Genetic polymorphisms in the 5'-flanking region change transcriptional regulation of the human cytochrome P450IIE1 gene.
We identified genetic polymorphisms in the 5'-flanking region of the human cytochrome P450IIE1 gene and investigated the effect of these polymorphisms on the transcriptional regulation of the gene. PCR direct sequencing of the two homozygous alleles [types A (c1/c1) and C (c2/c2)] revealed the existence of several point mutations in the distal 5'-flanking region of the gene, but no differences in the proximal promoter region. The DNA segment (-1372 to -960) placed upstream of SV40 promoter and the chloramphenicol acetyltransferase (CAT) gene enhanced the expression of the gene, and the enhancement of expression by type C DNA was about 10 times that by its type A counterpart. DNase I footprinting analysis showed at least one protected region in which one of the polymorphic loci (RsaI polymorphism) was located. The DNase I sensitivities and protection profiles of the two genotypes were different. The protected region had high homology to the consensus sequence of the binding region of liver specific transcription factor HNF1 (LF-B1), and this was confirmed by gel retardation assay. These results indicate that genetic polymorphisms in the 5'-flanking region of the human P450IIE1 gene affect its binding of trans-acting factor and change its transcriptional regulation. This may lead to inter-individual differences of microsomal drug oxidation activity.
Hayashi
S
S
Department of Biochemistry, Saitama Cancer Center Research Institute.
Watanabe
J
J
Kawajiri
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000097:blood / Q000235:genetics*; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D010089:Oxidoreductases, N-Demethylating / Q000097:blood / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D051381:Rats; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496489:Hnf1a protein, rat; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I