Functional analysis of the human cytochrome P4501A1 (CYP1A1) gene enhancer

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) induces gene transcription, a process that requires binding of the activated aryl hydrocarbon receptor (AhR) to dioxin responsive elements (DR Es) within the enhancer region of responsive genes. Most of what is known a bout the molecular mechanism of AhR-dependent gene activation results from studies on the murine prototype TCDD-responsive gene cytochrome P4501A1 (CY P1A1). Much less is known, however, about the regulation of human TCDD-resp onsive genes. We have therefore conducted a detailed analysis of the enhanc er region of the human CYP1A1 gene. From the ten DRE core motifs investigat ed within a stretch of 1400 bp in two human tumor cell lines using a ligati on-mediated PCR technique, five motifs displayed a TCDD-inducible in vivo f ootprint. Four of these sites were functional enhancer sequences as demonst rated by a transient expression assay. Based on these data, a distinct func tional consensus sequence for DRE motifs within the human CYP1A1 gene is su ggested. After introduction of the four functional sites into various mouse hepatoma cell lines, only three exhibited a functional response, suggestin g some species differences in CYP1A1 gene regulation. In addition to the fo otprints at DRE sites, we also detected protein-DNA interactions at three G -rich domains located within the enhancer region of the human CYP1A1 gene. Our data show that, besides some similarities in the regulation of the huma n and mouse CYP1A1 genes, there also exist some distinct differences, inclu ding number, location, and functional consensus sequences of DRE motifs, as well as quantity and location of footprinted G-rich domains.