As cells transition from the point of fertilization through the process of embryonic development, many molecular changes occur that affect cell fate. At the blastocyst stage, the earliest distinction, two separate cell populations arise. The trophectoderm cells will generate all of the extraembryonic tissues while the inner cell mass will yield all of the embryonic tissues. These cells, which generate the organism, are termed pluripotent at this stage and found within the inner cell mass (ICM). A variety of genetic mechanisms that regulate this event have been characterized. Here, we examined the effect of cJun expression in regulating Oct4, a gene known to be a master regulator of pluripotency. Luciferase assays were performed to validate our bioinformatics analysis of the 3 kb Oct4 promoter, which identified a cJun binding site approximately 2500 bp upstream of the transcription start site. These experiments demonstrate transient expression of cJun off a reporter plasmid significantly increases luciferase activity of the Oct4-luciferase reporter construct, while expression of the transcriptionally inactive cJun mutant L40/42A represses it. Additionally, we corroborate previous reports that cJun can synergize with Beta-catenin, a member of the Wnt pathway, to significantly increase the promoter activity of Oct4. A role for regulation of endogenous Oct4 expression was
evaluated by immunoblot analysis of murine embryonic stem cells transiently transfected with GFP cJun, which demonstrated increased OCT4A expression 36 hours post-transfection. To assess the developmental implications of this regulatory relationship, we evaluated the effect of cJun overexpression on early differentiation by transiently transfecting mESCs and initiating embryoid body formation. When evaluated for germ layer marker expression by immunoblot analysis, it was revealed that an increase in cJun correlated with an increase in GATA4 expression, suggesting cJun expression could lead to selection toward the endodermal germ layer. In light of this, we carried out directed differentiation into cardiomyocytes, using a hanging drop intermediate, and found that an increase in cJun led to a significant increase in cardiomyocyte formation and beating. Overall, this suggests a new role for cJun in the regulation of potency and the formation of the endoderm during those early cell fate choices
