DNA hypomethylation and gene expression in mice

Behnam, Yousif Toma (1992) DNA hypomethylation and gene expression in mice. University of Southampton, Doctoral Thesis.

Abstract

In the present study, DNA methylation was examined as a potential regulatory mechanism of alphafetoprotein (AFP) and ribosomal DNA genes (rDNA) expression in C3H mouse strain. DNA methylation status was studied in both genes by Southern blot analysis using restriction endonuclease digestion of DNA extracted from various tissues of 1-4 weeks old mice and from adults treated with 300μg of demethylating agents 5-azacytidine (AZC) or 50μg of its analog 5-aza-2'deoxycytidine (dAZC). Restricted fragments were blotted onto nylon membrane and hybridized to a ³²P labelled genomic or cDNA probes.

HPLC elution profiles of the mouse genomic DNA five nucleosides, their percentages and level of the total 5mC in seven different tissue of different ages and treatments were determined following enzymatic hydrolyses.

Incorporation rate of ³H labelled amino acid in the newly synthesised proteins in the sera and livers of AZC treated mice was higher when compared with controls.

Significant increase in AFP levels was detected immunologically in sera from mice pretreated with a single dose of liver regeneration inducer CCl₄ (25μl i.p.) 24 hrs prior to the treatment with AZC or dAZC.

No rearrangement of the methylation pattern was detected in the coding sequences in both genes compared with exclusive changes in the promoter reigon for AFP and massive hypomethylation for the rDNA genes.

A steady increase in DNA methylation was found to be associated with development. 5-methylcytosine (5mC) was found in its lowest levels in various tissue of 1 week old mice whereas the highest levels were detected in adults (4 weeks old). Animals treated with AZC or dAZC following CCl_4 pretreatment showed massive hypomethylation in all tissue studied with substantial reduction of 5mC in spleen and brain.

These results suggest that cytosine methylation in certain class I and class II genes is likely to be a regulatory mechanism of gene expression and critical regulatory role for DNA hypomethylation as an explanation for the differences observed in AFP expression following the treatment with AZC or dAZC.

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