| 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.
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