Patterns of hybrid loss of imprinting reveal tissue- and cluster-specific regulation

Abstract

Background: Crosses between natural populations of two species of deer mice, Peromyscus maniculatus (BW), and P. polionotus (PO), produce parent-of-origin effects on growth and development. BW females mated to PO males (bwxpo) produce growth-retarded but otherwise healthy offspring. In contrast, PO females mated to BW males (POxBW) produce overgrown and severely defective offspring. The hybrid phenotypes are pronounced in the placenta and include POxBW conceptuses which lack embryonic structures. Evidence to date links variation in control of genomic imprinting with the hybrid defects, particularly in the POxBW offspring. Establishment of genomic imprinting is typically mediated by gametic DNA methylation at sites known as gDMRs. However, imprinted gene clusters vary in their regulation by gDMR sequences.

Methodology/principal findings: Here we further assess imprinted gene expression and DNA methylation at different cluster types in order to discern patterns. These data reveal POxBW misexpression at the Kcnq1ot1 and Peg3 clusters, both of which lose ICR methylation in placental tissues. In contrast, some embryonic transcripts (Peg10, Kcnq1ot1) reactivated the silenced allele with little or no loss of DNA methylation. Hybrid brains also display different patterns of imprinting perturbations. Several cluster pairs thought to use analogous regulatory mechanisms are differentially affected in the hybrids.

Conclusions/significance: These data reinforce the hypothesis that placental and somatic gene regulation differs significantly, as does that between imprinted gene clusters and between species. That such epigenetic regulatory variation exists in recently diverged species suggests a role in reproductive isolation, and that this variation is likely to be adaptive.

Figure 1. Disruptions in imprinting and DNA methylation at the Kcnq1ot1 domain.

A. Allelic expression of the Kcnq1ot1 and Cd81 genes. An RT-PCR/RFLP assay is shown. Arrows indicate allele-specific bands. B. Domain structure and DNA methylation status as assessed by bisulfite sequencing. See text for details. Mus domain and imprinting status is shown at top. Peromyscus genes shown on same scale; complete genomic sequence was not available at the time of writing. Maternal allele expression indicated in red above line, paternal expression in blue below line. Grey–gene not examined in Peromyscus. gDMR is shown as a black (methylated) or white (unmethylated) box. Reactivated alleles are shown in their original color on the opposite allelic position. Sequenced clones from bisulfite-treated DNA shown at bottom. Each line represents an individual clone. Filled circles = methylated cytosines, open circles = unmethylated cytosines.

Figure 2. Loss of methylation at the Peg3-Usp29 locus.

Top–Locus structure/expression in Mus and reciprocal hybrids. All symbols are as described in Figure 1. Bottom–Bisulfite sequencing of the ICR in Peromyscus crosses. No polymorphism was available to determine parental origin in the bisulfite reads.

Figure 3. Imprinting and methylation analysis of the Peg10-Sgce domain.

A. Allelic expression of the Peg10 transcript. Arrows indicate allele-specific bands. B. Domain structure and DNA methylation status as assessed by bisulfite sequencing. The region assayed starts in the intergenic region and extends several hundred base-pairs into Peg10 intron 1. All symbols are as described in Fig. 1. C. Methyl-specific PCR (MSP) assay. Primers were designed to amplify either methylated or unmethylated bisulfite-treated DNA. Methylated and unmethylated products are indicated with M and U (respectively) below each lane. The amplicons are from sequences ∼200 bp 5′ of the bisulfite assay results shown (∼300 bp 5′ of the Peg10 transcription start site).

Figure 4. Imprinting analysis of the Dlk1-Meg3 imprinted domain.

A. Allelic usage assays for Meg3 and Dio3. F1 DNA is included to demonstrate an allelic amplification bias in biallelic samples of Dio3. Gtl2 is ubiquitously imprinted in both hybrids, while Dio3 displays embryo-specific imprinting. B. Structure of Igf2-H19 and Dlk1- Meg3 domains and differences in imprinting perturbations in PO×BW hybrids. C. Bisulfite sequencing of the domain gDMR. Symbols as in Fig. 1.

Figure 5. Allelic expression of the Dcn (A) and Plagl1 (B) genes.

The patterns for the PO, BW and a mix of the two alleles are shown at left. Lane identity at top; plc–placenta, emb–embryo. Note that other PO×BW samples displayed reactivation of the Plagl1 maternal allele.

Figure 6. Allelic expression in the brains of Peromyscus hybrids.

Placental expression is shown for comparison. Arrows indicate allele-specific bands. A–H19; B–Peg10; C–Kcnq1ot1; D–Meg3.