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Differential methylation of imprinting control regions in mammals is essential for distinguishing the parental alleles from each other and regulating their expression accordingly. To ensure parent of origin-specific expression of imprinted genes and thereby normal developmental progression, the differentially methylated states that are inherited at fertilization must be stably maintained by DNA methyltransferase 1 throughout subsequent somatic cell division. Further epigenetic modifications, such as the acquisition of secondary regions of differential methylation, are dependent on the methylation status of imprinting control regions and are important for achieving the monoallelic expression of imprinted genes, but little is known about how imprinting control regions direct the acquisition and maintenance of methylation at these secondary sites. Recent analysis has identified mutations that reduce DNA methyltransferase 1 fidelity at some genomic sequences but not at others, suggesting that it may function differently at different loci. We examined the impact of the mutant DNA methyltransferase 1 P allele on methylation at imprinting control regions as well as at secondary differentially methylated regions and non-imprinted sequences. We found that while the P allele results in a major reduction in DNA methylation levels across the mouse genome, methylation is specifically maintained at imprinting control regions but not at their corresponding secondary DMRs. This result suggests that DNA methyltransferase 1 may work differently at imprinting control regions or that there is an alternate mechanism for maintaining methylation at these critical regulatory regions and that maintenance of methylation at secondary DMRs is not solely dependent on the methylation status of the ICR.
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A Resource for the Allele-Specific Analysis of DNA Methylation at Multiple Genomically Imprinted Loci in Mice
Abstract Genomically imprinted loci are expressed mono-allelically, dependent upon the parent of origin. Their regulation not only illuminates how chromatin regulates gene expression but also how chromatin can be reprogrammed every generation. Because of their distinct parent-of-origin regulation, analysis of imprinted loci can be difficult. Single nucleotide polymorphisms (SNPs) are required to accurately assess these elements allele specifically. However, publicly available SNP databases lack robust verification, making analysis of imprinting difficult. In addition, the allele-specific imprinting assays that have been developed employ different mouse strains, making it difficult to systemically analyze these loci. Here, we have generated a resource that will allow the allele-specific analysis of many significant imprinted loci in a single hybrid strain of Mus musculus. This resource includes verification of SNPs present within 10 of the most widely used imprinting control regions and allele-specific DNA methylation assays for each gene in a C57BL/6J and CAST/EiJ hybrid strain background.
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- Award ID(s):
- 1931697
- PAR ID:
- 10472310
- Publisher / Repository:
- G3
- Date Published:
- Journal Name:
- G3 Genes|Genomes|Genetics
- Volume:
- 8
- Issue:
- 1
- ISSN:
- 2160-1836
- Page Range / eLocation ID:
- 91 to 103
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)Abstract 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.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1534/g3.117.300417
