Trans allele methylation and paramutation-like effects in mice

Abstract

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...

Meiotic silencing and the epigenetics of sex

“…Amazingly, the repressed allele is further able to induce a similar state when passaged in a heterozygous state opposite another allele with identical (and normal) sequence, albeit with decreasing efficiency (Rassoulzadegan et al 2002, Herman et al 2003. This is highly reminiscent of the paramutation phenomenon in plants (Chandler et al 2000), which was recently shown to require a putative RdRP (Alleman et al 2006).…”

“…A number of transvective processes have been identified in diverse species Y operating either within or outside of meiosis. In many cases these processes have either been determined, or are predicted to have a mechanistic link to RNAi (Wu & Morris 1999, Herman et al 2003Alleman et al 2006, Rassoulzadegan et al 2006. Genomic DNA elimination in Tetrahymena is a dramatic example of a trans-sensing process that involves RNAi (for a more informative review of thei complex process, see Mochizuki & Gorovsky 2004).…”

RNA-mediated non-mendelian inheritance of an epigenetic change in the mouse

“…Mouse testes and epididymis were fixed immediately after dissection in 1.6 % glutaraldehyde in 0.1M phosphate buffer (1h, 4°C). They were rinsed with buffer and free aldehyde groups were blocked with 50 mM NH 4 Cl in PBS for 30 min at 4°C. Specimens were dehydrated with acetone and embedded in Epon.…”

“…Often referred to as an exception to the law of Mendel, which states that genetic factors segregate unchanged from heterozygotes, paramutation is meiotically stable and inherited in the absence of the inducing allele. To date, the closest observations in an animal species were changes in the DNA methylation profiles directed by the allelic locus in the mouse that we and others described as "transvection" or "paramutation-like" effects 3,4 . We now report a modification in the phenotypic expression of the wild type allele of the Kit receptor gene in the progeny of heterozygotes with a null insertion mutant.…”

Paramutation: The Chromatin Connection

“…A recent publication on paramutation in maize now provides convincing evidence that chromatin-level regulation underlies the phenotypic differences between alleles that participate in paramutation (Stam et al, 2002a). Two additional articles-one detailing paramutation-like effects associated with mammalian imprinting and the other describing paramutation after a change in ploidy in Arabidopsis-challenge us to understand how these cases fit into the developing paradigm explaining the molecular basis for paramutation (Herman et al, 2003;Mittelsten Scheid et al, 2003). Together, these studies suggest a mechanistic link between paramutation and other types of chromatin-level epigenetic regulation of gene expression.…”