Figure 6

Foxm1 promoter occupancy by Nanog. (A) Schematic of the Foxm1 promoter showing putative Nanog-responsive elements (s1; s2 - s3 and s4). (B) qPCR-ChIP assay of endogenous Nanog occupancy of the Foxm1 promoter region in NSCs and Diff-NSCs. Immunoprecipitation with IgG was performed as control. Anti-acetyl-H3 antibodies was used to identify Foxm1 transcriptional activation. Eluted DNA was PCR-amplified with primers for Nanog binding sites s1, s2-s3, s4. Results are expressed as fold induction values relative to input controls. B-actin was utilized as unrelated chromatin control and is presented in Supplementary Figure 7A. Bars represent means (SD) of three independent experiments. P values vs. Diff-NSCs (Mann-Whitney U test): **P < 0.01: 0.002572 (s2-3, AcH3); ****P < 0.0001: 0.00000718 (s2-3, Nanog), 0.0001051 (s1, AcH3), 0.00004356 (s4, AcH3); NS: 0.4531 (s1, Nanog), 0.7118 (s4, Nanog). (C) Luciferase activity induced by ectopic expression of Nanog and Mock (negative control, PCDNA) in NSCs transfected with luciferase vector carrying the wild-type Foxm1 promoter (wt) and its mutant lacking the Nanog binding sites s2 and s3 (mutants s2, s3). Bars represent means (SD) of at least three independent experiments, each performed in triplicate. P values vs. indicated controls (Mann–Whitney U test). ***P < 0.001: 0. 000937 (Nanog wt); NS: 0.097 (Mut Nanog s2), 0.18 (Mut Nanog s3).