The human T-cell transcription factor-4 gene: Structure, extensive characterization of alternative splicings, and mutational analysis in colorectal cancer cell lines

The human T cell transcription factor-4 (hTCF-4) interacts functionally wit h beta-catenin in the Wnt signaling pathway, which regulates many developme ntal processes. Moreover, inappropriate reactivation of this pathway attrib utable to APC or beta-catenin mutations has been described in colorectal ca ncers, Because only the human TCF-4 cDNA sequence was known, we determined its genomic structure. A total of 17 exons, of which 5 were alternative, we re identified. Moreover, four alternative splice sites were observed either experimentally or ill silico by a BLAST approach in expressed sequence tag databases. The alternative use of three consecutive exons localized in the 3' part of the hTCF-4 gene changes the reading frames used in the last exo n, leading to the synthesis of a number of hTCF-4 isoforms with short, medi um, or long-size COOH-terminal ends, We next screened the entire hTCF-4 gen e for mutations in a series of 24 colorectal cancer cell lines by denaturin g gradient gel electrophoresis and/or direct sequencing. Besides an already described deletion of an A in an (A)9 coding repeat in four cases, we foun d DNA variants in eight cases for a total of 12 variants, of which 8 were c oding. These include one frameshift mutation in the beta-catenin binding do main (exon 1), and one missense mutation in exon 4. In the remaining six ca ses, nonsense or frameshift mutations were localized in the 3' part of the gene. These latter alterations have as a common consequence to decrease the proportion of the long COOH-terminal hTCF-4 isoform, which contains two bi nding domains for c-terminal binding protein, a protein implicated in the r epression of the TCF family transcriptional activity. Thus, loss of the TCF -4 capacity to interact with COOH-terminal binding protein could be an impo rtant event during colorectal carcinogenesis by modifying Wnt signaling.