FIG. 2.

Direct activation of the IL1R2 promoter by combinatorial GLI1/EGF signaling. Bs, binding site; mBs, mutated binding site; RLU, relative light units. (A) Illustration of the localization of three putative GLI binding sites in the 255-bp promoter (prom) fragment of the IL1R2 upstream regulatory region. The sequence of the three binding sites is shown in the table (right panel). For mutated binding sites used in EMSA (mBs1, mBs2, and mBs3), two essential C nucleotides (underlined) were changed to two G nucleotides. (B) EMSA analysis of the three GLI binding sites showing that all three sites are bound by recombinant DNA binding domain of GLI1. To demonstrate specific binding, competition experiments (comp) with increasing amounts of oligonucleotide corresponding either to wild-type or mutated binding site sequences were done. Furthermore, poly(dI-dC) · poly(dI-dC) (dIdC) was added to the binding reactions to test for unspecific binding. (C) Luciferase reporter assays showing that both GLI1 (left) and active GLI2* (right) efficiently stimulate reporter activity from the 255-bp IL1R2 promoter fragment. Note that mutation of any of the three GLI binding sites is sufficient to abrogate reporter activation. (D) Activation of the IL1R2 promoter by GLI1 and active GLI2* is enhanced by simultaneous activation of EGF signaling. Mutation of Bs1 in the IL1R2 promoter (IL1R2-mBs1) inhibits the stimulatory effect of EGF signaling. cont, control. Promoter (prom) activation of the known direct GLI target PTCH is unaffected by EGF signaling. Reporter assays shown in panels C and D were done in HaCaT keratinocytes.