Embryonic stem cell differentiation is regulated by SET through interactions with p53 and ß-catenin

The multifunctional histone chaperone, SET, is essential for embryonic development in mouse. Previously, we identified SET as a factor that is rapidly downregulated during embryonic stem cell (ESC) differentiation, suggesting a possible role in the maintenance of pluripotency. Here, we set out to explore SET's function and its mechanism in early differentiation. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we uncover the factors and complexes, including P53 and ß-catenin, by which SET regulates lineage-specification. SET knockout (KO) and shRNA-knockdown experiments for both P53 and ß-catenin in SET-KO ESCs reveal that P53 knockdown partially rescues lineage-marker misregulation during differentiation. ß-catenin depletion, on the other hand, rescues the elevated Wnt gene expression observed in SET-KO ESCs. Integration of multiple ChIP-seq and RNA-seq datasets reveals a predominantly activating function of SET in gene regulation. Moreover, further analysis reveals a co-regulatory relationship between SET and TCF3, a downstream effector of Wnt-signalling. Overall, we discover a role for both P53 and ß-catenin in SET-regulated early differentiation and uncover a potential mechanism for SET function at the ß-catenin-TCF regulatory axis. Overall design: RNA-seq of Wt and SET KO ESCs and before and after 4d RA treatment in R1 background. In addition, RNA-seq performed with and without addition of Dox in KH2 ESCs with Dox-inducible SETa expression.