Gastrin is a downstream target of the APC/β-catenin pathway. (a) Schematic representation of a 1,300-bp human gastrin promoter construct () containing several potential TCF-4–binding sites. TCF-4–binding sites with high homology to the known TCF/Lef-HMG box transcription factor consensus motifs [CC(A/T)TTG(A/T)(A/T)(T/C); ref. , ] were identified in the gastrin promoter using Lasergene99 DNA-analysis software. Homologous bases are printed as capital letters: –1166 tCTTTGgca-1156, –1000 GATGAAACC-990, –822 aCTTTGTcT-812, –516 CCATTGcTC –506, –103 CCATTccTC-93. (b) Northern blot analysis of RNA taken from HT-29-APC and HT-29-GAL cells that were induced with zinc to express either wild-type APC or β-galactosidase. The blot was probed with either a riboprobe to human gastrin exon 2 or GAPDH. (c) HeLa cells were transfected with a series of gastrin promoter-luciferase reporter deletion constructs (number reflects length of promoter from the transcriptional start site) and then cotransfected with a constitutively active β-catenin expression construct alone (filled bars) or with both the constitutively active β-catenin expression construct with a dominant-negative TCF-4 construct (open bars). The basal promoter construct Pxp2 was used as a control. The results are taken from three independent experiments. (d) Sequences containing either the wild-type TCF-4–binding site from –103 to –90 on the gastrin promoter, a mutated TCF-4–binding site, or a corresponding perfect TCF-4 consensus sequence (Con) are shown. These sequences were used to generate heterologous promoter constructs by cloning them upstream of the thymidine kinase promoter in the luciferase reporter construct pT81. (e) HeLa cells were then transfected with the wild-type TCF-4-pT81 construct, the mutant TCF-4-pT81 construct, the consensus TCF-4-pT81 construct, or an empty pT81 construct and a constitutively active β-catenin expression construct (filled bars) or both the constitutively active β-catenin expression construct and a dominant-negative TCF-4 construct (open bars).