Steroid hormones regulate genome-wide epigenetic programming and gene transcription in human endometrial cells with marked aberrancies in endometriosis

Abstract

Programmed cellular responses to cycling ovarian-derived steroid hormones are central to normal endometrial function. Abnormalities therein, as in the estrogen-dependent, progesterone-"resistant" disorder, endometriosis, predispose to infertility and poor pregnancy outcomes. The endometrial stromal fibroblast (eSF) is a master regulator of pregnancy success. However, the complex hormone-epigenome-transcriptome interplay in eSF by each individual steroid hormone, estradiol (E2) and/or progesterone (P4), under physiologic and pathophysiologic conditions, is poorly understood and was investigated herein. Genome-wide analysis in normal, early and late stage eutopic eSF revealed: i) In contrast to P4, E2 extensively affected the eSF DNA methylome and transcriptome. Importantly, E2 resulted in a more open versus closed chromatin, confirmed by histone modification analysis. Combined E2 with P4 affected a totally different landscape than E2 or P4 alone. ii) P4 responses were aberrant in early and late stage endometriosis, and mapping differentially methylated CpG sites with progesterone receptor targets from the literature revealed different but not decreased P4-targets, leading to question the P4-"resistant" phenotype in endometriosis. Interestingly, an aberrant E2-response was noted in eSF from endometriosis women; iii) Steroid hormones affected specific genomic contexts and locations, significantly enriching enhancers and intergenic regions and minimally involving proximal promoters and CpG islands, regardless of hormone type and eSF disease state. iv) In eSF from women with endometriosis, aberrant hormone-induced methylation signatures were mainly due to existing DNA methylation marks prior to hormone treatments and involved known endometriosis genes and pathways. v) Distinct DNA methylation and transcriptomic signatures revealed early and late stage endometriosis comprise unique disease subtypes. Taken together, the data herein, for the first time, provide significant insight into the hormone-epigenome-transcriptome interplay of each steroid hormone in normal eSF, and aberrant E2 response, distinct disease subtypes, and pre-existing epigenetic aberrancies in the setting of endometriosis, provide mechanistic insights into how endometriosis affects endometrial function/dysfunction.

Fig 1. Hormone induced differentially methylated CpG sites in normal eSF (NUP).

1A. Differentially methylated CpG sites induced by E₂, P₄ and E₂+P₄ versus vehicle. Each heatmap reflects differential methylation of each sample in each hormone treatment versus its corresponding non-treated vehicle control (Δβ: Hormone treated minus vehicle control). Yellow heatmaps above the X-axis reflect gain of methylation vs. vehicle; blue heatmaps below the X-axis reflect Δβ loss of methylation. In each heatmap, rows show Δβ of differentially methylated loci, columns indicate samples. Y-axis shows the number of differentially methylated loci for either gain or loss of methylation for each hormone treatment. 1B. Number of differentially methylated CpG sites and in gain/loss of methylation for each hormone treatment. 1C. Venn diagram of unique and common differentially methylated CpG sites for each hormone shows little overlap between differentially methylated loci in each hormone treatment 1D. Enrichment of intergenic regions in % in each hormone treatment for all differentially methylated loci (All Loci), those with gain or loss of methylation (Gain, Loss) and by individual hormones (E₂, P₄, E₂+P₄). Enrichment is assessed by Z-test and p<0.05 are shown in parentheses. Black bar represents percentage of intergenic loci of total interrogated CpG sites. 1E. Statistically significant involvement of enhancers by hormones and gain or loss of methylation. Enrichment is assessed by Z-test and p<0.05 are shown in parentheses. Black bar represents percentage of enhancers of total interrogated CpG sites. 1F. Genomic distribution of all differentially methylated CpG sites in each hormone and by gain or loss of methylation, assessed at TSS1500, TSS200, 5’UTR, 1^st exon, gene body, 3’UTR, and intergenic regions. Black line represents the percentage of interrogated CpG site at each location, green line (top panel) shows all differentially methylated loci in NUP for all hormones, yellow line (middle panel) shows all loci with gain of methylation in NUP, and blue line (bottom panel) shows all loci with loss of methylation for all hormones. Enrichment is assessed by Z-test and p<0.05 are shown in parentheses for each genomic location. 1G. Distribution of differentially methylated CpGs by CpG islands (CGI), CGI north/south shores and shelves for all loci with gain of methylation in all hormone treatments (orange lines) or with loss of methylation (blue line) in comparison to the distribution of the interrogated CpG sites in each of these genomic locations (black line). N Shelf: North Shelf; S Shelf: South Shelf; N Shore: North Shore; S Shore: South Shore. NUP: normal (no uterine pathology).

Fig 2. Hormone induced differentially methylated CpG sites in stage I eSF (Endo I) and stage IV eSF (Endo IV).

2A. Differentially methylated CpG sites induced by E₂, P₄ and E₂+P₄ versus vehicle. Heatmaps reflect the differential methylation of each sample in each hormone treatment versus its corresponding non-treated vehicle control (Δβ: Hormone treated minus vehicle control) (see Fig 1A legend for details). 2B. Number of differentially methylated CpG sites and in gain/loss of methylation for each hormone treatment in Endo I and Endo IV. C. Unique and common differentially methylated CpG sites for each hormone in Endo I (left) and Endo IV (right) indicating mostly unique loci for each hormone. 2D. Unique and common differentially methylated CpG sites across normal (NUP), Endo I and Endo IV, for each hormone: E₂: left, P₄: middle and E₂+P₄: right. 2E. Enrichment of intergenic regions; Endo I, top and Endo IV bottom charts (see Fig 1D legend for details). 2F. Enrichment of enhancers for each hormone and based on loss or gain of methylation in Endo I (left panel) and Endo IV (right panel) (see Fig 1E legend for details). 2G. Genomic distribution of all differentially methylated CpG sites in each group (Endo I, left, Endo IV right panel) and by gain or loss of methylation (see Fig 1F legend for details). 2H. Distribution of differentially methylated CpGs by CpG islands (CGI), CGI north/south shores and shelves for Endo I (left) and Endo IV (right) based on gain or loss of methylation for all hormones. For details see Fig 1G legend. N Shelf: North Shelf; S Shelf: South Shelf; N Shore: North Shore; S Shore: South Shore. Endo I: stage I; Endo IV: stage IV.