Fig. 5. Pax2 and Pax6 directly bind to and activate the MITF-A promoter in vitro. (A) Organization of the human MITF-gene (after Hallsson et al., 2000), position upstream promoter of exon A and of the predicted Pax2 and/or Pax6 binding sites (red boxes) in the MITF-A promoter construct, which is coupled to the luciferase reporter gene (Udono et al., 2000). (B) Sequences of the oligonucleotides used in the EMSAs in C and D. Red letters indicate the Pax6 or Pax2 consensus sequence (Epstein et al., 1994) in the Mitf promoter sequences. RE2 represents the Pax2 and Pax6 binding site (Schwarz et al., 2000). (C) EMSA with the sequences A1-A5 from (B), the control sequence RE2 and Pax2, Pax6 and `null' proteins (+Pax2, +Pax6, -Pax). Arrowheads indicate nonspecific binding. (D) The specificity of Pax2 and Pax6 binding to sequence A5 (lane 1 and 4, -ab) is confirmed by the addition of anti-Pax2 antibody (lane 2, +P2-ab) and anti-Pax6 antibody (lane 5, +P6-ab), which inhibits the formation of the complex. The addition of the reciprocal antibodies did not inhibit the binding (lanes 3 and 6). Without an overexpressed protein, only a faint band appears (lane 7, -Pax). The red arrowhead indicates the binding of Pax2 and of Pax6 by oligo A5. The green arrowhead marks the unbound oligos. (E) Pax2 and/or Pax6 activate the MITF-A promoter in cell culture experiments. Co-transfection of Cos-7 cells with the luciferase-coupled human MITF-A promoter and CMV-Pax2-cDNA and/or CMV-Pax6-cDNA lead to a strong increase in luciferase activity. CMV-Pax1-cDNA activates the MITF-A promoter only moderately.