C/EBPdelta (CCAAT/enhancer-binding protein delta) is a member of the C/EBP family of nuclear proteins that function in the control of cell growth, survival, differentiation and apoptosis. We previously demonstrated that C/EBPdelta gene transcription is highly induced in G(0) growth-arrested mammary epithelial cells but the C/EBPdelta protein exhibits a t(1/2) of only approximately 120 min. The goal of the present study was to investigate the role of C/EBPdelta modification by ubiquitin and C/EBPdelta proteasome-mediated degradation. Structural and mutational analyses demonstrate that an intact leucine zipper is required for C/EBPdelta ubiquitination; however, the leucine zipper does not provide lysine residues for ubiquitin conjugation. C/EBPdelta ubiquitination is not required for proteasome-mediated C/EBPdelta degradation and the presence of ubiquitin does not increase C/EBPdelta degradation by the proteasome. Instead, the leucine zipper stabilizes the C/EBPdelta protein by forming homodimers that are poor substrates for proteasome degradation. To investigate the cellular conditions associated with C/EBPdelta ubiquitination we treated G(0) growth-arrested mammary epithelial cells with DNA-damage- and oxidative-stress-inducing agents and found that C/EBPdelta ubiquitination is induced in response to H2O2. However, C/EBPdelta protein stability is not influenced by H2O2 treatment. In conclusion, our results demonstrate that proteasome-mediated protein degradation of C/EBPdelta is ubiquitin-independent.
Reprogramming somatic cells to become induced pluripotent stem cells (iPSCs) by using defined factors represents an important breakthrough in biology and medicine, yet remains inefficient and poorly understood. We therefore devised synthetic factors by fusing the VP16 transactivation domain to OCT4 (also known as Pou5f1), NANOG and SOX2, respectively. These synthetic factors could reprogramme both mouse and human fibroblasts with enhanced efficiency and accelerated kinetics. Remarkably, Oct4-VP16 alone could efficiently reprogramme mouse embryonic fibroblasts (MEFs) into germline-competent iPSCs. Furthermore, episomally delivered synthetic factors could reproducibly generate integration-free iPSCs from MEFs with enhanced efficiency. Our results not only demonstrate the feasibility of engineering more potent reprogramming factors, but also suggest that transcriptional reactivation of OCT4 target genes might be a rate-limiting step in the conversion of somatic cells to pluripotent cells. Synthetic factor-based reprogramming might lead to a paradigm shift in reprogramming research.
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