PEST motif sequence regulating human NANOG for proteasomal degradation

A number of transcriptional factors are required for pluripotency of stem cells. NANOG, a homeobox transcription factor, plays a critical role in regulating embryonic stem cell (ESC) pluripotency. The expression level of NANOG is tightly regulated, and perturbation in its expression level can lead to significant difference in the morphology, expression of cell surface markers, and growth factor dependence of human and mouse ESCs. Here, we demonstrate that the proteolysis of human NANOG is regulated by the ubiquitin-proteasomal pathway. The inhibition of proteasome activity by proteasome inhibitor MG132 showed increase in protein levels of endogenous NANOG in a dose-dependent manner in human ESCs (hESCS). We demonstrated that the inhibition of the proteasome activity and cotransfection with exogenous ubiquitin promotes endogenous ubiquitination of NANOG by coimmunoprecipitation assay. In addition, we showed that both K48- and K63-branched polyubiquitin chains can conjugate with NANOG in vivo. Moreover, NANOG was an unstable protein and exhibited relatively short half-life of about 120 min in hESCs. Pretreatment of hESCs with proteasome inhibitor MG132 inhibits NANOG protein degradation and extends its half-life. Finally, we found that a PEST motif sequence (rich in proline, glutamine, serine, and threonine) from amino acid 47 to 72 located toward the N-terminus of NANOG was shown to target the protein for degradation. Deletion of the PEST motif reduced ubiquitination of NANOG, leading to NANOG stabilization. Collectively, these results indicate that the expression level, stability, and activity of NANOG are modulated by post-translational mechanisms.