Active gene sequences are undermethylated

Abstract

The degree of methylation of active regions of the chromosome has been investigated by several techniques. DNase I (deoxyribonuclease I, EC 3.1.21.1) was used to introduce nicks in the active regions of the nucleus and thereby specifically label these areas. By using the methylation-specific restriction enzymes Hpa II and Hha I it could be shown that active genes are more sensitive to these probes than are other parts of the genome. In order to measure the amount of methylation at all CpG residues, DNA was nick-translated in the presence of [alpha-32P]dGTP as the sole nucleotide source and the methylated cytosine was detected by the standard nearest-neighbor analysis. Using this assay, we found that about 70% of all CpG sequences in animal cell DNA are methylated. In active nuclear regions that are sensitive to DNase I, only 30-40% of the CpG residues are methylated. This method was also employed to study the gene sequences that are complementary to cellular RNA. By this criterion expressed gene sequences are only 20-30% methylated. These data suggest that undermethylation is a general phenomenon in all actively transcribed genes.