Fig. 6. Impairment of Snail-induced repression of the claudin-7 promoter by mutations of the E-boxes. (A) A short fragment of the mouse claudin-7 promoter region (–110 to +190; Fig. 4). This short fragment includes five E-boxes (E1-E5) and showed the epithelium-specific promoter activity (data not shown). Double-stranded oligonucleotides corresponding to the E4-containing sequence (underlined sequence) were used in the electrophoretic mobility shift/oligonucleotide precipitation assays in Fig. 7. (B,C) Mutational analyses. The core sequence, 5'-CA(G/C)(G/C)TG-3', of E-boxes (E1E5) was mutated to 5'-AA(G/C)(G/C)TA-3' in various combinations (shadowed boxes). Luciferase reporter constructs carrying wild-type or these mutated claudin-7 promoters were transfected into Eph4 cells together with a mouse Snail expression vector (mSnail) or an empty vector (pCAG). (B) Luciferase activity found in cells co-transfected with a wild-type reporter construct and pCAG empty vector was defined as 1.0. (C) The same set of data expressed as the Snail-induced repression ratio (+mSnail/+pCAG in B) for individual reporter constructs. As the number of mutated E-boxes increased, the claudin-7 promoter became less sensitive to Snail. For no known reason, even when all five E-boxes were mutagenized, the Snail-induced repression ratio did not reach 1.0. The arrowhead shows the putative transcription start point. All results correspond to the average of three independent experiments.