Methylation differences in the murine P 1450 and P 3450 genes in wild-type and mutant hepatoma cell culture

Abstract

The murine P 1450 and P 3450 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 P 1450 mRNA and believed to carry a mutation in the P 1450 structural gene; c2, having negligible levels of Ah receptor; and c4, having a defect in nuclear translocation of the inducer-receptor complex. The P 3450 gene was not expressed constitutively or after treatment of these four cell lines with the P 1450 inducer 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) and, correspondingly, the P 3450 Msp I sites remained methylated. Treatment of all four cell lines with TCDD did not alter the P 1450 methylation pattern, nor was there any evidence of P 1450 gene amplification. Treatment of all four lines with 5-azacytidine caused demethylation of the P 1450 Msp I sites but did not change the usual P 1450 catalytic activity pattern found in each of the lines. The only detectable difference in the P 1450 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 P 3450 gene.