Bryn Mawr College Scholarship, Research, and Creative Work at Bryn Mawr College Biology Faculty Research and Scholarship Biology 2017 Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse Megan Guntrum Ekaterina Vlasova Tamara L. Davis [email protected] Let us know how access to this document benefits ouy . Follow this and additional works at: http://repository.brynmawr.edu/bio_pubs Part of the Biology Commons Custom Citation M. Guntrum, E. Vlasova and T.L. Davis 2017. "Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse." Epigenetics & Chromatin 10:31. This paper is posted at Scholarship, Research, and Creative Work at Bryn Mawr College. http://repository.brynmawr.edu/bio_pubs/24 For more information, please contact [email protected]. Guntrum et al. Epigenetics & Chromatin (2017) 10:31 DOI 10.1186/s13072-017-0138-0 Epigenetics & Chromatin RESEARCH Open Access Asymmetric DNA methylation of CpG dyads is a feature of secondary DMRs associated with the Dlk1/Gtl2 imprinting cluster in mouse Megan Guntrum, Ekaterina Vlasova and Tamara L. Davis* Abstract Background: Diferential DNA methylation plays a critical role in the regulation of imprinted genes. The diferentially methylated state of the imprinting control region is inherited via the gametes at fertilization, and is stably maintained in somatic cells throughout development, infuencing the expression of genes across the imprinting cluster. In con- trast, DNA methylation patterns are more labile at secondary diferentially methylated regions which are established at imprinted loci during post-implantation development. To investigate the nature of these more variably methylated secondary diferentially methylated regions, we adopted a hairpin linker bisulfte mutagenesis approach to examine CpG dyad methylation at diferentially 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 methyl- ated CpG dyads at the secondary diferentially methylated regions; the remaining 22–33% of methylated CpG dyads exhibited hemimethylation. Conclusions: We propose that this high degree of hemimethylation could explain the variability in DNA methylation patterns at secondary diferentially methylated regions associated with imprinted loci. We further suggest that the presence of 5-hydroxymethylation at secondary diferentially methylated regions may result in hemimethylation and methylation variability as a result of passive and/or active demethylation mechanisms. Keywords: Genomic imprinting, DNA methylation, 5-Hydroxymethylcytosine, Gtl2, Gametic DMR, Somatic DMR, Epigenetics Background imprinted genes are found within clusters that contain a Genomic imprinting in mammals results in the parent CpG-rich imprinting control region (ICR) that functions of origin-specifc monoallelic expression of a subset of both to specify parental origin and to regulate imprinted genes. Achieving the appropriate balance of gene expres- expression of the genes within the cluster [3, 4]. Monoal- sion from the maternally and paternally contributed lelic expression of imprinted genes is achieved via multi- genomes via the establishment of parental allele-spe- ple mechanisms, including epigenetic modifcations such cifc imprinting marks is crucial for normal growth and DNA methylation and histone modifcations, as well as development. Terefore, it is critical to understand the the activity of long noncoding RNAs [3, 4]. mechanisms responsible for controlling the expression Diferential DNA methylation at imprinted loci has of imprinted genes. To date, approximately 150 mamma- been shown to play an important role in distinguishing lian genes have been identifed as imprinted [1, 2]. Most the parental alleles and regulating their expression [5–9]. Diferentially methylated regions (DMRs) associated *Correspondence: [email protected] with imprinted genes fall into two categories: primary Department of Biology, Bryn Mawr College, 101 N. Merion Avenue, and secondary DMRs. Primary, or gametic, DMRs serve Bryn Mawr, PA 19010‑2899, USA © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guntrum et al. Epigenetics & Chromatin (2017) 10:31 Page 2 of 14 as imprinting control regions (ICRs), functioning both at two additional DMRs associated with the Dlk1/Gtl2 to specify parental origin and as a shared regulatory ele- imprinting cluster: the IG-DMR, a primary DMR, and ment that controls the expression of genes throughout the Gtl2-DMR, a secondary DMR [14, 28]. We also quan- the associated imprinting cluster. Primary DMRs acquire tifed the level of 5-hydroxymethylation (5-hmC) at the DNA methylation on one of the two parental alleles dur- IG-, Gtl2- and Dlk1-DMRs to determine whether there is ing gametogenesis and remain diferentially methyl- a correlation between high levels of hemimethylation and ated from fertilization throughout development, thereby high levels of 5-hydroxymethylation. Our results support marking parental origin [3]. Te diferentially methyl- the hypothesis that high levels of 5-hmC may contribute ated state of primary DMRs can afect expression in a to methylation instability at secondary DMRs associated variety of ways. For example, primary DMRs can regu- with imprinted genes. late gene expression through their diferential ability to bind enhancer blocking proteins, thereby infuencing the Results activity of an insulator [10, 11]. In other cases, primary CpG dyads within the Gtl2‑DMR display high levels DMRs are located at promoters, where they have been of hemimethylation shown to directly infuence the expression of long non- To determine whether asymmetric methylation is coding RNAs that subsequently regulate the expression unique to the Dlk1-DMR or is a feature of other second- of other genes in the imprinting cluster [12–15]. In con- ary DMRs associated with imprinted loci, we examined trast, secondary DMRs acquire parent of origin-specifc CpG dyad methylation at the linked Gtl2-DMR. We had DNA methylation after implantation [16–19]. Secondary previously analyzed DNA methylation on the coding DMRs are generally located at promoters or within gene strand of the Gtl2-DMR and observed moderate vari- bodies, and the acquisition of parental allele-specifc ability in the methylation status, with the 5′ half of the DNA methylation at these sequences is dependent on region analyzed displaying lower average DNA methyla- diferential methylation of the associated ICR, while the tion levels than the 3′ half [28]. We therefore assessed converse is not true [8, 9, 20]. While secondary DMRs do the DNA methylation status of cytosines located in 22 not function as primary imprinting marks, methylation pairs of complementary CpG dinucleotides spanning of these regions frequently corresponds with gene silenc- this region to determine whether these CpG dyads were ing and may play a role in maintaining imprinted expres- homomethylated versus hemimethylated (Fig. 1). All of sion [17, 21–23]. our experiments were conducted using F1 hybrid tissues Te DNA methylation associated with primary DMRs collected from crosses between C57BL/6 (B6) and a spe- is very stable, with the methylated allele displaying cially derived strain containing Mus musculus castaneus 90–100% methylation at the cytosines located in CpG (CAST)-derived sequences from chromosome 12 on an dinucleotides throughout development [5, 19, 24–27]. otherwise C57BL/6 genetic background (CAST12) [18, DNA methylation at secondary DMRs is less consistent. 28], allowing us to distinguish paternally inherited alleles For example, methylation at secondary DMRs located from maternally inherited alleles based on sequence pol- at the H19 and Gtl2 promoters average 70 and 78.9%, ymorphisms (detailed in the “Methods”). respectively, as compared to methylation at their respec- We analyzed CpG dyad methylation in DNA derived tive primary DMRs, which average ~90 and 95.8% [5, from four developmental stages: 7.5 d.p.c. embryo, 14.5 28]. We recently illustrated that the highly variable DNA d.p.c. embryo, 5 d.p.p. liver and adult liver. Te DNA methylation pattern at the secondary DMR associated methylation patterns on each parental allele were con- with the imprinted Dlk1 gene is asymmetric, with 35% sistent throughout development and were also similar of the methylated CpG dyads displaying hemimethyla- in tissues obtained from reciprocal crosses (Figs. 2, 3). tion [18]. Te trend that DNA methylation is more sta- Across all four developmental stages, cytosines in CpG ble at primary DMRs than at secondary DMRs associated dinucleotides were methylated 80-93% of the time on with imprinted genes has also been observed at human paternal alleles