Fig. 3. Prediction of TCF/Lef target genes in differentiating epithelial progenitors. (A) Real-time RT-PCR confirmation of an upregulation of putative TCF/Lef target genes during epithelial differentiation induced by Lif. Expression levels peak on either day 5 (class B genes) or day 7 (class C genes) of organ culture. Maximal expression levels were set to 100%. Prediction is based on either published evidence or computational identification of conserved TCF/Lef consensus sites in the promoter region (see below). ^*, P<0.05 versus Lif 2 days (n=3). (B) Example of the prediction of a TCF/Lef binding site 99 bp upstream of the transcriptional start site of the cyclin D1 (Ccnd1) gene. Mouse, rat and human sequences are aligned and reveal a high degree of conservation in the region surrounding the TCF/Lef consensus motif (red). (C) Overrepresentation of conserved TCF/Lef binding sites in promoters of genes induced during epithelial differentiation. Identification of genes containing at least one conserved TCF/Lef binding site in different intervals preceding the transcriptional start site consistently reveals a statistically significant overrepresentation in classes B and C compared with control genes (² test, P values for different intervals are indicated in orange). (D) Specific overrepresentation of the TCF/Lef core motif as measured by an overrepresentation index (see Materials and methods) is demonstrated by comparison with 4096 control motifs of equal dimension. The TCF/Lef matrix (red) scores in the 97.1st percentile of all examined matrices.