USF family members interact with <i>CSA</i> and <i>HR23A</i> proximal promoters.

<p>(A) Graphic representation of human, mouse, dog and zebrafish <i>CSA</i> proximal promoter. Conserved E-boxes are represented in dark grey. (B) <i>In vivo</i> chromatin immunoprecipitation assays (ChIP) with HaCaT cells using USF-1, USF-2 antibodies or non-specific IgG. Recovered DNA under basal or UV-irradiation conditions was subjected to PCR or quantitative PCR using specific primers of both proximal and distal region (negative control) of the <i>CSA</i> promoter. (C) <i>In vitro</i> Electrophoretic Mobility Shift Assay (EMSA) experiments were performed using HaCaT nuclear extract and radiolabeled probes centered on the E-box motif present in the <i>CSA</i> proximal promoter (−246) (shifted complex (→)). Competition assays were performed in the presence or not of cold competitors (WT or mutated cold probe). Supershift assays were obtained in the presence of anti-USF-1, anti-USF-2, and anti-TBX2 antibodies or IgG as non-specific controls ( = >). (D) Graphic representation of human, mouse and dog <i>HR23A</i> and human <i>HR23B</i> proximal promoters. Conserved E-box motifs are represented in dark grey and GC-rich regions in light grey. (E) ChIP assays were performed as in (B) targeting proximal <i>HR23A</i> or <i>HR23B</i> promoters and the distal region of <i>HR23A</i> promoter (−3 kb). (F) EMSA experiments were performed as described in (C) using radiolabeled probes centered on each E-box motif (−154 and −36) present in the <i>HR23A</i> proximal promoter. (G) ChIP assay using SP3 antibody or non-specific IgG were performed as previously described for <i>HR23A</i> and <i>HR23B</i> promoter occupancy. (H) EMSA experiments were performed as previously with HaCaT nuclear extract and radiolabelled probes centered on the GC box present in the <i>HR23A</i> proximal promoter (−131) (shifted complex (→)).