Fig 1.
Gene-specific alterations in DNA methylation in primary ovarian endometriosis stromal cells (OESC) compared to control endometrial stromal cells (CESC), chromatin state enrichment analysis of DM genes in endometriosis, and bi-seq validation for DAPK1.
A) Heat maps of gene-specific methylation changes in endometriotic stroma cells vs. controls. Supervised hierarchical clustering of the 450K methylation BeadChip data analyzed at the CpG level (FDR q-values < .05 and absolute difference in fractional methylation (Δmethyl.)>.15) and at the element-level (q-values < .05 and Δmethyl.>.15 for multiple CpG segment and>0.1 for single CpG segment) are shown. Biological samples are on the x-axis and differentially methylated loci are on the y-axis, with relative hypermethylation and hypomethylation indicated by the color scale. The fractional methylation values for each CpG are centered and standardized to have mean 0 and standard deviation 1. The red color represents a methylation level above the mean methylation of the CpG across all samples, the white color represents mean methylation and the blue color represents methylation lower than the mean. B) DM CpGs are enriched in enhancers but depleted in promoter regions. For each chromatin state, enrichment and under-representation are symmetrically visualized using log2(OR). C) Validation and mapping of the DMR in DAPK1 using bis-seq. A map of the DAPK1 gene showing hypomethylation in the promoter region is given on the top. The DMR overlaps an active promoter region (color coded in red) flanked by a strong enhancer (yellow). Bis-seq amplicons for validation and mapping of the DMR are indicated by the numbered rectangles. The bis-seq data (bottom panel) is visualized by the circles representing consecutive CpGs with black circles indicating methylated CpGs and white circles unmethylated CpGs, with each line being a unique DNA clone.
Table 1.
Examples of genes with DM and DE in OESC versus CESC.
Fig 2.
Gain of DNA methylation in the promoter and gene body of JAZF1.
A) Map showing multiple clusters of hyper-methylated CpGs in the promoter and gene body of JAZF1. The dashed and plain rectangles indicate clusters of CpGs with DM overlapping multiple individual enhancer regions. B) Validation and mapping of the DMR (d) in JAZF1 gene using bis-seq. The results from the Bis-seq for the amplicons indicated with number on the higher resolution gene map (top) are shown as QUMA blots (bottom). Every circle represents a single CpG. Black circles indicate methylated CpG and the white free of methylation CpG. The number of the amplicon and each individual sample ID are indicated on the top of every QUMA. The bis-seq data identify this DMR as a large 2.75 KB region spanning amplicons 5, 6 and 7.
Fig 3.
Analysis of 5mC and 5hmC in endometriosis lesions and stroma cells.
A) Immunofluorescence analysis of the levels of expression of 5mC (green) and 5hmC (red) in tissue samples of women with endometriosis and controls, showing disease-dependent loss of 5hmC in the epithelial but not in the stromal cell compartment. Representative photos from a total number of n = 2 control and n = 2 endometriotic tissue are shown. B) The results of the standard and oxidative bis-seq for BDNF in endometriotic and control stromal cells represented as QUMA plots (left) and as bar graph (right). The QUMA plot and bar-graph show the percent methylation assessed by standard bis-seq (which scores indistinctly 5hmC and 5mC) and by modified bis-seq (which scores 5mC only) in control stroma and endometriotic stroma cells. The contribution of 5hmC in endometriotic and control stroma is inferred from the difference between the percent methylation at each CpG in the standard bis-seq reactions (5hmC + 5mC) and the percent methylation in the modified bis-seq reactions (5mC). C) The data of JAZF1 standard and oxidative bis-seq in endometriotic and control stromal cells represented as QUMA plots (left) and bar-graph (right) are shown.
Fig 4.
DNA methylation changes at regulatory DNA elements in ROR1 and OSR2 correlate with gene expression.
A) Map and XY graph of the DMRs in ROR1. Multiple hyper-methylated CpGs are identified in the gene body. Differences in fractional methylation between OESC and ESC are indicated for all CpGs with nominal p-value<0.05 in the bar graph. Chromatin state in NHLF and H1-ESC cell lines (ENCODE project) are color coded as described in the USCS browser. A positive correlation between methylation and expression is observed at cg19267457 (rectangle), overlapping an enhancer region (color coded in yellow). The XY graph shows expression levels, assessed by RNA-Seq and estimated by FPKM values, as a function of the fractional methylation assessed by Illumina BeadChip arrays at cg19267457. B) Map and XY graph of the DMRs in OSR2. Strong hyper-methylation is observed in and upstream OSR2. The DMRs overlapped a dynamic promoter, active in differentiated cells and poised in embryonic stem cells (color coded in red or purple, respectively). The cluster of DM CpGs in the gene (rectangle) shows a strong negative correlation between methylation and expression. The XY graph shows OSR2 expression level against the fractional methylation of the CpG with the highest absolute rho correlation coefficient.
Fig 5.
Validation of RNA-sequencing by Q-PCR and effects of 5Aza-dC on a subset of DM and DE genes in CESC and OESC.
This figure shows average gene expression levels in CESC and OESC, from two or three biological replicates, after treatment with 5aza-dC for 72 hrs. All values were normalized to the mean of the non-treated CESC controls (0) set to 1. A) The relative expression of SLC22A23 under treatment with 5aza-dC increases in CESC as well as in OESC. In CESC, the increase in expression is dose dependent. B) The relative expression levels of OSR2 are decreased in CESC as well as in OESC under all tested doses of the demethylating agent. C) Treatment with 5aza-dC is associated with decreased relative expression levels of JAZF1 in both control and endometriotic stromal cells. D) Although in both CESC and OESC the ROR1 expression levels decrease under 5aza-dC treatment, in CESC the effect on gene expression is dose dependent and more pronounced compared to the endometriotic cells. E) While the HAND2 gene relative expression levels decrease after demethylation treatment as shown in the left bar graph, the HAND2as levels change in the opposite direction; the expression levels are increased in both CESC and OESC, especially at the highest level of 5aza-dC. DAPK1 (F) and TGFBR1 (G) expression levels are not influenced by the drug in CESC or in both CESC and OESCs, respectively. For direct comparison of Q-PCR and RNA-seq data, the differences in expression between CESC and OESC, identified by RNA-seq method are indicated below each graph. CESC, control endometrial stroma cells, OESC, ovarian ectopic endometriosis stroma cells. In each graph, † indicates conditions with biological duplicates.
Table 2.
Validation of RNA-sequencing by Q-PCR for seven DM and DE genes and effects of the hypomethylating drug 5aza-dC on their expression.
Fig 6.
WNT5A protein expression at the whole tissue level.
Immunostaining for the WNT5A protein was done on FFPE sections of endometriotic (n = 14) and normal endometrial tissue (n = 14) samples. Representative pictures from control endometrium and endometriotic lesions at proliferative and secretory phase of the menstrual cycle are shown. The stromal = S and epithelial compartments = E are indicated. The results indicate down-regulation of the levels of WNT5A in the stroma of endometriosis vs. control endometrium, independent of the cycle phase.