【作者】 潘光锦；

【导师】 裴端卿；

【作者基本信息】 清华大学 ， 生物学， 2005， 博士

【摘要】 胚胎干细胞(ES 细胞)具有发育的全能性。Oct4 与 Nanog 都是在ES 细胞中特异性表达的转录因子,对 ES 细胞多能性的维持起关键作用。Oct4 的功能主要是阻止 ES 细胞向滋养层细胞方向发生分化并维持 ES 细胞的自我复制能力。Nanog 能够使 ES 细胞在培养过程中不依赖于 LIF 而维持其多能性。1,本研究中我们利用生物学软件分析了 Oct4 的蛋白序列,发现在其 POUdomain 中有一段保守的核定位序列(RKRKR)可能参与其在细胞内的运输及调节机制。我们进一步的研究发现,该序列突变后不仅影响 Oct4 在细胞内的运输,还直接破坏了 Oct4 行使其转录激活调控基因的功能。更有趣的是,该突变体还能与野生型 Oct4 形成二聚体并进一步抑制其正常功能,也就是形成一种具有抑制性功能的突变体(dominant negative mutant)。将该突变体导入 P19 多能细胞后能进一步通过抑制内源性 Oct4 的功能导致其分化。2,我们研究和探讨了 Nanog 本身各个结构域的功能。利用 Gal4 及其报告基因系统及通过构建 Nanog 本身的报告基因。我们发现,Nanog 是一个拥有多功能结构域的具有基因激活功能的转录因子。在其 C domain 中存在两个结构独特,功能强大的转录激活区 WR 和 CD2。这两个转录激活区具有独特的结构特点及很强的保守性,迄今尚未在其它的转录因子的激活区中发现。这两个激活子 WR 和 CD2 分别在 Nanog 发挥功能时起着不同的作用。这些发现为进一步阐释拥有多功能结构域的转录因子——Nanog 维持 ES 细胞多能性的机制打下了基础。3,为揭示 Nanog 在 ES 多能性细胞中的表达调控机制,我们克隆并研究了Nanog 基因上游 promoter 的功能。我们克隆了 Nanog 基因上游 1.0k 左右的promoter 区,通过报告基因技术我们发现该 promoter 能够很好反映内源性 Nanog的表达模式。进一步通过构建不同长度的 Nanog promoter 的缺失体,我们发现位于 Nanog promoter 上游-676~-733 的一段序列介导了 Nanog 的细胞特异性表达。这段序列中,含有一段潜在的 homeobox protein 的结合位点,提示在多能细胞中可能存在一种特异性的 homeobox protein 调控 Nanog 的表达。另外,我们通过凝胶阻滞实验证明在高表达 Nanog 的多能细胞中确实村一种特异性的蛋白能够结合该位点。该发现有助于进一步鉴定和识别结合 Nanog promoter 的Homeobox 转录因子,以利于发现新的调控 ES 细胞多能性的转录因子并阐明 ES细胞多能性的分子机制。更多还原

【Abstract】 Embryonic stem (ES) cells can undergo self renewal indefinitely whilemaintaining pluripotency, the ability to differentiate into all types of cells. Oct4 isidentified to maintain the pluripotency both in vitro and in vivo. Nanog has also beendemonstrated to play a key role in maintaining the pluripotency of ES cells. It canmaintain the self-renewal and pluripotency of mouse ES cells independent ofLIF-STAT3 pathway.1, Oct4 plays a critical role in maintaining the pluripotency of ES cells, but theprecise mechanisms by which Oct4 achieve these biological function remainsunknown. Here we identify and characterize the RKRKR as a nuclear localizationsignal responsible for its nuclear localization and required for its transcriptionalfunction. Mutation of this site yielded a mutant fail to localize in the nuclei and lossthe trans-activation. We further demonstrated that this mutant is competent indimerization not only with itself but also wild type Oct4 and can interfere with theactivity of wild type of Oct4, thus behaved as dominant negative mutant. Indeed, thismutant can induce the differentiation of P19 cells by suppress the function ofendogenous Oct4. These data suggested new mechanisms through which Oct4achieves its tremendous function in maintaining ES cell pluripotency.2, Nanog, an homeodomain gene, has been proposed to play a key role inmaintaining stem cell pluripotency presumably by regulating the expression of genescritical to stem cell renewal and differentiation. Here, though harness the wellestablished Gal4 reporter system and construction of the reporter gene of Nanog itself,we defined the function of various domains of Nanog. Based on the evidence weobtained, Nanog behaves as a transcription activator with two unusually strongactivation domains embedded in its C-terminus-WR and CD2. These two activationmodules were very conserved and unique to the Nanog and have not been found toexist in other known transcription factors. Deletion of both WR and CD2 from nanogcompletely eliminated its transactivation function. These data suggest that either WRor CD2 is sufficient for nanog to function as a transactivator, and both domains arerequired to regulate the proper expression of genes necessary for stem cellpluripotency.3, As a pluripotent factor, the expression of nanog mRNA was restricted topluripotent stem cells and absent in differentiated cells. However, the molecularmechanisms that regulate Nanog gene expression are largely unknown. To addressthis important issue, we cloned and characterized the 5’promoter of Nanog gene byusing reporter gene-luciferase and GFP. We found that the expression patterns ofreporter gene controlled by the 1k Nanog promoter were well recapitulate that ofendogenous Nanog in pluripotent cells, such as F9, ES, suggesting that this promotercontained the cis regulatory elements contribute to the cell specific expression ofNanog. Through further constructing the deletions of 5’ flanking region, we foundthat the -676~-733 fragment directed the Nanog expression in pluripotent cells. Thisfragment contains a conserved homeodomain binding site, suggesting an unknownhomeobox protein may bind the Nanog promoter and regulate its expression.Electrophonic mobility shift assay using this element with whole cell extracts of ES,F9, P19, NIH, and MEF revealed that there indeed exists a specific protein which canbind it in ES and F9 cells but not in other cells. Further study need to identify thisunknown homeobox protein and define its role in Nanog regulation in pluripotentcells.更多还原