Parent-of-origin specific QTL--a possibility towards understanding reciprocal effects in chicken and the origin of imprinting

Reciprocal effects for sexual maturity, egg production, egg quality traits and viability are well known in poultry crosses. They have been used in an optimal way to form profitable production hybrids. These effects have been hypothesized to originate from sex-linked genes, maternal effects or a combination of both. However, these may not be the only explanations for reciprocal effects. Recent mapping of quantitative trait loci (QTL) has revealed autosomal areas with parent-of-origin specific effects in the chicken. In mammals, parental imprinting, i.e. the specifically regulated expression of either maternal or paternal allele in the offspring, is the main cause of such effects. The most commonly accepted hypothesis for the origin of imprinting, the conflict hypothesis, assumes a genetic conflict of interest between the maternal and paternal genomes regarding the allocation of resources to the offspring. It also intrinsically implies that imprinting should not occur in oviparous taxa. However, new molecular genetic information has raised a need to review the possible involvement of imprinting or some related phenomena as a putative cause of reciprocal effects in poultry. Comparative mapping provides strong evidence for the conservation of orthologous imprinted gene clusters on chicken macrochromosomes. Furthermore, these gene clusters exhibit asynchronous DNA replication, an epigenetic mark specific for all imprinted regions. It has been proposed that these intrinsic chromosomal properties have been important for the evolution of imprinted gene expression in the mammalian lineage. Many of the mapped parent-of-origin specific QTL effects in chicken locate in or close to these conserved regions that show some of the basic features involved in monoallelic expression. If monoallelic expression in these regions would be observed in birds, the actual mechanism and cause may be different from the imprinting that evolved later in the mammalian lineage. In this review we discuss recent molecular genetic results that may provide tools for understanding of reciprocal differences in poultry breeding and the evolution of imprinting.     

A genome-wide survey of imprinted genes in rice seeds reveals imprinting primarily occurs in the endosperm

Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.     

Sequential gene activation and gene imprinting during early embryo development in maize

Gene imprinting is a widely observed epigenetic phenomenon in maize endosperm; however, whether it also occurs in the maize embryo remains controversial. Here, we used high‐throughput RNA sequencing on laser capture microdissected and manually dissected maize embryos from reciprocal crosses between inbred lines B73 and Mo17 at six time points (3–13 days after pollination, DAP) to analyze allelic gene expression patterns. Co‐expression analysis revealed sequential gene activation during maize embryo development. Gene imprinting was observed in maize embryos, and a greater number of imprinted genes were identified at early embryo stages. Sixty‐four strongly imprinted genes were identified (at the threshold of 9:1) on manually dissected embryos 5–13 DAP (more imprinted genes at 5 DAP). Forty‐one strongly imprinted genes were identified from laser capture microdissected embryos at 3 and 5 DAP (more imprinted genes at 3 DAP). Furthermore, of the 56 genes that were completely imprinted (at the threshold of 99:1), 36 were not previously identified as imprinted genes in endosperm or embryos. In situ hybridization demonstrated that most of the imprinted genes were expressed abundantly in maize embryonic tissue. Our results shed lights on early maize embryo development and provide evidence to support that gene imprinting occurs in maize embryos.     

Imprint status of M6P/IGF2R and IGF2 in chickens

Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.     

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.     

Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.     

A survey for novel imprinted genes in the mouse placenta by mRNA-seq

Many questions about the regulation, functional specialization, computational prediction, and evolution of genomic imprinting would be better addressed by having an exhaustive genome-wide catalog of genes that display parent-of-origin differential expression. As a first-pass scan for novel imprinted genes, we performed mRNA-seq experiments on embryonic day 17.5 (E17.5) mouse placenta cDNA samples from reciprocal cross F1 progeny of AKR and PWD mouse strains and quantified the allele-specific expression and the degree of parent-of-origin allelic imbalance. We confirmed the imprinting status of 23 known imprinted genes in the placenta and found that 12 genes reported previously to be imprinted in other tissues are also imprinted in mouse placenta. Through a well-replicated design using an orthogonal allelic-expression technology, we verified 5 novel imprinted genes that were not previously known to be imprinted in mouse (Pde10, Phf17, Phactr2, Zfp64, and Htra3). Our data suggest that most of the strongly imprinted genes have already been identified, at least in the placenta, and that evidence supports perhaps 100 additional weakly imprinted genes. Despite previous appearance that the placenta tends to display an excess of maternally expressed imprinted genes, with the addition of our validated set of placenta-imprinted genes, this maternal bias has disappeared.     

Brain-expressed imprinted genes and adult behaviour: the example of Nesp and Grb10

Imprinted genes are defined by their parent-of-origin-specific monoallelic expression. Although the epigenetic mechanisms regulating imprinted gene expression have been widely studied, their functional importance is still unclear. Imprinted genes are associated with a number of physiologies, including placental function and foetal growth, energy homeostasis, and brain and behaviour. This review focuses on genomic imprinting in the brain and on two imprinted genes in particular, Nesp and paternal Grb10, which, when manipulated in animals, have been shown to influence adult behaviour. These two genes are of particular interest as they are expressed in discrete and overlapping neural regions, recognised as key “imprinting hot spots” in the brain. Furthermore, these two genes do not appear to influence placental function and/or maternal provisioning of offspring. Consequently, by understanding their behavioural function we may begin to shed light on the evolutionary significance of imprinted genes in the adult brain, independent of the recognised role in maternal care. In addition, we discuss the potential future directions of research investigating the function of these two genes and the behavioural role of imprinted genes more generally.     

Monoalleleic transcription of the insulin-like growth factor-II gene (Igf2) in chick embryos

A polymorphism in the igf2 gene of chickens was identified using NlaIII (GenBank accession number AF218827). In some embryos, the igf2 alleles were expressed monoallelically from either maternal or paternal alleles. These data demonstrate that genomic imprinting is not confined to mammalian vertebrates and suggest that genomic imprinting evolved at an early stage of vertebrate evolution. The observations that the igf2 gene is imprinted in a minority of embryos suggest that the imprinting in birds is unrelated to embryonic growth. Genome imprinting may provide opportunities for evolution of genes in a nonexpressed state. In poultry breeding, the presence of imprinted genes may make a major contribution to unequal performance in reciprocal matings between commercial lines.     

Comparison of multimarker logistic regression models, with application to a genomewide scan of schizophrenia

Background

Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue.

Results

Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%).

Conclusions

Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.     

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.     

Establishment of paternal allele-specific DNA methylation at the imprinted mouse Gtl2 locus

The monoallelic expression of imprinted genes is controlled by epigenetic factors including DNA methylation and histone modifications. In mouse, the imprinted gene Gtl2 is associated with two differentially methylated regions: the IG-DMR, which serves as a gametic imprinting mark at which paternal allele-specific DNA methylation is inherited from sperm, and the Gtl2-DMR, which acquires DNA methylation on the paternal allele after fertilization. The timeframe during which DNA methylation is acquired at secondary DMRs during post-fertilization development and the relationship between secondary DMRs and imprinted expression have not been well established. In order to better understand the role of secondary DMRs in imprinting, we examined the methylation status of the Gtl2-DMR in pre- and post-implantation embryos. Paternal allele-specific DNA methylation of this region correlates with imprinted expression of Gtl2 during post-implantation development but is not required to implement imprinted expression during pre-implantation development, suggesting that this secondary DMR may play a role in maintaining imprinted expression. Furthermore, our developmental profile of DNA methylation patterns at the Cdkn1c- and Gtl2-DMRs illustrates that the temporal acquisition of DNA methylation at imprinted genes during post-fertilization development is not universally controlled.Key words: genomic imprinting, DNA methylation, Gtl2, secondary DMR, epigenetics     

Molecular cloning, mRNA expression and imprinting status of PEG3, NAP1L5 and PPP1R9A genes in pig

Imprinted genes are expressed monoallelically depending on their parental origin, and play important roles in the regulation of fetal growth, development, and postnatal behavior. Most genes known to be imprinted have been identified and studied in the human and the mouse. However, there are only a small number of reported imprinted genes in pigs. Therefore, identification and characterization of more imprinted genes in pigs is useful for comparative analysis of genomic imprinting across species. In this study, we cloned the porcine PEG3, NAP1L5 and PPP1R9A genes. The imprinting status of these genes was determined using sequencing directly and single nucleotide polymorphisms (SNPs) identified in individuals from reciprocal cross of Meishan and Large White pigs. Imprinting analysis was carried out in 13 different tissues (skeletal muscle, fat, pituitary gland, heart, lung, liver, kidney, spleen, stomach, small intestine, uterus, ovary and testis) from twelve 2-month-old piglets. Imprinting analysis showed that PEG3 and NAP1L5 were exclusively expressed from the paternal allele whereas PPP1R9A was biallelically expressed in all tissues tested where the genes were expressed. The study is of interest to understand the conservation of genomic imprinting among mammals at the 3 loci.     

Genomic imprinting: A missing piece of the Multiple Sclerosis puzzle?

Evidence for parent-of-origin effects in complex diseases such as Multiple Sclerosis (MS) strongly suggests a role for epigenetic mechanisms in their pathogenesis. In this review, we describe the importance of accounting for parent-of-origin when identifying new risk variants for complex diseases and discuss how genomic imprinting, one of the best-characterized epigenetic mechanisms causing parent-of-origin effects, may impact etiology of complex diseases. While the role of imprinted genes in growth and development is well established, the contribution and molecular mechanisms underlying the impact of genomic imprinting in immune functions and inflammatory diseases are still largely unknown. Here we discuss emerging roles of imprinted genes in the regulation of inflammatory responses with a particular focus on the Dlk1 cluster that has been implicated in etiology of experimental MS-like disease and Type 1 Diabetes. Moreover, we speculate on the potential wider impact of imprinting via the action of imprinted microRNAs, which are abundantly present in the Dlk1 locus and predicted to fine-tune important immune functions. Finally, we reflect on how unrelated imprinted genes or imprinted genes together with non-imprinted genes can interact in so-called imprinted gene networks (IGN) and suggest that IGNs could partly explain observed parent-of-origin effects in complex diseases. Unveiling the mechanisms of parent-of-origin effects is therefore likely to teach us not only about the etiology of complex diseases but also about the unknown roles of this fascinating phenomenon underlying uneven genetic contribution from our parents.This article is part of a Directed Issue entitled: Epigenetics dynamics in development and disease.     

A reassessment of imprinting regions and phenotypes on mouse chromosome 6: Nap1l5 locates within the currently defined sub-proximal imprinting region

Previous studies (Beechey, 2000) have shown that mouse proximal chromosome (Chr) 6 has two imprinting regions. An early embryonic lethality is associated with two maternal copies of the more proximal imprinting region, while mice with two maternal copies of the sub-proximal imprinting region are growth retarded at birth, the weight reduction remaining similar to adulthood. No detectable postnatal imprinting phenotype was seen in these earlier studies with two paternal copies of either region. The sub-proximal imprinting region locates distal to the T77H reciprocal translocation breakpoint in G-band 6A3.2 and results reported here show that it does not extend beyond the breakpoint of the more distal T6Ad translocation in 6C2. It has been confirmed that the postnatal growth retardation observed with two maternal copies of the sub-proximal region is established in utero, although placental size was normal. A new finding is that 16.5-18.5-dpc embryos, with two paternal copies of the sub-proximal imprinting region, were larger than their normal sibs, although placental size was normal. As no postnatal growth differences have been observed in these mice, the fetal overgrowth must normalize by birth. The imprinted genes Peg1/Mest, Copg2, Copg2as and Mit1/Lb9 map to the sub-proximal imprinting region and are thus candidates for the observed imprinting phenotypes. Another candidate is the recently reported imprinted gene Nap1l5. Expression studies of Nap1l5 in mice with two maternal or two paternal copies of different regions of Chr 6 have demonstrated that the gene locates within the sub-proximal imprinting region. FISH has mapped Nap1l5 to G-band 6C1, within the sub-proximal imprinting region but several G-bands distal to the Peg1/Mest cluster. This location, and the 30-Mb separation of these loci on the sequence map, makes it probable that Nap1l5 defines a new imprinting domain within the currently defined sub-proximal imprinting region.