SAT-199 A Hypomethylated GATA6 Gene Body May Function as an Active Enhancer in Endometriotic Cells

[Background]Endometriosis is an estrogen-dependent, inflammatory disease characterized by the presence of endometrium-like tissue primarily on the pelvic peritoneum and ovaries. There is accumulating evidence suggesting epigenetic aberrations play a role in the pathogenesis and/or the pathophysiology in endometriosis. A number of aberrant gene expressions have been demonstrated in endometriosis, and these aberrations may be related to epigenetic aberrations. Our Genome-wide DNA methylation analysis showed a GATA6 gene body highly hypomethylated in endometrial cells. From our observation that GATA6 was highly expressed in endometriotic cells, we hypothesized the presence of cis-acting element within the gene body.[Objective]We examined a potential function of hypomethylated GATA6 gene body as a cis-acting element in endometriotic cells.[Patients]The Institutional Review Boards approved this project. We obtained the informed consent from all patients. The chocolate cyst lining in ovaries of patients with endometriosis was the source of endometriotic tissue. As the control, the eutopic endometrial tissues were obtained from uteri of cycling premenopausal women who had uterine leiomyoma. These patients had received no hormonal treatment before tissues were removed. [Methods]Stromal cells were prepared from endometriotic and endometrial tissues. GATA6 mRNA expressions was examined using RT-PCR. Immunocytochemical analysis was performed using anti-GATA6 antibody. The potential function of hypomethylated gene body as a cis-acting element was evaluated by ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies and eRNA expression analysis.[Results]We hypothesized the presence of CpG hypomethylation, which is responsible for increased gene expression in endometriotic cells. 1) Using endometrial cells as control, differentially methylated CpGs endometriotic cells were extracted. 2) We finally extracted a stretch of 14 CpGs within GATA6 gene body. 3) GATA6 mRNAexpression in endometriotic cells was 50-fold higher than that in endometrial cells. The expression depended on 1a promoter. 4) Wild type GATA6 expression was observed in endometriotic cells, but not in endometrial cells. 5) Immunocytochemistry showed positive staining of GATA6 in endometriotic cell nuclei. 6) ChIP analysis using anti-H3K4me1 and anti-H3K27ac antibodies predicted the presence of an active enhancer within the gene body sequence in endometriotic cells. 7) Supporting this observation, putative eRNA was transcribed from the region.[Conclusion]Our results suggest that the gene body sequence of GATA6 may function as an active enhancer under the control of DNA methylation in endometriotic cells. This is the first implication showing a link between an aberrant DNA methylation of cis element and gene expression in endometriosis.