The following is the abstract from a paper recently accepted for publication in Epigenetics & Chromatin.
Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse.
Megan Guntrum (’16), Ekaterina Vlasova (’15) and Tamara L. Davis
Background: 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. Results: 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. Conclusion: 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.
The following is the abstract for a poster presented at the Gordon Research Conference on Epigenetics, July 30 – August 4, 2017.
Secondary DMRs associated with imprinted loci are characterized by high levels of hemimethylation and 5-hydroxymethylcytosine
Julianna Nechin (’18), Emma Tunstall (’17), Nicole Hamagami (’16), Chris Pathmanabhan (’20), Samantha Forestier (’20) and Tamara L. Davis
Differential distribution of DNA methylation on the alleles of imprinted genes functions both to distinguish the alleles based on their parental origin and regulate them to achieve monoallelic expression. DNA methylation at primary differentially methylated regions (DMRs) that function as imprinting control regions (ICRs) is inherited from the gametes, is consistently maintained on one parental allele throughout development, and functions to modulate imprinted expression. In contrast, parent of origin-specific DNA methylation at secondary DMRs is acquired during post-fertilization development; while DNA methylation of these regions is believe to play a role in maintaining imprinted expression at individual imprinted loci, they lack the stability of DNA methylation that is inherited via the gamete. Our previous analysis of DNA methylation patterns within the mouse Dlk1/Gtl2 imprinting cluster illustrated that the secondary DMRs associated with the Dlk1 and Gtl2 genes exhibit significantly higher levels of hemimethylation that the primary imprinting control region, IG-DMR. We have recently extended this analysis by investigating hemimethylation profiles at both paternally and maternally methylated primary and secondary DMRs located at other genomic regions. Our data illustrate that hemimethylation levels are low at both paternally and maternally methylated primary DMRs (H19 and Snrpn, respectively), while hemimethylation levels are significantly higher at both paternally and maternally methylated secondary DMRs across the genome (H19, Cdkn1c and Ndn). We further show that secondary DMRs have higher levels of 5-hydroxymethylcytosine than primary DMRs, and suggest that the increase in 5hmC may be responsible for the high level of hemimethylation at these loci due to decreases in DNA methylation fidelity and/or loss of methylation via TET enzyme activity followed by base excision repair. This poster will describe our current results.