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Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse.

ABSTRACT: Differential DNA methylation plays a critical role in the regulation of imprinted genes. The differentially methylated state of the imprinting control region is inherited via the gametes at fertilization, and is stably maintained in somatic cells throughout development, influencing the expression of genes across the imprinting cluster. In contrast, DNA methylation patterns are more labile at secondary differentially methylated regions which are established at imprinted loci during post-implantation development. To investigate the nature of these more variably methylated secondary differentially methylated regions, we adopted a hairpin linker bisulfite mutagenesis approach to examine CpG dyad methylation at differentially methylated regions associated with the murine Dlk1/Gtl2 imprinting cluster on both complementary strands.We observed homomethylation at greater than 90% of the methylated CpG dyads at the IG-DMR, which serves as the imprinting control element. In contrast, homomethylation was only observed at 67-78% of the methylated CpG dyads at the secondary differentially methylated regions; the remaining 22-33% of methylated CpG dyads exhibited hemimethylation.We propose that this high degree of hemimethylation could explain the variability in DNA methylation patterns at secondary differentially methylated regions associated with imprinted loci. We further suggest that the presence of 5-hydroxymethylation at secondary differentially methylated regions may result in hemimethylation and methylation variability as a result of passive and/or active demethylation mechanisms.

SUBMITTER: Guntrum M

PROVIDER: S-EPMC5480104 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the <i>Dlk1</i>/<i>Gtl2</i> imprinting cluster in mouse.

Guntrum Megan M Vlasova Ekaterina E Davis Tamara L TL

Epigenetics & chromatin 20170621

<h4>Background</h4>Differential DNA methylation plays a critical role in the regulation of imprinted genes. The differentially methylated state of the imprinting control region is inherited via the gametes at fertilization, and is stably maintained in somatic cells throughout development, influencing the expression of genes across the imprinting cluster. In contrast, DNA methylation patterns are more labile at secondary differentially methylated regions which are established at imprinted loci du ...[more]

PMID: 28649282

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Project description:BackgroundThe Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.ResultsA previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Delta5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Delta5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Delta5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.ConclusionThese data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Delta5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.

Project description:BackgroundMicroRNAs play an important functional role in post-transcriptional gene regulation. One of the largest known microRNA clusters is located within the imprinted Dlk1/Gtl2 region on human chromosome 14 and mouse chromosome 12. This cluster contains more than 40 microRNA genes that are expressed only from the maternal chromosome in mouse.ResultsTo shed light on the function of these microRNAs and possible crosstalk between microRNA-based gene regulation and genomic imprinting, we performed extensive in silico analyses of the microRNAs in this imprinted region and their predicted target genes.Bioinformatic analysis reveals that these microRNAs are highly conserved in both human and mouse. Whereas the microRNA precursors at this locus mostly belong to large sequence families, the mature microRNAs sequences are highly divergent. We developed a target gene prediction approach that combines three widely used prediction methods and achieved a sufficiently high prediction accuracy. Target gene sets predicted for individual microRNAs derived from the imprinted region show little overlap and do not differ significantly in their properties from target genes predicted for a group of randomly selected microRNAs. The target genes are enriched with long and GC-rich 3' UTR sequences and are preferentially annotated to development, regulation processes and cell communication. Furthermore, among all analyzed human and mouse genes, the predicted target genes are characterized by consistently higher expression levels in all tissues considered.ConclusionOur results suggest a complex evolutionary history for microRNA genes in this imprinted region, including an amplification of microRNA precursors in a mammalian ancestor, and a rapid subsequent divergence of the mature sequences. This produced a broad spectrum of target genes. Further, our analyses did not uncover a functional relation between imprinted gene regulation of this microRNA-encoding region, expression patterns or functions of predicted target genes. Specifically, our results indicate that these microRNAs do not regulate a particular set of genes. We conclude that these imprinted microRNAs do not regulate a particular set of genes. Rather, they seem to stabilize expression of a variety of genes, thereby being an integral part of the genome-wide microRNA gene regulatory network.

Project description:BACKGROUND:In mammals, the regulation of imprinted genes is controlled by differential methylation at imprinting control regions which acquire parent of origin-specific methylation patterns during gametogenesis and retain differences in allelic methylation status throughout fertilization and subsequent somatic cell divisions. In addition, many imprinted genes acquire differential methylation during post-implantation development; these secondary differentially methylated regions appear necessary to maintain the imprinted expression state of individual genes. Despite the requirement for both types of differentially methylated sequence elements to achieve proper expression across imprinting clusters, methylation patterns are more labile at secondary differentially methylated regions. To understand the nature of this variability, we analyzed CpG dyad methylation patterns at both paternally and maternally methylated imprinted loci within multiple imprinting clusters. RESULTS:We determined that both paternally and maternally methylated secondary differentially methylated regions associated with imprinted genes display high levels of hemimethylation, 29-49%, in comparison to imprinting control regions which exhibited 8-12% hemimethylation. To explore how hemimethylation could arise, we assessed the differentially methylated regions for the presence of 5-hydroxymethylcytosine which could cause methylation to be lost via either passive and/or active demethylation mechanisms. We found enrichment of 5-hydroxymethylcytosine at paternally methylated secondary differentially methylated regions, but not at the maternally methylated sites we analyzed in this study. CONCLUSIONS:We found high levels of hemimethylation to be a generalizable characteristic of secondary differentially methylated regions associated with imprinted genes. We propose that 5-hydroxymethylcytosine enrichment may be responsible for the variability in methylation status at paternally methylated secondary differentially methylated regions associated with imprinted genes. We further suggest that the high incidence of hemimethylation at secondary differentially methylated regions must be counteracted by continuous methylation acquisition at these loci.

Dataset Information

Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse.

Publications

Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the <i>Dlk1</i>/<i>Gtl2</i> imprinting cluster in mouse.

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