Hypomethylation of GATA6 Gene Body May Cause the up-Regulation in Endometriotic Cells

Presentation Number: SUN-0035
Date of Presentation: June 22nd, 2014

Masao Izawa*1, Fuminori Taniguchi2, Naoki Terakawa2 and Tasuku Harada2
1Tottori University Faculty of Medicine, Yonago, Japan, 2Tottori Univ/Fac of Med, Yonago, Japan

Abstract

Background: DNA methylation provides a layer of epigenetic controls that has important implications for diseases including endometriosis. A marked up-regulation of aromatase gene associates with aberrant DNA hypomethylation leading to a high estrogen environment in endometriotic tissues (1, 2). However, it is unknown whether global alterations in DNA methylation profiles occur in endometriosis, and to what extent they are involved in the estrogen-dependent pathophysiology (3).

Objective: Using a genome-wide profiling of DNA methylation, we challenged an extraction of genes having aberrant DNA hypomethylation, and then evaluated their expressions in endometriotic cells.

 Patients: The Institutional Review Boards of Tottori University Faculty of Medicine 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 control, endometrial tissues were obtained from uteri of cycling premenopausal women who had uterine leiomyoma. These patients had received no hormonal treatment before surgery.

Methods: Stromal cells were prepared from endometrial and endometriotic tissues. DNA samples were isolated from 4 endometrial and 4 endometriotic cells. DNA methylation profiles were assayed using Illumina Infinium HumanMethylation450 BeadChip Array and GeneSpring GX 11.5.  Gene expression was evaluated using qRT-PCR, Western blots and immunocytochemistry.

Results:  1) The 857 GpG sites, which were differentially hypomethylated more than 10-fold in endometriotic cells, were extracted from 485,512 CpGs on an Array. 2) From these CpGs, the 317 CpGs were further extracted depending on the β cut-off value of 0.25. 3) We finally extracted GATA6 gene, which showed more than 90% hypomethylation within the gene body. 4) GATA6 mRNA was highly expressed in endometriotic cells, while in endometrial cells the expression was at a marginal level. 5) As anticipated, GATA4, which is under the control of GATA6, was highly expressed in endometriotic cells. 6) Within GATA4 gene, aberrant CpG methylation profile was not observed. 7) GATA6 and GATA4 expression were also demonstrated in endometriotic tissues on the peritoneum.

Conclusion: Although the overall methylation profile in endometriotic cells was highly similar to that in endometrial cells, we successfully extracted a series of hypomethylated CpGs within GATA6 gene body in endometriotic cells. GATA6 was highly expressed in endometriotic cells. The present finding along with our previous observations (1, 2) provides a facet of molecular basis of epigenetic disorder in endometriosis.

 

Nothing to Disclose: MI, FT, NT, TH