H19 imprinting control region methylation requires an imprinted environment only in the male germ line.

The 2.4-kb H19 imprinting control region (H19ICR) is required to establish parent-of-origin-specific epigenetic marks and expression patterns at the Igf2/H19 locus. H19ICR activity is regulated by DNA methylation. The ICR is methylated in sperm but not in oocytes, and this paternal chromosome-specific methylation is maintained throughout development. We recently showed that the H19ICR can work as an ICR even when inserted into the normally nonimprinted alpha fetoprotein locus. Paternal but not maternal copies of the ICR become methylated in somatic tissue. However, the ectopic ICR remains unmethylated in sperm. To extend these findings and investigate the mechanisms that lead to methylation of the H19ICR in the male germ line, we characterized novel mouse knock-in lines. Our data confirm that the 2.4-kb element is an autonomously acting ICR whose function is not dependent on germ line methylation. Ectopic ICRs become methylated in the male germ line, but the timing of methylation is influenced by the insertion site and by additional genetic information. Our results support the idea that DNA methylation is not the primary genomic imprint and that the H19ICR insertion is sufficient to transmit parent-of-origin-dependent DNA methylation patterns independent of its methylation status in sperm.