Epigenetic profiling at mouse imprinted gene clusters reveals novel epigenetic and genetic features at differentially methylated regions

Abstract

Genomic imprinting arises from allele-specific epigenetic modifications that are established during gametogenesis and that are maintained throughout somatic development. These parental-specific modifications include DNA methylation and post-translational modifications to histones, which create allele-specific active and repressive domains at imprinted regions. Through the use of a high-density genomic tiling array, we generated DNA and histone methylation profiles at 11 imprinted gene clusters in the mouse from DNA and from chromatin immunoprecipitated from sperm, heart, and cerebellum. Our analysis revealed that despite high levels of differential DNA methylation at non-CpG islands within these regions, imprinting control regions (ICRs) and secondary differentially methylated regions (DMRs) were identified by an overlapping pattern of H3K4 trimethylation (active chromatin) and H3K9 trimethylation (repressive chromatin) modifications in somatic tissue, and a sperm differentially methylated region (sDMR; sperm not equal somatic tissue). Using these features as a common signature of DMRs, we identified 11 unique regions that mapped to known imprinted genes, to uncharacterized genes, and to intergenic regions flanking known imprinted genes. A common feature among these regions was the presence of a CpG island and an array of tandem repeats. Collectively, this study provides a comprehensive analysis of DNA methylation and histone H3K4me3 and H3K9me3 modifications at imprinted gene clusters, and identifies common epigenetic and genetic features of regions regulating genomic imprinting.

Figure 1.

DNA methylation patterns at imprinted gene clusters. (A) MeDIP was performed on DNA isolated from sperm, heart, and cerebellum and the enriched (positive log₂ IP/IN) values are plotted for each imprinted gene cluster. Chromosomal regions are marked as black boxes with corresponding chromosome numbers. (B) Sequence characterization of methylated regions in sperm, heart, and cerebellum showed that the imprinted gene clusters were primarily methylated at repetitive DNA based on repeat masker. (C) Plots showing the genomic location of methylated regions relative to the RefSeq gene annotation. (D) CpG island methylation was less frequent in sperm samples compared to the heart and cerebellum, particularly for the promoter and intragenic regions. (E) The majority of methylated CpG islands in heart and cerebellum were present in the promoters of known imprinted genes.

Figure 2.

Characterization of sperm-specific and tissue-specific differentially methylated regions at imprinted gene clusters. (A) A sliding window t-test was performed between sperm and heart (Sp vs. Ht; sDMR), sperm and cerebellum (Sp vs. Cb; sDMR), and heart and cerebellum (Ht vs. Cb; tDMR) using the log₂ IP/IN ratios obtained from MeDIP to determine sperm and tissue-specific differentially methylated regions (sDMR and tDMR). The y-axis indicates regions that have significantly different (P < 0.00001) methylation profiles between samples and the direction of methylation. (B) The mean log₂ IP/IN of probes was calculated over each known DMR in cerebellum, heart, and sperm. Box plots show less methylation in sperm DNA at each DMR, except at the H19 ICR and IG-DMR, which are methylated in sperm and differentially methylated in heart and cerebellum. Box plots represent the 25th and 75th percentile for each averaged sample. Whiskers show 10th and 90th percentiles. (C) An example showing the different patterns observed between sperm DNA methylation (Sperm DNAme) and heart DNA methylation (Heart DNAme) at the Mest DMR. (D) Sequence characterization of sDMR and tDMR showed that nonrepetitive DNA is primarily differentially methylated at imprinted gene clusters. (E) Genomic location of sDMRs and tDMRs relative to the RefSeq gene annotation. (F) An example of a tDMR at the 3′ end of the Igf2 gene. (G) Bisulfite sequencing confirmed loss of methylation in the cerebellum DNA at the 3′ end of the Igf2 gene. Arrows in F indicate regions amplified for bisulfite sequencing.

Figure 3.

H3K4me3 and H3K9me3 enriched regions at imprinted gene clusters. (A) ChIP-chip profiles of histone modifications for H3K4me3 and H3K9me3 in heart and cerebellum. Y-axis values represent log₂ IP/IN. (B) Sequence characterization of regions enriched for H3K4me3 and H3K9me3 showed that each modification was present primarily at repetitive sequences. (C) Genomic location of H3K4me3 and H3K4me3 modifications relative to the RefSeq gene annotation. (D) Venn diagram showing overlapping H3K4me3 and H3K9me3 regions in both heart and cerebellum. (E) Genomic locations of overlapping H3K4me3 and H3K9me3 modifications relative to the RefSeq gene annotations.

Figure 4.

Overlapping patterns of H3K4me3 and H3K9me3 at sDMRs identified imprinting control regions. (A) An example of the Igf2r DMR in intron 2 of the Igf2r locus. The CpG island (green box) indicates the DMR, which contains both H3K4me3 and H3K9me3 modifications and differentially methylated DNA between sperm and heart. (B) Examples of the Ndn and Magel2 DMR showing overlapping H3K4me3 and H3K9me3, as well as sDMR between sperm and heart. (C) Genomic locations of regions with a sDMR and overlapping H3K4me3 and H3K9me3. (D) Genomic locations of CpG islands with a sDMR and overlapping H3K4me3 and H3K9me3. (E) Sequence characterization of regions with a sDMR and overlapping H3K4me3 and H3K9me3 indicated regions are highly repetitive and (F) are comprised mostly of simple repeats, SINES, and low complexity repeats. (G) Examples of sequence alignments for known ICRs (e.g., Igf2r DMR, Meg3 DMR, H19 ICR, and Tsix) and putative ICRs (e.g., Olfr1349, Upstream Ndn, 5730403M16Rik, and Xlr5C) showing repeated elements.