Fig. 5. cyclopamine changes binding of GLI protein species to 5' flanking regions in Shh target genes. (A) Identification of GLI consensus binding sequences in the 5' flanking region of mouse Pax2, Sall1, cyclin D1 and Mycn. Nucleotides represented in upper case are exact matches to those identified previously in GLI consensus binding sequences (see text). Arrows indicate promoter segments amplified by PCR during chromatin immunoprecipitation (see below). (B) PCR products identified by agarose electrophoresis after chromatin immunoprecipitation using E11.5 wild-type kidney tissue cultured for 4 days in the presence of drug vehicle or cyclopamine. In vehicle-treated embryonic kidneys, GLI1 and/or GLI2 bound 5' flanking regions containing GLI consensus binding regions in Pax2, Sall1, cyclin D1 and Mycn. No binding of GLI3 with these regions was detectable. Cyclopamine decreased GLI1 and GLI2 binding most markedly in Pax2 and Sall1, and induced binding of GLI3 to each promoter region. Neg, DNA amplified after immunoprecipitation with non-immune serum; Pos, DNA amplified using genomic DNA and specific primers; Input, DNA amplified from cross-linked DNA before immunoprecipitation.