Increased transcriptional activity of the CYP3A4* 1B promoter variant

Numerous single nucleotide polymorphisms (SNPs) have been identified in the human genome, yet the functional significance of most is unknown. CYP3A4 is a key enzyme in the metabolism of numerous compounds. An A-->G substitution 290 by upstream of the CYP3A4 transcription start site (CYP3A4* 1B) has been associated with cancer phenotypes, but its phenotypic effects are unclear. To investigate the functional significance of CYP3A4* 1B, we generated two luciferase reporter constructs: 1-kb (denoted L, long) and 0.5-kb (denoted S, short) promoter fragments containing either the variant (V-L,V-S) or the wild-type (W-L, W-S) sequences. We evaluated the effect of the variant sequence in the HepG2 and MCF-7 cell lines, and in primary human hepatocytes from three donors. Reporter constructs with the variant sequence had 1.2- to 1.9-fold higher luciferase activity than constructs with wild-type sequence in the cell lines (P < 0.0001) and hepatocytes (P = 0.021, P = 0.027, P = 0.061). The ratio of transcriptional activity for V-S:W-S was similar to the V-L:W-L ratio in HepG2 cells, but the V-S:W-S ratio was consistently less than the V-L:W-L ratio in MCF-7 cells. This suggests that CYP3A4 expression is higher from the variant promoter and that a repressor sequence may exist in the longer constructs. Electrophoretic mobility shift assays demonstrated specific binding of a component of HepG2 nuclear extract to both wild-type and variant promoters with consistently higher binding affinities to the wild-type sequence. This suggests the existence of a transcriptional repressor responsible for the lower CYP3A4* 1 A activity. Therefore, the phenotypic effects of the variant CYP3A4* 1 B may be associated with enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor. (C) 2003 Wiley-Liss, Inc.