In mammals, DNA is methylated at cytosines within CpG dinucleotides. Properly regulated methylation is crucial for normal development. Inappropriate methylation may contribute to tumorigenesis by silencing tumor-suppressor genes or by activating growth-stimulating genes. Although many genes have been identified that acquire methylation and whose expression is methylation-sensitive, little is known about how DNA methylation is controlled. We have identified a DNA sequence that regulates establishment of DNA methylation in the male germ line at Rasgrf1. In mice, the imprinted Rasgrf1 locus is methylated on the paternal allele within a differentially methylated domain (DMD) 30 kbp 5' of the promoter. Expression is exclusively from the paternal allele in neonatal brain. Methylation is regulated by a repeated sequence, consisting of a 41-mer repeated 40 times, found immediately 3' of the DMD. This sequence is present in organisms in which Rasgrf1 is imprinted. In addition, DMD methylation is required for imprinted Rasgrf1 expression. Together the DMD and repeat element constitute a binary switch that regulates imprinting at the locus.

In mammals, imprinted genes have parent-of-origin-specific patterns of DNA methylation that cause allele-specific expression. At Rasgrf1 (encoding RAS protein-specific guanine nucleotide-releasing factor 1), a repeated DNA element is needed to establish methylation and expression of the active paternal allele 1 . At Igf2r (encoding insulin-like growth factor 2 receptor), a sequence called region 2 is needed for methylation of the active maternal allele 2,3 . Here we show that replacing the Rasgrf1 repeats on the paternal allele with region 2 allows both methylation and expression of the paternal copy of Rasgrf1, indicating that sequences that control methylation can function ectopically. Paternal transmission of the mutated allele also induced methylation and expression in trans of the normally unmethylated and silent wild-type maternal allele. Once activated, the wild-type maternal Rasgrf1 allele maintained its activated state in the next generation independently of the paternal allele. These results recapitulate in mice several features in common with paramutation described in plants 4 .Rasgrf1 is methylated on the paternal allele in a differentially methylated domain (DMD) 30 kb 5′ of the promoter. Expression is from the paternal allele in neonatal brain 5 . This imprinting requires a 1.6-kb repeated element located immediately downstream of the DMD consisting of a 41-mer repeated 40 times that regulates establishment of methylation at the DMD 1,6 . The DMD is a methylation-sensitive enhancer-blocking element, which, together with the repeats, functions as a binary switch that regulates imprinting. Sequences regulating DNA methylation have been identified for one other locus, Igf2r. In intron 2 of Igf2r, region 2 controls methylation and allele-specific expression 2,3,7 .We generated mice containing Igf2r region 2 in place of the Rasgrf1 repeats to determine if their activities overlap. Reciprocal crosses were done between mice heterozygous with respect to this allele (Rasgrf1 tm3.1Pds , Fig. 1) and PWK mates to monitor expression from the two alleles in neonatal brain 5 . Similar crosses were done with C57BL/6 mates to evaluate changes in methylation of the Rasgrf1 DMD.Maternal transmission of the Rasgrf1 tm3.1Pds allele (Rasgrf1 −/+ ) had no effect on methylation or expression of the locus, which remained paternal allele-specific and was expressed at levels COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. Fig. 2a and data not shown). Mice with a paternally transmitted repeat deletion lacked both methylation and expression of the paternal allele 1 , but paternal transmission of the Rasgrf1 tm3.1Pds mutation (Rasgrf1 +/− ) permitted expression of the locus, albeit at lower levels than in wild-type mice (Fig. 2b). The paternal Rasgrf1 tm3.1Pds allele also caused derepression of the normally silent wild-type maternal allele. Expression of the mutated paternal allele in Rasgrf1 +/− mice showed that region 2 was able, in part, to replace the function of t...