Genomic imprinting is an epigenetic phenomenon characterized by the monoallelic expression of a gene in a parent-of-origin dependent manner
. Its discovery in mammals showed that parental genomes are functionally non-equivalent, and that the sex of the parent, not of the resulting progeny, is critical in determining expression of a particular allele
In vertebrates, imprinting is believed to have originated approximately 150 million years ago, in the common ancestor of the two Therian infra-classes: eutherians (placental mammals like humans and mice), and metatherians (marsupials like the opossum and tammar wallaby which have a less invasive placenta)
. The evidence comes from approximately 98 imprinted genes that have been discovered to date in eutherian mammals, and the six known to be imprinted in the metatherians
[6–12]. Imprinted genes have not been identified in prototherian (i.e. platypus and echidna) and avian (i.e. chicken) species
[6, 13]. To explain this unique phylogenetic distribution of imprinted genes, the “Conflict Hypothesis” proposes that genomic imprinting evolved in placental mammals in response to polygamy, viviparity, and multiple births
[14, 15]. This theory is based on maximizing competitive fitness of the father’s progeny while preserving the ability of the mother to equally provide care to all her offspring, regardless of paternity. A corollary to this hypothesis is that paternally and maternally expressed genes will enhance and inhibit growth, respectively.
An unusual feature of imprinted genes in eutherians is that they tend to occur in clusters throughout the genome. This suggests that shared regulatory elements play a role in epigenetic control of these clusters. In many imprinted gene clusters, imprint control regions (ICRs) function as discrete cis-acting DNA elements that are characterized by heritable epigenetic marks that distinguish the two parental alleles
. These ICRs simultaneously and often reciprocally regulate two or more imprinted genes.
The imprint mark in eutherians includes germ-line derived cytosine methylation patterns wherein the two alleles exhibit opposite methylation states
. A group of such differentially methylated CpG sites at a locus constitute a differentially methylated region (DMR). The methylation marks are completely erased in the germ cells and then re-established in the gametes, based on the sex of the individual now carrying the DNA. Surprisingly, of the six genes known to be imprinted in metatherians, DMRs are only present at PEG10 and IGF2. Moreover, the presence of a DMR is not sufficient to confer imprinted expression in eutherians, since IGF2R is biallelically expressed
 in humans despite having a DMR in intron 2
Differential chromatin states of the parental alleles resulting from covalent modification of histone tails also plays a prominent role in regulating expression and imprinting patterns. Repressive modifications, including H3 Lysine 9 (H3K9) and H3 Lysine 27 (H3K27) methylation, function in silencing of an allele whereas active marks, including H3 Lysine 4 (H3K4) methylation and histone acetylation are associated with the expressed allele. These histone modifications at the promoter region of imprinted genes are better correlated with expression than the methylation marks
[20–22]. As a case in point, human IGF2R has an intronic DMR in peripheral tissues, but lacks the promoter-restricted histone code present in the mouse, and is biallelically expressed. Thus the histone code may be the “primordial imprint mark,” and mechanisms of DNA methylation were probably added later to assist in stabilizing the expression status
Understanding the molecular mechanisms by which imprinting is established in all mammals is critical to furthering knowledge about imprint gene regulation in humans. Although differential methylation of the parental alleles is involved in controlling genomic imprinting in eutherians, there is no clear evidence supporting a role for DMRs in metatherian imprinting. The DNA methyltransferases necessary for conferring imprinting are present and functional in the metatherians
[24, 25], yet DMRs are present at only two of the six currently known imprinted genes. The lack of DMR identification at the other imprinted loci may reflect an inability to thoroughly investigate the sequence content associated with these regions in metatherians due to incomplete sequence availability at the time these studies were done. Alternatively, it may be due to the true absence of DMRs in these regions if imprinting is indeed controlled primarily by histone marks in metatherians. The sequencing of the genome of the gray short-tailed opossum Monodelphis domestica
(M. domestica) now allows for the comprehensive investigation of such imprint regulatory regions.
It is presently unknown if chromatin modifications exist at imprinted loci in metatherians despite the knowledge that histone modifications, and not DNA methylation, are involved in paternal X chromosome silencing in metatherians
[26, 27]. Since imprinted X-inactivation appears to have co-evolved with genomic imprinting in Therians, it is possible that the same molecular mechanism is used to delineate the active and inactive regions for both of these epigenetic phenomena
Here we exploited the availability of the genomic sequence of the gray short-tailed opossum, M. domestica, to identify orthologues of known eutherian imprinted genes and then determined their imprint status in this metatherian. CpG rich regions in close proximity to these genes were also identified and examined for the presence of differential methylation. We then assessed active H3K4 dimethylation and repressive H3K9 trimethylation in these regions. We identified a novel DMR within IGF2R, but DMRs were not present near or within the other genes imprinted in the opossum. The active alleles of the imprinted genes were enriched for the active mark H3K4 dimethylation. The discovery of the novel IGF2R DMR demonstrates that differential methylation might be present at imprinted loci in metatherians at non-conserved locations, but these have been missed in previous analyses due to incomplete sequence availability. Moreover, the presence of histone modifications at imprinted loci also shows that genomic imprinting arose in certain loci before the eutherian-metatherian split, but the mechanisms governing retention of the imprint have diverged in each lineage over the past 150 million years.