PRC2 Promotes Canalisation During Endodermal Differentiation [4sU-seq]

In this study we employed a mouse embryonic stem cell (ESCs) differentiation system to convert pluripotent ESCs into Anterior Definitive Endoderm (ADE) to model primitive streak formation and early gastrulation. To monitor the progression of the differentiation we utilised a dual reporter mESC line (B6) bearing fluorescent reporters knocked in to the Gsc (marker of the PS/mesendoderm tagged with GFP) and Hhex (marker of definitive endoderm lineages tagged with Redstar) loci. To establish the transcriptional changes which occurred during a phase of differentiation that resembles the epithelial to mesenchymal transition (EMT) at the primitive streak (PS), we performed 4sU-seq analysis on FACS sorted GSC negative and positive populations from day 3 and day 4 of differentiation. Using this approach, in combination with genome-wide gene expression analysis and ChIP-seq data, we: i). Found that changes in the gene expression where primarily regulated at the levels of transcription; ii). Identified putative enhancer elements with bi-directional transcription signatures proximal to developmentally regulated genes; iii). Determined that transcriptional upregulation of genes did not require the prior loss of H3K27me3 at gene TSSs. 4sU-seq was performed on GSC negative and positive FACS sorted populations form days 3 and 4 of ADE differentiation. Three independent experiments were performed for each condition.