Variability of DNA methylation patterns during serial passage of human diploid fibroblasts

We have examined DNA methylation in diploid human fibroblasts, early and late in their replicative life-span. The extent of methylation of -C-C-G-G- was measured by comparison of fragment sizes after digestion with methylation-specific restriction enzyme Hpa II or Msp I, or both. Methylation of -C-C-G-G- sites in total DNA, occurring predominantly at internal (3') cytosines, increased from 59% to 64% of sites in one cell strain at late passage, remained constant in another, and decreased in four other strains (54% to 48%, 58.5% to 48%, 55% to 51.5%, and 52% to 44.5%). Base composition analysis confirmed a substantial loss of total DNA 5-methylcytosine (mC) in one strain. Seven clonal isolates, examined at middle to late passage, ranged from 33% to 51% methylation of 3' cytosines in -C-C-G-G- sites. Three discrete classes of highly repetitive DNA were found which contained Msp I sites at intervals of 45, 110, and 175 base pairs. These repeat families consistently had 70-80% of sites methylated at 3' cytosines, in all clones and in all strains examined both at early and at late passage. Thus, altered methylation of repetitive sequences is unlikely to account for the variable -C-C-G-G- methylation observed in total DNA. When DNA from one fibroblast strain and from eight pure clones isolated from that parental culture was digested with Msp I or Hpa II followed by EcoRI and probed for gamma-globin gene sequences, considerable interclonal and intraclonal heterogeneity was observed for methylation at four -C-C-G-G- sites in the gamma-globin coding region of DNA. Therefore, the pattern of methylation in endogenous gene regions appears to undergo random drift during replication of diploid fibroblasts.