Figure 4

Activation of TGM3 in response to GLI requires two low affinity GLI binding sites. (A) The human TGM3 upstream regulatory region contains two non-consensus GLI binding sites (7A) (BS2, BS3). Lines below represent DNA amplified by qPCR in ChIP (*;, *;*;). (B) Luciferase reporter assay with the wild type TGM3 promoter fragment (TGM3prom) and constructs with mutated putative GLI binding sites (BS1 Mut, BS2 Mut, BS3 Mut). HaCaT cells were co-transfected with promoter constructs as indicated and GLI2act expression constructs or the empty expression vector pcDNA4/to. Data shown are mean values of relative light units (RLU) of three independent experiments. Mutation of either BS2 or BS3 completely abolishes reporter activation while mutation of the consensus sequence has no effect. (C) Chromatin immunoprecipitation (ChIP) demonstrates specific binding of GLI2act to a region of the TGM3 promoter containing BS2 and BS3 (*;*; in A). No amplification was observed for the region containing BS1, the GLI consensus binding site (*; in A). Chromatin isolated from GLI2actHaCaT cells was precipitated with either specific antibody (GLI2 N-20) or species matched normal IgG (nIgG). As positive control the region of the human PTCH promoter containing the functional consensus site (PTCH BS2) was used. Data shown are fold enrichment of specifically precipitated DNA (GLI2 N-20) compared to samples using species matched normal IgG for unspecific precipitation.