FIGURE 7.

Analysis of C/EBPα and HNF4α binding to the CYP2B6 5′-flanking region in human livers. A, total RNA from six liver donors showing high (n = 3) and low (n = 3) CYP2B6 expression was purified, and the mRNA levels of CYP2B6, C/EBPα, HNF4α, and CAR were determined by real-time quantitative RT-PCR analysis. In parallel, we also analyzed the mRNA concentration of the housekeeping porphobilinogen deaminase for normalization. Data represent the mean ± S.D. (error bars) expressed as -fold increase/decrease compared with a reference human liver cDNA pool (n = 12). B, formaldehyde cross-linked chromatin from human liver tissues was immunoprecipitated with antibodies against C/EBPα and HNF4α or with preimmune IgG. Quantitative PCR was performed on immunoprecipitated, purified DNA (bound DNA fraction) using primers specific to eight CYP2B6 5′-flanking regions (as depicted in Fig. 6A). Parallel PCRs were performed with both input and IgG-immunoprecipitated DNA (background DNA fraction). Data represent the mean ± S.D. of three different livers and are expressed as the difference between the recoveries in the bound and in the background fractions. Lower panel, PCR aliquots (10 μl) from a representative amplification were subjected to electrophoresis on 1.5% agarose gel and stained with ethidium bromide. IN, input; C, anti-C/EBPα; H, anti-HNF4α; M, 100-bp DNA ladder.