Convergent and divergent evolution of genomic imprinting in the marsupial Monodelphis domestica

Abstract

Background: Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin specific monoallelic gene expression. It is postulated to have evolved in placental mammals to modulate intrauterine resource allocation to the offspring. In this study, we determined the imprint status of metatherian orthologues of eutherian imprinted genes.

Results: L3MBTL and HTR2A were shown to be imprinted in Monodelphis domestica (the gray short-tailed opossum). MEST expressed a monoallelic and a biallelic transcript, as in eutherians. In contrast, IMPACT, COPG2, and PLAGL1 were not imprinted in the opossum. Differentially methylated regions (DMRs) involved in regulating imprinting in eutherians were not found at any of the new imprinted loci in the opossum. Interestingly, a novel DMR was identified in intron 11 of the imprinted IGF2R gene, but this was not conserved in eutherians. The promoter regions of the imprinted genes in the opossum were enriched for the activating histone modification H3 Lysine 4 dimethylation.

Conclusions: The phenomenon of genomic imprinting is conserved in Therians, but the marked difference in the number and location of imprinted genes and DMRs between metatherians and eutherians indicates that imprinting is not fully conserved between the two Therian infra-classes. The identification of a novel DMR at a non-conserved location as well as the first demonstration of histone modifications at imprinted loci in the opossum suggest that genomic imprinting may have evolved in a common ancestor of these two Therian infra-classes with subsequent divergence of regulatory mechanisms in the two lineages.

Figure 1

Imprinting analysis of metatherian orthologues of eutherian imprinted genes in M. domestica. The five gray short-tailed opossum genes investigated are (A) PLAGL1, (B) COPG2, (C) IMPACT, (D) L3MBTL and (E) HTR2A. The arrow denotes the polymorphism used to determine the imprint status of the gene. PLAGL1 (n = 2), COPG2 (n = 2), and IMPACT (n = 2) are not imprinted since the polymorphism is present in both the genomic DNA and the cDNA. In contrast, L3MBTL (n = 4) and HTR2A (n = 3) are imprinted since the genomic polymorphism is absent in the cDNA, demonstrating that only one allele is expressed. Using those samples where the offspring are heterozygous and the mothers are homozygous at the same polymorphic site, it was determined that L3MBTL and HTR2A were expressed from the paternal and maternal alleles, respectively.

Figure 2

Analysis of imprinting at the MEST locus in M. domestica. (A) 3′ RACE distinguished two alternative MEST transcripts. The vertical arrows indicate the polymorphic sites used to analyze the imprint status of the two transcripts. (B) Transcript 1 (n = 2) is imprinted since the genomic A/G polymorphism is absent in the cDNA, demonstrating that only one allele is expressed. The parental origin of expression for transcript 1 could not be determined because a heterozygous offspring with a mother homozygous at the A/G polymorphic site was not found. In contrast, transcript 2 (n = 1) is not imprinted since the C/A polymorphism is present in both the genomic DNA and the cDNA.

Figure 3

Methylation profiles of M. domestica CpG-rich regions close to orthologues of eutherian imprinted genes. Bisulfite-modified genomic DNA was PCR amplified and cloned. Each line denotes an individual cloned allele. Representative CpG-rich regions analyzed are (A) putative promoter region of HTR2A, (B) 5^′ upstream region of L3MBTL, (C) 5^′ upstream region of MEST, (D) 5^′ upstream region of IMPACT, (E) 5^′ upstream region of COPG2, and (F) putative promoter region of IGF2R. Unfilled circles depict unmethylated cytosines at CpG sites, while filled circles depict methylated cytosines. The genomic co-ordinates of these regions are listed in Additional file 1: Table S3.

Figure 4

Methylation profile and antisense transcript analysis for the novel DMR identified in intron 11 of M. domestica IGF2R.(A) IGF2R is represented graphically. The three sets of primers used in strand-specific RT-PCR analysis are labeled as 1, 2 and 3. The reverse transcription primers for anti-sense strand detection are labeled as 1a, 2a, and 3a while those for sense-strand detection are labeled as 1b, 2b, and 3b. (B) Parent-of-origin analysis was performed at the DMR by analyzing methylation of cloned alleles for an individual bearing an (A/T) SNP at chr2: 442,443,695 with a mother homozygous for A at the same location. Only the allele inherited from the mother is methylated. (C) Strand-specific RT-PCR analysis using primers described above shows a PCR product only by reverse transcription utilizing 1b, 2b band 3b; L represents the 100 bp DNA ladder. This demonstrates that the gene is expressed at this locus but there is no associated anti-sense transcript.

Figure 5

Analysis of histone modifications at the promoter regions of M. domestica orthologues of eutherian imprinted genes.(A) Chromatin immunoprecipitation with H3K4me2 antibody followed by PCR amplification is shown for the IGF2R promoter region; L denotes the 100 bp ladder, I the input, R the sample immunoprecipitated with rabbit serum albumin (i.e. non-specific pull-down), and K4 the sample immunoprecipitated with H3K4me2 antibody. Analysis of enrichment was performed by real-time PCR following chromatin immunoprecipitation with H3K4me2 and H3K9me3 antibodies at the promoter regions of (B) IGF2R, (C) HTR2A, and (D) L3MBTL. The fold change is plotted on the Y-axis, and the distance from the putative transcription start site (0) on the X-axis. The black bars represent H3K4me2, and the grey bars represent H3K9me3.