Research resource: genome-wide profiling of methylated promoters in endometriosis reveals a subtelomeric location of hypermethylation

Abstract

Several lines of evidence indicate that endometriosis could be partially due to selective epigenetic deregulations. Promoter hypermethylation of some key genes, such as progesterone receptor and aromatase, has been associated with the silencing of these genes and might contribute to the disease. However, it is unknown whether global alterations in DNA methylation patterns occur in endometriosis and to what extent they are involved in its pathogenesis. We conducted a whole-genome scanning of methylation status in more than 25,000 promoters, using methylated DNA immunoprecipitation with hybridization to promoter microarrays. We detailed the methylation profiles for each subtype of the disease (superficial endometriosis, endometriomas, and deep infiltrating endometriosis) and compared them with the profile obtained for the eutopic endometrium. In line with the current theory of the endometrial origin of endometriosis, the overall methylation profile was highly similar between the endometrium and the lesions. It showed promoter regions consistently hypomethylated or hypermethylated (more than 1.5-times, as compared with endometrium) and others specific to one given subtype. Albeit there was no systematic correlation between promoter methylation and expression of nearby genes, 35 genes had both methylation and expressional alterations in the lesions. These genes, reported here for the first time, might be of interest in the development of endometriosis. In addition, hypermethylated regions were located at the ends of the chromosomes, whereas hypomethylated regions were randomly distributed all along the chromosomes. We postulated that this original observation might participate to the chromosomal stability and protect the endometriotic lesion against malignancy.

Fig. 1.

Global comparison of promoter methylation patterns between SUP, OMA, DIE, and EE. Pair-wise correlation comparisons were made between all groups to establish the similarity of promoter methylation. R values were compared for significant correlation both within endometriotic subtypes (SUP, OMA, DIE) and between ectopic endometrium and EE.

Fig. 2.

Promoter methylation profiles in SUP, OMA, and DIE. The Venn diagram compares the number of active regions (in bold) identified as hypomethylated (in italic characters) or hypermethylated (in roman characters) in the three subtypes.

Fig. 3.

Quantitative RT-PCR results obtained for McrBC assay. McrBC is a methylation-specific endonuclease, which cleaves DNA containing 5′-methylcytosine residues but will not act on unmethylated DNA. Thus, a lesser PCR product recovery is indicative of hypermethylation. The black horizontal line represents the reference level and corresponds to EE; above this line, the region is hypermethylated; below, the region is hypomethylated. * and ** denote a significant difference in methylation level between OMA and EE (P < 0.05 and P < 0.01, respectively).

Fig. 4.

Methylation analysis of the LAMA5 region by bisulfite treatment and direct sequencing. The peak heights in the sequencing chromatogram were visually evaluated to quantify the respective amount of methylated C vs. unmethylated C at each CpG position. A, Average values of methylation at each of the six analyzed CpGs. B, Ratios of methylation between ectopic and eutopic endometrium at each CpG position. *, P < 0.05; **, P < 0.01; ****, P < 0.001.

Fig. 5.

Chromosomal distribution of DNA methylation in endometriosis. Plotted is the methylation level (Log2 ratio) relative to chromosomal position for SUP (in green), OMA (in red), and DIE (in blue). Regions of significant differences are above 0.4 or below −0.4.