Post-translational Regulatory Mechanisms Acting on KLF4 Mediate Pluripotency Exit in Naïve Mouse Embryonic Stem Cells

Pluripotent embryonic stem (ES) cells have the potential to self-renew and differentiate to generate all adult tissues. Pluripotency is regulated by an interconnected network of transcription factors including OCT4, SOX2, NANOG and KLF4. This transcriptional network is integrated with extracellular signaling pathways regulating pluripotency and differentiation. Mouse ES cells can be maintained in a state known as naïve pluripotency by culture in leukemia inhibitory factor (LIF) and two signaling pathway inhibitors (LIF/2i). A previous study observed that a reduction in Klf4 transcript, due to LIF withdrawal, was the first change in pluripotency gene expression during differentiation. Based on this finding I hypothesized that removal of KLF4 protein from transcriptional network would be required for exit from pluripotency. To investigate this I examined the levels of pluripotency associated transcription factors in the nucleus of ES cells as they exit the pluripotent state. This investigation revealed that a reduction in KLF4 protein levels did not occur immediately following the reduction in Klf4 transcription; instead I identified nuclear export of KLF4 protein was required for the reduction in Klf4 transcription and pluripotency exit. Next, I investigated Klf4 gene and protein regulation and found that LIF/2i maintains Klf4 expression through both transcriptional and post-translational mechanisms. Specifically, KLF4 protein is highly stable in LIF/2i and this stability is maintained by physical interaction with active transcriptional complexes which ensure nuclear anchoring of the KLF4 protein. Surprisingly, KLF4 protein stability is so high (t½ >24 hr) that protein levels change byPh.D