Targeting of the TFAP2B promoter by PAX3/FKHR. (a) mRNA levels of TFAP2B and CB1 were measured by qRT–PCR in Rh4 cells at indicated time points after RNAi-mediated PAX3/FKHR downregulation. The ratio of siRNA to scRNA treatment is shown in %. (b) mRNA levels of TFAP2B and CB1 in 293T cells, either untransfected or transfected with the indicated PAX3/FKHR construct, as measured by qRT–PCR. PAX3hd and PAX3pd are homeodomain- and paired domain-specific mutants of PAX3/FKHR. (c) Deletion analysis of the TFAP2B promoter. A luciferase reporter construct containing the 3200 bp upstream of the TFAP2B transcriptional start site (TFAP2B_full) or deletion constructs thereof, containing remaining parts of 1592 bp (TFAP2B_1) or 806 bp (TFAP2B_2), respectively, are shown schematically in the upper panel. Transactivation of PAX3/FKHR on the different constructs as measured by luciferase assays is shown in the lower panel. The means±s.d. (error bars) from two independent duplicate experiments are shown (RLU, relative light units). (d) Transactivation of the indicated PAX3/FKHR constructs on deletion construct TFAP2B_1 as measured by luciferase assay. (e) Sequences of three potential PAX3/FKHR binding sites within the TFAP2B promoter region. Commonly described motifs for paired domain binding are indicated by bold cursive type. Sequences for the design of oligonucleotides used for EMSA are underlined. (f) Binding of PAX3 to the promoter regions of the TFAP2B_1 and TFAP2B_2 deletion constructs as detected by chromatin immunoprecipitation (ChIP). His-tagged PAX3 protein/DNA complexes were immunoprecipitated with α-His antibody or unspecific mouse IgG. qRT–PCR quantification of precipitated DNA of each experiment (n=3) was performed in triplicates and values from the anti-His immunoprecipitation were normalized with the values from the control immunoprecipitation with IgG. (g) Schematic representation of single deletions of possible binding sites of PAX3/FKHR in TFAP2B_1 as introduced by site-directed mutagenesis is shown in the upper panel. Luciferase activity of the indicated deletion constructs as measured after cotransfection with PAX3/FKHR in 293T cells. Each bar represents means±s.d. from three independent duplicate experiments. (h) EMSA used for measurement of DNA-binding properties of PAX3 against the deletion site 2 in the promoter region of TFAP2B. The assay was performed with 8 μl of nuclear protein extracted after transfection of PAX3-His in 293T cells. As control, mock-transfected 293T cells and mismatch oligonucleotides were used. EMSA, electrophoretic mobility shift assay; qRT–PCR, quantitative reverse transcription-PCR; scRNA, scrambled siRNA; siRNA, small interfering RNA.