The imprinted expression of the mouse Igf2/H19 locus is governed by the differential methylation of the imprinting control region (ICR), which is established initially in germ cells and subsequently maintained in somatic cells, depending on its parental origin. By grafting a 2.9-kbp H19 ICR fragment into a human -globin yeast artificial chromosome in transgenic mice, we previously showed that the ICR could recapitulate imprinted methylation and expression at a heterologous locus, suggesting that the H19 ICR in the -globin locus contained sufficient information to maintain the methylation mark (K. Tanimoto Curiously, however, the transgenic H19 ICR was not methylated in sperm, which was distinct from that seen in the endogenous locus. Here, we reevaluated the ability of the H19 ICR to mark the parental origin using more rigid criteria. In the testis, the methylation levels of the solitary 2.9-kbp transgenic ICR fragment varied significantly between six transgenic mouse lines. However, in somatic cells, the paternally inherited ICR fragment exhibited consistently higher methylation levels at five out of six randomly integrated sites in the mouse genome. These results clearly demonstrated that the H19 ICR could acquire parent-of-origin-dependent methylation after fertilization independently of the chromosomal integration site or the prerequisite methylation acquisition in male germ cells.
Genomic imprinting is a major monoallelic gene expression regulatory mechanism in mammals, and depends on gametespecific DNA methylation of specialized cis-regulatory elements called imprinting control regions (ICRs). Allele-specific DNA methylation of the ICRs is faithfully maintained at the imprinted loci throughout development, even in early embryos where genomes undergo extensive epigenetic reprogramming, including DNA demethylation, to acquire totipotency. We previously found that an ectopically introduced H19 ICR fragment in transgenic mice acquired paternal allele-specific methylation in the somatic cells of offspring, whereas it was not methylated in sperm, suggesting that its gametic and postfertilization modifications were separable events. We hypothesized that this latter activity might contribute to maintenance of the methylation imprint in early embryos. Here, we demonstrate that methylation of the paternally inherited transgenic H19 ICR commences soon after fertilization in a maternal DNMT3A-and DNMT3L-dependent manner. When its germline methylation was partially obstructed by insertion of insulator sequences, the endogenous paternal H19 ICR also exhibited postfertilization methylation. Finally, we refined the responsible sequences for this activity in transgenic mice and found that deletion of the 5′ segment of the endogenous paternal H19 ICR decreased its methylation after fertilization and attenuated Igf2 gene expression. These results demonstrate that this segment of the H19 ICR is essential for its de novo postfertilization DNA methylation, and that this activity contributes to the maintenance of imprinted methylation at the endogenous H19 ICR during early embryogenesis.
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.
Imprinted expression of the mouse Igf2/H19 locus is controlled by parent-of-origin-specific methylation of the imprinting control region (ICR). We previously demonstrated that when placed in a heterologous genomic context, the H19 ICR fragment contains an intrinsic activity that allows it to acquire differential methylation in somatic cells but not in germ cells. In the present study, we investigated the requirements for the CTCF-binding sites of the ICR in the acquisition of post-fertilization methylation. To this end, two mutant ICR fragments were introduced into the human beta-globin locus in a yeast artificial chromosome transgenic mouse (TgM) model: 4xMut had mutations in all four ICR CTCF-binding sites that prevented CTCF binding but retained the methylation target CpG motifs, and -9CG harbored mutations in the CpG motifs within the CTCF-binding sites but each site retained constitutive CTCF-binding activity. In TgM germ cells and pre-implantation blastocysts, the absence of CTCF-binding sites (4xMut) did not lead to hypermethylation of the transgenic H19 ICR. However, after implantation, the mutations of CTCF sites (4xMut and -9CG) affected the maintenance of methylation. These results demonstrated that although the CTCF-binding sites are indispensable for maintenance of the unmethylated state of the maternal ICR in post-implantation embryos, they are not required to establish paternal-allele-specific methylation of the transgenic H19 ICR in pre-implantation embryos.
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.
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