FIG. 2.
Oct4 and Sox2 bind to the distal enhancer of Pou5f1 in living mouse ESCs. (A) Specificity of the antibodies used in the ChIP assays was confirmed by Western blotting of mouse ESC nuclear extracts. (B) The locations of the amplified products (black boxes) of the primer sets used to detect the ChIP-enriched DNA fragments, shown within the context of the genomic structure of mouse Pou5f1. The locations of the conserved regions within the promoter are indicated. The composite sox-oct element is within CR4. Amplicons are numbered in order relative to their sites along the gene. Open boxes represent exons. (C to E) High-resolution mapping of Oct4 (C), Sox2 (D), and control (glutathione S-transferase antibody [GST]) (E) binding sites across the Pou5f1 promoter in mouse ESCs by ChIP analysis. Fold enrichment is the relative abundance of DNA fragments at the indicated regions (see panel B) over a control region as quantified by real-time PCR. Standard deviations are shown. (F to I) A similar analysis of Oct4 (G) and Sox2 (H) occupancy on the Pou5f1 promoter in undifferentiated mouse ESCs and in ESCs induced to differentiate with RA for 3 and 6 days. Oct4 and Sox2 levels are shown to decrease in these differentiating conditions as identified by Western blotting (F) with a histone deacetylase 1 antibody (αHDAC1) used as a loading control. ChIP analysis was used as a subset (4, 6, and 7) of the amplicons described in panel B. (I) A control ChIP assay using an anti-MLL antibody.