Figure 4

C/EBPα/β and NeuroD are bound to the Fos, Egr1, and Egr2 promoters in vivo. (a,d,g) In vivo detection of Fos, Egr1, or Egr2 promoter occupancy by C/EBPα/β and NeuroD using ChIP analysis. The schemes illustrate the Fos, Egr1, and Egr2 promoter regions amplified by PCR in their 5' UTR (nucleotides -453 to -126, 1220 to 1361, and -96 to 72, respectively). The large black boxes indicate the predicted DNA binding sites (bs) for C/EBP and NeuroD. The small boxes indicate the position of the primers used for PCR amplification. ChIP assays were performed using E16.5 wild-type forebrain lysates and antibodies against NeuroD, C/EBPα and β, and demonstrated that these proteins are recruited to the three promoters. Input corresponds to PCR reactions containing 0.5% of the total amount of chromatin used in IP reactions. (b,e) To confirm the specificity of the results, IPs from each antibody were also analyzed by PCR with primers specific for a region of the Fos gene located in the 3' UTR, or for a region of the Egr1 gene located in exon 4 as indicated in the schemes. (c,f,h) qPCR analysis was used to quantify the relative amounts of DNA obtained by ChIP for the Fos (c), Egr1 (f), and Egr2 (h) promoters. DNA quantities (expressed as percentages of input) were compared for specific IPs versus Ig IP samples (see Materials and methods). The corresponding p values are indicated: *p < 0.002, **p < 0.02 for Egr1; *p < 0.0001, **p < 0.005 for Fos; *p < 0.0001, **p < 0.002 for Egr2. Error bars represent the standard error based on three independent experiments.