Methylation of mouse adenine phosphoribosyltransferase gene is altered upon cellular differentiation and loss of phenotypic expression

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Morphologically differentiated cell lines were previously isolated from a mouse teratocarcinoma stem cell line exhibiting an unstable heterozygous deficiency for adenine phosphoribosyltransferase (APRT) expression. In this study, the methylation sensitive and insensitive isoschizomer restriction endonucleases HpaII and MspI, respectively, were used to demonstrate that the aprt gene in the heterozygous deficient stem cell line was hypomethylated. Loss of APRT activity in this stem cell line was not associated with DNA methylation change. However, differentiation of this stem cell line was associated with hypermethylation of three consecutive HpaII/MspI sites that were located in the second intron and the third exon of the aprt gene. A total of 15 independent APRT homozygous deficient cell lines were isolated from three differentiated heterozygous deficient cell lines, and in all 15 cell lines this differentiation-related methylation pattern was altered. Two classes of alterations were noted: (1) hypomethylation of a site located in the second intron or (2) the apparent spreading of methylation to downstream methylation sites. The CpG-rich promoter region remained hypomethylated in the APRT homozygous deficient differentiated cell lines and a methylation change affecting a specific CpG site upstream of the promoter region was noted in only two of the 15 homozygous deficient cell lines. It is proposed that methylation of the mouse aprt gene may be involved in controlling phenotypic expression in the differentiated cell lines, but not in the stem cell line they were derived from.