CHARACTERIZATION OF THE HUMAN CYTOCHROME P4502D6 PROMOTER - A POTENTIAL ROLE FOR ANTAGONISTIC INTERACTIONS BETWEEN MEMBERS OF THE NUCLEAR RECEPTOR FAMILY

The functional mapping of the human cytochrome P4502D6 (CYP2D6) promot er in HepG2 cells revealed the presence of both positive and negative regulatory elements. One of these regulatory elements overlapped a seq uence that is highly conserved in most members of the CYP2 family, Thi s element, which consists of a degenerate AGGTCA direct repeat spaced by 1 base pair (DR1) and is known to be a target for members of the st eroid receptor superfamily, was found to bind in vitro translated hepa tocyte nuclear factor 4 (HNF4) in gel retardation analysis. Using HepG 2 nuclear extracts, three protein-DNA complexes were formed on the DR1 element, one of which was confirmed to be dependent on the binding of HNF4. The other DR1 complexes were shown to be due to the interaction of the orphan receptor chicken ovalbumin upstream promoter transcript ion factor I (COUP-TFI). Experiments in COS-7 cells showed that HNF4 c ould activate the CYP2D6 promoter 30-fold. Surprisingly, mutation of t he DR1 element produced a relatively minor 23% decrease in activity in HepG2 cells. Additionally, COUP-TFI was shown to inhibit HNF4 stimula tion of the CYP2D6 promoter in COS-7 cells, suggesting that COUP-TFI c ould attenuate the effect of HNF4 in HepG2 cells, However, when HNF4 l evels were increased in HepG2 cells by co-transfection, it resulted in the enhancement of CYP2D6 promoter activity, indicating that HNF4 cou ld overcome the repressive effect of COUP-TFI. Therefore, the contribu tion of the DR1 element in controlling the transcription of the CYP2D6 gene depends on the balance between positively and negatively acting transcription factors.