EOMES establishes mesoderm and endoderm differentiation potential through SWI/SNF-mediated global enhancer remodeling [ATAC-Seq]

In this study we analysed the dynamic changes in the epigenetic landscape during first embryonic lineage decision following pluripotency exit. Here, mouse pluripotent epiblast cells segregate towards mesoderm and endoderm (ME) or neuroectoderm (NE). The analysis of chromatin accessibility, histone modifications and accompanied gene expression confirmed a bias of the epigenetic landscape towards NE fate while ME cis regulatory elements (CREs) are becoming accessible and active during lineage specification. We show that the default state of NE differentiation is actively overcome by the activity of the Tbx transcription factor (TF) EOMES binding to closed ME CREs. We found the ATP-dependent chromatin remodelling complex SWI/SNF is recruited by EOMES to target ME enhancers. The recruitment of the complex by EOMES leads to the remodelling of the enhancer landscape at ME genes. The ME enhancer landscape is remodelled by the activity of EOMES independent on gene expression genome wide. Overall design: This study involves mouse embryonic stem cells (mESCs) of the following genotypes: wild type (WT), cells deficient for both T-box TFs Eomes and Tbxt (dKO) and cells expressing Eomes:GFP, Eomes:ER or Tbxt:GFP fusion protein under doxycycline treatment in dKO background (dKOEoGFP, dKOEoER, dKOTbxtGFP). mESCs were differentiated for 3.5 days as embryoid bodies under Activin A treatment. Samples for ATAC-seq, histone modification (H3K4me3, H3K4me1, H3K27ac) ChIP-seq, EOMES ChIP-seq and SWI/SNF subunit SMARCA4 (BRG1) ChIP-seq were collected at different time points during differentiation. ATAC-seq and ChIP-seq experiments were done in biological duplicates. Total RNA was isolated from embryoid bodies at different time points for RNA-seq experiments in 3-4 biological replicates.