signatures as condensed profiles from the  <dig> medoids profiles for each clusters in breast normal  or cancerous  tissues.
the two orange dashed lines represent the normalized 1kb long cgi region while the two blue lines represent the 2kb limit between shores and shelves regions
performing the same unsupervised classification independently on signatures obtained from the three types of cancerous tissues leads to different results, with the apparition of a third stable cluster .
comparing the clusters of normal and cancerous tissues shows that, for all types of tissues, the first two clusters found in cancerous tissues are mostly composed of cgi + ss of the corresponding clusters in normal tissues, while the cgi + ss in the third cluster, specifically found in cancerous tissues, tend to come evenly from both clusters in normal tissues .
a look at representative signatures of each cluster  confirms that clusters  <dig> and  <dig> contain respectively hypo- and hyper-methylated profiles, just like the respective clusters in normal tissues, while cluster  <dig> contains cgi + ss signatures which are hemi-methylated .
separating the cgi + ss in cluster  <dig> into sub-clusters “3up” and “3down”, depending on whether they are in cluster  <dig> or  <dig> in normal tissues, we further see that the level of methylation of cgi + ss signatures in the “3up” sub-cluster tends to be lower than the level of methylation of cgi + ss signatures in the “3down” sub-cluster .
interestingly, cluster  <dig> is mostly conserved between tissues , suggesting that these epigenetic variations might be associated with a tissue-independent carcinogenesis process.
fig.
<dig> stability of the cancerous-specific cluster between tissues.
venn diagram representing the intersection of the cgi + ss associated with cancerous-specific cluster  <dig> for each tissue
in summary, this global analysis of methylation signatures suggests the existence of four types of cgi + ss largely conserved across tissues: the majority of them remains hypo-methylated on the cgi and hyper-methylated on the shores and shelves in normal and cancerous tissues ; a minority is hyper-methylated in normal and cancerous tissues ; finally, a fraction of cgi + ss signatures is hypo-methylated in normal tissues and partly methylated in cancerous tissues , while another fraction is hyper-methylated in normal tissues and partly methylated in cancerous ones .
to clarify whether these four categories or cgi + ss are associated to particular biological functions, we performed a gene functional enrichment analysis  <cit>  of the genes associated to the cgi + ss in each of the four categories, for each tissue.
results are shown inadditional file  <dig>  restricting ourselves to gene ontology  biological processes associated to at least  <dig> genes, we found that the large cluster  <dig> is mostly enriched in genes involved in metabolic processes, while the cancer-specific cluster 3up is enriched in genes involved in developmental processes.
there was no significant functional enrichment for genes in cluster  <dig> and 3down.
cancer-specific methylation does not repress gene expression but instead targets genes lowly expressed in normal tissues
cgi methylation is often associated with gene expression silencing.
we therefore assess whether the cgi + ss clusters defined above, corresponding roughly to lowly methylated , highly methylated  or partially methylated in cancer  cgi + ss, are associated with different mean levels of gene expression.
in normal breast tissues, we indeed observe that genes near hypo-methylated islands in cluster  <dig> are slightly but significantly less expressed than genes near an hyper-methylated islands in cluster  <dig> .
there is however no significant difference between the two clusters in normal lung tissues , and we could not test the hypothesis on normal colon tissues since we have none with both methylation and expression data .
in cancerous samples, we observe that genes near a cgi + ss in the cancer-specific cluster  <dig> have a significantly lower expression than other genes , particularly for the genes near a cgi + ss in the “3up” cluster.
as genes in the “3up” cluster are hypo-methylated in normal tissues, this could suggest that their cancer-specific methylation is a way to repress their expression in cancer.
however, a closer look at the expression of these genes in normal tissues  shows that they are already lowly expressed in normal tissues.
this suggests that instead of activating cgi methylation to silence to genes, cancer cells instead activates cgi methylation of hypo-methylated genes which are already lowly expressed in normal tissues.
fig.
<dig> distribution of gene expression in different clusters for in breast tissues.
gene expression distribution for genes based on the cluster assignment of their associated cgi + ss.
panel a gene expression distribution in normal breast tissues shows a slight repression for genes associated with cluster  <dig> .
“ref” represents the genome-wide gene expression distribution panel b gene expression profiles in cancerous breast tissues shows high repression for genes associated with cluster  <dig> and specifically cluster “3up” .
panel c gene expression profiles in both normal and cancerous breast tissues using the cluster assignement in cancerous tissues shows that genes associated with cluster “3up” in cancerous tissues define a cluster of genes already repressed in normal tissues
cancer-specific methylation is an independent predictor of patient survival in breast cancer
our analysis so far compares cgi + ss in terms of their mean methylation across a set of samples and does not take into account between-sample variations.
cgi + ss associated with cluster  <dig>  are hypo-  methylated on average, which indicates that there is little to no variations between samples.
however, signatures of cgis in the cancer-specific cluster  <dig> are partly methylated, which can either hide the fact that they are hemi-methylated for most cancerous samples, or that they are highly variable between samples.
we therefore assess whether the partial methylation of cgi + ss signatures in cluster  <dig> is related to an overall increase  or decrease  in methylation for all or most of the patients, or if this it is caused by a subset of patients that become hyper- methylated for these cgi + ss.
for that purpose, we first summarize the methylation of each cgi + ss on each breast cancer sample by a single value, the average methylation of the probes in the cgi + ss.
we then represent each sample by the vector of methylation values of the cgi + ss in cluster 3up, and perform a ward hierarchical clustering of the cancerous samples based on this representation.
the resulting clustering is shown in fig.
5a, where in addition we indicate the er +, her <dig> and survival information for each patient.
we observe that the distribution methylation values is very bimodal, and that the hyper-methylation of a given cgi + ss from cluster 3up generally happens in a subset of patients only.
interestingly, we see that the same subset of patients tends to be simultaneously hyper-methylated for all cgi + ss from cluster 3up, suggesting that hyper-methylation of these islands is a characteristics of a subset of the tumors.
this allows us to divide the set of breast cancer patients in three clusters given the level of methylation in cluster 3up as either “low”, “intermediate”, or “high” .
interestingly, distinguishing patients given the level of methylation from the cgi + ss in cluster 3up is significantly predictive of the patient survival .
surprisingly, the cluster with the lowest survival is the “intermediate” cluster encompassing a portion but not all of the triple negative breast cancers .
a multivariate cox proportional hazards regression model fitted with available clinical parameters  further shows that this stratification of patients based on the methylation level of genes in cluster 3up adds prognostic value independently of other clinical features .
these results support the existence of a cpg island methylator phenotype  as introduced by toyota et al.
<cit>  that is clinically relevant to assess the survival of patients.
more importantly, they suggest that low survival might not be associated with a positive or negative cimp, but with an intermediate phenotype termed as cimp-low  <cit> .
fig.
<dig> cluster 3up methylation is a predictive factor for survival of patients in breast cancer patients.
panel a hierarchical clustering of breast cancer patients given the average methylation level of all the cgi + ss associated with cluster 3up.
the row color bar represents the average methylation level for the same cgi + ss in healthy breast tissues.
the column color bar gives clinical information about the patients such as er and her <dig> statuses , survival information .
the top row of the column color bar represents the three classes distinguished by methylation profiles in cluster 3up .
panel b kaplan-meier estimate of breast cancer patient survival given the cluster 3up class  shows that cluster 3up “intermediate” patients have a significantly higher risk of death within  <dig> years than either cluster 3up “low” or “high” patients
a similar analysis on cgi + ss associated with cluster 3down is less conclusive, and does not clearly cluster patients in separate clusters .
a lack of sufficient survival data for colon and lung tissues prevented a similar analysis for these tissues.
methylation of cpg in the 3’ region outside the cgi is the most correlated with gene expression
our analysis so far compares cgi + ss to one another, by looking at their average methylation profiles across collections of samples.
we found no clear evidence for a correlation between mean methylation level of a cgi and mean expression level of the corresponding genes, but this may be due to the fact that many other factors impact the expression level of a gene, including biological and technical ones.
another way to assess how methylation impacts expression is to look, for each given gene, how variation in expression across samples correlates with variations in methylation of nearby cgis.
for each set of samples , we measure the strength of association between methylation and expression for each gene by computing a predictive goodness of fit r <dig> which represents the level of gene expression variation explained by cgi + ss methylation variation .
this coefficient is calculated either when the cgi + ss methylation status is summarized by the mean methylation values of all the probes, or by using the full cgi + ss methylation information of each probe.
we observe that the full cgi + ss methylation profile is predictive of gene expression for a subset of genes in each dataset, and that this predictive power is significantly higher than using only the average cgi + ss methylation .
we provide in table  <dig> the list of the top  <dig> genes based on their predictive score in cancerous breast tissues and similar lists for normal breast, lung and colon tissues in additional file  <dig>  among the  <dig>  genes studied,  <dig> genes are associated with more than one cgi + ss.
for these genes, the predictive power is computed using the cgi + ss closest to the tss.
using all the cgi + ss for these genes do not yield significant improvement over taking only the cgi + ss closest to the tss except for breast tissues .
we also observe no association between the predictive goodness of fit r <dig> and the cgi + ss clusters described above .
fig.
<dig> impact of dna methylation in gene expression prediction.
predictive power distribution of dna methylation for gene expression using either the average cgi methylation and least squares  or the full cgi + ss profile and lasso regression  shows that a more complex model allows to better predict gene expression variations in both normal and cancerous tissues
dqx1
irs2
gpsm3
foxc1
†
psmb9
hoxc10
†
ndrg2
mapt
stc2
znf502
†
ptprcap
scand3
slc1a4
tap1
dbndd2
otx1
†
tcf7
†
ly6g6c
fermt3
zic4
†
hla-b
gdf9
sox9
†
celsr1
sys1-dbndd2
hla-e
cyp1b1
runx3
†
kiaa1949
ripk4
tppp2
hla-f
ppp1r3c
hoxb5
†
celsr3
b3gnt5
me3
tmc8
aif1
slc39a6
hoxc11
†
erbb2
tbc1d10c
sim2
camk2n1
rgma
loc100132215
pax6
†
vangl2
ddhd2
gene: top scoring genes ranked by the predictive power of methylation to predict gene expression variation.
score: r
<dig> score associated.
transcription factors are highlighted with a †
since the predictive power of multivariate models based on all cpg probes in a cgi + ss is larger than the predictive power of the mean methylation value only, we now investigate which cpg in a cgi + ss are particularly important predictors of expression.
for that purpose, we measure the correlation between the methylation of individual cpg and gene expression for the  <dig> genes with the largest predictive r <dig>  and summarize the correlation values based on the position of the probe in the cgi + ss in fig.
<dig> for breast samples .
as expected, we observe overall a negative correlation between methylation and gene expression, and notice that this association is stronger in cgi shores and shelves located in the 3’ region than in the cgi itself.
this is coherent with results in  <cit>  stating that variations in the cgi are less critical than variations in proximity regions of the cgi.
performing the same analysis by varying the number of genes selected to compute correlations from  <dig> to  <dig> gave similar results.
fig.
<dig> correlation between individual cpg methylation and gene expression for breast cancer.
distribution of the correlation between individual cpg methylation and gene expression for normal  and cancerous  breast samples.
the analysis is restricted to the  <dig> genes with the largest r
<dig> predictive value.
correlations are aggregated by regions in the cgi + ss.
we see the strongest association for probes located outside of the cgi, particularly in shores regions, in both normal and cancerous tissues
correlation between gene expression and promoter methylation is tissue-specific, and is modified in cancer tissues but overall targets transcription factors
results in the previous section suggest that for a subset of genes, a significant correlation between promoter methylation and gene expression is observed, which may be due for example to a direct regulation of gene expression by promoter methylation.
to assess whether this correlation is conserved across tissues, we compare the predictive powers of methylation for each gene when it is computed on normal or cancerous samples from different tissues.
as shown on additional file  <dig>  however, we observe little correlation between the predictive power across tissues in normal and in cancer samples, suggesting that the association between promoter methylation and gene expression is tissue-specific .
we also observe very little correlation between predictive powers in normal and cancerous tissues, which could suggests a shift of the epigenetic regulation mechanism during cancer development .
fig.
<dig> shift of epigenetic regulation in cancer.
scatterplot between the predictive power of dna methylation for gene expression in normal and cancerous breast tissues shows little correlation
many mechanisms besides dna methylation are involved in gene expression regulation.
in particular, transcription factors  play a critical role in the recruitment of rna polymerase that allows gene transcription  <cit> .
we noticed that the list of the  <dig> genes with the largest predictive r <dig> score in each tissue is significantly enriched in tfs as collected from  <cit> , suggesting that methylation may play an important role in the gene regulatory process of transcription factors .
using the tf list obtained from  <cit>  yields similar conclusions, as well as varying the number of genes selected from  <dig> to  <dig>
copy number variations in cancer is an independent factor correlated with gene expression
in cancer, aberrant dna copy number variations  can have an important impact on gene expression phenotypes  <cit> .
since genome-wide dna copy number information is available for all samples analyzed in this study, we now perform an integrated analysis combining methylation, dna copy number and gene expression.
we compute a predictive goodness of fit r <dig> to represents the power of dna copy number information alone to predict gene expression, on the one hand, and a multidimensional regression model combining both the full cgi + ss dna methylation information and the dna copy number information, on the other hand.
we observe that combining methylation and copy number information leads to significantly better results in predicting gene expression than taking each information separately .
moreover, correlation analysis between predictive scores using dna methylation only, on the one hand, and predictive scores using cnvs only, on the other hand, shows very little correlation .
this suggests that both methylation and dna cnvs are important and non-redundant predictors of gene expression variation.
fig.
<dig> association between predictive power of methylation and cnvs.
panel a predictive power distribution using either cnv data only with least squares, dna methylation data only with lasso regression or both cnv and dna methylation data with lasso regression.
combined methylation and cnv information yield significantly higher predictive power .
panel b scatterplot of predictive power using dna methylation only and copy number information only shows that both regulation mechanisms operate exclusively on genes
discussion
dna methylation is a well-described process in normal development and is critical in specific gene expression regulations such as x-chromosome inactivation, genomic imprinting and tissue developpment .
since aberrant hyper- and hypo-methylation have also been frequently observed in cancer, it has been often argued that activation of oncogenes or repression of tumor suppressor genes could be caused by these epigenetic variations  <cit> .
in the present study, we assessed the existence of characteristic cgi + ss dna methylation signatures in normal tissues and showed a weak association between the hyper-methylated signature and gene expression repression.
a similar study in cancerous tissues showed the existence of a cancer-specific signature highly associated with repressed genes.
however, the corresponding genes are already highly repressed in normal tissues, questioning the causal impact of methylation in gene expression regulation, as already observed  <cit> .
using regression methods we analyzed whether differences between cgi + ss methylation across samples - independently of signatures - are predictive of gene expression variation.
we showed that for certain genes, expression variations across samples can be well predicted from dna methylation and that these genes are not associated with cancer-specific methylation patterns.
we also showed that using the full cgi + ss methylation profiles in a multidimensional regression framework yields better predictive power than summarizing the methylation of a cpg island by one mean value, as done in previous studies  <cit> .
looking at probewise methylation correlation with gene expression for the top scoring genes, we observed that the impact of a cpg methylation on gene expression is largely dependent on its location in or near the island, and that cpgs located outside of cgis have a bigger impact on gene expression variations than cpg located within the cgi, in accordance with  <cit> .
the impact of cgis located outside of promoter regions, such as intragenic cgis is still unclear as it does not seem to contribute significantly to global gene expression regulation.
yet, a few studies point at their potential role in modulating alternative promoters  <cit>  or in long-range regulation  <cit> .
reproducing this methodology on different datasets allowed us to compare the variations of gene expression regulation by methylation in normal and cancerous tissues but also between different types of tissues.
our results suggest that genes targeted by methylation are not only very different between different normal tissues, but more importantly that they are very different between normal and cancerous samples of a given tissue suggesting a shift of epigenetic regulation between normal and cancerous tissues.
recently, hydroxymethylation of cytosines  has been shown to be significantly present in mammalians cells  <cit>  and methylation data generated with illumina arrays, as done here, are not able to distinguish methylation  from hmc  <cit> .
however, hydroxymethylation is significantly less observed in cancer tissues  <cit> .
it is therefore likely that the epigenetic information measured here is indeed cytosine methylation.
in addition, the association between dna methylation and other important regulation mechanisms widens our understanding of the role of methylation in the whole gene expression regulation process.
while tfs are essential for controlling gene expression, we showed that their activation itself is significantly associated with dna methylation markers, highlighting the critical role of methylation in the regulatory process.
cnvs have been widely analyzed as a source of genetic variation that plays an important role in complex phenotypes such as cancer  <cit> .
while cnv contribution has been characterized on a genome-wide scale, the link with other regulation mechanisms, particularly dna methylation, is still unclear  <cit> .
we showed that the impact of both processes in gene expression regulation seems to be non-redundant.
the relatively large dataset size gives us confidence in the statistical validity of the results, which are however limited to a fraction of the total genes because of uneven coverage.
methylome sequencing has already been performed and also supports the complexity of methylation patterns but is still limited to very small datasets  <cit> .
undoubtedly, larger methylome datasets available in the near future will further improve our understanding of the role of dna methylation in gene expression regulation.
CONCLUSIONS
in summary, this study suggests that promoter methylation profiles can be summarized with a few characteristic profiles that we refer to as cgi+ss methylation signatures.
in cancer, we observe an epigenetic reprogramming that leads to the apparition of a cancer-specific cgi+ss methylation signa- ture.
however, this epigenetic reprogramming is not associated changes in gene expression, suggesting that this mechanism does not contribute to cancer development via direct inhibition of gene expression through promoter hypermethylation.
on the other hand, we observe that genes which demonstrate high correlation between methylation variations and gene expression variations differ from normal to cancer- ous tissues.
this suggests that in cancer, the association between gene expression and promoter dna methylation is modified.
materials and methods
patients selection
all data were retrieved from the tcga data portal.
we selected samples from breast, colon and lung adenocarcinomas because large matched datasets were available for methylation, gene expression and copy number profiles.
the datasets are detailed in table  <dig> and the different institutions that released the data are mentioned in the acknowledgement section.
methylation profiling
methylation profiles were retrieved from level  <dig> tcga data.
they were obtained with the illumina humanmethylation450k dna analysis beadchip assay, which is based on genotyping of bisulfite-converted genomic dna at individual cpg-sites to provide a quantitative measure of dna methylation  <cit> .
following hybridization, the methylation value for a specific probe was calculated as the ratio m/ where m is the methylated signal intensity and u is the unmethylated signal intensity.
<dig>  cpg methylation levels, associated with  <dig>  cgis and  <dig>  genes, were measured as such accross the genome.
following  <cit> , we considered not only the cgi methylation profile but also included in the analysis proximal regions in the near vicinity , namely the cgi shores and shelves regions in a general cgi + ss methylation profile.
as we were interested in the coordinated variations of methylation, we restricted the analysis to cgi + ss profiles containing at least  <dig> probes which reduced the analysis to  <dig> cgi+ss associated with  <dig>  genes from the original dataset.
gene expression profiling
gene expression profiles were retrieved from level  <dig> tcga data.
they were obtained from the illumina hiseq rnaseq technology and processed following  <cit> .
cnv processing
cnvs were retrieved from the level  <dig> tcga data infered from affymetrix snp <dig>  data files in genepattern following  <cit> .
for each gene, we then obtained the log ratio copy number score as the segmented log ratio score for the interval containing its transcription start site.
combined cpg island, shores and shelves pattern analysis
cgi + ss patterns were compared using dynamic time warping   <cit>  as it is less sensitive to small variations than the fréchet distance  <cit> .
dynamic time warping was originally applied as a speech signal similarity measure and has been applied with success in several other fields including computer vision  <cit> , protein structure matching  <cit>  and time series analysis  <cit> .
a cgi + ss profile i can be represented as a couple of vector =,…,) where xki represents the position of the kth cpg associated with the cgi + ss and yki∈ represents the mean methylation level for this probe accross a given dataset.
for two cgi + ss profiles with respectively m and n probes, we compute the distance between the two profiles as:
dtw=minw∈path∑k=1length|yw1k1−yw2k2| <dig>   where a path w of length k is a pair of vectors k∈ in × that verifies:
∈{1}×∪×{1}
∀i∈{1;2}, wik+1=wik or wik+1=wik+ <dig>
∈{n}×∪×{n}
the algorithm is applied for each pair of cgi + ss patterns to obtain a dissimilarity matrix.
ward hierarchical clustering is then performed on this dissimilarity matrix to assess the existence of characteristic patterns amongst the different datasets.
the number of significant clusters is assessed through bootstrapping  on a random subset of cgi + ss of the initial dataset  following ben-hur et al  <cit> .
r code for analysis is available upon request.
survival analysis
overall survival was estimated using the kaplan-meier method  <cit>  to compare the survival between the group of patients with a lower level of methylation in the hemi-methylated cgi + ss compared to the group of patients with a higher level of methylation.
a multivariate cox proportional hazards regression model  <cit>  was also fitted to estimate the additional value of this classification as a predictive factor for survival compared to other clinical parameters such as age, tumor size, lymph node status, receptor status and her2/neu status.
computing gene expression susceptibility to dna methylation changes
we apply ridge  <cit>  and lasso  <cit>  multivariate regression methods to predict gene expression using the full cgi + ss methylation profiles as well as univariate least square regression when using only the averaged methylation from the whole cgi + ss profile.
following acharjee et al.
<cit> , we assess the predictive power of the methylation using the predictive goodness of fit r <dig> which represents the squared pearson correlation between observed and fitted values on an independent dataset.
the estimation of the predictive power for each gene is obtained through 3-fold cross-validation averaged over  <dig> repeats.
parameters for both lasso and ridge regression methods were obtained by minimizing the mean squared error function using nested 3-fold cross-validation on the training dataset.
the use of the predictive goodness of fit instead of the classic mean squared error as a score allows to compute a comparable score between different predictions.
in particular, the mean squared error is highly affected by the absolute level of gene expression while the r <dig> is invariant to scaling.
it is also important to note that in this case the r <dig> computed for least square regression is a prediction r <dig> and not just a goodness-of-fit of the given dataset and therefore provides confidence on the generalization of the score on independent datasets.
r code for analysis is available upon request.
additional files
additional file  <dig>
hierarchical clustering of average cgi + ss patterns for lung tissues.
additional file  <dig>
hierarchical clustering of average cgi + ss patterns for colon tissues.
additional file  <dig>
characteristic profiles of cgi + ss clusters in lung tissues.
additional file  <dig>
characteristic profiles of cgi + ss clusters in colon tissues.
additional file  <dig>
example of cgis with different methylation patterns between normal and tumor samples .
the black vertical bars represent the position of the cpg island.
additional file  <dig>
gene ontology analysis for the genes associated with cancer-specific cluster  <dig>
additional file  <dig>
gene expression patterns for each cgi + ss clusters in lung tissues.
additional file  <dig>
gene expression patterns for each cgi + ss clusters in colon tissues.
additional file  <dig>
clinical impact of cluster “3up”.
multivariate cox regression analysis including the level of methylation in the cancer-specific cluster “3up” in addition to all clinical variables available for breast cancer.
additional file  <dig>
hierarchical clustering of breast cancer patients based on the average methylation level of cgi + ss associated with cluster 3down.
additional file  <dig>
impact of dna methylation on gene expression prediction.
additional file  <dig>
top genes regulated by methylation for breast normal tissues, lung normal and cancerous tissues and colon cancerous tissues.
additional file  <dig>
methylation association with gene expression by regions.
panel a. colon cancerous samples.
panel b. lung normal samples.
panel c. lung cancerous samples.
additional file  <dig>
tissue-specificity of methylation for gene expression regulation in normal and cancerous tissues.
panel a. normal breast and lung samples.
panel b. cancerous breast and colon samples.
panel c. cancerous colon and lung samples.
panel d. cancerous lung and breast samples.
additional file  <dig>
shift of epigenetic regulation between normal and cancerous lung tissues.
additional file  <dig>
specific increase of predictive power for aberrant cnv by including copy number profiles in lung tissues.
additional file  <dig>
specific increase of predictive power for aberrant cnv by including copy number profiles in colon tissues.
abbreviations
cgicpg island
cgi + sscombined cpg island, cgi shores and shelves
cnvcopy number variation
grmgene regulated by methylation
tftranscription factor
dtwdynamic time warping
competing interests
the authors declare that they have no competing interests.
authors’ contributions
mm and vb participated in the statistical analyses and the writing of the manuscript.
jpv and fr conceived the study and participated in its design and coordination.
all authors read and approved the final manuscript.

 dna methylation is one of the main epigenetic mechanisms, alongside histone modifications, that plays a significant role in gene silencing, tissue differentiation, cellular development, x-chromosome inactivation, or genetic imprinting. aberrant hyper-methylation of high-density cpg regions known as cpg islands and genome-wide hypo-methylation have often been associated with cancer and there has been an increasing effort to understand the specific epigenetic modifications that contribute to carcinogenesis. in addition to promoter cgis themselves, their surrounding area called shores and shores have also a cancer- and tissue-specific methylation, while even larger cancer-specific methylation variations were reported in so called open sea regions, far from cgis. in this study, we focus on methylation in promoter cgis and surrounding regions only, in order to investigate its association in cis with gene expression. the possibility to quantify dna methylation genome-wide on normal and cancer tissues, with microarray or sequencing technologies, has triggered a lot of data-driven research to clarify the role of methylation in gene regulation and cancer. several studies have highlighted a correlation between differentially methylated regions near promoter regions and gene expression changes. however, it has also been reported that aberrant over-methylation occurs mostly in normally down-regulated genes, questioning the role of methylation as a causal mechanism for gene repression. more recently, timp et al. have proposed a model where epigenetic aberrations contribute to carcinogenesis by dysregulating the functions of specific genes that regulate the epigenome itself. reddington et al. speculate that epigenetic reprogramming might lead to an altered polycomb binding landscape which could impact genome regulation. to gain further insight into the role of dna methylation in cancer, we perform a large-scale meta-analysis of methylation profiles of normal and cancerous samples from multiple tissues from the cancer genome atlas. for each cgi and surrounding area, we focus on its average methylation profile and the association between variations of its methylation profile and variations in the expression of the target gene. comparing these parameters between normal and cancerous samples of different tissues suggests that the interplay between promoter methylation and gene expression, and how they are modified in cancer, is not simple. on the one hand, while each promoter cgi tends to be either hypo- or hypermethylated in all normal samples, we observe hypermethylation of a common subset of cgis in several cancer samples of different tissues, supporting the existence of a cpg island methylator phenotype with prognostic value, as introduced by toyota et al.. however, we did not find evidence that the genes associated with hypermethylated promoter cgis were less expressed in the cancer samples, as most of the genes concerned are already lowly expressed in normal tissues, as already observed by. on the other hand, looking more precisely at associations between promoter methylation level and gene expression within a set of samples, we observe for each tissue and each normal or cancerous sample set a subset of genes, different from the genes hypermethylated in the cimp phenotype, for which this association is important and strongest outside of the cgis, namely in the n-shores and n-shelves. this subset of genes varies across tissues but also whether we consider healthy or cancerous samples. however, transcription factors are over-represented in all subsets. this suggests that epigenetic reprogramming might contribute to carcinogenesis in part by modifying gene expression susceptibility to changes in dna methylation.  classification of genes based on their cgi methylation profiles in normal and cancerous tissues we first assess how promoter methylation profiles differ between genes, when for each gene we consider the average methylation profile across normal or cancerous samples. for that purpose, we collected high-density methylation datasets from the cancer genome atlas data portal providing more than 485k cpg methylation levels for normal and cancerous samples from three tissues of origin: breast, colon and lung. for each cgi, we combine the probes in the cgi and in the shore and shelves of the cgi, defined as the regions up to 4kb outside of the cgi, in a unique cgi, shores and shelves methylation profile. we restrict our analysis to the cgi + ss where at least cpg probes are measured by the technology in order to have high enough coverage to measure the methylation variation within each cgi + ss. for each of the three tissue of origin, and each normal or cancerous set of tissues, we compute the average methylation profile of each cgi + ss by averaging the methylation values of each cpg across the samples. hence we compute 3×2= average profile for each cgi + ss, with we refer to below as cgi+ss signatures. to assess the diversity of cgi + ss signatures across genes, we perform an unsupervised classification of all signatures for each of the types of samples, using ward hierarchical clustering. since different cgi + ss may contain a different number of gpg probes, we use a specific distance based on dynamic time warping to compare signatures of different lengths. figure 1a shows the cgi + ss clustering obtained for signatures measured on normal samples from breast samples. similar figures were obtained for lung and colon samples. we observe two clusters, which are largely conserved across the tissues of origin. to clarify the types of signatures captured by each cluster, we represent on a standardized cgi + ss x-axis the medoid cgi + ss signatures for each cluster and each tissue. we clearly observe that the large cluster which contains about % of all cgi + ss, corresponds to hypo-methylated islands with hemi-methylated cgi shores and hyper-methylated cgi shelves, while the smaller cluster contains about % of cgi + ss which are fully hyper-methylated. a closer look at cluster shows that, in some cases, the variation of methylation between islands and shores is unclear, in the sense that some shores are fully hypo-methylated. as cgis, shores and shelves regions are delimited based on somehow arbitrary criteria, a systematic analysis of these signatures could lead to a refinement of currently accepted boundaries. fig. cgi + ss patterns in breast tissues. hierarchical clustering of cgi + ss dna methylation patterns for breast normal tissues and breast cancerous tissues using dtw as a distance metric and a “ward” linkage. the colorbar represents the clusters association fig. characteristic profiles for each clusters. visualization of the cgi + ss dna methylation signatures as condensed profiles from the medoids profiles for each clusters in breast normal or cancerous tissues. the two orange dashed lines represent the normalized 1kb long cgi region while the two blue lines represent the 2kb limit between shores and shelves regions performing the same unsupervised classification independently on signatures obtained from the three types of cancerous tissues leads to different results, with the apparition of a third stable cluster. comparing the clusters of normal and cancerous tissues shows that, for all types of tissues, the first two clusters found in cancerous tissues are mostly composed of cgi + ss of the corresponding clusters in normal tissues, while the cgi + ss in the third cluster, specifically found in cancerous tissues, tend to come evenly from both clusters in normal tissues. a look at representative signatures of each cluster confirms that clusters and contain respectively hypo- and hyper-methylated profiles, just like the respective clusters in normal tissues, while cluster contains cgi + ss signatures which are hemi-methylated. separating the cgi + ss in cluster into sub-clusters “3up” and “3down”, depending on whether they are in cluster or in normal tissues, we further see that the level of methylation of cgi + ss signatures in the “3up” sub-cluster tends to be lower than the level of methylation of cgi + ss signatures in the “3down” sub-cluster. interestingly, cluster is mostly conserved between tissues, suggesting that these epigenetic variations might be associated with a tissue-independent carcinogenesis process. fig. stability of the cancerous-specific cluster between tissues. venn diagram representing the intersection of the cgi + ss associated with cancerous-specific cluster for each tissue in summary, this global analysis of methylation signatures suggests the existence of four types of cgi + ss largely conserved across tissues: the majority of them remains hypo-methylated on the cgi and hyper-methylated on the shores and shelves in normal and cancerous tissues; a minority is hyper-methylated in normal and cancerous tissues; finally, a fraction of cgi + ss signatures is hypo-methylated in normal tissues and partly methylated in cancerous tissues, while another fraction is hyper-methylated in normal tissues and partly methylated in cancerous ones. to clarify whether these four categories or cgi + ss are associated to particular biological functions, we performed a gene functional enrichment analysis of the genes associated to the cgi + ss in each of the four categories, for each tissue. results are shown inadditional file restricting ourselves to gene ontology biological processes associated to at least genes, we found that the large cluster is mostly enriched in genes involved in metabolic processes, while the cancer-specific cluster 3up is enriched in genes involved in developmental processes. there was no significant functional enrichment for genes in cluster and 3down. cancer-specific methylation does not repress gene expression but instead targets genes lowly expressed in normal tissues cgi methylation is often associated with gene expression silencing. we therefore assess whether the cgi + ss clusters defined above, corresponding roughly to lowly methylated, highly methylated or partially methylated in cancer cgi + ss, are associated with different mean levels of gene expression. in normal breast tissues, we indeed observe that genes near hypo-methylated islands in cluster are slightly but significantly less expressed than genes near an hyper-methylated islands in cluster. there is however no significant difference between the two clusters in normal lung tissues, and we could not test the hypothesis on normal colon tissues since we have none with both methylation and expression data. in cancerous samples, we observe that genes near a cgi + ss in the cancer-specific cluster have a significantly lower expression than other genes, particularly for the genes near a cgi + ss in the “3up” cluster. as genes in the “3up” cluster are hypo-methylated in normal tissues, this could suggest that their cancer-specific methylation is a way to repress their expression in cancer. however, a closer look at the expression of these genes in normal tissues shows that they are already lowly expressed in normal tissues. this suggests that instead of activating cgi methylation to silence to genes, cancer cells instead activates cgi methylation of hypo-methylated genes which are already lowly expressed in normal tissues. fig. distribution of gene expression in different clusters for in breast tissues. gene expression distribution for genes based on the cluster assignment of their associated cgi + ss. panel a gene expression distribution in normal breast tissues shows a slight repression for genes associated with cluster. “ref” represents the genome-wide gene expression distribution panel b gene expression profiles in cancerous breast tissues shows high repression for genes associated with cluster and specifically cluster “3up”. panel c gene expression profiles in both normal and cancerous breast tissues using the cluster assignement in cancerous tissues shows that genes associated with cluster “3up” in cancerous tissues define a cluster of genes already repressed in normal tissues cancer-specific methylation is an independent predictor of patient survival in breast cancer our analysis so far compares cgi + ss in terms of their mean methylation across a set of samples and does not take into account between-sample variations. cgi + ss associated with cluster are hypo- methylated on average, which indicates that there is little to no variations between samples. however, signatures of cgis in the cancer-specific cluster are partly methylated, which can either hide the fact that they are hemi-methylated for most cancerous samples, or that they are highly variable between samples. we therefore assess whether the partial methylation of cgi + ss signatures in cluster is related to an overall increase or decrease in methylation for all or most of the patients, or if this it is caused by a subset of patients that become hyper- methylated for these cgi + ss. for that purpose, we first summarize the methylation of each cgi + ss on each breast cancer sample by a single value, the average methylation of the probes in the cgi + ss. we then represent each sample by the vector of methylation values of the cgi + ss in cluster 3up, and perform a ward hierarchical clustering of the cancerous samples based on this representation. the resulting clustering is shown in fig. 5a, where in addition we indicate the er +, her and survival information for each patient. we observe that the distribution methylation values is very bimodal, and that the hyper-methylation of a given cgi + ss from cluster 3up generally happens in a subset of patients only. interestingly, we see that the same subset of patients tends to be simultaneously hyper-methylated for all cgi + ss from cluster 3up, suggesting that hyper-methylation of these islands is a characteristics of a subset of the tumors. this allows us to divide the set of breast cancer patients in three clusters given the level of methylation in cluster 3up as either “low”, “intermediate”, or “high”. interestingly, distinguishing patients given the level of methylation from the cgi + ss in cluster 3up is significantly predictive of the patient survival. surprisingly, the cluster with the lowest survival is the “intermediate” cluster encompassing a portion but not all of the triple negative breast cancers. a multivariate cox proportional hazards regression model fitted with available clinical parameters further shows that this stratification of patients based on the methylation level of genes in cluster 3up adds prognostic value independently of other clinical features. these results support the existence of a cpg island methylator phenotype as introduced by toyota et al. that is clinically relevant to assess the survival of patients. more importantly, they suggest that low survival might not be associated with a positive or negative cimp, but with an intermediate phenotype termed as cimp-low. fig. cluster 3up methylation is a predictive factor for survival of patients in breast cancer patients. panel a hierarchical clustering of breast cancer patients given the average methylation level of all the cgi + ss associated with cluster 3up. the row color bar represents the average methylation level for the same cgi + ss in healthy breast tissues. the column color bar gives clinical information about the patients such as er and her statuses, survival information. the top row of the column color bar represents the three classes distinguished by methylation profiles in cluster 3up. panel b kaplan-meier estimate of breast cancer patient survival given the cluster 3up class shows that cluster 3up “intermediate” patients have a significantly higher risk of death within years than either cluster 3up “low” or “high” patients a similar analysis on cgi + ss associated with cluster 3down is less conclusive, and does not clearly cluster patients in separate clusters. a lack of sufficient survival data for colon and lung tissues prevented a similar analysis for these tissues. methylation of cpg in the 3’ region outside the cgi is the most correlated with gene expression our analysis so far compares cgi + ss to one another, by looking at their average methylation profiles across collections of samples. we found no clear evidence for a correlation between mean methylation level of a cgi and mean expression level of the corresponding genes, but this may be due to the fact that many other factors impact the expression level of a gene, including biological and technical ones. another way to assess how methylation impacts expression is to look, for each given gene, how variation in expression across samples correlates with variations in methylation of nearby cgis. for each set of samples, we measure the strength of association between methylation and expression for each gene by computing a predictive goodness of fit r which represents the level of gene expression variation explained by cgi + ss methylation variation. this coefficient is calculated either when the cgi + ss methylation status is summarized by the mean methylation values of all the probes, or by using the full cgi + ss methylation information of each probe. we observe that the full cgi + ss methylation profile is predictive of gene expression for a subset of genes in each dataset, and that this predictive power is significantly higher than using only the average cgi + ss methylation. we provide in table the list of the top genes based on their predictive score in cancerous breast tissues and similar lists for normal breast, lung and colon tissues in additional file among the genes studied, genes are associated with more than one cgi + ss. for these genes, the predictive power is computed using the cgi + ss closest to the tss. using all the cgi + ss for these genes do not yield significant improvement over taking only the cgi + ss closest to the tss except for breast tissues. we also observe no association between the predictive goodness of fit r and the cgi + ss clusters described above. fig. impact of dna methylation in gene expression prediction. predictive power distribution of dna methylation for gene expression using either the average cgi methylation and least squares or the full cgi + ss profile and lasso regression shows that a more complex model allows to better predict gene expression variations in both normal and cancerous tissues dqx1 irs2 gpsm3 foxc1 † psmb9 hoxc10 † ndrg2 mapt stc2 znf502 † ptprcap scand3 slc1a4 tap1 dbndd2 otx1 † tcf7 † ly6g6c fermt3 zic4 † hla-b gdf9 sox9 † celsr1 sys1-dbndd2 hla-e cyp1b1 runx3 † kiaa1949 ripk4 tppp2 hla-f ppp1r3c hoxb5 † celsr3 b3gnt5 me3 tmc8 aif1 slc39a6 hoxc11 † erbb2 tbc1d10c sim2 camk2n1 rgma loc100132215 pax6 † vangl2 ddhd2 gene: top scoring genes ranked by the predictive power of methylation to predict gene expression variation. score: r score associated. transcription factors are highlighted with a † since the predictive power of multivariate models based on all cpg probes in a cgi + ss is larger than the predictive power of the mean methylation value only, we now investigate which cpg in a cgi + ss are particularly important predictors of expression. for that purpose, we measure the correlation between the methylation of individual cpg and gene expression for the genes with the largest predictive r and summarize the correlation values based on the position of the probe in the cgi + ss in fig. for breast samples. as expected, we observe overall a negative correlation between methylation and gene expression, and notice that this association is stronger in cgi shores and shelves located in the 3’ region than in the cgi itself. this is coherent with results in stating that variations in the cgi are less critical than variations in proximity regions of the cgi. performing the same analysis by varying the number of genes selected to compute correlations from to gave similar results. fig. correlation between individual cpg methylation and gene expression for breast cancer. distribution of the correlation between individual cpg methylation and gene expression for normal and cancerous breast samples. the analysis is restricted to the genes with the largest r predictive value. correlations are aggregated by regions in the cgi + ss. we see the strongest association for probes located outside of the cgi, particularly in shores regions, in both normal and cancerous tissues correlation between gene expression and promoter methylation is tissue-specific, and is modified in cancer tissues but overall targets transcription factors results in the previous section suggest that for a subset of genes, a significant correlation between promoter methylation and gene expression is observed, which may be due for example to a direct regulation of gene expression by promoter methylation. to assess whether this correlation is conserved across tissues, we compare the predictive powers of methylation for each gene when it is computed on normal or cancerous samples from different tissues. as shown on additional file however, we observe little correlation between the predictive power across tissues in normal and in cancer samples, suggesting that the association between promoter methylation and gene expression is tissue-specific. we also observe very little correlation between predictive powers in normal and cancerous tissues, which could suggests a shift of the epigenetic regulation mechanism during cancer development. fig. shift of epigenetic regulation in cancer. scatterplot between the predictive power of dna methylation for gene expression in normal and cancerous breast tissues shows little correlation many mechanisms besides dna methylation are involved in gene expression regulation. in particular, transcription factors play a critical role in the recruitment of rna polymerase that allows gene transcription. we noticed that the list of the genes with the largest predictive r score in each tissue is significantly enriched in tfs as collected from, suggesting that methylation may play an important role in the gene regulatory process of transcription factors. using the tf list obtained from yields similar conclusions, as well as varying the number of genes selected from to copy number variations in cancer is an independent factor correlated with gene expression in cancer, aberrant dna copy number variations can have an important impact on gene expression phenotypes. since genome-wide dna copy number information is available for all samples analyzed in this study, we now perform an integrated analysis combining methylation, dna copy number and gene expression. we compute a predictive goodness of fit r to represents the power of dna copy number information alone to predict gene expression, on the one hand, and a multidimensional regression model combining both the full cgi + ss dna methylation information and the dna copy number information, on the other hand. we observe that combining methylation and copy number information leads to significantly better results in predicting gene expression than taking each information separately. moreover, correlation analysis between predictive scores using dna methylation only, on the one hand, and predictive scores using cnvs only, on the other hand, shows very little correlation. this suggests that both methylation and dna cnvs are important and non-redundant predictors of gene expression variation. fig. association between predictive power of methylation and cnvs. panel a predictive power distribution using either cnv data only with least squares, dna methylation data only with lasso regression or both cnv and dna methylation data with lasso regression. combined methylation and cnv information yield significantly higher predictive power. panel b scatterplot of predictive power using dna methylation only and copy number information only shows that both regulation mechanisms operate exclusively on genes discussion dna methylation is a well-described process in normal development and is critical in specific gene expression regulations such as x-chromosome inactivation, genomic imprinting and tissue developpment. since aberrant hyper- and hypo-methylation have also been frequently observed in cancer, it has been often argued that activation of oncogenes or repression of tumor suppressor genes could be caused by these epigenetic variations. in the present study, we assessed the existence of characteristic cgi + ss dna methylation signatures in normal tissues and showed a weak association between the hyper-methylated signature and gene expression repression. a similar study in cancerous tissues showed the existence of a cancer-specific signature highly associated with repressed genes. however, the corresponding genes are already highly repressed in normal tissues, questioning the causal impact of methylation in gene expression regulation, as already observed. using regression methods we analyzed whether differences between cgi + ss methylation across samples - independently of signatures - are predictive of gene expression variation. we showed that for certain genes, expression variations across samples can be well predicted from dna methylation and that these genes are not associated with cancer-specific methylation patterns. we also showed that using the full cgi + ss methylation profiles in a multidimensional regression framework yields better predictive power than summarizing the methylation of a cpg island by one mean value, as done in previous studies. looking at probewise methylation correlation with gene expression for the top scoring genes, we observed that the impact of a cpg methylation on gene expression is largely dependent on its location in or near the island, and that cpgs located outside of cgis have a bigger impact on gene expression variations than cpg located within the cgi, in accordance with. the impact of cgis located outside of promoter regions, such as intragenic cgis is still unclear as it does not seem to contribute significantly to global gene expression regulation. yet, a few studies point at their potential role in modulating alternative promoters or in long-range regulation. reproducing this methodology on different datasets allowed us to compare the variations of gene expression regulation by methylation in normal and cancerous tissues but also between different types of tissues. our results suggest that genes targeted by methylation are not only very different between different normal tissues, but more importantly that they are very different between normal and cancerous samples of a given tissue suggesting a shift of epigenetic regulation between normal and cancerous tissues. recently, hydroxymethylation of cytosines has been shown to be significantly present in mammalians cells and methylation data generated with illumina arrays, as done here, are not able to distinguish methylation from hmc. however, hydroxymethylation is significantly less observed in cancer tissues. it is therefore likely that the epigenetic information measured here is indeed cytosine methylation. in addition, the association between dna methylation and other important regulation mechanisms widens our understanding of the role of methylation in the whole gene expression regulation process. while tfs are essential for controlling gene expression, we showed that their activation itself is significantly associated with dna methylation markers, highlighting the critical role of methylation in the regulatory process. cnvs have been widely analyzed as a source of genetic variation that plays an important role in complex phenotypes such as cancer. while cnv contribution has been characterized on a genome-wide scale, the link with other regulation mechanisms, particularly dna methylation, is still unclear. we showed that the impact of both processes in gene expression regulation seems to be non-redundant. the relatively large dataset size gives us confidence in the statistical validity of the results, which are however limited to a fraction of the total genes because of uneven coverage. methylome sequencing has already been performed and also supports the complexity of methylation patterns but is still limited to very small datasets. undoubtedly, larger methylome datasets available in the near future will further improve our understanding of the role of dna methylation in gene expression regulation.  in summary, this study suggests that promoter methylation profiles can be summarized with a few characteristic profiles that we refer to as cgi+ss methylation signatures. in cancer, we observe an epigenetic reprogramming that leads to the apparition of a cancer-specific cgi+ss methylation signa- ture. however, this epigenetic reprogramming is not associated changes in gene expression, suggesting that this mechanism does not contribute to cancer development via direct inhibition of gene expression through promoter hypermethylation. on the other hand, we observe that genes which demonstrate high correlation between methylation variations and gene expression variations differ from normal to cancer- ous tissues. this suggests that in cancer, the association between gene expression and promoter dna methylation is modified. materials and methods patients selection all data were retrieved from the tcga data portal. we selected samples from breast, colon and lung adenocarcinomas because large matched datasets were available for methylation, gene expression and copy number profiles. the datasets are detailed in table and the different institutions that released the data are mentioned in the acknowledgement section. methylation profiling methylation profiles were retrieved from level tcga data. they were obtained with the illumina humanmethylation450k dna analysis beadchip assay, which is based on genotyping of bisulfite-converted genomic dna at individual cpg-sites to provide a quantitative measure of dna methylation. following hybridization, the methylation value for a specific probe was calculated as the ratio m/ where m is the methylated signal intensity and u is the unmethylated signal intensity. cpg methylation levels, associated with cgis and genes, were measured as such accross the genome. following, we considered not only the cgi methylation profile but also included in the analysis proximal regions in the near vicinity, namely the cgi shores and shelves regions in a general cgi + ss methylation profile. as we were interested in the coordinated variations of methylation, we restricted the analysis to cgi + ss profiles containing at least probes which reduced the analysis to cgi+ss associated with genes from the original dataset. gene expression profiling gene expression profiles were retrieved from level tcga data. they were obtained from the illumina hiseq rnaseq technology and processed following. cnv processing cnvs were retrieved from the level tcga data infered from affymetrix snp data files in genepattern following. for each gene, we then obtained the log ratio copy number score as the segmented log ratio score for the interval containing its transcription start site. combined cpg island, shores and shelves pattern analysis cgi + ss patterns were compared using dynamic time warping as it is less sensitive to small variations than the fréchet distance. dynamic time warping was originally applied as a speech signal similarity measure and has been applied with success in several other fields including computer vision, protein structure matching and time series analysis. a cgi + ss profile i can be represented as a couple of vector =,…,) where xki represents the position of the kth cpg associated with the cgi + ss and yki∈ represents the mean methylation level for this probe accross a given dataset. for two cgi + ss profiles with respectively m and n probes, we compute the distance between the two profiles as: dtw=minw∈path∑k=1length|yw1k1−yw2k2| where a path w of length k is a pair of vectors k∈ in × that verifies: ∈{1}×∪×{1} ∀i∈{1;2}, wik+1=wik or wik+1=wik+ ∈{n}×∪×{n} the algorithm is applied for each pair of cgi + ss patterns to obtain a dissimilarity matrix. ward hierarchical clustering is then performed on this dissimilarity matrix to assess the existence of characteristic patterns amongst the different datasets. the number of significant clusters is assessed through bootstrapping on a random subset of cgi + ss of the initial dataset following ben-hur et al. r code for analysis is available upon request. survival analysis overall survival was estimated using the kaplan-meier method to compare the survival between the group of patients with a lower level of methylation in the hemi-methylated cgi + ss compared to the group of patients with a higher level of methylation. a multivariate cox proportional hazards regression model was also fitted to estimate the additional value of this classification as a predictive factor for survival compared to other clinical parameters such as age, tumor size, lymph node status, receptor status and her2/neu status. computing gene expression susceptibility to dna methylation changes we apply ridge and lasso multivariate regression methods to predict gene expression using the full cgi + ss methylation profiles as well as univariate least square regression when using only the averaged methylation from the whole cgi + ss profile. following acharjee et al., we assess the predictive power of the methylation using the predictive goodness of fit r which represents the squared pearson correlation between observed and fitted values on an independent dataset. the estimation of the predictive power for each gene is obtained through 3-fold cross-validation averaged over repeats. parameters for both lasso and ridge regression methods were obtained by minimizing the mean squared error function using nested 3-fold cross-validation on the training dataset. the use of the predictive goodness of fit instead of the classic mean squared error as a score allows to compute a comparable score between different predictions. in particular, the mean squared error is highly affected by the absolute level of gene expression while the r is invariant to scaling. it is also important to note that in this case the r computed for least square regression is a prediction r and not just a goodness-of-fit of the given dataset and therefore provides confidence on the generalization of the score on independent datasets. r code for analysis is available upon request. additional files additional file hierarchical clustering of average cgi + ss patterns for lung tissues. additional file hierarchical clustering of average cgi + ss patterns for colon tissues. additional file characteristic profiles of cgi + ss clusters in lung tissues. additional file characteristic profiles of cgi + ss clusters in colon tissues. additional file example of cgis with different methylation patterns between normal and tumor samples. the black vertical bars represent the position of the cpg island. additional file gene ontology analysis for the genes associated with cancer-specific cluster additional file gene expression patterns for each cgi + ss clusters in lung tissues. additional file gene expression patterns for each cgi + ss clusters in colon tissues. additional file clinical impact of cluster “3up”. multivariate cox regression analysis including the level of methylation in the cancer-specific cluster “3up” in addition to all clinical variables available for breast cancer. additional file hierarchical clustering of breast cancer patients based on the average methylation level of cgi + ss associated with cluster 3down. additional file impact of dna methylation on gene expression prediction. additional file top genes regulated by methylation for breast normal tissues, lung normal and cancerous tissues and colon cancerous tissues. additional file methylation association with gene expression by regions. panel a. colon cancerous samples. panel b. lung normal samples. panel c. lung cancerous samples. additional file tissue-specificity of methylation for gene expression regulation in normal and cancerous tissues. panel a. normal breast and lung samples. panel b. cancerous breast and colon samples. panel c. cancerous colon and lung samples. panel d. cancerous lung and breast samples. additional file shift of epigenetic regulation between normal and cancerous lung tissues. additional file specific increase of predictive power for aberrant cnv by including copy number profiles in lung tissues. additional file specific increase of predictive power for aberrant cnv by including copy number profiles in colon tissues. abbreviations cgicpg island cgi + sscombined cpg island, cgi shores and shelves cnvcopy number variation grmgene regulated by methylation tftranscription factor dtwdynamic time warping competing interests the authors declare that they have no competing interests. authors’ contributions mm and vb participated in the statistical analyses and the writing of the manuscript. jpv and fr conceived the study and participated in its design and coordination. all authors read and approved the final manuscript. 
methylation of high-density cpg regions known as cpg islands has been widely described as a mechanism associated with gene expression regulation. these subsets of genes are not the same in the different tissues, nor between normal and cancerous samples, but transcription factors are over-represented in all subsets. our results suggest that epigenetic reprogramming in cancer does not contribute to cancer development via direct inhibition of gene expression through promoter hypermethylation. on the one hand, we recover the existence of a cpg island methylator phenotype with prognostic value in a subset of breast, colon and lung cancer samples, where a common subset of promoter cgis hypomethylated in normal samples become hypermethylated. on the other hand, we identify tissue-specific sets of genes, different between normal and cancer samples, whose inter-individual variation in expression is significantly correlated with the variation in methylation of the 3’ flanking regions of the promoter cgis. however, recent studies have also challenged the simple model of gene expression control by promoter methylation in cancer, and the precise mechanism of and role played by changes in dna methylation in carcinogenesis remains elusive. using a large dataset of matched cancerous and healthy methylomes, gene expression, and copy number profiles accross types of tissues from the cancer genome atlas, we perform a detailed meta-analysis to clarify the interplay between promoter methylation and gene expression in normal and cancer samples. aberrant promoter methylation is considered a hallmark of cancer involved in silencing of tumor suppressor genes and activation of oncogenes. it may instead modify how the expression of a few specific genes, particularly transcription factors, are associated with dna methylation variations in a tissue-dependent manner. epigenetic regulationcancercpg island methylator phenotypeissue-copyright-statement© the author 2015 however, this hypermethylation is not accompanied by a decrease in expression of the corresponding genes, which are already lowly expressed in the normal genes.
 methylation of high-density cpg regions known as cpg islands has been widely described as a mechanism associated with gene expression regulation. aberrant promoter methylation is considered a hallmark of cancer involved in silencing of tumor suppressor genes and activation of oncogenes. however, recent studies have also challenged the simple model of gene expression control by promoter methylation in cancer, and the precise mechanism of and role played by changes in dna methylation in carcinogenesis remains elusive.  using a large dataset of matched cancerous and healthy methylomes, gene expression, and copy number profiles accross types of tissues from the cancer genome atlas, we perform a detailed meta-analysis to clarify the interplay between promoter methylation and gene expression in normal and cancer samples. on the one hand, we recover the existence of a cpg island methylator phenotype with prognostic value in a subset of breast, colon and lung cancer samples, where a common subset of promoter cgis hypomethylated in normal samples become hypermethylated. however, this hypermethylation is not accompanied by a decrease in expression of the corresponding genes, which are already lowly expressed in the normal genes. on the other hand, we identify tissue-specific sets of genes, different between normal and cancer samples, whose inter-individual variation in expression is significantly correlated with the variation in methylation of the 3’ flanking regions of the promoter cgis. these subsets of genes are not the same in the different tissues, nor between normal and cancerous samples, but transcription factors are over-represented in all subsets.  our results suggest that epigenetic reprogramming in cancer does not contribute to cancer development via direct inhibition of gene expression through promoter hypermethylation. it may instead modify how the expression of a few specific genes, particularly transcription factors, are associated with dna methylation variations in a tissue-dependent manner. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords epigenetic regulationcancercpg island methylator phenotypeissue-copyright-statement© the author 2015 