The following is the abstract from a paper accepted at Epigenetics & Chromatin in July 2023.

Methylation is maintained specifically at imprinting control regions but not other DMRs associated with imprinted genes in mice bearing a mutation in the Dnmt1 intrinsically disordered domain

Shaili Regmi (’21), Lana Giha (’24), Ahado Ali (’25), Christine Siebels-Lindquist (’20) and Tamara L. Davis

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 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.

The following are abstracts for two posters presented at the Allied Genetic Conference (TAGC24), March 6-10, 2024.

Determining the cause of hemimethylation at secondary DMRs associated with imprinted genes

Jordan Ellis-Pugh (’21), Jacqueline Saulnier (’24), Jaclyn Lo (’22), Courtney Link (’20), Sophia Gibson (’20), Isabel Oalican (’23), Julia Kesack (’23), Naideline Raymond (’19) and Tamara L. Davis

The monoallelic expression of imprinted genes is regulated by parent of origin-specific differential DNA methylation. Imprinting control regions, or primary DMRs, regulate epigenetic profiles and expression patterns across imprinting clusters. In contrast, differential methylation at secondary DMRs influences the expression of individual imprinted genes. Unlike primary DMRs, secondary DMRs display considerable variability in their DNA methylation patterns and have high levels of hemimethylation. We hypothesize that the high level of hemimethylation is the result of 5-hydroxymethylcytosine enrichment at these loci. We tested this hypothesis by subjecting mouse genomic DNA to parallel bisulfite and oxidative bisulfite mutagenesis reactions. Loci associated with imprinted and non-imprinted genes were amplified from the mutagenized DNA and subjected to NextGeneration amplicon sequencing. This approach allowed us to determine the amount of 5hmC at each locus through a subtractive method as the bisulfite reaction identifies 5-methylcytosine and 5-hydroxymethylcytosine while the oxidative bisulfite reaction only identifies 5-methylcytosine. Our preliminary results illustrate low levels of 5-hmC at primary DMRs and non-imprinted loci. Secondary DMRs show less consistent results, but generally indicate that there may be 5-hmC enrichment at paternally methylated secondary DMRs. Additional studies are underway to better understand the distribution of 5-hmC at differentially methylated sequences associated with imprinted genes.

Increased hemimethylation levels correlate with methylation reductions in DNA methyltransferase mutant mouse embryos

Chloe Tang (’24) and Tamara L. Davis

Mammals have evolved to have many forms of gene regulation. Genomic imprinting is one form that only allows the expression of one parental allele from an organism’s genome. Imprinted genes are regulated epigenetically through DNA methylation, facilitated by DNA methyltransferase (DNMT1), where a methyl group (CH3) is added to one parental allele to generate a differentially methylated region (DMR). Differential methylation results in differential gene expression of the parental alleles and the proper establishment of methylation patterns is crucial for embryonic development as abnormal methylation patterns can lead to inappropriate gene expression, developmental disorders, and diseases such as cancer. Methylation at primary DMRs, which is inherited at fertilization, is stable and symmetric throughout development whereas methylation at secondary DMRs, which is acquired during early embryogenesis, is less stable and asymmetric. In addition, previous research suggested that methylation is well maintained at primary DMRs in mouse embryos with a hypomorphic mutation of Dnmt1 but is dramatically reduced at secondary DMRs and non-imprinted loci. These pieces of evidence suggest that DNMT1 might function differently at different loci. We hypothesize that reduced DNMT1 fidelity leads to an increase in hemimethylation, resulting to the observed methylation loss at secondary DMRs and non-imprinted loci. To test this hypothesis, I am analyzing the methylation patterns at both non-imprinted and imprinted loci in 12.5 days post conception (dpc) wild-type and Dnmt1 mutant mouse embryos. Analysis of methylation patterns at three paternally methylated loci associated with imprinted genes indicated that the paternally methylated primary DMR was minimally affected but methylation was dramatically reduced at both secondary DMRs in Dnmt1 mutant vs. wild-type embryos. In addition, the hemimethylation level at secondary DMRs was significantly higher in mutants than in wild type but this difference was less pronounced at the primary DMR. This inverse correlation between methylation and hemimethylation levels suggests that DNMT1 is essential in maintaining methylation patterns, especially regarding methylation symmetry during fetal development and that its inconsistent function leads to a loss of methylation and increase in hemimethylation. I’m currently extending my analysis to maternally methylated DMRs and non-imprinted loci to see if the pattern is generalizable.