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Methylation differences in the murine P1450 and P3450 genes in wild-type and mutant hepatoma cell culture
Abstract:
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.
Insights
Methylation differences in mouse P1450 and P3450 genes were investigated. A specific hypomethylation at the M9 site in the P1450 gene was observed in a mutant cell line (c1).
Area of Science:
- Molecular Biology
- Genetics
- Epigenetics
Background:
- The study investigates methylation patterns of murine P1450 and P3450 genes in Hepa-1 wild-type and mutant cell lines.
- Mutant cell lines c1, c2, and c4 were selected based on P1450 mRNA levels, Ah receptor presence, and inducer-receptor complex translocation.
- The P3450 gene showed no expression, and its Msp I sites remained methylated across all tested cell lines.
Purpose of the Study:
- To analyze methylation differences in the P1450 and P3450 genes using Msp I and Hpa II isoschizomers.
- To investigate the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 5-azacytidine on gene methylation and expression.
- To identify specific methylation variations associated with distinct cellular phenotypes.
Main Methods:
- Restriction digestion with isoschizomers Msp I and Hpa II to detect methylation at specific sites (M1-M15).
- Analysis of P1450 and P3450 gene methylation patterns in wild-type and three mutant mouse Hepa-1 cell lines.
- Treatment with TCDD (inducer) and 5-azacytidine (demethylating agent) to assess their effects on gene methylation and activity.
Main Results:
- TCDD treatment did not alter P1450 methylation patterns or cause gene amplification.
- 5-azacytidine treatment induced demethylation of P1450 Msp I sites but did not affect catalytic activity.
- The c1 mutant cell line exhibited hypomethylation at the M9 site within the P1450 gene, distinct from wild-type and other mutants.
Conclusions:
- The P1450 gene's M9 site hypomethylation in the c1 mutant may be linked to its high constitutive P1450 mRNA levels.
- The P3450 gene remains methylated and unexpressed under the tested conditions.
- Epigenetic modifications, specifically DNA methylation, play a role in regulating P1450 gene expression in mouse liver cells.