Inhibiting Smad2/3 Signaling in Pluripotent Mouse Embryonic Stem Cells Enhances Endoderm Formation by Increasing Transcriptional Priming of Lineage-Specifying Target Genes

Background: Pluripotent embryonic stem cells (ESCs) offer great potential for regenerative medicine. However, efficient in vitro generation of specific desired cell types is still a challenge. We previously established that Smad2/3 signaling, essential for endoderm formation, regulates target gene expression by counteracting epigenetic repression mediated by Polycomb Repressive Complex 2 (PRC2). Although this mechanism has been demonstrated during differentiation and reprogramming, little is known of its role in pluripotent cells. Results: Chromatin immunoprecipitation-deep sequencing of undifferentiated mouse ESCs inhibited for Smad2/3 signaling identified Prdm14, important for protecting pluripotency, as a target gene. Although Prdm14 accumulates the normally repressive PRC2 deposited histone modification H3K27me3 under these conditions, surprisingly, expression increases. Analysis indicates that increased H3K27me3 leads to increased binding of PRC2 accessory component Jarid2 and recruitment of RNA polymerase II. Similar increases were found at the Nodal endoderm target gene Eomes but it remained unexpressed in pluripotent cells as normal. Upon differentiation, however, Eomes expression was significantly higher than in cells that had not been inhibited for signaling before differentiation. In addition, endoderm formation was markedly increased. Conclusions: Blocking Smad2/3 signaling in pluripotent stem cells results in epigenetic changes that enhance the capacity for endoderm differentiation. (C) 2016 Wiley Periodicals, Inc.