Abnormal methylation at the maternally inherited H19 imprinted control region (H19 ICR) is one of the causative alterations leading to pathogenesis of Beckwith-Wiedemann syndrome (BWS). Recently, it was shown in human BWS patients, as well as mouse cell culture experiments, that Sox-Oct motifs (SOM) in the H19 ICR might play a role in protecting the maternal ICR from de novo DNA methylation. By grafting a mouse H19 ICR fragment into a human β-globin yeast artificial chromosome (YAC) followed by analysis in transgenic mice (TgM), we showed previously that the fragment carried sufficient information to establish and maintain differential methylation after fertilization. To examine possible functions of the SOM in the establishment and/or maintenance of differential methylation, two kinds of YAC-TgM were generated in this study. In the ΔSOM TgM, carrying the mouse H19 ICR bearing an SOM deletion, a maternally inherited transgenic ICR exhibited increased levels of methylation around the deletion site, in comparison to the wild-type control, after implantation. In the λ + CTCF + b (LCb) TgM, carrying a 2.3 kb λ DNA fragment supplemented with the fragment b including the SOM and four CTCF binding sites, maternally and some of the paternally inherited LCb fragments were significantly less methylated when compared with a control λ + CTCF fragment that was supplemented only with additional CTCF sites; the λ + CTCF was substantially methylated regardless of the parent of origin after implantation. These results demonstrated that the SOM in the maternal H19 ICR was required for maintaining surrounding sequences in the unmethylated state in vivo.

cIn 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 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 of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited 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.