Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
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.
Overall design
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
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