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.
Highlights
Genomic 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
Two cis elements, CTCF-binding sites and Sox-Oct motifs within the H19 ICR, have been shown to be essential for this process, since mutation of these elements causes aberrant methylation of the maternal ICR after implantation [18,19,20,21]. These results collectively suggested that the differentially methylated state of the H19 ICR is governed by distinct processes during gametogenesis, preimplantation, and postimplantation, but among which the mechanisms after fertilization are more decisive in determination of imprinting
We demonstrate that a reconstituted DNA fragment, composed of multiple cis regulatory sequences found in the H19 ICR, are capable of recapitulating appropriate imprinted methylation dynamics after fertilization
Summary
Genomic 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. Using transgenic mice (TgM), we previously demonstrated that the H19 ICR possesses autonomous activity to acquire paternal-allele-specific DNA methylation after fertilization. This activity is indispensable for the maintenance of imprinted methylation at the endogenous H19 ICR during the preimplantation period. The imprinted genes are marked by epigenetic modifications, among which allele-specific DNA methylation at the imprinting control regions (ICRs) plays a pivotal role in their unique expression pattern, as demonstrated in DNA methyltransferase deficient mice [3,4,5] and ICR-knockout mice [6,7,8]. We assume that a specific mechanism, by which allelic methylation is maintained at restricted sequences against genome-wide epigenetic reprogramming during preimplantation development, defines imprinted genomic loci [14]
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