FIG. 2.

Brn-2 expression is activated by β-catenin in vitro and in vivo. (A) Sequence of a region of the Brn-2 promoter required for activity in melanocytes and melanoma, with the consensus Lef1/Tcf factor binding site indicated (−241 to −247). The sequence of the mutation in the binding site is indicated, and the underlined region was used as a probe for the DNA-binding assays presented below. (B) Lef1 binds the Brn-2 promoter in vitro. Band shift assay with a probe corresponding to the sequence underlined in panel A together with bacterially expressed Lef1. The indicated competitor oligonucleotides were used at 2, 5, 10, 20, 50, and 250 ng. The sequence of the Lef1 consensus oligonucleotide was 5′-CTAGAAGGGCACCCTTTGAAGCTCT-3′. (C) β-Catenin activates the Brn-2 promoter. The 501 mel cells were transfected with a Brn-2 promoter-luciferase reporter construct extending to either −2.3 kb (wild type) or deleted to −266, −184, or −111 or a reporter, −266/Lef.m1, in which the Lef1/Tcf site has been mutated, as illustrated in the inset. The indicated reporters were transfected alone or together with a β-catenin expression vector, and luciferase activity was determined.