Fig. 6. Hoxa2 binds the Six2 promoter. (A) Schematic representation of the 900Six2 promoter: the black rectangle corresponds to the TATA box. Sequences showing 95% conservation between mouse and human are underlined in red, probes used in bindshift, in blue. (B) Hoxa2-HA binds to BstEII-SmaI probe, and the two retarded complexes (arrows) are supershifted by the anti-HA antibody (arrowhead). (C,D) Hoxa2-HA binds to probes 1 (C) and 2 (D). The addition of cold wild-type double-stranded oligonucleotides (wt1, wt2), but not of mutant oligonucleotides (m1, m2), competes the formation of the complexes (arrows). The sequence of the wild-type and mutant oligonucleotides and their relative position on the promoter are shown; red lowercase letters indicate the introduced nucleotide changes. Cold oligonucleotides were added at 250-fold (3,6) and 500-fold (4,5,7,8,) molar excess. (E) The incubation of Hoxa2-HA with labeled wild-type oligonucleotides results in the formation of the same specific retarded complexes (arrows, 2, 7), recognized by anti-HA antibody (arrowheads, 3, 8). No protein/DNA interaction is observed when Hoxa2-HA is incubated with mutant oligonucleotides (5, 10). (F) Pbx1a and Meis1 cooperate in binding to the proximal Six2 promoter. Pbx1a, Meis1 and Hoxa2 were incubated, separately or in combination, with the BstEII-SspI probe. Hoxa2-HA/DNA complex, black arrows; Pbx1a/DNA complex, arrowhead; Pbx1a/Meis1/DNA complex, red arrow. The position and the sequence of the putative Hoxa2 (blue rectangles) and Pbx/Meis (red rectangle) sites are indicated. The Pbx/Meis site was identified using Patch search at Biobase (www.gene-regulation.com).