FIGURE 5.

Identification and characterization of Cdx binding sites in Pax3 NCE2. A, sequence comparisons of all three putative Cdx-binding sites (CdxBS1, CdxBS2, and CdxBS3) relative to a consensus CdxBS sequence. Mismatches are denoted by lowercase letters. B, analysis of Cdx1 binding to CdxBS1 and CdxBS3 via electrophoretic mobility shift assay. All in vitro binding reactions were performed in parallel under identical conditions. M, mock. The presence of Cdx1 in the shifted bands was confirmed by the addition of 1 μg of anti-FLAG antibody to the binding reaction mix. Cdx1 binding and anti-FLAG supershifts (SS) are indicated by arrows. A probe containing a consensus CdxBS present in the Hoxb8 promoter was used as a positive control. Specificity of Cdx1 binding was assessed by preincubation of FLAG-Cdx1-containing nuclear extracts with a 100-fold excess of wild type (wt) or mutated (mut) cold probes. Note that preincubation with wild type unlabeled probes leads to inhibition of Cdx1 binding to the radiolabeled probes, whereas preincubation with mutated probes did not. C, impact of CdxBS mutation on Cdx2-mediated activation of the NCE2 reporter in cell culture. Wild type or CdxBS mutant versions of a Pax3NCE2-luciferase reporter were generated and assessed for Cdx2-mediated transactivation in P19 and N2a cells. Cells were transiently transfected with the NCE2-Luc reporter alone or with a Cdx2 expression vector. The results (mean ± S.E. (error bars) of seven or eight independent experiments performed in triplicate) are expressed as -fold induction compared with the relevant reporter vector alone. m1, m2, and m3, Pax3NCE2-luciferase reporter constructs containing point mutations in the CdxBS1, CdxBS2, and CdxBS3, respectively; m1,2, m1,3, and m2,3, double mutations of CdxBS; m1,2,3, triple mutation. Note that concomitant mutation of all three CdxBS is required to almost completely abolish the transactivation of Pax3 NCE2 by Cdx2.