DNA methylation contributes to tissue- and allele-specific expression of the T-cell differentiation marker RT6

We investigated the role of DNA methylation in gene regulation of the rat T -cell differentiation marker RT6. Analysis of the methylation status of var ious tissues revealed that the RT6 promoter was hypomethylated in RT6-expre ssing tissues, and methylated in nonexpressing ones. Remarkably, among RT6- nonexpressing tissues, the extent of methylated regions varied greatly betw een lymphatic tissues, where regions larger than 23 kb were methylated, and nonlymphatic tissues, where methylation was restricted to a 3- to 4-kb reg ion surrounding the promoter. We have previously shown that cis-regulatory elements determine differential expression of the two RT6 alleles in a subp opulation of T cells. We now show that the RT6 alleles in these cells diffe red in their methylation status. The promoter region of the silent allele w as methylated, while that of the transcribed allele was not. Upon treatment of RT6-nonexpressing thymoma cells with the methyltransferase inhibitor 5- azacytidine, RT6 expression was induced. In RT6 heterozygous hybridoma cell s, expressing only one RT6 allele, induction of the silent, methylated RT6 allele was observed. Sensitivity of the RT6 promoter to DNA methylation was demonstrated by promoter-specific in vitro methylation, which inhibited RT 6 promoter activity, while that of the SV40 promoter was not influenced. Ou r findings indicate that DNA methylation plays an important role in the con trol of monoallelic and tissue-specific RT6 expression.