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, 12 (1), 64

Hemimethylation of CpG Dyads Is Characteristic of Secondary DMRs Associated With Imprinted Loci and Correlates With 5-hydroxymethylcytosine at Paternally Methylated Sequences

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Hemimethylation of CpG Dyads Is Characteristic of Secondary DMRs Associated With Imprinted Loci and Correlates With 5-hydroxymethylcytosine at Paternally Methylated Sequences

Julianna Nechin et al. Epigenetics Chromatin.

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.

Keywords: Cdkn1c; DNA methylation; Epigenetics; Genomic imprinting; H19; Ndn; Peg12; Secondary DMR; Snrpn.

Conflict of interest statement

The authors declare they have no competing interests.

Figures

Fig. 1
Fig. 1
Imprinting clusters on mouse chromosome 7. a Location of proximal, central and distal imprinting clusters. Detail of central (b) and distal (c) imprinting clusters. Red and blue rectangles correspond to maternally and paternally expressed genes, respectively. Genes located above and below the line have + and − strand orientation, respectively. Pound signs and asterisks indicate the location, respectively, of primary and secondary DMRs analyzed in this study. Snrpn, maternally methylated primary DMR; Ndn and Peg12, maternally methylated secondary DMRs; H19 ICR, paternally methylated primary DMR; H19-pp and Cdkn1c, paternally methylated secondary DMRs
Fig. 2
Fig. 2
The paternally methylated secondary DMRs associated with H19 and Cdkn1c display high levels of hemimethylation. Bisulfite mutagenesis and sequencing of F1 hybrid DNA derived from 7.5 dpc BxC embryos and 5 dpp BxC liver. Individual circles in each row represent one of the potentially methylated CpG dinucleotides analyzed at the H19-ppDMR (a) or the Cdkn1c DMR (b), and each paired row of circles represents the complementary strands of an individual subclone; semi-circles to the right or left indicate the location of the linker connecting the complementary strands. Filled circles represent methylated cytosines, open circles represent unmethylated cytosines, absent circles represent ambiguous data. Alphanumeric labels identify subclones analyzed; letters represent independent amplification reactions, while numbers represent individual subclones. Subclones derived from the same amplification that have identical sequence and methylation patterns are grouped together, as it was not possible to determine if these amplicons were derived from the same or different template molecules. Data obtained from 14.5 dpc BxC embryos and adult BxC liver are shown in Additional file 7: Figure S1. Reciprocal cross-data obtained from 13.5 dpc CxB embryos are shown in Additional file 10: Figure S4
Fig. 3
Fig. 3
The maternally methylated secondary DMRs associated with Ndn and Peg12 display high levels of hemimethylation. a Methylation status at the Ndn DMR; F1 hybrid DNA derived from 7.5 dpc BxC embryos and 5 dpp BxC liver and brain. b Methylation status at the Peg12 DMR; F1 hybrid DNA derived from 5 dpp liver. Other details as described in Fig. 2. Data obtained from 7.5 and 14.5 dpc BxC embryos and adult BxC liver and brain are shown in Additional file 8: Figure S2. Reciprocal cross-data obtained from 13.5 dpc CxB embryos are shown in Additional file 10: Figure S4
Fig. 4
Fig. 4
The primary DMRs associated with H19 and Snrpn display low levels of hemimethylation. Details as described in Fig. 2. Data shown are from DNA derived from 5 dpp BxC liver. Data obtained from 7.5 and 14.5 dpc BxC embryos and adult BxC liver are shown in Additional file 9: Figure S3. Reciprocal cross-data obtained from 13.5 dpc CxB embryos are shown in Additional file 10: Figure S4
Fig. 5
Fig. 5
Hemimethylation levels at primary vs. secondary DMRs are significantly different. a Hemimethylation levels at primary and secondary DMRs. Chi square tests of independence reveal that hemimethylation levels at primary DMRs are not significantly different from each other (b), hemimethylation levels at secondary DMRs show some significant differences (c), and that the differences in hemimethylation levels at primary vs. secondary DMRs are highly significant (d)
Fig. 6
Fig. 6
5-hydroxymethylcytosine is enriched at paternally methylated secondary DMRs. Average 5hmC levels and standard deviations for primary DMRs associated with the paternally methylated H19-ICR, the maternally methylated Snrpn DMR, the paternally methylated H19-pp and Cdkn1c DMRs and the maternally methylated Ndn and Peg12 DMRs in DNA derived from 9.5 and 14.5 d.p.c. embryos and from 5 d.p.p. and adult liver

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