PTCH1 reporter constructs. (A) For PTCH1_WT a 1.3 kb fragment (-1022 to +211 relative to the main TSS) was cloned upstream of the firefly luciferase gene of the pGL3b vector. GLI binding sites BS1 and BS2 are represented by boxes, arrowheads indicate the orientation of the binding site. For PTCH1_VAR, binding site BS2 was replaced by a 29 bp linker. All variant GLI binding sites were inserted between the NsiI and XhoI restriction sites of the linker (consensus sequence is shown). (B) PTCH1_WT and PTCH1_VAR luciferase reporters with the GLI consensus binding site were co-transfected with GLI2act expression vector into HaCaT cells. Variants 6G7G (G in position 6 and 7 of consensus) were used as inactive controls. PTCH1_WT and PTCH1_VAR(Cons) show comparable activation, no activation was observed for 6G7G and the empty PTCH1_VAR construct. (C) BS2 is absolutely required for activation of the PTCH1 wild type promoter PTCH1_WT. PTCH1_WT or mutagenized variants (mBS1, mBS2) were cotransfected with GLI2act into HaCaT cells. Mutagenesis of BS2 (GACCACCCA) to 6G7G completely abolishes activation of the PTCH1_WT reporter construct (mBS2), while substitution of BS1 (GACCTCCCA) with 6G7G only reduces the luciferase signal (mBS1).
Winklmayr et al. BMC Molecular Biology 2010 11:2 doi:10.1186/1471-2199-11-2