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.     

The distinguishing sequence characteristics of mouse imprinted genes

Sequence comparison analysis has been carried out for 31 imprinted mouse genes and a set of 150 control genes. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable elements (SINEs), in particular SINE Alu repeats. This is similar to recent analyses of human imprinted genes and supports the suggestion that there is either active selection against SINE elements in imprinted regions or a reduced rate of insertion of these elements. The reduction in numbers of SINEs was more consistent in paternally expressed genes, whereas for maternally expressed genes significantly reduced numbers of SINE-B2 elements were coupled with increased numbers of SINE-B4 and SINE-ID elements. Paternally expressed genes were also found to be associated with a lower GC content. Discriminant analysis revealed that the two sub-groups of imprinted genes can be cleanly separated from each other on the basis of their genomic sequence characteristics and that they tend to localize to different genomic compartments. The differences between the sequence characteristics of imprinted and control genes have also enabled us to develop a discriminant function that can be used in a genome-wide screen to identify candidate imprinted genes.     

A phylogenetic approach to test for evidence of parental conflict or gene duplications associated with protein-encoding imprinted orthologous genes in placental mammals

There are multiple theories on the evolution of genomic imprinting. We investigated whether the molecular evolution of true orthologs of known imprinted genes provides support for theories based on gene duplication or parental conflicts (where mediated by amino-acid changes). Our analysis of 34 orthologous genes demonstrates that the vast majority of mammalian imprinted genes have not undergone any subsequent significant gene duplication within placental species, suggesting that selection pressures against gene duplication events could be operating for imprinted loci. As antagonistic co-evolution between imprinted genes can regulate offspring growth, proteins mediating this interaction could be subject to rapid evolution via positive selection. Supporting this, we detect evidence of site specific positive selection for the imprinted genes OSBPL5 (and GNASXL), and detect lineage-specific positive selection for 14 imprinted genes where it is known that the gene is imprinted in a specific lineage, namely for: PLAGL1, IGF2, SLC22A18, OSBPL5, DCN, DLK1, RASGRF1, IGF2R, IMPACT, GRB10, NAPIL4, UBE3A, GATM and GABRG3. However, there is an overall lack of concordance between the known imprinting status of each gene (i.e. whether the gene is imprinted or biallelically expressed in a particular mammalian lineage) and positive selection. While only a small number of orthologs of imprinted loci display evidence of positive selection, we observe that the majority of orthologs of imprinted loci display high levels of micro-synteny conservation and have undergone very few cis- or trans-duplications in placental mammalian lineages.     

A novel approach for identifying candidate imprinted genes through sequence analysis of imprinted and control genes

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.     

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.     

Epigenetic states and expression of imprinted genes in human embryonic stem cells

AIM: To investigate the epigenetic states and expression of imprinted genes in five human embryonic stem cell (hESC) lines derived in Taiwan.METHODS: The heterozygous alleles of single nucleotide polymorphisms (SNPs) at imprinted genes were analyzed by sequencing genomic DNAs of hESC lines and the monoallelic expression of the imprinted genes were confirmed by sequencing the cDNAs. The expression profiles of 32 known imprinted genes of five hESC lines were determined using Affymetrix human genome U133 plus 2.0 DNA microarray.RESULTS: The heterozygous alleles of SNPs at seven imprinted genes, IPW, PEG10, NESP55, KCNQ1, ATP10A, TCEB3C and IGF2, were identified and the monoallelic expression of these imprinted genes except IGF2 were confirmed. The IGF2 gene was found to be imprinted in hESC line T2 but partially imprinted in line T3 and not imprinted in line T4 embryoid bodies. Ten imprinted genes, namely GRB10, PEG10, SGCE, MEST, SDHD, SNRPN, SNURF, NDN, IPW and NESP55, were found to be highly expressed in the undifferentiated hESC lines and down-regulated in differentiated derivatives. The UBE3A gene abundantly expressed in undifferentiated hESC lines and further up-regulated in differentiated tissues. The expression levels of other 21 imprinted genes were relatively low in undifferentiated hESC lines and five of these genes (TP73, COPG2, OSBPL5, IGF2 and ATP10A) were found to be up-regulated in differentiated tissues.CONCLUSION: The epigenetic states and expression of imprinted genes in hESC lines should be thoroughly studied after extended culture and upon differentiation in order to understand epigenetic stability in hESC lines before their clinical applications.     

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.     

Is it genomic imprinting or preferential expression?

Imprinted genes are monoallelically expressed in a parent-of-origin-specific manner, but for many genes reported to be imprinted, the occurrence of preferential expression--where both alleles are expressed but one is expressed more strongly than the other in a parent-of-origin-specific way--has been reported. This preferential expression found in genes described as imprinted has not been thoroughly addressed in genomic imprinting studies. To study this phenomenon, 50 genes, reported to be imprinted in the mouse, were chosen for investigation. Preferential expression was observed for 21 of 27 maternally expressed genes. However, only 5 of 23 paternally expressed genes showed preferential expression. Recently, it has been reported that a remarkable proportion of non-imprinted genes show differential allelic expression. If there is overlap between non-imprinted genes that are differentially expressed and imprinted genes that are preferentially expressed, we need to set new definitions of imprinted genes that, in turn, would probably lead to reassessments of the total number of imprinted genes in mammalian species.     

Computation method to identify differential allelic gene expression and novel imprinted genes

MOTIVATION: Genomic imprinting plays an important role in both normal development and diseases. Abnormal imprinting is strongly associated with several human diseases including cancers. Most of the imprinted genes were discovered in the neighborhood of the known imprinted genes. This approach is difficult to extend to analyze the whole genome. We have decided to take a computational approach to systematically search the whole genome for the presence of mono-allelic expressed genes and imprinted genes in human genome. RESULTS: A computational method was developed to identify novel imprinted or mono-allelic genes. Individuals represented in human cDNA libraries were genotyped using Bayesian statistics, and differential expression of polymorphic alleles was identified. A significant reduction in the number of libraries that expressed both alleles, measured by Z-statistics, is a strong indicator for an imprinted or a mono-allelic gene. AVAILABILITY: The data sets are available at http://leelab.nci.nih.gov/leelab/jsp/IGDM/IGDM.html     

Extra-embryonic-specific imprinted expression is restricted to defined lineages in the post-implantation embryo

A subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably, these so-called “placental-specific” imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue, and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar “extra-embryonic–lineage-specific” pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS “extra-embryonic–lineage-specific” imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic, and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic–lineage-specific imprinted expression.     

PHLDA2 is an imprinted gene in cattle

Genomic imprinting is an epigenetic non-Mendelian phenomenon found predominantly in placental mammals. Imprinted genes display differential expression in the offspring depending on whether the gene is maternally or paternally inherited. Currently, some 100 imprinted genes have been reported in mammals, and while some of these genes are imprinted across most mammalian species, others have been shown to be imprinted in only a few species. The PHLDA2 gene that codes for a pleckstrin homology-like domain, family A (member 2), protein has to date been shown to be a maternally expressed imprinted gene in humans, mice and pigs. Genes subject to imprinting can have major effects on mammalian growth, development and disease. For instance, disruption of imprinted genes can lead to aberrant growth syndromes in cloned domestic mammals, and it has been demonstrated that PHLDA2 mRNA expression levels are aberrant in the placenta of somatic clones of cattle. In this study, we demonstrate that PHLDA2 is expressed across a range of cattle foetal tissues and stages and provide the first evidence that PHLDA2 is a monoallelically expressed imprinted gene in cattle foetal tissues, and also in the bovine placenta.     

The role of imprinted genes in fetal growth abnormalities

Epigenetics, and in particular imprinted genes, have a critical role in the development and function of the placenta, which in turn has a central role in the regulation of fetal growth and development. A unique characteristic of imprinted genes is their expression from only one allele, maternal or paternal and dependent on parent of origin. This unique expression pattern may have arisen as a mechanism to control the flow of nutrients from the mother to the fetus, with maternally expressed imprinted genes reducing the flow of resources and paternally expressed genes increasing resources to the fetus. As a result, any epigenetic deregulation affecting this balance can result in fetal growth abnormalities. Imprinting-associated disorders in humans, such as Beckwith-Wiedemann and Angelman syndrome, support the role of imprinted genes in fetal growth. Similarly, assisted reproductive technologies in animals have been shown to affect the epigenome of the early embryo and the expression of imprinted genes. Their role in disorders such as intrauterine growth restriction appears to be more complex, in that imprinted gene expression can be seen as both causative and protective of fetal growth restriction. This protective or compensatory effect needs to be explored more fully.     

Do imprinted genes have few and small introns?

A gene is described as imprinted if its pattern of expression depends on whether it passed the previous generation in a male or female germ line. A recent paper⁽¹⁾ reports that imprinted genes have fewer and smaller introns than a control set of genes. The differences are striking but their interpretation is unclear. The loss of introns after a gene becomes imprinted is not sufficient to explain why imprinted genes have fewer introns than average, because related unimprinted genes also have few introns. Similarly, small introns appear to be a property of chromosomal region rather than of imprinting status itself, because neighboring unimprinted genes also have small introns.     

Reproductive barrier and genomic imprinting in the endosperm of flowering plants

In flowering plants, success or failure of seed development is determined by various genetic mechanisms. During sexual reproduction, double fertilization produces the embryo and endosperm, which both contain maternally and paternally derived genomes. In endosperm, a reproductive barrier is often observed in inter-specific crosses. Endosperm is a tissue that provides nourishment for the embryo within the seed, in a similar fashion to the placenta of mammals, and for the young seedling after germination. This review considers the relationship between the reproductive barrier in endosperm and genomic imprinting. Genomic imprinting is an epigenetic mechanism that results in mono-allelic gene expression that is parent-of-origin dependent. In Arabidopsis, recent studies of several imprinted gene loci have identified the epigenetic mechanisms that determine genomic imprinting. A crucial feature of genomic imprinting is that the maternally and paternally derived imprinted genes must carry some form of differential mark, usually DNA methylation and/or histone modification. Although the epigenetic marks should be complementary on maternally and paternally imprinted genes within a single species, it is possible that neither the patterns of epigenetic marks nor expression of imprinted genes are the same in different species. Moreover, in hybrid endosperm, the regulation of expression of imprinted genes can be affected by upstream regulatory mechanisms in the male and female gametophytes. Species-specific variations in epigenetic marks, the copy number of imprinted genes, and the epigenetic regulation of imprinted genes in hybrids might all play a role in the reproductive barriers observed in the endosperm of interspecific and interploidy crosses. These predicted molecular mechanisms might be related to earlier models such as the "endosperm balance number" (EBN) and "polar nuclei activation" (PNA) hypotheses.