The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

Abstract

In the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a λ DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited λ sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited λ sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.

ACKNOWLEDGMENTS

We thank Doug Engel (University of Michigan) and Jörg Bungert (University of Florida) for critically reading the manuscript and Y. Tanimoto for outstanding technical assistance.

This work was supported in part by a research grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research and a Grant-in-Aid for Young Scientists (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT; KAKENHI grant number 20678002) to K.T. E.O. is a research fellow of the Japan Society for the Promotion of Science (JSPS; KAKENHI grant number 11J00587).

We declare that we have no conflict of interest.