Identification of ENPEP as a crucial epigenetically regulated marker during early trophoblast differentiation

Abstract

<p>Early trophoblast differentiation is crucial for embryo implantation, placentation and fetal development. Previous studies revealed that dynamic changes in DNA methylation occurred during preimplantation embryo development and a few epigenetic gatekeepers were identified to safeguard the trophoblast fate. However, the underlying regulatory mechanism is poorly understood. Recently, we established human expanded potential stem cells (hEPSC-em) directly from preimplantation embryos, providing a powerful tool for studying early trophoblast differentiation. To investigate the roles of DNA methylation on trophoblast differentiation, trophoblastic spheroids derived from hEPSC-em were treated with Tet methylcytosine dioxygenase inhibitor (dimethyloxalylglycine, DMOG) and DNA methyltransferase (5-Azacytidine, 5-AzaC) inhibitor during differentiation. We found that DMOG treatment hindered trophoblast differentiation with reduced expression of trophoblast markers including KRT7, HLA-G, SDC1, ERVW-1 and CGB3, and reduced the attachment of trophoblastic spheroids onto receptive Ishikawa cells; while 5-AzaC promoted trophoblast differentiation. We further conducted integrative and bioinformatic analyses to identify new epigenetic determinants. We found that Aminopeptidase A (ENPEP) was hypermethylated in 8-cell and morula-stage embryos with promoter methylation levels of 57.5% and 70.8% respectively, but hypomethylated in trophectoderm (37.3%) and trophoblastic stem cells (TSC) (1.6%), which was negatively correlated with its expression. Concordantly, progressive demethylation of ENPEP was detected during trophoblast differentiation. Moreover, DMOG treatment delayed demethylation of ENPEP and suppressed its expression. We generated an ENPEP^-/- hEPSC-em line using CRISPR/Cas9 approach and found that ENPEP deletion delayed the downregulation of pluripotent markers and decreased the expression of trophoblast markers in hEPSC-em-derived trophoblastic spheroids. In addition, ENPEP^-/- hEPSC-em line exhibited lower efficiency of TSC derivation and impaired its competence in extra-villous trophoblast and syncytiotrophoblast differentiation. These results suggested that ENPEP was epigenetically regulated and played a vital role in trophoblast fate commitment.<br></p>