Nanog基因的功能与调控研究进展

胚胎干细胞的无限增殖能力和亚全能性决定了它在再生医学、新药开发及发育生物学基础研究中具有巨大的应用前景。探索维持胚胎干细胞亚全能性的因子及其网络的调控功能成为胚胎干细胞生物学研究的热点。已研究发现多个与维持胚胎干细胞亚全能性相关的基因如Oct4, Nanog, Sox2等,其中Nanog是2003年5月末发现的一个基因,它对维持胚胎干细胞亚全能性起关键性作用,能够独立于L1F/Stat3维持ICM和胚胎干细胞的亚全能性。几年来,Nanog的生物学功能及其与 Oct4, Sox2等亚全能性维持基因之间的相互作用关系已有较为深入的研究,并发现多个调控Nanog表达的转录因子,从而进一步明晰Nanog与已知调控胚胎发育的信号通路之间的关系。本文在综述Nanog基因的表达特征和功能的基础上、重点探讨Nanog基因表达调控以及Oct4, Sox2等亚全能性维持基因之间的相互作用关系,并对未来的研究趋势予以展望。     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways. This work was partly funded by the Millennium Research Fund National University of Ireland Galway.     

Nanog overcomes reprogramming barriers and induces pluripotency in minimal conditions

Induced pluripotency requires the expression of defined factors and culture conditions that support the self-renewal of embryonic stem (ES) cells. Small molecule inhibition of MAP kinase (MEK) and glycogen synthase kinase 3 (GSK3) with LIF (2i/LIF) provides an optimal culture environment for mouse ES cells and promotes transition to naive pluripotency in partially reprogrammed (pre-iPS) cells. Here we show that 2i/LIF treatment in clonal lines of pre-iPS cells results in the activation of endogenous Nanog and rapid downregulation of retroviral Oct4 expression. Nanog enables somatic cell reprogramming in serum-free medium supplemented with LIF, a culture condition which does not support induced pluripotency or the self-renewal of ES cells, and is sufficient to reprogram epiblast-derived stem cells to naive pluripotency in serum-free medium alone. Nanog also enhances reprogramming in cooperation with kinase inhibition or 5-aza-cytidine, a small molecule inhibitor of DNA methylation. These results highlight the capacity of Nanog to overcome multiple barriers to reprogramming and reveal a synergy between Nanog and chemical inhibitors that promote reprogramming. We conclude that Nanog induces pluripotency in minimal conditions. This provides a strategy for imposing naive pluripotency in mammalian cells independently of species-specific culture requirements.     

The pluripotency rheostat Nanog functions as a dimer

The defining activity of the homeodomain protein Nanog is the ability to confer cytokine-independent self-renewal upon ES (embryonic stem) cells in which it is overexpressed. However, the biochemical basis by which Nanog achieves this function remains unknown. In the present study, we show that Nanog dimerizes through a functionally critical domain. Co-immunoprecipitation of Nanog molecules tagged with distinct epitopes demonstrates that Nanog self-associates through a region in which every fifth residue is tryptophan. In vitro binding experiments establish that this region participates directly in self-association. Moreover, analytical ultracentrifugation indicates that, in solution, Nanog is in equilibrium between monomeric and dimeric forms with a K(d) of 3 muM. The functional importance of Nanog dimerization is established by ES cell colony-forming assays in which deletion of the tryptophan-repeat region eliminates the capacity of Nanog to direct LIF (leukaemia inhibitory factor)-independent self-renewal.     

The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells

Embryonic stem (ES) cells derived from the inner cell mass (ICM) of blastocysts grow infinitely while maintaining pluripotency. Leukemia inhibitory factor (LIF) can maintain self-renewal of mouse ES cells through activation of Stat3. However, LIF/Stat3 is dispensable for maintenance of ICM and human ES cells, suggesting that the pathway is not fundamental for pluripotency. In search of a critical factor(s) that underlies pluripotency in both ICM and ES cells, we performed in silico differential display and identified several genes specifically expressed in mouse ES cells and preimplantation embryos. We found that one of them, encoding the homeoprotein Nanog, was capable of maintaining ES cell self-renewal independently of LIF/Stat3. nanog-deficient ICM failed to generate epiblast and only produced parietal endoderm-like cells. nanog-deficient ES cells lost pluripotency and differentiated into extraembryonic endoderm lineage. These data demonstrate that Nanog is a critical factor underlying pluripotency in both ICM and ES cells.     

维持胚胎干细胞多能性的分子机制

胚胎干细胞（ES细胞）来源于早期发育的胚胎,具有分化为任何细胞类型的多能性,因此具有巨大的基础研究及潜在的应用前景.目前认为ES细胞主要通过一些外源性信号分子的作用及某些重要的内源性转录因子的表达共同起作用来达到其维持多能性的目的.外源性信号分子LIF、BMP4以及Wnt等介导的信号传导通路与内源性转录因子Oct4、Nanog、Sox2、FoxD3等共同起作用来抑制那些促进ES细胞分化的基因表达和激活那些有助于维持ES细胞多能性维持的基因表达,进而形成一个相互调控和依存的基因调控网络共同维持ES细胞的多能性.