Contribution of TETs and DNA Hydroxymethylation in the Endometrial-Epigenetic Machinery

Abstract

Ovarian steroid hormones govern the cyclic and complex changes of the endometrium predominantly through their receptors. An interplay between steroid hormones and epigenetic mechanisms controls the dynamic endometrial gene regulation. This includes genes associated with steroid hormone receptors and enzymes controlling hormonal signalling. Although DNA Methyltransferases(DNMT)-mediated methylation is a widely studied regulator of hormonal action in the endometrium, the inter-related, Ten Eleven Translocation Protein(TET)-mediated DNA hydroxymethylation’s role is relatively unknown. This thesis aims to explore the relationship between DNA methylation, hydroxymethylation and steroid hormone signalling in the endometrium. TET expression was characterised in endometrial tissues using immunohistochemistry. The role of steroid hormones in transcriptional, translational and epigenomic regulation of TETs and steroid hormone receptors was examined using real-time PCR, fluorescent western blotting, bisulphite/oxidised bisulphite conversion and next generation sequencing in endometrial tissues and in epithelial (HES), stromal (HESC) and cancer cell lines (AN3 and RL95-2), in vitro. TETs were generally upregulated during the secretory phase with a cell-specific, dynamic expression observed throughout the menstrual cycle. In HESCs, estrogen treatment decreased TET1 and AR transcription and increased TET3 and ERα transcription. Translationally, a decreased expression of TET3 and ERβ and increased PRA, PRB and AR expression, was seen with estrogen-progesterone combined treatment. TET1 and TET3 protein expression was cell-specific and differential in cancer cells. Consecutively, in response to estrogen-progesterone together, a decreased AR translation in AN3 and increased ER translation in RL95-2 cells was seen, while PR gene and protein expression was absent from both cancer cell-lines. Further, an upregulation of TET2 transcription and translation was seen in cancer cell-lines in response to steroid hormones. Overall, results imply that expressions of steroid hormones, steroid hormone receptors and TETs is co-regulated in endometrial cells. Preliminary data on promoter methylation analysis of specific CpG sites indicate that many sites were neither controlled by steroid hormones, nor involved in the transcriptional activation of steroid hormone receptor genes. A framework for undertaking DNA methylation/hydroxymethylation studies in the future is also presented. The data generated in this study adds novel insights into the role of TETs and DNA hydroxymethylation in the endometrial-epigenetic machinery and highlights the current limitations in technologies.