Synthetic DNA fragments bearing ICR cis elements become differentially methylated and recapitulate genomic imprinting in transgenic mice

Abstract

BackgroundGenomic imprinting is governed by allele-specific DNA methylation at imprinting control regions (ICRs), and the mechanism controlling its differential methylation establishment during gametogenesis has been a subject of intensive research interest. However, recent studies have reported that gamete methylation is not restricted at the ICRs, thus highlighting the significance of ICR methylation maintenance during the preimplantation period where genome-wide epigenetic reprogramming takes place. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. Furthermore, this activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. In addition, we showed that a specific 5′ fragment of the H19 ICR is required for its paternal methylation after fertilization, while CTCF and Sox-Oct motifs are essential for its maternal protection from undesirable methylation after implantation.ResultsTo ask whether specific cis elements are sufficient to reconstitute imprinted methylation status, we employed a TgM co-placement strategy for facilitating detection of postfertilization methylation activity and precise comparison of test sequences. Bacteriophage lambda DNA becomes highly methylated regardless of its parental origin and thus can be used as a neutral sequence bearing no inclination for differential DNA methylation. We previously showed that insertion of only CTCF and Sox-Oct binding motifs from the H19 ICR into a lambda DNA (LCb) decreased its methylation level after both paternal and maternal transmission. We therefore appended a 478-bp 5′ sequence from the H19 ICR into the LCb fragment and found that it acquired paternal-allele-specific methylation, the dynamics of which was identical to that of the H19 ICR, in TgM. Crucially, transgene expression also became imprinted. Although there are potential binding sites for ZFP57 (a candidate protein thought to control the methylation imprint) in the larger H19 ICR, they are not found in the 478-bp fragment, rendering the role of ZFP57 in postfertilization H19 ICR methylation a still open question.ConclusionsOur results demonstrate that a differentially methylated region can be reconstituted by combining the activities of specific imprinting elements and that these elements together determine the activity of a genomically imprinted region in vivo.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0207-z) contains supplementary material, which is available to authorized users.

Recapitulation of gametic DNA methylation and its post-fertilization maintenance with reassembled DNA elements at the mouse Igf2/H19 locus

“…First, ZFP57 binds to DNA in a CpG methylation-dependent manner, whereas the transgenic H19 ICR in sperm is unmethylated. Consistent with this, in our previous work [15], we failed to demonstrate ZFP57 binding to the 478-bp sequence in gel-shift assays. In addition, the DNA methylation status of the endogenous H19 ICR is not affected in Zfp57-knockout mice [16,18].…”

“…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]). Furthermore, we showed that postfertilization, allele-specific methylation was recapitulated in a λ-phage-based synthetic DNA fragment in the TgM only when the 478-bp sequence was included [15].…”

“…1a; [12,15]). Furthermore, we showed that postfertilization, allele-specific methylation was recapitulated in a λ-phage-based synthetic DNA fragment in the TgM only when the 478-bp sequence was included [15]. To further define the responsible sequence in this study, we generated H19 ICR fragments with a series of 5′-deletions of the 478-bp region (~ 60 bp intervals) and inserted them into the human β-globin YAC to generate TgMs (Fig.…”

“…The H19 ICR, located approximately at − 4 to − 2 kb relative to the transcription start site of H19 gene is contained within a 2.9-kb SacI (Sa)-BamHI (B) fragment. DNA sequence (478 bp) shown to be sufficient for acquiring paternal methylation in TgM [15] is marked in gray. Dots (1)(2)(3)(4) indicate CTCF-binding sites.…”

“…The regulatory sequences we have identified thus far (a 478-bp segment of the 765-bp fragment mentioned above, the CTCF-binding sites and Sox-Oct motifs) in the H19 ICR are capable of transforming a normally nonimprinted λ DNA sequence into the DMR, when they are assembled together on a λ DNA fragment and assayed in TgM [15]. However, it remains to be determined whether the synthetic fragment can fully reproduce the genomic imprinting phenomena at endogenous mouse Igf2/H19 gene locus, as allele-specific methylation of the fragment was observed only during the post-fertilization period in the transgenic β-globin gene locus.…”