Tcf ChIP_Seq analysis in cultured differentiated TSC cells at the presence of DMSO or CHIRGene expression in the placenta of WT and Ctnnb1 overexpression mice

Wnt signaling are essential for the maintenance and differentiation of stem/progenitor cells, including trophoblast stem cells during placental development. Hyper-activation of Wnt signaling has been shown to relate with human trophoblast diseases. However, litter is known about the impact and underlying mechanisms of excessive Wnt signaling during placental trophoblast development. In the present work, we found that Sfrp1,5 double mutant mouse exhibited disturbed trophoblast differentiation in the placental ectoplacental cone (EPC), where the precursors of secondary trophoblast giant cells (TGCs) and trophoblast cells in the spongiotrophoblast layer are located. Employing mouse models expressing a trunked β-catenin with exon 3 deletion globally and trophoblast-specifically, combining cultured trophoblast stem cells, we found that hyper-activation of canonical Wnt pathway exhausted the trophoblast precursor cells in the EPC, resulting in the overabundance of giant cells at the expense of spongiotrophoblast cells. Further examination uncovered that hyper-activation of canonical Wnt pathway disturbed trophoblast differentiation in the EPC via repressing Mash2 expression. Collectively, our findings demonstrate that appropriate canonical Wnt-β-catenin pathway is essential for EPC trophoblast differentiation during placental development. Our work also has high clinical relevance, since abnormal Wnt signaling are often associated with trophoblast-related diseases. To detect the direct target genes of Ctnnb1 in differentiated TSC cells, differentiated TSC cells at the presence of CHIR are collected and subjected to ChIP-Seq. After aligned to mouse mm10 by HISAT2, peaks are called by MACS2. Our results show that Ctnnb1 the key regulator for placentation by repressing Ascl2 expression as indicated by the presence of significant enrichment of TCF4 at the distal region of Aslc2. This specific enrichment was confirmed by ChIP-qPCR. Additionlly, there are also some other direct genes of Ctnnb1, indicating the essential role of Ctnnb1. This ChIP-Seq data provides fundamental information for our further physiological study of Ctnnb1.

Wnt信号通路（Wnt signaling）对于干细胞/祖细胞（包括胎盘发育过程中的滋养层干细胞（trophoblast stem cells））的维持与分化至关重要。已有研究证实，Wnt信号通路的过度激活与人类滋养层疾病存在关联。然而，目前关于胎盘滋养层发育过程中Wnt信号过度激活的影响及其潜在分子机制，人们的认知仍较为匮乏。本研究发现，Sfrp1与Sfrp5双突变小鼠的胎盘外胎盘锥（ectoplacental cone, EPC）出现滋养层分化紊乱，而外胎盘锥正是次级滋养层巨细胞（secondary trophoblast giant cells, TGCs）前体以及海绵滋养层层滋养层细胞的分布区域。通过构建全局过表达外显子3缺失的截短型β-连环蛋白（β-catenin）以及滋养层特异性表达该截短蛋白的小鼠模型，并结合体外培养的滋养层干细胞实验，我们证实经典Wnt通路（canonical Wnt pathway）的过度激活会耗竭外胎盘锥内的滋养层前体细胞，最终导致以海绵滋养层细胞数量减少为代价的巨细胞过度增殖。进一步研究揭示，经典Wnt通路的过度激活通过抑制Mash2的表达，扰乱了外胎盘锥内的滋养层分化进程。综上，本研究结果表明，适度激活的经典Wnt-β-连环蛋白通路对于胎盘发育过程中外胎盘锥的滋养层分化具有不可或缺的作用。本研究同时具备较高的临床相关性，因为异常的Wnt信号通路常与滋养层相关疾病密切相关。为了检测分化型滋养层干细胞中Ctnnb1的直接靶基因，我们收集了经CHIR处理的分化型滋养层干细胞，并开展染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-Seq）实验。测序数据通过HISAT2比对至小鼠mm10参考基因组，随后使用MACS2进行峰区调用。研究结果显示，Ctnnb1作为胎盘形成的关键调控因子，可通过抑制Ascl2的表达发挥功能——具体表现为转录因子4（TCF4）在Aslc2基因远端区域存在显著富集。该特异性富集现象经ChIP-qPCR（chromatin immunoprecipitation quantitative PCR）得以验证。此外，还存在其他若干Ctnnb1的直接靶基因，进一步印证了Ctnnb1的关键调控作用。本染色质免疫共沉淀测序数据为我们后续开展Ctnnb1的生理学功能研究提供了核心基础依据。