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 bp 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*1A activity. Therefore, the phenotypic effects of the variant CYP3A4*1B may be associated with enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor.
Amirimani
B
B
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6021, USA.
Ning
B
B
Deitz
A C
AC
Weber
B L
BL
Kadlubar
F F
FF
Rebbeck
T R
TR
D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017930:Genes, Reporter; D005822:Genetic Vectors; D015894:Genome, Human; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D003577:Cytochrome P-450 Enzyme System; D008156:Luciferases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D001616:beta-Galactosidase