Regulation Of Pluripotent Stem Cell Self-renewal And Differentiation By Post-translational Modifications Of Oct4

Embryonic stem cells (ESCs) and embryonal carcinoma cells (ECCs) are known as pluripotent stem cells that are characterized by their self-renewal and pluripotency. Such features are maintained by complex interplays among various transcription factors, epigenetic factors and intrinsic signaling pathways. The molecule mechanisms by which these cultured pluripotent stem cells (PSCs) differentiate under certain stimuli largely reflect those during the embryo development. Thus, PSCs were widely used as an invaluable tool for the studies of early embryogenesis. Gaining deeper understanding on how and what happens during cell differentiation would greatly propel future development of regenerative medicine.Oct4 was initially found as a master transcription factor during early embryogenesis. Since its discovery, Oct4 is widely recognized as one of the core pluripotency factors for both the induction and maintenance of cellular pluripotency. Recently, studies have been focused on its roles in priming cell fate and lineage-specific differentiation during embryonic development, and site-specific post-translational modifications (PTM, such as sumoylation, phosphorylation and ubiquitination) have begun to be appreciated as a key player in regulating Oct4 protein function. However, it remains unclear how specific extracellular/intracellular stimuli and signaling pathways trigger the PTMs of Oct4 and how the modifications affect downstream gene transcription.Here we demonstrated that the master protein kinase Akt, an emerging regulator of PSC self-renewal, phosphorylated Oct4 at threonine 235 in ECCs. Unphosphorylated Oct4 bound to the AKT1 promoter and repressed its transcription, while phosphorylation of Oct4 by Akt resulted in dissociation of Oct4 from the AKT1 promoter, which activated AKT1 transcription. Furthermore, the phosphorylated Oct4 promoted the self-renewal of ECCs and specifically suppressed their differentiation toward ectoderm.The Oct4-Sox2 complex is at the top of the regulatory hierarchy in maintaining the self-renewal and pluripotency of PSCs. Sumoylation of Oct4 has been shown to disrupt its interaction with Sox2. However, the specific sites mediating their interaction and the dynamic changes of this interaction during lineage-specific differentiation remain largely unknown. Here, we revealed that the highly-conserved Oct4-K156, by forming the intramolecular salt bridge with D212, critically stabilized the helical structure of the Oct4 POUs domain and hence the Oct4-Sox2 interaction. Post-translational modifications at Oct4-K156, and Oct4-K156N, a somatic mutation detected in bladder cancer patients, both impaired the K156-D212 salt bridge and the Oct4-Sox2 interaction. When produced as a recombinant protein or overexpressed in PSCs, Oct4-K156N, with reduced binding to Sox2, significantly down-regulated the sternness genes that are cooperatively controlled by the Oct4-Sox2 complex, and specifically up-regulated the mesendodermal genes and the SNAIL family genes that promote the epithelial-mesenchymal transition (EMT).Taken together, my thesis work provided evidence supporting the conclusion that site-specific PTMs of Oct4 coordintively control the self-renewal, differentiation and tumorigenesis of PSCs by regulating its transcriptional activity, protein stability and protein-protein interations.