Eomes and Brachyury control pluripotency exit and germ-layer segregation by changing the chromatin state

We aimed to elucidate molecular mechanisms of first lineage decision in the mouse pluripotent epiblast that segregates mesoderm and endoderm (ME) from neuroectoderm (NE). By analyzing mouse embryonic stem cells (ESCs) and embryos deficient for two T-box (Tbx) transcription factors Eomes and Brachyury we demonstrate that this process is controlled by the changes in the chromatin accessibility of ME gene enhancers and activation of the target genes, but also direct repression the opposing pluripotency and NE gene programs. We differentiated WT ESCs, cells deficient for Eomes and Brachyury individually (EoKO and BraKO cells) as well as in combination (dKO cells) for 4-5 days using embryoid bodies treated with Activin A. We tested different doxycycline inducible constructs for the ability to rescue the observed dKO phenotype, with GFP, VP16 and EnR fusions, as well as with cDNAs encoding Mesp1, Msgn1, Mixl1 and Foxa2. Generated cells were characterized by RNA-seq, ChIP-seq and ATAC-seq. We show that cells recapitulate the phenotype of mutant embryos by analyzing RNA-seq from embryonic day E7.5 EomesDEpi , T2J/2J and double mutant T2J/2J;EomesDEpi mouse epiblasts. For RNA-seq experiments, biological triplicates were always used, and ChiP-seq and ATAC-seq experiments were done in biological duplicates. As most of the processed data files were generated from multiple samples, they were linked as Series supplementary files and indicated both in the corresponding sample description field and processed_data_file_list.txt本研究旨在阐明小鼠多能上胚层中首个谱系决定的分子机制，该过程将中内胚层（mesoderm and endoderm, ME）与神经外胚层（neuroectoderm, NE）相互分离。本研究通过分析缺失两种T-box（Tbx）转录因子Eomes与Brachyury的小鼠胚胎干细胞（embryonic stem cells, ESCs）及胚胎，证实该过程不仅受中内胚层基因增强子的染色质可及性变化与靶基因激活调控，还可直接抑制与之拮抗的多能性及神经外胚层基因程序。本研究采用经激活素A（Activin A）处理的拟胚体，对野生型（wild-type, WT）胚胎干细胞、分别缺失Eomes或Brachyury的细胞（EoKO与BraKO细胞）以及同时缺失二者的双敲除细胞（double knockout, dKO细胞）进行了4-5天的分化培养。本研究测试了多种多西环素（doxycycline）诱导型载体挽救双敲除表型的能力，所用载体包括与GFP、VP16及EnR融合的序列，以及编码Mesp1、Msgn1、Mixl1与Foxa2的互补DNA（complementary DNA, cDNA）。我们通过RNA测序（RNA-sequencing, RNA-seq）、染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）以及转座酶可及性染色质测序（assay for transposase-accessible chromatin using sequencing, ATAC-seq）对分化得到的细胞进行了表征。通过对胚胎发育第7.5天（embryonic day E7.5）的EomesDEpi、T2J/2J以及双突变体T2J/2J;EomesDEpi小鼠上胚层进行RNA-seq分析，我们证实分化得到的细胞可重现突变体胚胎的表型。RNA-seq实验均采用生物学重复三次的设计，ChIP-seq与ATAC-seq实验则采用生物学重复两次的设计。由于多数已处理的数据文件由多个样本生成，因此这些文件被作为系列补充文件进行关联，并在对应的样本描述字段及processed_data_file_list.txt中均进行了标注。