Epigenetic Alterations Affecting Transcription Factors and Signaling Pathways in Stromal Cells of Endometriosis

Abstract

Endometriosis is characterized by growth of endometrial-like tissue outside the uterine cavity. Since its pathogenesis may involve epigenetic changes, we used Illumina 450K Methylation Beadchips to profile CpG methylation in endometriosis stromal cells compared to stromal cells from normal endometrium. We validated and extended the Beadchip data using bisulfite sequencing (bis-seq), and analyzed differential methylation (DM) at the CpG-level and by an element-level classification for groups of CpGs in chromatin domains. Genes found to have DM included examples encoding transporters (SLC22A23), signaling components (BDNF, DAPK1, ROR1, and WNT5A) and transcription factors (GATA family, HAND2, HOXA cluster, NR5A1, OSR2, TBX3). Intriguingly, among the TF genes with DM we also found JAZF1, a proto-oncogene affected by chromosomal translocations in endometrial stromal tumors. Using RNA-Seq we identified a subset of the DM genes showing differential expression (DE), with the likelihood of DE increasing with the extent of the DM and its location in enhancer elements. Supporting functional relevance, treatment of stromal cells with the hypomethylating drug 5aza-dC led to activation of DAPK1 and SLC22A23 and repression of HAND2, JAZF1, OSR2, and ROR1 mRNA expression. We found that global 5hmC is decreased in endometriotic versus normal epithelial but not stroma cells, and for JAZF1 and BDNF examined by oxidative bis-seq, found that when 5hmC is detected, patterns of 5hmC paralleled those of 5mC. Together with prior studies, these results define a consistent epigenetic signature in endometriosis stromal cells and nominate specific transcriptional and signaling pathways as therapeutic targets.

GATA6 expression promoted by an active enhancer may become a molecular marker in endometriosis lesions

“…[11][12][13] Epigenetic regulation, including DNA methylation and histone modifications, is responsible for a number of gene transcriptions that distinguish the various cell types and the states of diseases, including cancer and neurological disease. 19,20 Consistent with their analysis, we observed that the overall methylation profile in endometriotic cells was highly similar to that in endometrial cells. We hypothesized that genomewide profiling of DNA methylation could show a distinct profile of epigenetic backgrounds in endometriosis that can be used as molecular markers.…”

“…Genome-wide DNA methylation analysis in endometriotic cells reported the genome-wide DNA methylation profile. 19,20 Consistent with their analysis, we observed that the overall methylation profile in endometriotic cells was highly similar to that in endometrial cells. In our genome-wide DNA methylation analysis, we extracted only 1811 CpGs (0.38%), which showed more than 10-fold difference in the methylation rate, were extracted.…”

“…9 Among these CpGs extracted, we found a cluster of hypomethylated CpGs within GATA 6 gene body in endometriotic cells as reported. 22 From our observation that GATA6 was highly expressed in endometriotic cells as reported, 19,20 we hypothesized and searched for the presence of cis-acting element within the gene body. 21 Loss of GATA6 has been implicated in ovarian cancer development.…”

“…9 Among these CpGs extracted, we found a cluster of hypomethylated CpGs within GATA 6 gene body in endometriotic cells as reported. 19,20 GATA6 is a zinc finger-containing transcription factor that has been shown to play critical roles in cell lineage determination during early embryonic development and organ formation. 21 Loss of GATA6 has been implicated in ovarian cancer development.…”

“…21 Loss of GATA6 has been implicated in ovarian cancer development. 22 From our observation that GATA6 was highly expressed in endometriotic cells as reported, 19,20 we hypothesized and searched for the presence of cis-acting element within the gene body. Here, we show for the first time the presence of active enhancer within the hypopethylated gene body in endometriotic cells.…”