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

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 theH19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited sequence, normally methylated after implantation in the absence ofH19 ICR se-quences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the pater-nally inherited sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of theH19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNAmethylation. These results show that postfertilization methylation im-printing is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of theH19 ICR, while maternal allele-specific activities protect the allele from de novoDNAmethylation. The mouse insulin-like growth factor 2 (Igf2) and noncodingH19 genes, located about 90 kb apart on distal chromosome 7 (see Fig. 1A), are preferentially expressed from the paternal and maternal alleles, respectively. Monoallelic expression of thes