Abstract
BackgroundPaternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. The molecular mechanism by which differential methylation of the H19 ICR is established during gametogenesis and maintained after fertilization, however, is not fully understood. We previously showed that a 2.9-kb H19 ICR fragment in transgenic mice was differentially methylated only after fertilization, demonstrating that two separable events, gametic and post-fertilization methylation, occur at the H19 ICR. We then determined that CTCF/Sox-Oct motifs and the 478-bp sequence of the H19 ICR are essential for maintaining its maternal hypomethylation status and for acquisition of paternal methylation, respectively, during the post-fertilization period.ResultsUsing a series of 5′-truncated H19 ICR transgenes to dissect the 478-bp sequence, we identified a 118-bp region required for post-fertilization methylation activity. Deletion of the sequence from the paternal endogenous H19 ICR caused loss of methylation after fertilization, indicating that methylation activity of the sequence is required to protect endogenous H19 ICR from genome-wide reprogramming. We then reconstructed a synthetic DNA fragment in which the CTCF binding sites, Sox-Oct motifs, as well as the 118-bp sequence, were inserted into lambda DNA, and used it to replace the endogenous H19 ICR. The fragment was methylated during spermatogenesis; moreover, its allele-specific methylation status was faithfully maintained after fertilization, and imprinted expression of the both Igf2 and H19 genes was recapitulated.ConclusionsOur results identified a 118-bp region within the H19 ICR that is required for de novo DNA methylation of the paternally inherited H19 ICR during pre-implantation period. A lambda DNA-based artificial fragment that contains the 118-bp sequence, in addition to the previously identified cis elements, could fully replace the function of the H19 ICR in the mouse genome.
Highlights
Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes
Our results demonstrated that two separable methylation acquisition processes occurred at the H19 ICR: one during spermatogenesis that depends on the activity of the surrounding sequence, and another in post-fertilization embryos that is governed by its intrinsic activity
A 118‐bp sequence at the 5′‐segment of the H19 ICR is essential for acquisition of paternal methylation In previous work, we have narrowed down the sequence responsible for post-fertilization paternal methylation of the H19 ICR to a 478-bp region and demonstrated that it is required in vivo for normal development (Fig. 1a; [12, 15])
Summary
Paternal allele-specific DNA methylation of the H19 imprinting control region (ICR) regulates imprinted expression of the Igf2/H19 genes. In which a subset of genes is monoallelically expressed in a manner specific to the parent of origin, is a prominent epigenetic phenomenon in mammals. This form of regulation is essential for normal development; its dysregulation causes human diseases, including Beckwith–Wiedemann (BWS) and Silver–Russell syndromes (SRS) [1, 2]. The allelic methylation pattern is maintained after fertilization, throughout the lifespan: the germline-methylated ICRs on one of the alleles are resistant to genome-wide demethylation activity, which is associated with epigenetic reprogramming during the pre-implantation period, and non-methylated ICRs on the other allele are protected from allele-nonspecific de novo methylation during cell differentiation in post-implantation embryos. Differential methylation of the ICRs is regulated at three distinct stages: gametogenesis, pre-implantation, and post-implantation, ensuring monoallelic gene expression in somatic cells
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