Figure 3. USF family members interact with CSA and HR23A proximal promoters.

(A) Graphic representation of human, mouse, dog and zebrafish CSA proximal promoter. Conserved E-boxes are represented in dark grey. (B) In vivo 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 CSA promoter. (C) In vitro Electrophoretic Mobility Shift Assay (EMSA) experiments were performed using HaCaT nuclear extract and radiolabeled probes centered on the E-box motif present in the CSA 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 HR23A and human HR23B 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 HR23A or HR23B promoters and the distal region of HR23A 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 HR23A proximal promoter. (G) ChIP assay using SP3 antibody or non-specific IgG were performed as previously described for HR23A and HR23B promoter occupancy. (H) EMSA experiments were performed as previously with HaCaT nuclear extract and radiolabelled probes centered on the GC box present in the HR23A proximal promoter (−131) (shifted complex (→)).