Analysis of DNA methylation acquisition at the imprinted Dlk1 locus reveals asymmetry at CpG dyads
- Alyssa Gagne†1,
- Abigail Hochman†1,
- Mahvish Qureshi1,
- Celia Tong1,
- Jessica Arbon1,
- Kayla McDaniel1 and
- Tamara L Davis1Email author
© Gagne et al.; licensee BioMed Central Ltd. 2014
Received: 11 February 2014
Accepted: 20 May 2014
Published: 29 May 2014
Differential distribution of DNA methylation on the parental alleles of imprinted genes distinguishes the alleles from each other and dictates their parent of origin-specific expression patterns. While differential DNA methylation at primary imprinting control regions is inherited via the gametes, additional allele-specific DNA methylation is acquired at secondary sites during embryonic development and plays a role in the maintenance of genomic imprinting. The precise mechanisms by which this somatic DNA methylation is established at secondary sites are not well defined and may vary as methylation acquisition at these sites occurs at different times for genes in different imprinting clusters.
In this study, we show that there is also variability in the timing of somatic DNA methylation acquisition at multiple sites within a single imprinting cluster. Paternal allele-specific DNA methylation is initially acquired at similar stages of post-implantation development at the linked Dlk1 and Gtl2 differentially methylated regions (DMRs). In contrast, unlike the Gtl2-DMR, the maternal Dlk1-DMR acquires DNA methylation in adult tissues.
These data suggest that the acquisition of DNA methylation across the Dlk1/Gtl2 imprinting cluster is variable. We further found that the Dlk1 differentially methylated region displays low DNA methylation fidelity, as evidenced by the presence of hemimethylation at approximately one-third of the methylated CpG dyads. We hypothesize that the maintenance of DNA methylation may be less efficient at secondary differentially methylated sites than at primary imprinting control regions.
KeywordsGenomic imprinting DNA methylation Dlk1 Secondary DMR Epigenetics
Genomic imprinting in mammals results in the monoallelic expression of approximately 150 genes [1, 2]. The majority of these imprinted genes are found in clusters distributed throughout the mammalian genome, with each cluster containing two or more imprinted genes as well as an imprinting control region (ICR) . One common feature of the CpG-rich ICRs is the presence of a gametic, or primary, differentially methylated region (DMR) which generally functions both to identify parental origin and to regulate expression of the imprinted genes within the cluster, either directly or indirectly . Establishment of parent of origin-specific DNA methylation at the ICR occurs during gametogenesis and the zygote either inherits a methylated allele from its mother or from its father at fertilization. Differential methylation at the ICR is then maintained throughout development such that the parental alleles can be distinguished from each other and the expression of their adjacent imprinted genes regulated appropriately.
In addition to the differential methylation present at the ICR, some imprinted loci also acquire distinct secondary regions of differential methylation during post-implantation development [4–6]. It has been proposed that the establishment of differential DNA methylation at secondary DMRs could serve as a mechanism for maintaining imprinted expression at developmental times when the primary imprinting control region is no longer functioning [6, 7]. Support for this hypothesis comes from a recent study of DNA methylation and expression at the imprinted Gpr1-Zdbf2 locus at which the maternally methylated Gpr1 DMR functions as the gametic imprinting mark responsible for establishing paternal allele-specific expression while paternal allele-specific DNA methylation at the secondary Zdbf2 DMR is established after the onset of imprinted Zdbf2 expression . Paternal allele-specific expression of Zdbf2 is maintained after DNA methylation at the Gpr1 DMR becomes biallelic, suggesting that the paternally methylated secondary Zdbf2 DMR functions to maintain monoallelic expression at this locus. Furthermore, biallelic methylation at the Zdbf2 DMR in offspring derived from Dnmt3L mat-/- mothers correlated with biallelic expression of Zdbf2. While the exact mechanism responsible for the parental allele-specific acquisition of DNA methylation at secondary DMRs has not yet been determined, it is clear that there is a relationship between the epigenetic states at primary and secondary DMRs [9, 10].
The majority of secondary DMRs found at imprinted genes are methylated on the paternally-inherited allele, suggesting that there may be a common mechanism responsible for establishing secondary imprinting marks. At the same time, it is clear that not all secondary DMRs are acquired at the same developmental stage. Paternal allele-specific DNA methylation is established at Gtl2 prior to 6.5 days post coitum (d.p.c.), at Cdkn1c between 7.5 and 9.5 d.p.c. and at Igf2r region 1 during late embryogenesis [7, 11–13]. Gtl2, Cdkn1c, and Igf2r are located on mouse chromosomes 12, 7, and 17, respectively. DNA methylation at secondary DMRs has generally been shown to affect the expression of a single adjacent imprinted gene, rather than the expression of the entire imprinting cluster [6, 7]. Therefore, it is possible that the same molecular machinery is used to establish DNA methylation at these sites and that the difference in temporal acquisition reflects the time at which it becomes critical to maintain monoallelic expression for each imprinted gene.
The Dlk1-Dio3 cluster of imprinted genes spans 1 Mb on mouse chromosome 12 and contains three paternally expressed protein-coding genes (Dlk1, Rtl1, and Dio3), multiple maternally expressed untranslated RNAs (including Gtl2), and at least three DMRs that are methylated on the paternal allele [14–18]. The IG-DMR, located between Dlk1 and Gtl2, functions as the ICR on the unmethylated maternally inherited allele . Secondary DMRs have been identified at the promoter of Gtl2 and in exon 5 of Dlk1. Evidence suggests that the Gtl2-DMR has a functional role; studies of the mouse Gtl2-DMR and its human homolog, MEG3-DMR, indicate that methylation of this region directly influences expression in cis[10, 20, 21]. Although the functional role of differential methylation at Dlk1 has not been determined, both the Gtl2- and Dlk1-DMRs become methylated on the paternal allele following fertilization, and the Gtl2-DMR has been shown to acquire paternal allele-specific methylation during early post-implantation development, between embryonic days 3.5 and 6.5 [5, 11]. Since these two DMRs are located within the same imprinting cluster, we hypothesized that the acquisition of paternal allele-specific DNA methylation at these secondary DMRs would be coordinately controlled. We tested this hypothesis by examining the methylation status of the Dlk1-DMR throughout development. We found that the Dlk1-DMR acquires paternal allele-specific methylation during embryogenesis and that the methylation pattern remains dynamic in late embryonic development and into adulthood. Furthermore, our analysis of DNA methylation on the complementary strands of the Dlk1-DMR illustrates the unexpectedly fluid nature of DNA methylation at this locus.
The Dlk1-DMR acquires paternal allele-specific DNA methylation during post-fertilization development
Previous research illustrated that somatic mouse tissues exhibit paternal allele-specific DNA methylation at the Dlk1-DMR that is acquired after fertilization [5, 14, 15]. To elucidate the temporal acquisition of paternal allele-specific DNA methylation at the Dlk1-DMR following fertilization, we assessed the DNA methylation status on both the paternal and maternal Dlk1 alleles at various stages of mouse development.
Average levels of DNA methylation on the paternal and maternal Dlk1 -DMR alleles during development
Genomic DNA sample
% methylation, paternal alleles
% methylation, maternal alleles
B6xCAST adult sperm
3.5 d.p.c. B6xCAST12 embryo
6.5 d.p.c. B6xCAST12 embryo
7.5 d.p.c. B6xCAST12 embryo
8.5 d.p.c. B6xCAST12 embryo
9.5 d.p.c. B6xCAST12 embryo
14.5 d.p.c. B6xCAST12 embryo
14.5 d.p.c. CAST12xB6 embryo
17.5 d.p.c. CAST12xB6 liver
5 d.p.p. B6xCAST12 liver
5 d.p.p. CAST12xB6 liver
6 d.p.p. B6xCAST12 lung
5 d.p.p. CAST12xB6 lung
Adult B6xCAST12 liver
Adult CAST12xB6 liver
Adult B6xCAST12 lung
Adult CAST12xB6 lung
DNA methylation patterns at the Dlk1-DMR are dynamic during development
It had previously been reported that the extent of DNA methylation on the maternal and paternal alleles of Dlk1 varied in different tissues . We therefore examined the methylation status at the Dlk1-DMR in stage-matched neonatal liver and lung tissues derived from reciprocal crosses between B6 and CAST12 mice. We chose liver and lung as representative tissues for this analysis as these tissues exhibit low and high levels of Dlk1 expression during perinatal development, respectively . We found that the paternally inherited allele had a significantly higher level of DNA methylation than the maternally inherited allele in both B6xCAST12 and CAST12xB6 tissues (P = 0.001, liver; P <0.0001, lung; Figure 3C, D), consistent with previously obtained data derived from DNA methylation analyses of 18.5 d.p.c. uniparental disomic (UPD) 12 liver and lung tissues . In addition, the median levels of DNA methylation on paternal alleles derived from neonatal liver and lung were significantly higher than the median levels in 14.5 d.p.c. embryos (P = 0.0016 and 0.004, respectively), indicating that the DNA methylation level continues to increase on the paternal allele during development. However, we did not detect statistically significant differences in the DNA methylation patterns of neonatal liver versus lung, demonstrating that the methylation status of Dlk1 in these tissues is not different at this developmental stage.
Our statistical analyses indicate that the low level of DNA methylation observed on the maternal Dlk1-DMR of 6.5 d.p.c. embryos does not change significantly during post-implantation and perinatal development, although it acquired significantly higher levels of DNA methylation in some of the adult tissues analyzed. In contrast, the median level of DNA methylation on the paternal Dlk1-DMR is significantly different in early and mid-gestation embryos when compared either to late embryos or to adult liver, illustrating that the paternal Dlk1-DMR becomes incrementally more methylated over time. Therefore, although the onset of paternal allele-specific DNA methylation acquisition at the Dlk1- and Gtl2-DMRs occurs at a similar time during development, the DNA methylation pattern at the Dlk1-DMR is more labile (Table 1).
Placental tissue displays biallelic methylation at the Dlk1-DMR
Our analysis of methylation at the Dlk1-DMR was complicated by the fact that we did not separate the maternal component of the placenta from the embryonic component. Therefore, while paternal CAST12 alleles must be derived from the embryonic component of the placenta, B6 alleles could derive from the maternal allele in the embryonic component of the placenta or from either of the parental alleles in the maternal component. To assess the relative proportion of embryonic versus maternally-derived B6 DNA in our placental samples, we analyzed the methylation status at the IG-DMR, which has been shown to remain differentially methylated in extraembryonic tissue and placenta [12, 22]. As expected, we detected hypermethylation on the nine paternal IG-DMR alleles we analyzed. In contrast, six of the 11 maternal alleles analyzed were hypermethylated, suggesting that these hypermethylated alleles were derived from the maternal component of the placenta and that the level of DNA methylation we observed on the maternal alleles overestimates the true extent of methylation present on the maternal Dlk1 alleles in the placenta (Figure 5B). Interestingly, we observed differential methylation on the parental Gtl2-DMR alleles in the same 14.5 d.p.c. placental samples (Figure 5C); these data are in contrast to those obtained by Sato et al.. and Lin et al., who found similar moderate levels of DNA methylation on both the maternal and paternal Gtl2-DMR in 6.5, 7.5, and 16.5 d.p.c. extraembryonic tissue.
CpG dyads within the Dlk1-DMR display a high level of hemimethylation
Extent of homo- vs. hemimethylation at CpG dyads in densely methylated subclones
BxC9C12 adult liver
C9C12xB adult liver
Independent subclones analyzed (n)
Subclones with >65% methylation (n)
Methylated dyads (n)
Homomethylated dyads (n,%)
Hemimethylated dyads (n,%)
Proper regulation of imprinted genes is required for normal growth and development in mammals. Loss of imprinting has been shown to result in developmental disorders and disease such as Beckwith-Wiedemann syndrome, which is associated with fetal growth defects, and Prader-Willi and Angelman syndromes, both of which affect neurological development . The regulation of imprinted gene expression is complex and involves various factors, including epigenetic modifications, such as DNA methylation and histone modifications, as well as the activity of long non-coding RNAs and trans-acting factors such as CTCF . The Dlk1-Dio3 imprinting cluster does not contain CTCF binding sites, and while it does include a maternally expressed long non-coding RNA, Gtl2, it is unlikely that Gtl2 expression regulates the paternally expressed Dlk1, as there is limited overlap in the expression patterns of these genes [24, 25]. In contrast, differentially methylated regions have been shown to play an important role in the regulation of imprinted expression within the Dlk1-Dio3 cluster, highlighting the critical role epigenetic modifications play in the regulation of genomic imprinting. For example, deletion of the imprinting control region, IG-DMR, from the maternal chromosome results in its paternalization .
In addition to regulating the expression of imprinted genes in the Dlk1-Dio3 cluster, the IG-DMR also influences the acquisition of paternal allele-specific DNA methylation at the secondary Gtl2-DMR. It has been shown that the methylation status of the Gtl2/MEG3-DMR is dependent on the methylation status at the IG-DMR, and that inappropriate hypermethylation of the Gtl2/MEG3-DMR is concordant with loss of expression [10, 20, 21]. These data point to a direct role for secondary DMRs in the regulation of imprinted gene expression, although the observation that secondary DMRs acquire differential methylation after the onset of imprinted expression has led to the hypothesis that secondary DMRs play a role in the maintenance of imprinted expression rather than its establishment [6–8]. To date, this study is the first to examine the temporal acquisition of DNA methylation at multiple secondary DMRs within the same imprinting cluster. Our data illustrate that the timing of post-fertilization DNA methylation acquisition is coordinated across the Dlk1-Dio3 locus, although methylation at the Dlk1 locus appears more labile (data herein) .
Paternal allele-specific methylation at the Dlk1-DMR is more variable than at many other imprinted loci, in that the total level of methylation on an individual paternally-inherited allele ranges from 0% to close to 100% at essentially all developmental stages analyzed. Some of this variation may be attributed to the pattern of DNA methylation acquisition at this locus, which appears to be dynamic throughout development. It is also possible that tissue-specific differences result in the variable DNA methylation patterns we observed in whole embryos. For example, Dlk1 is expressed at high levels in skeletal muscle, a tissue in which imprinting is relaxed, which could correlate with reduced levels of DNA methylation [12, 25]. However, even in tissues that display high levels of total DNA methylation on some paternal alleles, such as adult liver, other paternal alleles show little to no methylation and the reason for these differences is not clear. Furthermore, although there are some correlations, there does not appear to be a direct relationship between the DNA methylation profile at the Dlk1-DMR and Dlk1 expression. In most tissues, Dlk1 expression is restricted to the paternal allele, although there is a relaxation of imprinting in 6.5 d.p.c. embryos and in skeletal muscle, in which 20% and 17% of the expression is derived from the maternal allele, respectively [5, 12, 15, 25]. Dlk1 is expressed at relatively low levels in early embryos, as compared to the high levels of expression detected in various mid- and late-gestation embryonic tissues such as the pituitary gland, skeletal muscle, liver, and lung [12, 25, 26]. Despite these differences in expression, our analyses illustrated that the median levels of DNA methylation on the paternal allele is not significantly different in 6.5 to 14.5 d.p.c. whole embryos (Figures 2, 3; Table 1). Finally, while Dlk1 expression is downregulated in most tissues during late embryogenesis, there was no direct correlation between DNA methylation and Dlk1 expression levels in tissues derived from 18.5 d.p.c. uniparental disomies , nor did we detect a direct correlation in this study. Together, these data suggest that the DNA methylation status at the Dlk1-DMR, located in exon 5, may not play an important role in the regulation of expression at this locus. In contrast, the methylation status of the Gtl2/MEG3-DMR has been shown to directly influence expression of Gtl2 in cis, consistent with its location at the Gtl2 promoter [5, 10, 20, 21]. The critical regulatory role of the Gtl2-DMR may explain the maintenance of high average DNA methylation levels at this locus once it has been established [5, 11, 12]. It is possible that DNA methylation at the Dlk1-DMR may reflect a broader, locus-wide epigenetic profile that encompasses both Gtl2 and Dlk1.
The Dlk1-DMR displays low methylation fidelity
The approach we utilized allowed us to analyze the methylation pattern for complementary CpG dinucleotides within the Dlk1-DMR. To the best of our knowledge, this is the first study to comprehensively examine the methylation status of complementary CpG dinucleotides at an imprinted gene during development. Of the 1,953 methylated CpG dyads, 1,272 (65.1%) were homomethylated, while 681 (34.9%) were hemimethylated. This result was unexpected, as the fidelity with which the maintenance DNA methyltransferase in mouse, Dnmt1, has been shown to be greater than 95% [27, 28]. There are several possible reasons to explain some of the hemimethylation we detected. It is likely that some of the hemimethylated sites we observed are a result of hybrid subclones, which have been shown to result as an artifact of PCR amplification following bisulfite mutagenesis . It is also possible that some of the observed hemimethylation is a result of Taq-induced PCR error during amplification. However, these artifacts are unlikely to account for the high level of hemimethylation we detected. Rather, the high level of hemimethylation we observed challenges the idea that Dnmt1 functions with high fidelity at all genomic locations.
A large-scale study analyzing the in vivo regulation of CpG methylation by DNA methyltransferases was recently conducted by Arand et al.. In this study, the authors found relatively high levels of hemimethylated CpGs in embryonic liver, ranging from 16.2% to 30.6% of the methylated CpG dyads. Interestingly, this work illustrated the relative stability of homomethylation at the imprinted Snprn and H19 genes, but demonstrated high levels of hemimethylation at the imprinted Igf2 gene (22%). Analyses of DNA methylation profiles in Dnmt-mutant embryonic stem cells indicated that the DNA methylation profiles at Snprn and H19 were dependent on the activity of Dnmt1 alone, while maintenance of DNA methylation at Igf2 required the coordinated activity of Dnmt1, Dnmt3a, and Dnmt3b, a possible consequence of 5-hydroxymethylcytosine enrichment at the Igf2 DMR . It is therefore possible that the high level of hemimethylation we observed at the Dlk1-DMR may be due to the presence of 5-hydroxymethylcytosine at this locus, preventing high levels of fidelity via Dnmt1. An analysis of methylcytosine versus 5-hydroxymethylcytosine levels at the Dlk1-DMR will address this possibility.
An alternative hypothesis to explain the high level of hemimethylation we observed at the Dlk1-DMR is that there may be a lower level of fidelity associated with the maintenance of DNA methylation at secondary DMRs. Consistent with this hypothesis, a study by Vu et al. examined DNA methylation on the top and bottom strands of the human Igf2/H19 imprinted region. Vu and colleagues analyzed DNA methylation on the top and bottom strands separately and found uniform levels of methylation present at the primary DMR. In contrast, they observed less uniformity in the methylation of the top and bottom strands at the H19 promoter, which is categorized as a secondary DMR as it loses and then regains paternal allele-specific methylation during pre- and post-implantation development, respectively [32, 33]. Additionally, a more recent survey of differentially methylated regions associated with imprinted genes in humans support this hypothesis. Woodfine et al. reported a higher level of stability for DNA methylation at gametic DMRs than at secondary DMRs. Further examination of CpG dyad methylation patterns at imprinted loci may provide additional insight into the mechanisms responsible for the acquisition and maintenance of DNA methylation at these sites.
Our analysis of DNA methylation at the mouse Dlk1-DMR illustrates that the acquisition of paternal allele-specific DNA methylation initiates between 3.5 and 6.5 d.p.c., suggesting that epigenetic modifications across the Dlk1-Dio3 imprinting cluster may be coordinately regulated during post-implantation development. The range of DNA methylation levels on individual alleles at the same developmental stage as well as the additional acquisition of DNA methylation on the maternal Dlk1 allele in adult tissues suggest that the DNA methylation profile of this secondary DMR is more variable than is commonly seen at imprinted loci. We further observed a high level of hemimethylation at the Dlk1-DMR: 35% of CpG dyads containing methylated residues were methylated on only one of the two complementary strands. This result is significant because it challenges the idea that Dnmt1 functions with high fidelity at all genomic locations. We hypothesize that the low DNA methylation fidelity we observed is related to the variable DNA methylation profiles at the Dlk1-DMR, and may be a consequence of high levels of 5-hydroxymethylcytosine at this locus. These data provide insight into a novel epigenetic profile that may distinguish primary DMRs from secondary DMRs.
C57BL/6 J (B6) and Mus musculus castaneus (CAST) mice were purchased from the Jackson Laboratory. To facilitate the isolation of F1 hybrid mice, a strain of mice that served as the source of the M. m. castaneus allele (CAST12) was constructed as previously described . Natural matings between B6 and CAST were used to generate F1 hybrid males for spermatozoa collection; all other F1 hybrid tissues used for bisulfite analyses were generated from natural matings between B6 and CAST12 mice. For all F1 hybrid tissues, the maternal allele is located on the left. Ethical approval for procedures involving animals was granted by the Bryn Mawr College Institutional Animal Care and Use Committee, PHS Welfare Assurance Number A3920-01.
DNA purification and bisulfite analysis
For bisulfite analysis of 3.5 and 6.5 d.p.c. DNA, two to four embryos were pooled prior to digestion with proteinase K. The resulting DNA was subjected to bisulfite mutagenesis using an EZ DNA methylation-direct kit (Zymo Research, cat# D5020). For all other tissues, genomic DNA extractions were performed either from a pool (four 7.5 d.p.c. embryos) or from single embryos, fetuses, or tissues according to the DNeasy protocol (Qiagen) or using a series of phenol/chloroform extractions as described previously , and the complementary strands were covalently attached prior to bisulfite mutagenesis as follows: 0.5 μg of genomic DNA was digested with 1 μL Bgl I (NEB, cat# R0143S) and ligated to 1 μg of a phosphorylated hairpin linker (5′-AGCGATGCGTTCGAGCATCGCTCCC-3′) . A total of 0.5 μg of hairpin linked-ligated DNA was denatured by incubating in freshly prepared 3 M NaOH for 20 min at 42°C, then subjected to bisulfite mutagenesis using an EZ DNA methylation-direct kit, as above. All mutagenized DNAs were subjected to multiple independent PCR amplifications to ensure analysis of different strands of DNA; subclones derived from independent PCR amplifications are distinguished by different letters of the alphabet. Data from multiple individuals at the same developmental stage were combined, as we did not detect variation between biological replicates. The following primer pairs were used for nested amplification of the mutagenized DNA, and were designed to incorporate both the SNP and at least 50% of the CpG dinucleotides within the CpG island. All base pair numbers are from GenBank Accession Number NC_000078.6. For the first round of amplification of mutagenized 3.5 and 6.5 d.p.c. DNA, two cycles of 94°C for 2 min, 52°C for 1 min, 72°C for 1 min followed by 30 cycles of 94°C for 30 s, 52°C for 1 min, 72°C for 1 min using primers RDlke5BF3 (5′-CCCCATCTAACTAATAACTTACA-3′)/RDlke5BR3 (5′-GTGTTTAGTATTATTAGGTTGGTG-3′). For the second round of amplification, 35 cycles of 94°C for 30 s, 52°C for 1 min, 72°C for 1 min using primers RDlke5BF4 (5′-ATTTCTACTACTCTATCCTAACCC-3′)/RDlke5BR4 (5′-TTAGGATGGTGAAGTAGATGGT-3′) yielded a 597 bp product. To amplify mutagenized DNA treated with the hairpin linker, the same reaction conditions were used with the following primers to yield a 464 bp product: first round, RDlke5BR4 (5′-TTAGGATGGTGAAGTAGATGGT-3′)/Dlk1e5BR1 (5′-AACTCTTTCATAAACACCTTCAA-3′); second round, HPDlk1e5F (5′-GTTTATTTGGGTGTGTTGGAGG-3′)/HPDlk1e5R (5′-AAACTCACCTAAATATACTAAAAAC-3′). The following primer pairs were used for nested or semi-nested amplification of IG- and Gtl2-DMRs, as previously described . All base pair numbers are from NC_000078.5. Gtl2 IG-DMR, with the first nucleotide of IG-BS-F1 corresponding to position 110,766,235: 30 cycles of 94°C for 30 s, 52°C for 1 min, 72°C for 1 min, using primers IG-BS-F1/IG-BS-R, followed by 35 cycles using IG-BS-F2/IG-BS-R and the same cycling conditions as above. Identical reaction conditions were used to amplify the Gtl2- DMR, with the first nucleotide of Gtl2BI4F1 corresponding to position 110,779,293: Gtl2BI4F1/Gtl2BI4R1 followed by Gtl2BI4F2/Gtl2BI4R2. Primer sequences follow. IG-BS-F1, 5′-GTATGTGTATAGAGATATGTTTATATGGTA-3′; IG-BS-F2, 5′-GTGTTAAGGTATATTATGTTAGTGTTAGGA-3′; IG-BS-R, 5′-GCTCCATTAACAAAATAATACAACCCTTCC-3′; Gtl2BI4F1, 5′-GAAGAATTTTTTATTTGGTGAGTGG-3′; Gtl2BI4F2, 5′-GTTTGAAAGGATGTGTAAAAATG-3′; Gtl2BI4R1, 5′-CAACACTCAAATCACCCCCC-3′; Gtl2BI4R2, 5′-GCCCCCCACATCTATTCTACC-3′. Subcloning of amplified products was achieved using a pGEM-T Easy vector (Promega Corporation, Madison, WI, USA). Sequencing reactions were performed using a Thermo Sequenase Cycle Sequencing Kit (USB Corporation, Cleveland, OH, USA), and reactions were analyzed on a 4300 DNA Analyzer (LI-COR Biosciences, Lincoln, NE, USA). Percent methylation was calculated based on data obtained from both complementary strands.
Identification of CpG island
The extent of the CpG island identified by Paulsen et al. was determined using the EMBOSS CpGPlot analyzer (http://www.ebi.ac.uk/Tools/emboss/cpgplot/index.html), with the following parameters: program = cpgplot, window = 200, step = 1, obs/exp = 0.6, MinPC = 50, length = 200. The position of the CpG island corresponds to nucleotides 109,459,650-109,460,035 (GenBank: NC_000078.6).
- C or CAST:
Mus musculus castaneus
- C12 or CAST12:
Mus musculus castaneus chromosome 12 on a C57BL/6 background
Differentially methylated region
Days post coitum
Days post partum
Imprinting control region
Polymerase chain reaction
We thank Jeanette Bates for her contributions towards this work, Joshua Shapiro for assistance with the statistical analyses, and Michelle Wien and Joshua Shapiro for thoughtful discussion. This work was supported by awards from the Bryn Mawr College Faculty Research Fund and National Science Foundation grant 1157819 to TLD. In addition, AG, AH, MQ, CT, JA, and KM were supported in part by the Bryn Mawr College Summer Science Research program.
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