Andrew J. Pask

Retrotransposon silencing by DNA methylation can drive mammalian genomic imprinting

Among mammals, only eutherians and marsupials are viviparous and have genomic imprinting that leads to parent-of-origin-specific differential gene expression. We used comparative analysis to investigate the origin of genomic imprinting in mammals. PEG10 (paternally expressed 10) is a retrotransposon-derived imprinted gene that has an essential role for the formation of the placenta of the mouse. Here, we show that an orthologue of PEG10 exists in another therian mammal, the marsupial tammar wallaby (Macropus eugenii), but not in a prototherian mammal, the egg-laying platypus (Ornithorhynchus anatinus), suggesting its close relationship to the origin of placentation in therian mammals. We have discovered a hitherto missing link of the imprinting mechanism between eutherians and marsupials because tammar PEG10 is the first example of a differentially methylated region (DMR) associated with genomic imprinting in marsupials. Surprisingly, the marsupial DMR was strictly limited to the 5′ region of PEG10, unlike the eutherian DMR, which covers the promoter regions of both PEG10 and the adjacent imprinted gene SGCE. These results not only demonstrate a common origin of the DMR-associated imprinting mechanism in therian mammals but provide the first demonstration that DMR-associated genomic imprinting in eutherians can originate from the repression of exogenous DNA sequences and/or retrotransposons by DNA methylation.

Conservation of the H19 noncoding RNA and H19-IGF2 imprinting mechanism in therians

Comparisons between eutherians and marsupials suggest limited conservation of the molecular mechanisms that control genomic imprinting in mammals. We have studied the evolution of the imprinted IGF2-H19 locus in therians. Although marsupial orthologs of protein-coding exons were easily identified, the use of evolutionarily conserved regions and low-stringency Bl2seq comparisons was required to delineate a candidate H19 noncoding RNA sequence. The therian H19 orthologs show miR-675 and exon structure conservation, suggesting functional selection on both features. Transcription start site sequences and poly(A) signals are also conserved. As in eutherians, marsupial H19 is maternally expressed and paternal methylation upstream of the gene originates in the male germline, encompasses a CTCF insulator, and spreads somatically into the H19 gene. The conservation in all therians of the mechanism controlling imprinting of the IGF2-H19 locus suggests a sequential model of imprinting evolution.

The Evolution of the DLK1-DIO3 Imprinted Domain in Mammals

A comprehensive, domain-wide comparative analysis of genomic imprinting between mammals that imprint and those that do not can provide valuable information about how and why imprinting evolved. The imprinting status, DNA methylation, and genomic landscape of the Dlk1-Dio3 cluster were determined in eutherian, metatherian, and prototherian mammals including tammar wallaby and platypus. Imprinting across the whole domain evolved after the divergence of eutherian from marsupial mammals and in eutherians is under strong purifying selection. The marsupial locus at 1.6 megabases, is double that of eutherians due to the accumulation of LINE repeats. Comparative sequence analysis of the domain in seven vertebrates determined evolutionary conserved regions common to particular sub-groups and to all vertebrates. The emergence of Dlk1-Dio3 imprinting in eutherians has occurred on the maternally inherited chromosome and is associated with region-specific resistance to expansion by repetitive elements and the local introduction of noncoding transcripts including microRNAs and C/D small nucleolar RNAs. A recent mammal-specific retrotransposition event led to the formation of a completely new gene only in the eutherian domain, which may have driven imprinting at the cluster.

Analysis of the platypus genome suggests a transposon origin for mammalian imprinting

BackgroundGenomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression. Many hypotheses have been advanced to explain why genomic imprinting evolved in mammals, but few have examined how it arose. The host defence hypothesis suggests that imprinting evolved from existing mechanisms within the cell that act to silence foreign DNA elements that insert into the genome. However, the changes to the mammalian genome that accompanied the evolution of imprinting have been hard to define due to the absence of large scale genomic resources between all extant classes. The recent release of the platypus genome has provided the first opportunity to perform comparisons between prototherian (monotreme; which appear to lack imprinting) and therian (marsupial and eutherian; which have imprinting) mammals.ResultsWe compared the distribution of repeat elements known to attract epigenetic silencing across the entire genome from monotremes and therian mammals, particularly focusing on the orthologous imprinted regions. There is a significant accumulation of certain repeat elements within imprinted regions of therian mammals compared to the platypus.ConclusionsOur analyses show that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. The accumulation of repeats, especially long terminal repeats and DNA elements, in therian imprinted genes and gene clusters is coincident with, and may have been a potential driving force in, the development of mammalian genomic imprinting. These data provide strong support for the host defence hypothesis.

Evolution of Genomic Imprinting: Insights from Marsupials and Monotremes

Parent-of-origin gene expression (genomic imprinting) is widespread among eutherian mammals and also occurs in marsupials. Most imprinted genes are expressed in the placenta, but the brain is also a favored site. Although imprinting evolved in therian mammals before the marsupial-eutherian split, the mechanisms have continued to evolve in each lineage to produce differences between the two groups in terms of the number and regulation of imprinted genes. As yet there is no evidence for genomic imprinting in the egg-laying monotreme mammals, although these mammals also form a placenta (albeit short-lived) and transfer nutrients from mother to embryo. Therefore, imprinting was not essential for the evolution of the placenta and its importance in nutrient transfer but the elaboration of imprinted genes in marsupials and eutherians is associated with viviparity. Here we review the recent analyses of imprinted gene clusters in marsupials and monotremes, which have served to shed light on the origin and evolution of imprinting mechanisms in mammals.

Genomic imprinting of IGF2, p57KIP2 and PEG1/MEST in a marsupial, the tammar wallaby

Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.

Recent Assembly of an Imprinted Domain from Non-Imprinted Components

Genomic imprinting, representing parent-specific expression of alleles at a locus, raises many questions about how—and especially why—epigenetic silencing of mammalian genes evolved. We present the first in-depth study of how a human imprinted domain evolved, analyzing a domain containing several imprinted genes that are involved in human disease. Using comparisons of orthologous genes in humans, marsupials, and the platypus, we discovered that the Prader-Willi/Angelman syndrome region on human Chromosome 15q was assembled only recently (105–180 million years ago). This imprinted domain arose after a region bearing UBE3A (Angelman syndrome) fused with an unlinked region bearing SNRPN (Prader-Willi syndrome), which had duplicated from the non-imprinted SNRPB/B′. This region independently acquired several retroposed gene copies and arrays of small nucleolar RNAs from different parts of the genome. In their original configurations, SNRPN and UBE3A are expressed from both alleles, implying that acquisition of imprinting occurred after their rearrangement and required the evolution of a control locus. Thus, the evolution of imprinting in viviparous mammals is ongoing.

The evolution of class V POU domain transcription factors in vertebrates and their characterisation in a marsupial

POU5F1 (OCT4) encodes a master regulator of pluripotency that is present in all mammals. A paralogue, POU2, is also present in the genomes of marsupials and monotremes and is an orthologue of zebrafish pou2 and chicken POUV. We explored the evolution of class V POU domain transcription factors and show that POU5F1 arose by gene duplication of pou2 early in the evolution of tetrapods and is not mammal-specific, as previously thought. Instead, either POU5F1 or POU2/POUV has become extinct independently in various lineages, although all gnathostomes appear to possess at least one or the other. In the tammar wallaby, POU5F1 expression is limited to pluripotent cell types (embryonic tissues and germ cells). POU2 is similarly expressed in pluripotent tissues but is also expressed in a broad range of adult tissues. Thus, unlike POU5F1, the role of POU2 may not be restricted to pluripotent cell types but could have a related function such as maintaining multipotency in adult stem cells.

Sex determination in platypus and echidna: autosomal location of SOX3 confirms the absence of SRY from monotremes

In eutherian (‘placental’ mammals, sex is determined by the presence or absence of the Y chromosome-borne gene SRY, which triggers testis determination. Marsupials also have a Y-borne SRY gene, implying that this mechanism is ancestral to therians, the SRY gene having diverged from its X-borne homologue SOX3 at least 180 million years ago. The rare exceptions have clearly lost and replaced the SRY mechanism recently. Other vertebrate classes have a variety of sex-determining mechanisms, but none shares the therian SRY-driven XX female:XY male system. In monotreme mammals (platypus and echidna), which branched from the therian lineage 210 million years ago, no orthologue of SRY has been found. In this study we show that its partner SOX3 is autosomal in platypus and echidna, mapping among human X chromosome orthologues to platypus chromosome 6, and to the homologous chromosome 16 in echidna. The autosomal localization of SOX3 in monotreme mammals, as well as non-mammal vertebrates, implies that SRY is absent in Prototheria and evolved later in the therian lineage 210-180 million years ago. Sex determination in platypus and echidna must therefore depend on another male-determining gene(s) on the Y chromosomes, or on the different dosage of a gene(s) on the X chromosomes.

Genomic imprinting in marsupial placentation

Genomic imprinting is a widespread epigenetic phenomenon in eutherian mammals, which regulates many aspects of growth and development. Parental conflict over the degree of maternal nutrient transfer is the favoured hypothesis for the evolution of imprinting. Marsupials, like eutherian mammals, are viviparous but deliver an altricial young after a short gestation supported by a fully functional placenta, so can shed light on the evolution and time of acquisition of genomic imprinting. All orthologues of eutherian imprinted genes examined have a conserved expression in the marsupial placenta regardless of their imprint status. Differentially methylated regions (DMRs) are the most common mechanism controlling genomic imprinting in eutherian mammals, but none were found in the marsupial imprinted orthologues of IGF2 receptor (IGF2R), INS or mesoderm-specific transcript (MEST). Instead, histone modification appears to be the mechanism used to silence these genes. At least three genes in marsupials have DMRs: H19, IGF2 and PEG10. PEG10 is particularly interesting as it is derived from a retrotransposon, providing the first direct evidence that retrotransposon insertion can drive the evolution of an imprinted region and of a DMR in mammals. The insertion occurred after the prototherian-therian mammal divergence, suggesting that there may have been strong selection for the retention of imprinted regions that arose during the evolution of placentation. There is currently no evidence for genomic imprinting in the egg-laying monotreme mammals. However, since these mammals do have a short-lived placenta, imprinting appears to be correlated with viviparity but not placentation.