Restoration of Dlk1 and Rtl1 Is Necessary but Insufficient to Rescue Lethality in Intergenic Differentially Methylated Region (IG-DMR)-deficient Mice

Nozomi Takahashi,Ryota Kobayashi,Tomohiro Kono

doi:10.1074/jbc.m109.075325

Nozomi Takahashi, Ryota Kobayashi + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m109.075325

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Abstract

In the Dlk1-Dio3 imprinted domain, an intergenic differentially methylated region (IG-DMR) regulates the parental allele-specific expression of imprinted genes. The maternally inherited deletion of IG-DMR (IG-DMR((-/+))) results in perinatal lethality because of the overexpression of paternally expressed genes and repression of maternally expressed noncoding RNAs (ncRNAs), including Gtl2. To better understand the possible contribution of paternally expressed genes to the lethality, we attempted to rescue the lethality of IG-DMR((-/+)) mutants by restoring the paternally expressed genes. Because the paternally inherited Gtl2 deletion (Gtl2((+/-))) induced a decrease in the expression of paternally expressed genes, we crossed female IG-DMR heterozygous mice and male Gtl2 heterozygous mutant mice. The resultant IG-DMR((-/+))/Gtl2((+/-)) double mutant mice had normal expression levels of paternally expressed genes, and none of them showed perinatal lethality; however, most mice showed postnatal lethality with decreased expression of the maternally expressed ncRNAs. Thus, we inferred that paternally expressed genes are necessary for perinatal survivability and that maternally expressed ncRNAs are involved in postnatal lethality.

Highlights

The Dlk1-Dio3 domain on the distal arm of mouse chromosome 12 contains the paternally expressed genes Dlk1, Rtl1, and
Imprinting on the Dlk1-Dio3 domain is regulated primarily by an intergenic differentially methylated regions (DMRs) (IG-DMR) that is methylated during spermatogenesis [5]
The double mutants showed no lethality at the perinatal stage; most of them died at the postnatal stage. These results indicate that the restoration of Dlk1 and Rtl1 was necessary but insufficient for normal development in the IG-DMR(Ϫ/ϩ) mutants

Summary

Introduction

The Dlk1-Dio3 domain on the distal arm of mouse chromosome 12 contains the paternally expressed genes Dlk1, Rtl1, and. The paternally inherited Gtl2/Gtl2 DMR deletion (Gtl2(ϩ/Ϫ)) leads to a decrease in the expression levels of Dlk1 and Rtl1; the double mutant mice (IG-DMR(Ϫ/ϩ)/ Gtl2(ϩ/Ϫ)) would have normal expression levels of Dlk1 and Rtl1 (Fig. 1) [19]. These skeletal defects were thesized using the SuperScript III reverse transcriptase absent in all the IG-DMR(Ϫ/ϩ)/Gtl2(ϩ/Ϫ) double mutants (Invitrogen) in a reaction solution (20 ␮l) containing total RNA (four embryos) (Fig. 3).

Results

Conclusion