Defining key transcriptional regulators of extravillous trophoblast differentiation [ChIP-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. Overall design: ChIP-seq of H3K27ac was performed in human trophoblast stem cells (TSC), EVT differentiating cells (day 3, EVT D3), and fully differentiated EVT (day 8, EVT D8). ATAC-seq and H3K4me3 ChIP-seq were performed in TSC and EVT D8. Time-course ChIP-seq of TFAP2C and DLX6 were performed in TSC, EVT D2, EVT D5, and EVT D8. To map genomic targets of DLX5 and ASCL2, ChIP-seq using native antibodies was conducted, and NRIP1 targets were identified by biotin-mediated ChIP-seq (bioChIP-seq). Inputs were sequenced for a control.

在人类早期妊娠期间，绒毛外滋养层细胞（extravillous trophoblasts, EVT）在胎盘锚定与血管重塑过程中发挥核心作用。尽管其在妊娠过程中发挥不可或缺的作用，但EVT分化背后的关键调控因子与分子机制仍未完全阐明。本研究通过监测EVT分化过程中的转录组动态与增强子使用模式，鉴定得到关键转录因子（transcription factors, TFs），并验证了所鉴定转录因子的必要性，同时阐明了其作用机制。
实验整体设计：
针对人滋养层干细胞（trophoblast stem cells, TSC）、EVT分化细胞（第3天，EVT D3）以及完全分化的EVT（第8天，EVT D8），开展了H3K27ac的染色质免疫共沉淀测序（ChIP-seq）。针对TSC与EVT D8样本，开展了转座酶可及性染色质测序（ATAC-seq）与H3K4me3 ChIP-seq实验。针对TSC、EVT D2、EVT D5以及EVT D8样本，开展了TFAP2C与DLX6的时间进程ChIP-seq实验。为绘制DLX5与ASCL2的基因组靶标图谱，研究使用天然抗体开展了ChIP-seq实验；同时通过生物素介导的ChIP-seq（bioChIP-seq）鉴定了NRIP1的靶标基因。所有Input对照样本均进行了测序。