High-performance liquid chromatographic analysis of methylation changes of CCGG sequence in brain and liver DNA of...

The change of the methylation of CpG in the CCGG sequence of brain and liver DNAs of mice during late fetal and suckling periods was determined by high-performance liquid chromatography using a reversed-phase column and 0.1 M phosphate buffer (pH 6.0) as the mobile phase. The tissue DNA was digested with the restriction enzyme, MspI, and was labeled at the 5'-end with [gamma-32P]ATP. The cpm% of deoxycytidine 5'-monophosphate (5mdCMP) in total CpG dinucleotides was calculated from the equation 5mdCMP/total CCGG (cpm%) = (5mdCMP)MspI,cpm/[(5mdCMP)MspI,cpm + (dCMP)MspI,cpm] x 100. The brain DNA exhibited a significant decrease in CpG methylation at prenatal day 18 but little change after birth. This marked decline of 5mdCMP in the CCGG sequence may be associated with the increase of enzymes before birth. The liver DNA showed considerable change during the late prenatal period. The observed changes of CpG methylation in liver DNA are indicative of the corresponding alterations of enzymes, multinucleate cells and hepatocytes. The results obtained indicate that both brain and liver cells have the development-associated changes in the conformation and transition of DNA around the time of birth.