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• _4 value " A 926 bp fragment upstream the transcription start site was cloned into a reporter vector, pGL-basic, as shown in Fig. 1C . As shown in Fig 1C , Nanog promoter functioned as expected with high activities in F9 and ES cells and low and no activities in P19 and NIH3T3, respectively, paralleling the endogenous levels of Nanog in these cells. To see if Oct4 can suppress Nanog promoter directly, we cotransfected Oct4 with this Nanog reporter into F9 cells. As a control, we also included a reporter that carries six copies of Oct4-binding sites. As shown in Fig. 1D , Oct4 suppressed the activity of Nanog promoter in a dose-dependent fashion, while it activated the control reporter bearing Oct4 binding sites. As these assays were performed transiently within 48 h before the expected differentiation triggered by Oct4 over-expression, these data suggested that over-expressed Oct4 act as a repressor of Nanog, consistent with our prediction from Fig. 1A and B . There is one Oct4 binding site at ?181 position within the Nanog promoter (12 13 14) , and we subsequently confirmed its occupation by Oct4 by ChiP as shown in Fig. 1E . Two deletion constructs were made, one deleting sequences upstream of ?554 position and another deleting upstream of the ?181 position but all retaining the Oct4 binding site. As expected, both constructs were repressed by Oct4 in non pluripotent NIH3T3 cells (Fig. 1F ). However, a mutation at the Oct4 binding site rendered the 926 bp promoter insensitive to coexpressed Oct4 or Oct4 siRNA, suggesting that Oct4 regulates Nanog promoter specifically (Fig. 1G ). These data strongly suggested a negative role of Oct4 in regulating Nanog. Indeed, the concentration of Nanog in ES cells over-expressing Oct4 is also reduced significantly as monitored by Western blot to detect endogenous Nanog (Fig. 1H , upper panel) and transfected Oct4 (Fig. 1H , middle panel). Together, these data demonstrate that elevated Oct4 induces ES cell differentiation by suppressing Nanog." provenance.
• _4 wasQuotedFrom 16790525 provenance.