Fig. 7. Pax1 and Pax9 bind to regulatory sequences of the Bapx1 promoter. (A) PIP analysis of an 8550 bp mouse Bapx1 genome sequence when compared with an 8600 bp human BAPX1 sequence. Exons are outlined in black and coding parts are gray. Numbers at the top indicate nucleotide positions as defined in the text, and they correspond to 5' ends of intervals included in the series of deletion constructs used in the transactivation assay. The interval between -880 and -748 is outlined in green. PIP scores in % are shown on the right, with 50% identity at the bottom level. Note the presence of several CNS segments outside the coding region. Many of them are located in the interval between -880 and +109. (B) Sequence of the interval between -880 and -731. The different oligonucleotides (S1, S2, B4, B5 and S4) employed in EMSA assays are indicated by lines. Sequence in green (-880 to -748) corresponds to the segment included in the plasmid p0.9Bp-luc, but not in the plasmid p0.7Bp-luc. A potential Pax6-binding site predicted by TFSEARCH and MatInspector is in the region of S1 and indicated in red. (C-F) Results of EMSA experiments. The indicated labeled oligonucleotides (Probe, marked with an asterisk) were incubated with the in vitro translated protein (Prot.) Pax1, Pax9 or luciferase as a control (C), separated and visualized as described in the Materials and Methods. The specific Pax1 (black arrows) and Pax9 (gray arrow in F) complexes are indicated. (C) Pax1 binds to oligonucleotide B4. Cold oligonucleotides e5-5 or e5-3 were added at 250-fold (lanes 2, 4) or 500-fold (lanes 3, 5) molar excess, as indicated. e5-5 specifically competes with B4 for Pax1 binding, whereas mutated e5-3 does not. (D) Specific inhibition of Pax1-B4 complex formation by anti-Pax1 antibody. The indicated antibodies were incubated in the binding reactions. P1, anti-Pax1 goat antibody; C, anti-mouse goat antibody, used as control. The anti-Pax1 antibody abrogates the formation of the larger complex. (E) Competition experiments employing a 250-fold (lanes 2, 4, 6) or 500-fold (lanes 3, 5, 7) molar excess of cold oligonucleotides B4, S1 or S2. Note that oligonucleotides B4 and S1 compete with B4 for Pax1 binding, whereas oligonucleotide S2 does not. (F) Binding of Pax1 and Pax9 to oligonucleotide S1. S1 or S2 labeled oligonucleotides were incubated with Pax1, Pax9 or luciferase control protein as indicated. Both Pax1 and Pax9 interact with S1 oligonucleotide, but not with S2 oligonucleotide.