Comparative genomics approach toward critical determinants for the imprinting of an evolutionarily conserved gene Impact

The Impact is an evolutionarily conserved gene subjected to genomic imprinting in mouse but not in human. A characteristic tandem repeat similar to those found in many other imprinted genes and an elevated expression level, both observed only for the mouse gene, are implicated in the evolution of imprinting, to which the repeat might have contributed via enhancement of the expression. To pursue the possibility further, we examined the correlation among the repeat, expression level, and imprinting of Impact in various mammals ranging from rodents, lagomorphs, carnivores, artiodactyls to primates. Intriguingly, rabbit Impact is abundantly expressed and imprinted like those of rodents, but is missing the repeat from its first intron like those of other mammals that express both alleles weakly. It thus seems that lineage-specific enhancement of gene expression rather than the tandem repeat per se played a critical role in the evolution of imprinting of Impact.     

Comparative analysis of sequence characteristics of imprinted genes in human, mouse, and cattle

Genomic imprinting is an epigenetic mechanism that results in monoallelic expression of genes depending on parent-of-origin of the allele. Although the conservation of genomic imprinting among mammalian species has been widely reported for many genes, there is accumulating evidence that some genes escape this conservation. Most known imprinted genes have been identified in the mouse and human, with few imprinted genes reported in cattle. Comparative analysis of genomic imprinting across mammalian species would provide a powerful tool for elucidating the mechanisms regulating the unique expression of imprinted genes. In this study we analyzed the imprinting of 22 genes in human, mouse, and cattle and found that in only 11 was imprinting conserved across the three species. In addition, we analyzed the occurrence of the sequence elements CpG islands, C + G content, tandem repeats, and retrotransposable elements in imprinted and in nonimprinted (control) cattle genes. We found that imprinted genes have a higher G + C content and more CpG islands and tandem repeats. Short interspersed nuclear elements (SINEs) were notably fewer in number in imprinted cattle genes compared to control genes, which is in agreement with previous reports for human and mouse imprinted regions. Long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) were found to be significantly underrepresented in imprinted genes compared to control genes, contrary to reports on human and mouse. Of considerable significance was the finding of highly conserved tandem repeats in nine of the genes imprinted in all three species. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Conserved synteny of mammalian imprinted genes in chicken, frog, and fish genomes

Conservation of synteny of mammalian imprinted genes between chicken and human suggested that highly conserved gene clusters were selected long before these genes were recruited for genomic imprinting in mammals. Here we have applied in silico mapping of orthologous genes in pipid frog, zebrafish, spotted green and Japanese pufferfish to show considerable conservation of synteny in lower vertebrates. More than 400 million years ago in a common ancestor of teleost fish and tetrapods, 'preimprinted' chromosome regions homologous to human 6q25, 7q21, 7q32, 11p15, and 15q11-->q12 already contained most present-day mammalian imprinted genes. Interestingly, some imprinted gene orthologues which are isolated from imprinted clusters in mouse and human could be linked to preimprinted regions in lower vertebrates, indicating that separation occurred during mammalian evolution. On the contrary, newly arisen genes by segmental duplication in the mammalian lineage, i.e. SNRPN and FRAT3, were transposed or translocated to imprinted clusters and recruited for parent-specific activity. By analysis of currently available sequences of non-mammalian vertebrates, the imprinted gene clusters homologous to human chromosomes 14q32 and 19q12 are only poorly conserved in chicken, frog, and fish and, therefore, may not have evolved from ancestral preimprinted gene arrays. Evidently, evolution of imprinted gene clusters is an ongoing and dynamic process in mammals. In general, imprinted gene orthologues do not show a higher degree of synteny conservation in vertebrates than non-imprinted genes interspersed with or adjacent to an imprinted cluster.     

The origin and evolution of genomic imprinting and viviparity in mammals

Genomic imprinting is widespread in eutherian mammals. Marsupial mammals also have genomic imprinting, but in fewer loci. It has long been thought that genomic imprinting is somehow related to placentation and/or viviparity in mammals, although neither is restricted to mammals. Most imprinted genes are expressed in the placenta. There is no evidence for genomic imprinting in the egg-laying monotreme mammals, despite their short-lived placenta that transfers nutrients from mother to embryo. Post natal genomic imprinting also occurs, especially in the brain. However, little attention has been paid to the primary source of nutrition in the neonate in all mammals, the mammary gland. Differentially methylated regions (DMRs) play an important role as imprinting control centres in each imprinted region which usually comprises both paternally and maternally expressed genes (PEGs and MEGs). The DMR is established in the male or female germline (the gDMR). Comprehensive comparative genome studies demonstrated that two imprinted regions, PEG10 and IGF2-H19, are conserved in both marsupials and eutherians and that PEG10 and H19 DMRs emerged in the therian ancestor at least 160 Ma, indicating the ancestral origin of genomic imprinting during therian mammal evolution. Importantly, these regions are known to be deeply involved in placental and embryonic growth. It appears that most maternal gDMRs are always associated with imprinting in eutherian mammals, but emerged at differing times during mammalian evolution. Thus, genomic imprinting could evolve from a defence mechanism against transposable elements that depended on DNA methylation established in germ cells.     

Comparative sequence analysis of imprinted genes between human and mouse to reveal imprinting signatures

We performed a comparative genomic sequence analysis between human and mouse for 24 imprinted genes on human chromosomes 1, 6, 7, 11, 13, 14, 15, 18, 19, and 20. The MEME program was used to search for motifs within conserved sequences among the imprinted genes and we then used the MAST program to analyze for the presence or absence of motifs in the imprinted genes and 128 nonimprinted genes. Our analysis identified 15 motifs that were significantly enriched in the imprinted genes. We generated a logistic regression model by combining multiple motifs as input variables and the 24 imprinted genes and the 128 nonimprinted genes as a training set. The accuracy, sensitivity, and specificity of our model were 98, 92, and 99%, respectively. The model was further validated by an open test on 12 additional imprinted genes. The motifs identified in this study are novel imprinting signatures, which should improve our understanding of genomic imprinting and the role of genomic imprinting in human diseases.     

Genomic imprinting in ruminants: allele-specific gene expression in parthenogenetic sheep

Studies in the mouse have established that both parental genomes are essential for normal embryonic development. Parthenogenetic mouse embryos (which have two maternal genomes and no paternal genome), for example, are growth-retarded and die at early postimplantation stages. The distinct maternal and paternal contributions are mediated by genomic imprinting, an epigenetic mechanism by which the expression of certain genes is dependent on whether they are inherited from mother or father. Although comparative studies have established that many imprinted mouse (and rat) genes are allele-specifically expressed in humans as well (and vice versa), so far imprinting studies have not been performed in other mammalian species. When considering evolutionary theories of genomic imprinting, it would be important to know how widely it is conserved among placental mammals. We have investigated its conservation in a bovid ruminant, the domestic sheep, by comparing parthenogenetic and normal control embryos. Our study establishes that, like in the mouse, parthenogenetic development in sheep is associated with growth-retardation and does not proceed beyond early fetal stages. These developmental abnormalities are most likely caused by imprinted genes. We demonstrate that, indeed, like in mice and humans, the growth-related PEG1/MEST and Insulin-like Growth Factor 2 (IGF2) genes are expressed from the paternal chromosome in sheep. These observations suggest that genomic imprinting is conserved in a third, evolutionarily rather diverged group of placental mammals, the ruminants. Received: 13 May 1998 / Accepted: 16 July 1998     

Imprinting evolution and the price of silence

In contrast to the biallelic expression of most genes, expression of genes subject to genomic imprinting is monoallelic and based on the sex of the transmitting parent. Possession of only a single active allele can lead to deleterious health consequences in humans. Aberrant expression of imprinted genes, through either genetic or epigenetic alterations, can result in developmental failures, neurodevelopmental and neurobehavioral disorders and cancer. The evolutionary emergence of imprinting occurred in a common ancestor to viviparous mammals after divergence from the egg-laying monotremes. Current evidence indicates that imprinting regulation in metatherian mammals differs from that in eutherian mammals. This suggests that imprinting mechanisms are evolving from those that were established 150 million years ago. Therefore, comparing genomic sequence of imprinted domains from marsupials and eutherians with those of orthologous regions in monotremes offers a potentially powerful bioinformatics approach for identifying novel imprinted genes and their regulatory elements. Such comparative studies will also further our understanding of the molecular evolution and phylogenetic distribution of imprinted genes.     

An evolutionary scenario for genomic imprinting of Impact lying between nonimprinted neighbors.

Mouse Impact is the sole imprinted gene mapped to chromosome 18 to date. Despite its remarkable evolutionary conservation, human IMPACT was shown to escape genomic imprinting. Here we identified Hrh4 and Osbpl1 as the distal and proximal nearest neighbors of Impact, respectively, and found that both genes are expressed biallelically. Thus, in contrast with most imprinted genes, Impact fails to show apparent physical clustering with other imprinted genes. Since Impact not only lies in an intergenic region but also consists of 11 exons, it does not seem to be an imprinted gene generated by a retrotransposition. Hazardous effects of overexpressed Impact, a genomic segment containing paralogues of Hrh4 and Osbpl1 but not of Impact, and enhanced promoter activity in the mouse led us to propose an alternative model. This model assumes that segmental duplication followed by enhancement of the promoter activity in the lineage to mouse is responsible for the species-specific imprinting of Impact.     

Non-coding RNAs and the acquisition of genomic imprinting in mammals

Genomic imprinting, representing parent-specific expression of alleles at a locus, is mainly evident in flowering plants and placental mammals. Most imprinted genes, including numerous non-coding RNAs, are located in clusters regulated by imprinting control regions (ICRs). The acquisition and evolution of genomic imprinting is among the most fundamental genetic questions. Discoveries about the transition of mammalian imprinted gene domains from their non-imprinted ancestors, especially recent studies undertaken on the most ancient mammalian clades — the marsupials and monotremes from which model species genomes have recently been sequenced, are of high value. By reviewing and analyzing these studies, a close connection between non-coding RNAs and the acquisition of genomic imprinting in mammals is demonstrated. The evidence comes from two observations accompanied with the acquisition of the imprinting: (i) many novel non-coding RNA genes emerged in imprinted regions; (ii) the expressions of some conserved non-coding RNAs have changed dramatically. Furthermore, a systematical analysis of imprinted snoRNA (small nucleolar RNA) genes from 15 vertebrates suggests that the origination of imprinted snoRNAs occurred after the divergence between eutherians and marsupials, followed by a rapid expansion leading to the fixation of major gene families in the eutherian ancestor prior to the radiation of modern placental mammals. Involved in the regulation of imprinted silencing and mediating the chromatins epigenetic modification may be the major roles that non-coding RNAs play during the acquisition of genomic imprinting in mammals. Supported by National Natural Science Foundation of China (Grant No. 30830066), the Ministry of Education of China and Natural Science Foundation of Guangdong Province (Grant No. IRT0447, NSF-05200303) and National Key Basic Research and Development Program of China (Grant No. 2005CB724600)     

Human and mouse ZFP57 proteins are functionally interchangeable in maintaining genomic imprinting at multiple imprinted regions in mouse ES cells

Genomic imprinting is a common epigenetic phenomenon in mammals. Dysregulation of genomic imprinting has been implicated in a variety of human diseases. ZFP57 is a master regulator in genomic imprinting. Loss of ZFP57 causes loss of DNA methylation imprint at multiple imprinted regions in mouse embryos, as well as in embryonic stem (ES) cells. Similarly, mutations in human ZFP57 result in hypomethylation at many imprinted regions and are associated with transient neonatal diabetes and other human diseases. Mouse and human Zfp57 genes are located in the same syntenic block. However, mouse and human ZFP57 proteins only display about 50% sequence identity with different number of zinc fingers. It is not clear if they share similar mechanisms in maintaining genomic imprinting. Here we report that mouse and human ZFP57 proteins are functionally interchangeable. Expression of exogenous wild-type human ZFP57 could maintain DNA methylation imprint at three imprinted regions in mouse ES cells in the absence of endogenous mouse ZFP57. However, mutant human ZFP57 proteins containing the mutations found in human patients could not substitute for endogenous mouse ZFP57 in maintaining genomic imprinting in ES cells. Like mouse ZFP57, human ZFP57 and its mutant proteins could bind to mouse KAP1, the universal cofactor for KRAB zinc finger proteins, in mouse ES cells. Thus, we conclude that mouse and human ZFP57 are orthologs despite relatively low sequence identity and mouse ES cell system that we had established before is a valuable system for functional analyses of wild-type and mutant human ZFP57 proteins.     

Chicken orthologues of mammalian imprinted genes are clustered on macrochromosomes and replicate asynchronously

In the chicken genome, most orthologues of mouse imprinted genes are clustered on macrochromosomes. Only a few orthologues are located in the microchromosome complement. Macrochromosomal and, to a lesser extent, microchromosomal regions containing imprinted gene orthologues exhibit asynchronous DNA replication. We conclude that highly conserved arrays of imprinted gene orthologues were selected during vertebrate evolution, long before these genes were recruited for parent-specific gene expression by genomic imprinting mechanisms. Evidently, the macrochromosome complement provides a better chromatin environment for the establishment of asynchronous DNA replication and imprinted gene expression later in evolution than microchromosomes.     

The continuing quest to comprehend genomic imprinting

The discovery of the phenomenon of genomic imprinting in mammals showed that the parental genomes are functionally non-equivalent. Considerable advances have occurred in the field over the past 20 years, which has resulted in the identification and functional analysis of a number of imprinted genes the expression of which is determined by their parental origin. These genes belong to many diverse categories and they have been shown to regulate growth, complex aspects of mammalian physiology and behavior. Many aspects of the mechanism of imprinting have also been elucidated. However, the reasons for the evolution of genomic imprinting remain enigmatic. Further research is needed to determine if there is any relationship between the apparently diverse functions of imprinted genes in mammals, and their role in human diseases. It also remains to be seen what common features exist amongst the diverse imprinting control elements. The mechanisms involved in the erasure and re-establishment of imprints should provide deeper insights into epigenetic mechanisms of wide general interest.     

Conserved features of imprinted differentially methylated domains

Genomic imprinting is a conserved epigenetic phenomenon in eutherian mammals, with regards both to the genes that are imprinted and the mechanism underlying the expression of just one of the parental alleles. Epigenetic modifications of alleles of imprinted genes are established during oogenesis and spermatogenesis, and these modifications are then inherited. Differentially methylated domains (DMDs) of imprinted genes are the genomic sites of these inherited epigenetic imprints. We previously showed that CpG-rich imperfect tandem direct repeats within three different mouse DMDs (Snurf/Snrpn, Kcnq1 and Igf2r), each with a unique sequence, play a central role in maintaining the differential methylation. This finding implicates repeat-related DNA structure, not sequence, in the imprinting mechanism. To better define the important features of this signal, we compared sequences of these three DMD tandem repeats among mammalian species. All DMD repeats contain short indirect repeats, many of which are organized into larger unit repeats. Even though the larger repeat units undergo deletion and addition during evolution (most likely through unequal crossovers during meiosis), the size of DMD tandem repeated regions has remained remarkably stable during mammalian evolution. Moreover, all three DMD tandem repeats have a high-CpG content, an ordered arrangement of CpG dinucleotides, and similar predicted secondary structures. These observations suggest that a structural feature or features of these DMD tandem repeats is the conserved DMD imprinting signal.     

Genomic imprinting of IGF2, p57(KIP2) 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.     

The regulation and biological significance of genomic imprinting in mammals

Genomic imprinting is a system of non-Mendelian inheritance that is unique to mammals. Two types of imprinted genes show parent-of-origin-specific expression patterns: the paternally expressed genes (Pegs), and the maternally expressed genes (Megs). Parental genomic imprinting memory is maintained in the somatic cell lineage and regulates the expression of Pegs and Megs, while it is erased and re-established in the germ cell lineage according to the sex of the individual. The paternal and maternal imprinting mechanisms, which regulate different sets of Pegs and Megs, are essential for establishing the parental expression profiles of imprinted genes that are observed in sperms and eggs. Based on recent evidence, we outline the relationship between parental imprinting and the expression profiles of Pegs and Megs and discuss a novel view of the regulation of genomic imprinting. We also discuss the biological significance of genomic imprinting and propose hypotheses on the essential nature of genomic imprinting and the close relationship between genomic imprinting and the acquisition of placental tissues during mammalian evolution.     

Complementation hypothesis: the necessity of a monoallelic gene expression mechanism in mammalian development

Gene expression from both parental alleles (biallelic expression) is beneficial in minimizing the occurrence of recessive genetic disorders in diploid organisms. However, imprinted genes in mammals display parent of origin-specific monoallelic expression. As some imprinted genes play essential roles in mammalian development, the reason why mammals adopted the genomic imprinting mechanism has been a mystery since its discovery. In this review, based on the recent studies on imprinted gene regulation we discuss several advantageous features of a monoallelic expression mechanism and the necessity of genomic imprinting in the current mammalian developmental system. We further speculate how the present genomic imprinting system has been established during mammalian evolution by the mechanism of complementation between paternal and maternal genomes under evolutionary pressure predicted by the genetic conflict hypothesis.     

Convergent evolution of genomic imprinting in plants and mammals

Parental genomic imprinting is characterized by the expression of a selected panel of genes from one of the two parental alleles. Recent evidence shows that DNA methylation and histone modifications are responsible for this parent-of-origin-dependent expression of imprinted genes. Because similar epigenetic marks have been recruited independently in plants and mammals, the only organisms in which imprinted gene loci have been identified so far, this phenomenon represents a case for convergent evolution. Here we discuss the emerging parallels in imprinting in both taxa. We also describe the significance of imprinting for reproduction and discuss potential models for its evolution.