Abstract
CYP3A4 is the most abundant P450 enzyme expressed in the human liver and it is responsible for the metabolism of approximately 50% of all clinically administrated drugs. The CYP3A4 gene is transcriptionally regulated by xenobiotics and previous work has demonstrated the first 300bp of proximal promoter to be the minimal requirement for such activation. Several nuclear receptors (CAR, SXR) have been shown to be involved in the induction of the CYP3A4 gene. The aim of this work was to further delineate the molecular basis of CYP3A4 gene expression. In vitro DNase I footprinting was carried out using HepG2 nuclear extracts to map the sites of DNA-protein interaction within the -301/+7 region of the CYP3A4 gene. Putative protein assignment for these sites using in silico analysis revealed the potential binding of transcription factors previously shown to be involved in the regulation of other CYP genes (Sp1, HNF3 and C/EBP?) at the identified protein-DNA interaction sites. These regulatory regions were then disrupted by mutagenesis and their functional effect assessed by transient transfections of reporter gene plasmids into HuH7 hepatoma cells. Statistically significant reductions of reporter gene expression were observed when putative C/EBPa and HNF sites were altered, in both the basal and rifampicin (SXR ligand) induced states. This finding suggests the involvement of proteins binding at these sites in the regulation of the CYP3A4 gene expression. An examination of the CYP3A4 promoter (-1056/+7) from 11 human DNA samples exhibiting a 14.3 fold variability in CYP3A mediated metabolism failed to show the presence of mutations. Protein-DNA interaction analysis were carried out within the newly identified CYP3A4*IE and CYP3A4*1F alleles as well as the CYP3A4 *1B allele. The results implicate the Spl transcription factor in the regulation of the CYP3A4 gene, albeit at a more distal binding site. The findings described in this thesis suggest a substantial involvement of transcription factors other than SXR/CAR in expression of CYP3A4. Because of the polymorphic expression of several liver- and hormone-dependent transcription factors their role in CYP3A4 regulation must be taken into account to understand drug-induction mechanisms and assess variability in inter-individual drug response.