Abstract
A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated β-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27KIP1 (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27KIP1 gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene.
Highlights
Nanog protein transduction resulted in growth of more than 250 colonies in a 6-cm dish, 20% of them exceeding a diameter of 200 μm, whereas only few colonies were observed in case of the control (Fig. 1E,F)
We found that Nanog protein transduction enhanced proliferation of both NIH 3T3 cells and primary mouse and human fibroblasts
Nanog has been reported to enhance proliferation and/or self-renewal in other somatic and stem cell lines by regulating molecules involved in stemness, cell cycle and senescence machinery [Cao et al, 2010; Choi et al, 2012; Go et al, 2008; Shan et al, 2012; Tanaka et al, 2007]
Summary
A thorough understanding of the molecular network of cellular factors, extracellular and intracellular signaling pathways, cell cycle regulation and microenvironment that establish and maintain selfrenewal and pluripotency is key for the development of biomedical applications of stem cells (Boyer et al, 2005; Cox et al, 2011; Loh et al, 2006; Lowry and Quan, 2010). Establishment and maintenance of stem cell identity, pluripotency, is regulated by a core network of transcription factors. In concert with Oct and Sox, Nanog governs pluripotent features in mouse and human cells (Boyer et al, 2005; Loh et al, 2006) by occupying the promoters of active genes encoding transcription factors, signal transduction components and chromatin-modifying enzymes. Expression of Oct and Sox is relatively homogeneous in pluripotent cells whereas Nanog exhibits a heterogeneous expression (Singh et al, 2007), with cells having elevated levels of Nanog exhibiting efficient self-renewal
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