Allele-specific DNA methylation at differentially methylated regions (DMRs) is critical for controlling imprinted genes expression and early embryogenesis in mammals. We previously demonstrated that the H19 imprinting control region (ICR), a DMR in the Igf2/H19 locus, was capable of acquiring paternal-allele-specific DNA methylation (imprinted methylation) when ectopically introduced into mouse genome (transgenic mouse; TgM). By using this genetic approach, we have dissected the H19 ICR activity and identified a couple of cis elements essential for protecting the fragment from undesired methylation on the maternal allele. To test if these elements, when combined and introduced into heterologous CG-rich sequences, are sufficient to generate imprinted methylation status, we conducted reconstitution experiments. We employed a bacteriophage lambda DNA fragment as a scratch sequence, generated its TgM, and found that the fragment acquired DNA methylation regardless of its parental origin. Then, we introduced CTCF binding sites and Sox-Oct motifs of the mouse H19 ICR into the lambda DNA (termed "LCb") and found that transgenic LCb fragment in mouse exhibited unmethylated state irrespective of their parental origin.
In this study, to search for cis elements essential for depositing paternal-allele specific methylation, we generated multiple TgM lines carrying a series of 5’-truncated H19 ICR fragments and identified the 118-bp region as a candidate element carrying such an activity. When these sequences were internally deleted from the intact H19 ICR transgene (2.9-kb) in mouse, the mutant fragment failed to acquire DNA methylation after paternal transmission. Finally, we appended the 118-bp sequences to the LCb fragment (termed “LCb118”) and tested its ability to instruct imprinted methylation in TgM. The fragment became capable of acquiring DNA methylation only when paternally inherited. These results demonstrated that the 118-bp region was necessary and sufficient for acquisition of paternal allele-specific DNA methylation in TgM. Furthermore, combination of specific DNA sequences, rather than general property of the local DNA sequence, such as GC-content, determines identity of a DMR.