Transient RUNX1 Expression during Early Mesendodermal Differentiation of hESCs Promotes Epithelial to Mesenchymal Transition through TGFB2 Signaling

摘要

class="head no_bottom_margin" id="sec1title">IntroductionHuman embryonic stem cells (hESCs) have unlimited replicative potential and are capable of differentiating into cell types from each of the three germ layers (). While much is known about the maintenance of pluripotency (, , , , ), how differentiation signals regulate the dissolution of pluripotency and the establishment of phenotype is not well understood. Studies in hESCs have shown that many genes responsible for early developmental events are poised for either activation or repression by epigenetic mechanisms (, , ). Once a differentiation signal has been introduced, early factors are expressed that prime the gene expression program of cells for lineage acquisition (). In this study, we investigated whether phenotype-associated transcription factors may play an initial role in differentiation prior to their established function in specifying lineage identity. A candidate screen of phenotypic transcription factors identified RUNX1 as selectively and transiently upregulated as early as 8 hr during mesendodermal differentiation of hESCs.Members of the RUNX family of transcription factors have known roles in development (): RUNX1 is necessary for definitive hematopoiesis (), RUNX2 for bone formation (), and RUNX3 for gastrointestinal and nervous system development (, , href="#bib30" rid="bib30" class=" bibr popnode">Li et al., 2002). When genetically deleted in mice, Runx1 causes embryonic lethality due to major defects in the formation of the fetal liver and hemorrhaging in the CNS (href="#bib37" rid="bib37" class=" bibr popnode">Okuda et al., 1996, href="#bib53" rid="bib53" class=" bibr popnode">Wang et al., 1996). Emerging evidence suggests that RUNX1 has roles in non-hematopoietic lineages (href="#bib40" rid="bib40" class=" bibr popnode">Osorio et al., 2008, href="#bib44" rid="bib44" class=" bibr popnode">Scheitz and Tumbar, 2013, href="#bib46" rid="bib46" class=" bibr popnode">Stifani et al., 2008). Our discovery of transient upregulation of RUNX1 points to a role for RUNX1 during early mesendodermal differentiation of hESCs.We investigated what role the early expressed phenotypic transcription factor RUNX1 might play during differentiation in addition to its known role in association with hematopoietic lineage identity. Genome-wide transcriptome analysis of RUNX1-depleted hESCs revealed that RUNX1 positively regulates transforming growth factor β2 (TGFB2) signaling and the motility of the differentiating hESCs. Importantly, genes associated with the epithelial to mesenchymal transition (EMT) were affected, with epithelial gene expression increasing in the absence of RUNX1. Exogenous reintroduction of TGFB2, but not TGFB1, ameliorates the effect of RUNX1 depletion on cell motility and the EMT process. Taken together, our results show that the selective and transient expression of RUNX1 during early hESC differentiation to mesendoderm promotes EMT and motility through regulation of the TGFB2 signaling pathway.