Poster Presentation ESA-SRB-APEG-NZSE 2022

Investigating steroid hormone regulation of TETs and developing a framework for hydroxymethylation studies in human endometrium (#375)

Abbey C Lissaman 1 , Vishakha Mahajan 2 , Lynsey Cree 3 , Jane Girling 4 , Anna Ponnampalam 5
  1. Department of Physiology, University of Auckland, Auckland, New Zealand
  2. Liggins Institute, University of Auckland, Auckland, New Zealand
  3. Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand
  4. Department of Anatomy, School of Biomedical Sciences, University of Otago, Dunedin, New Zealand
  5. Manaaki Manawa - The Center for Heart Research, Department of Physiology, University of Auckland, Auckland, New Zealand

Cyclic changes in DNA methylation machinery occur in the endometrium, and deregulation of DNA methylation could lead to aberrant gene expression, including steroid hormone receptors. Although methylation (5mc), mediated by DNA Methyltransferases (DNMTs) is widely studied in the endometrium, the role of the inter-related process hydroxymethylation (5hmc), mediated by Ten Eleven Translocation enzymes (TETs), is relatively unknown. We have reported temporal changes in endometrial TET expression, and recent studies show deregulation in endometrial pathologies. Measuring levels of 5hmc is challenging as bisulfite conversion, often used to study 5mc, does not discriminate between the two DNA modifications. A robust method to detect/study 5hmc in the endometrium is needed. Here we report further analysis of TET regulation by steroid hormones in endometrial stromal cells and evolving work on developing a framework for measuring 5hmc. 

 

Endometrial stromal cells (HESCs) were primed in vitro with 24h estrogen (E) followed by combined estrogen-progesterone (EP) for 24h, 48h and 72h. DNA from HESCs and normal proliferative/secretory endometrial tissues underwent both bisulfite sequencing (BS-seq) and oxidative bisulfite sequencing (oxBS-seq). The methylation/hydroxymethylation status of the CpG island 105 of estrogen receptor alpha (ERα) was assessed using Ion Torrent Next-Generation Sequencing or Sanger Sequencing. Real-time PCR was used to assess gene expression. 


In HESCs, TET1 transcription increased and TET3 decreased following 48h and 72h combined treatment, respectively. No significant changes were observed in TET2 expression. 24h and 48h EP treatment upregulated ERα expression. Consistent methylation levels were observed at CpG105 in both HESCs and tissue samples. Neither sequencing method used to date has detected any hydroxymethylation. BS-seq and oxBS-seq remain inefficient, low DNA concentrations prevent successful sequencing in some cases. Further optimisation using restriction enzymes is in progress. Data implies that CpG site-specific differential methylation/hydroxymethylation might regulate ERa expression.