Defining key transcriptional regulators of extravillous trophoblast differentiation [RNA-seq]

During early human pregnancy, extravillous trophoblasts (EVT) play a central role in placental anchorage and blood vessel remodeling. Despite the essential roles in pregnancy, key factors and mechanisms underlying EVT differentiation remain largely unknown. Here, we defined key transcription factors (TFs) by monitoring the dynamics of transcriptome and enhancer usage during EVT differentiation. We confirmed the requirements of the defined TFs and identified their action mechanisms. Transcriptome profiling of human trophoblast stem cells (TSC) and EVT differentiating cells (multiple time-points) was conducted to monitor dynamics in the gene expression program during EVT differentiation. To investigate the requirements of the defined TFs (DLX5, DLX6, ASCL2, ZNF439, NRIP1, and TFAP2C) in proper EVT differentiation, we knocked down (KD) the individual TF in TSC and differentiated the cells to EVT. RNA-seq was performed on the individual key TF-KD EVT and non-targeting shRNA-expressing EVT (control). We also conducted RNA-seq of EVT overexpressing TFAP2C during days 2 to 8 of differentiation to monitor the effects of sustained expression of TFAP2C on EVT differentiation. The non-DOX-treated EVT was used as a control.