Toward the evaluation of function in genetic variability: Characterizing human SNP frequencies and establishing BAC‐transgenic mice carrying the humanCYP1A1_CYP1A2locus

Abstract

Interindividual differences in human CYP1A1 and CYP1A2 expression appear to be associated with variability in risk toward various types of environmental toxicity and cancer. These two genes are oriented head-to-head on human chromosome 15; the 23.3-kb spacer region might contain distinct regulatory regions for CYP1A1 and distinct regulatory regions for CYP1A2, or the regulatory regions for the two genes might overlap one another. From 24 unrelated subjects of five major, geographically-isolated subgroups, we resequenced both genes (all exons and all introns) plus some 3' flanking sequences and the entire spacer region (39.6 kb total); 85 SNPs were found, 49 of which were not currently in the National Center for Biotechnology Information (NCBI) database. Of the 57 double-hit SNPs, we carried out SNP-typing in 94 Africans, 96 Asians, and 83 Caucasians and found striking ethnic differences in SNP frequencies and haplotype evolution; the two CYP1A1 SNPs and the one CYP1A2 SNP that are most commonly used in epidemiological studies were shown not to be representative haplotype tag SNPs across these three human subgroups. Four BAC-transgenic mouse lines, carrying the human CYP1A2 and 15,190 bp of 5' flank, expressed only negligible basal or inducible CYP1A2 mRNA. A fifth BAC-transgenic mouse line, carrying both the human CYP1A1 and CYP1A2 genes and ample amounts of 3' flanking sequences, plus all of the spacer region--in the absence of the mouse Cyp1a1 or Cyp1a2 genes--expressed the human CYP1A1 and CYP1A2 mRNA, protein and enzyme activities in liver and nonhepatic tissues very similar to that of the mouse. Comparison of this hCYP1A1_1A2 transgenic line with hCYP1A1_1A2 lines carrying other common human haplotypes will enable us to evaluate function in human CYP1A1_CYP1A2 locus variability, with regard to toxicity and cancer caused by combustion products.

Generation of ‘humanized’ hCYP1A1_1A2_Cyp1a1/1a2(−/−) mouse line

“…Both the Cyp1a1/1a2(−/−) double-knockout and this humanized line are viable, fertile, and show normal longevity. Herein we have demonstrated the presence of human CYP1A1 and CYP1A2 mRNAs and proteins, and absence of mouse CYP1A1 and CYP1A2 mRNAs and proteins; presence of human CYP1A1 and CYP1A2 mRNAs, proteins and enzyme activities in the hCYP1A1_1A2 line were also shown previously [5,15].Although the human CYP1A1 in place of mouse CYP1A1 only partially "rescues" the animal from oral BaP-induced immunosuppression, it appears that human CYP1A1 does as well as its mouse counterpart in eliminating BaP (Table 1). In liver oral BaP-induced human CYP1A1 is as high as mouse CYP1A1; this conclusion is counter to the toxicological data, showing systemic effects of BaP in the humanized mice.…”

“…This "humanized" hCYP1A1_1A2 line, theoretically on a >99.8% B6 background, had been bred separately with Cyp1a1(−/−) or Cyp1a2(−/−) mice. By Northern hybridization, Western immunoblot analysis, and three enzyme assays, human dioxin-inducible CYP1A1 and both human basal and dioxin-inducible CYP1A2 levels of expression (mRNA, protein, and enzyme activity)-in every one of nine tissues examinedparalleled very closely those seen with the mouse homologs [5]. The hCYP1A1_1A2(+/−) mouse, containing one copy of the human BAC, was bred with the Cyp1a1/1a2(−/−) mouse (described above), to produce the hCYP1A1_1A2_Cyp1a1/1a2(−/−) mouse line.…”

“…2). These data indicate that posttranslational stabilization is not a major contributory factor in determining the ultimate level of enzyme activity, but rather that protein levels closely reflect mRNA levels for the two mouse genes and the two human genes in these mice.As we had previously found [5], it is difficult to resolve the human CYP1A1 and CYP1A2 proteins [5]; in fact, both of these bands run in the gap between mouse CYP1A1 and CYP1A2. The antibody used here was raised against rat CYP1A1/1A2, and therefore is expected to react better with mouse than with human CYP1A1/1A2 proteins.…”

“…Thus, land animals (including fowl) carry both CYP1A1 and CYP1A2; sea animals do not have the CYP1A2 gene [4]. Accordingly, the CYP1A1 and CYP1A2 genes are located at human chromosome 15q24.1, in head-to-head orientation, 23,306 bases from one transcription initiation start-site to the other [5]. Of three mammalian genomes studied, estimates are that about 10% of gene duplication pairs share bidirectional promoters [6].…”

Functional characterization of eight human cytochrome P450 1A2 gene variants by recombinant protein expression

“…Their genes are located on chromosome 15 and are oriented head-to-head, 23.3 kb apart. 9 The spacer sequence may contain distinct regulatory regions of each gene or, alternatively, the regulatory regions of both genes. 10 CYP1A1 is mainly expressed in extrahepatic tissues, whereas CYP1A2 is almost exclusively expressed in the liver, representing B15% of its total CYP content.…”