Methylation differences in the murine P₁450 and P₃450 genes in wild-type and mutant hepatoma cell culture

The murine P₁450 and P₃450 genes and flanking regions contain 14 and 15 Msp I sites (C-C-G-G-), respectively, designated M1 through M14 or M15. These two genes from mouse Hepa-1 wild-type (wt) parent and three mutant cell lines were studied for methylation differences with use of the isoschizomers Msp I and Hpa II. The mutant lines included: cl, having high constitutive P₁450 mRNA and believed to carry a mutation in the P₁450 structural gene; c2, having negligible levels of Ah receptor; and c4, having a defect in nuclear translocation of the inducer-receptor complex. The P₃450 gene was not expressed constitutively or after treatment of these four cell lines with the P₁450 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and, correspondingly, the P₃450 Msp I sites remained methylated. Treatment of all four cell lines with TCDD did not alter the P₁450 methylation pattern, nor was there any evidence of P₁450 gene amplification. Treatment of all four lines with 5-azacytidine caused demethylation of the P₁450 Msp I sites but did not change the usual P₁450 catalytic activity pattern found in each of the lines. The only detectable difference in the P₁450 gene among the four lines was hypomethylation of the M9 site in c1 that was not seen in wt, c2 and c4 cells. The M9 site is part of a 9-bp box (5′-C-C-G-G-G-A-C-A-T-3′), located near the beginning of exon 3. It is of interest that the same nine bases are found in intron 2 about 80 bp upstream from the 5′ end of exon 3 in the homologous P₃450 gene.