Human Papillomavirus Type 8 Interferes with a Novel C/EBPβ-Mediated Mechanism of Keratinocyte CCL20 Chemokine Expression and Langerhans Cell Migration
Figure 2
C/EBPβ binds to the enhancer region of CCL20 in vivo.
(A) Nucleotide sequence of the human CCL20 promoter region with twelve putative C/EBP binding sites (underlined). Numbers below the underlined C/EBP binding sites mark the sequences, which display C/EBP DNA binding activity in EMSA. In bold is the DNA sequence tested for C/EBP binding in ChIP assay. (B) 32P-labeled oligonucleotides containing the respective C/EBP binding sites (nt 294–308, nt 574–584, nt 652–667, nt 716–724, nt 734–748) of the CCL20 promoter were incubated with 5 µg GST, GST-C/EBPα or GST-C/EBPβ fusion proteins and analyzed by EMSA. The arrow indicates complexes corresponding to C/EBP DNA binding activity. (C) Chromatin immunoprecipitation assay was performed using RTS3b cells transfected with the C/EBPβ expression vector. For precipitation anti-C/EBPβ (H-7) antibody was used. Genomic DNA was isolated, amplified by real-time PCR with primers specific for the nt 638–677 region of the CCL20 promoter (in bold). The amplicon was quantified (left panel) and visualized on an agarose gel (right panel). The amount of target DNA precipitated with the control antibody was set at 1. Shown are mean values ± SD from four experiments. The asterisk represents statistical significance, p = 0.02.