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. 2012 Sep 12;287(45):38295–38304. doi: 10.1074/jbc.M112.373092

FIGURE 5.

FIGURE 5.

Ncoa3 binds to and activates the Nanog promoter. A, GO term analysis of genes regulated by Ncoa3 knockdown in ESCs. B, chromatin immunoprecipitation in V6.5 ESCs with anti-Ncoa3 antibody demonstrated that Ncoa3 binds to the Nanog promoter but not the promoters of Sox2 or Oct4. C, overexpression of Ncoa3 enhanced endogenous Nanog expression. V6.5 ESCs were transfected by empty pCAGIPuro vector or pCAGIPuro-Ncoa3 overexpression plasmid. Twenty-four hours after transfection, cells were harvested. Nanog expression was measured by quantitative RT-PCR. D, luciferase assay showed that overexpression of Ncoa3 increased the Nanog promoter activity. Empty pCAGIPuro vector or pCAGIPuro-Ncoa3 overexpression plasmid, together with pRV-SV40 and the Nanog promoter reporter plasmids, were cotransfected into V6.5 ESCs. Luciferase activities were measured 24 h after transfection. Means ± S.D. from three independent experiments were plotted. E, knockdown of Ncoa3 suppresses the Nanog promoter activity. A luciferase assay was performed as described in B, except that shGFP control or two shNcoa3 plasmids (shN3-1 and shN3-2) were used. Means ± S.D. from three independent experiments were plotted. *, p < 0.05; **, p < 0.01; ***, p < 0.001.