YY1 antagonizes the positive effect of C/EBP-LAP through mutually exclusive DNA binding.

<p>(A) Beas2B cells were cotransfected with a fixed amount of both <i>CFTR</i> (0.072 µg) reporter and C/EBPβ (0.04 µg) expression vectors and increasing amount of plasmid encoding YY1 (0.004 to 0.08 µg) as indicated (left panel). Endogenous <i>CFTR</i> mRNA level following C/EBPβ (0.04 µg) expression vectors and increasing amounts of plasmid encoding YY1 (0.004 and 0.08 µg) (right panel). *P<0.05. (B) Left panel: ChIP experiment was performed on cells transfected or not with either C/EBPβ plasmid or respective siRNA. Protein extracts were immunoprecipitated IP with the indicated antibody. Input (IN) corresponds to total lysate used as a control for PCR amplification of total DNA. CFTR, represents <i>CFTR</i> promoter amplification and negative control, ChIP analysis of <i>CFTR</i> sequence which lacks the YY1 binding motif. Right panel: DNA from immunoprecipitates and input DNA (which represents 5% of total chromatin) were analyzed by quantitative PCR using primers amplifying the minimal <i>CFTR</i> promoter. Data are defined as fold enrichment of DNA associated with indicated immunoprecipitated antibody relative to input chromatin and specific binding was expressed as a function of non-sepcific (NS) antibody binding set as 1. (C) Functional interplay between C/EBPβ and YY1. Mutually exclusive DNA-binding activity of YY1 and C/EBPβ at the C/EBPb3 binding site. The labelled b3WT probe was incubated with C/EBPβ-transfected Beas2B nuclear extracts in the presence of increasing amounts of purified C/EBPβ. Arrows indicate the position of C/EBPβ and YY1.