Interaction of upstream stimulatory factor proteins with an E-box located within the human CYP1A2 5′-flanking gene contributes to basal transcriptional gene activation

Abstract

Cytochrome P450 (CYP)1A2 is abundantly expressed in the liver of all vertebrate species. In most, its expression is restricted to the liver. Sequence analysis of the human CYP1A2 5′-flanking region from +3 to −3201 identified six E-box motifs within the 3-methylcholanthrene (MC) enhancer element (−1987 to −3201). The E-box motif is recognized by members of the basic helix-loop-helix (bHLH) family of transcription factors. Gel mobility shift and antibody supershift assays were used to examine each of the six upstream E-box motifs for their ability to bind nuclear proteins and to compete with the ubiquitously expressed bHLH protein, upstream stimulatory factor (USF), for binding. We found that USF-1 and USF-2 proteins bind to the upstream E-box motifs EB2, EB3, and EB4. Transient transfection assays in HepG2 cells were performed with different segments of the human CYP1A2 5′-flanking region linked to a luciferase reporter gene. Site-directed mutagenesis of one of the E-box motifs, EB2, resulted in a 60% reduction in basal reporter gene activity. Mutations in EB3 and EB4 had no effect. We found that transfection of expression vectors containing USF-1 or USF-2 cDNAs activated CYP1A2 reporter gene activity, while a dominant-negative USF-2 expression vector blocked such activity. Chromatin immunoprecipitation assays confirmed that the interaction of USF proteins with the CYP1A2 EB2 site occurs in vivo. These data support the role of USF as a constitutive transcriptional activator of human CYP1A2.