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: c1, 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.