PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (970432)

Clipboard (0)
None

Related Articles

1.  DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation 
BMC Genomics  2013;14:685.
Background
Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes.
Results
Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse.
Conclusions
Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development.
doi:10.1186/1471-2164-14-685
PMCID: PMC3829101  PMID: 24094292
Genomic imprinting; Placenta; Next generation sequencing; Differentially Methylated Region (DMR); DNMT1; AIM1
2.  EXTRA-EMBRYONIC-SPECIFIC IMPRINTED EXPRESSION IS RESTRICTED TO DEFINED LINEAGES IN THE POST-IMPLANTATION EMBRYO 
Developmental biology  2011;353(2):420-431.
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.
doi:10.1016/j.ydbio.2011.02.017
PMCID: PMC3081948  PMID: 21354127
genomic imprinting; placenta; yolk sac; non-coding RNA; insulator
3.  Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse 
PLoS Genetics  2013;9(4):e1003401.
Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.
Author Summary
BPA is a widely used compound to which humans are exposed, and recent studies have demonstrated the association between exposure and adverse developmental outcomes in both animal models and humans. Unfortunately, exact mechanisms of BPA–induced health abnormalities are unclear, and elucidation of these relevant biological pathways is critical for understanding the public health implication of exposure. Recently, increasing data have demonstrated the ability of BPA to induce changes in DNA methylation, suggesting that epigenetic mechanisms are relevant. In this work, we study effects of BPA exposure on expression and regulation of imprinted genes in the mouse. Imprinted genes are regulated by differential DNA methylation, and they play critical roles during fetal, placental, and postnatal development. We have found that fetal exposure to BPA at physiologically relevant doses alters expression and methylation status of imprinted genes in the mouse embryo and placenta, with the latter tissue exhibiting the more significant changes. Additionally, abnormal imprinting is associated with defective placental development. Our data demonstrate that BPA exposure may perturb fetal and postnatal health through epigenetic changes in the embryo as well as through alterations in placental development.
doi:10.1371/journal.pgen.1003401
PMCID: PMC3616904  PMID: 23593014
4.  Genome-wide mapping of imprinted differentially methylated regions by DNA methylation profiling of human placentas from triploidies 
Background
Genomic imprinting is an important epigenetic process involved in regulating placental and foetal growth. Imprinted genes are typically associated with differentially methylated regions (DMRs) whereby one of the two alleles is DNA methylated depending on the parent of origin. Identifying imprinted DMRs in humans is complicated by species- and tissue-specific differences in imprinting status and the presence of multiple regulatory regions associated with a particular gene, only some of which may be imprinted. In this study, we have taken advantage of the unbalanced parental genomic constitutions in triploidies to further characterize human DMRs associated with known imprinted genes and identify novel imprinted DMRs.
Results
By comparing the promoter methylation status of over 14,000 genes in human placentas from ten diandries (extra paternal haploid set) and ten digynies (extra maternal haploid set) and using 6 complete hydatidiform moles (paternal origin) and ten chromosomally normal placentas for comparison, we identified 62 genes with apparently imprinted DMRs (false discovery rate <0.1%). Of these 62 genes, 11 have been reported previously as DMRs that act as imprinting control regions, and the observed parental methylation patterns were concordant with those previously reported. We demonstrated that novel imprinted genes, such as FAM50B, as well as novel imprinted DMRs associated with known imprinted genes (for example, CDKN1C and RASGRF1) can be identified by using this approach. Furthermore, we have demonstrated how comparison of DNA methylation for known imprinted genes (for example, GNAS and CDKN1C) between placentas of different gestations and other somatic tissues (brain, kidney, muscle and blood) provides a detailed analysis of specific CpG sites associated with tissue-specific imprinting and gestational age-specific methylation.
Conclusions
DNA methylation profiling of triploidies in different tissues and developmental ages can be a powerful and effective way to map and characterize imprinted regions in the genome.
doi:10.1186/1756-8935-4-10
PMCID: PMC3154142  PMID: 21749726
5.  Comparative analysis of sequence characteristics of imprinted genes in human, mouse, and cattle 
Mammalian Genome  2007;18(6-7):538-547.
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: 10.1007/s00335-007-9039-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00335-007-9039-z
PMCID: PMC2000230  PMID: 17653590
6.  Assessment of genomic imprinting of SLC38A4, NNAT, NAP1L5, and H19 in cattle 
BMC Genetics  2006;7:49.
Background
At present, few imprinted genes have been reported in cattle compared to human and mouse. Comparative expression analysis and imprinting status are powerful tools for investigating the biological significance of genomic imprinting and studying the regulation mechanisms of imprinted genes. The objective of this study was to assess the imprinting status and pattern of expression of the SLC38A4, NNAT, NAP1L5, and H19 genes in bovine tissues.
Results
A polymorphism-based approach was used to assess the imprinting status of four bovine genes in a total of 75 tissue types obtained from 12 fetuses and their dams. In contrast to mouse Slc38a4, which is imprinted in a tissue-specific manner, we found that SLC38A4 is not imprinted in cattle, and we found it expressed in all adult tissues examined. Two single nucleotide polymorphisms (SNPs) were identified in NNAT and used to distinguish between monoallelic and biallelic expression in fetal and adult tissues. The two transcripts of NNAT showed paternal expression like their orthologues in human and mouse. However, in contrast to human and mouse, NNAT was expressed in a wide range of tissues, both fetal and adult. Expression analysis of NAP1L5 in five heterozygous fetuses showed that the gene was paternally expressed in all examined tissues, in contrast to mouse where imprinting is tissue-specific. H19 was found to be maternally expressed like its orthologues in human, sheep, and mouse.
Conclusion
This is the first report on the imprinting status of SLC38A4, NAP1L5, and on the expression patterns of the two transcripts of NNAT in cattle. It is of interest that the imprinting of NAP1L5, NNAT, and H19 appears to be conserved between mouse and cow, although the tissue distribution of expression differs. In contrast, the imprinting of SLC38A4 appears to be species-specific.
doi:10.1186/1471-2156-7-49
PMCID: PMC1629023  PMID: 17064418
7.  Chromosome-wide identification of novel imprinted genes using microarrays and uniparental disomies 
Nucleic Acids Research  2006;34(12):e88.
Genomic imprinting refers to a specialized form of epigenetic gene regulation whereby the expression of a given allele is dictated by parental origin. Defining the extent and distribution of imprinting across genomes will be crucial for understanding the roles played by imprinting in normal mammalian growth and development. Using mice carrying uniparental disomies or duplications, microarray screening and stringent bioinformatics, we have developed the first large-scale tissue-specific screen for imprinted gene detection. We quantify the stringency of our methodology and relate it to previous non-tissue-specific large-scale studies. We report the identification in mouse of four brain-specific novel paternally expressed transcripts and an additional three genes that show maternal expression in the placenta. The regions of conserved linkage in the human genome are associated with the Prader–Willi Syndrome (PWS) and Beckwith–Wiedemann Syndrome (BWS) where imprinting is known to be a contributing factor. We conclude that large-scale systematic analyses of this genre are necessary for the full impact of genomic imprinting on mammalian gene expression and phenotype to be elucidated.
doi:10.1093/nar/gkl461
PMCID: PMC1524921  PMID: 16855283
8.  Identification of Novel Imprinted Differentially Methylated Regions by Global Analysis of Human-Parthenogenetic-Induced Pluripotent Stem Cells 
Stem Cell Reports  2013;1(1):79-89.
Parental imprinting is an epigenetic phenomenon by which genes are expressed in a monoallelic fashion, according to their parent of origin. DNA methylation is considered the hallmark mechanism regulating parental imprinting. To identify imprinted differentially methylated regions (DMRs), we compared the DNA methylation status between multiple normal and parthenogenetic human pluripotent stem cells (PSCs) by performing reduced representation bisulfite sequencing. Our analysis identified over 20 previously unknown imprinted DMRs in addition to the known DMRs. These include DMRs in loci associated with human disorders, and a class of intergenic DMRs that do not seem to be related to gene expression. Furthermore, the study showed some DMRs to be unstable, liable to differentiation or reprogramming. A comprehensive comparison between mouse and human DMRs identified almost half of the imprinted DMRs to be species specific. Taken together, our data map novel DMRs in the human genome, their evolutionary conservation, and relation to gene expression.
Highlights
•Imprinted DMRs were identified by comparing normal and parthenogenetic human PSCs•The study showed some DMRs to be unstable, liable to differentiation or reprogramming•Over 20 imprinted DMRs, including a class of intergenic DMRs, were found•Comparison between mouse and human DMRs identified about half to be species specific
doi:10.1016/j.stemcr.2013.03.005
PMCID: PMC3757747  PMID: 24052944
9.  Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome 
PLoS ONE  2010;5(6):e10947.
Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.
doi:10.1371/journal.pone.0010947
PMCID: PMC2881032  PMID: 20532033
10.  Differential methylation persists at the mouse Rasgrf1 DMR in tissues displaying monoallelic and biallelic expression 
A subset of mammalian genes exhibits genomic imprinting, whereby one parental allele is preferentially expressed. Differential DNA methylation at imprinted loci serves both to mark the parental origin of the alleles and to regulate their expression. In mouse, the imprinted gene Rasgrf1 is associated with a paternally methylated imprinting control region which functions as an enhancer blocker in its unmethylated state. Because Rasgrf1 is imprinted in a tissue-specific manner, we investigated the methylation pattern in monoallelic and biallelic tissues to determine if methylation of this region is required for both imprinted and non-imprinted expression. Our analysis indicates that DNA methylation is restricted to the paternal allele in both monoallelic and biallelic tissues of somatic and extraembryonic lineages. Therefore, methylation serves to mark the paternal Rasgrf1 allele throughout development, but additional factors are required for appropriate tissue-specific regulation of expression at this locus.
PMCID: PMC2872782  PMID: 19502804
genomic imprinting; Rasgrf1; DNA methylation; epigenetics
11.  Imprinted genes show unique patterns of sequence conservation 
BMC Genomics  2010;11:649.
Background
Genomic imprinting is an evolutionary conserved mechanism of epigenetic gene regulation in placental mammals that results in silencing of one of the parental alleles. In order to decipher interactions between allele-specific DNA methylation of imprinted genes and evolutionary conservation, we performed a genome-wide comparative investigation of genomic sequences and highly conserved elements of imprinted genes in human and mouse.
Results
Evolutionarily conserved elements in imprinted regions differ from those associated with autosomal genes in various ways. Whereas for maternally expressed genes strong divergence of protein-encoding sequences is most prominent, paternally expressed genes exhibit substantial conservation of coding and noncoding sequences. Conserved elements in imprinted regions are marked by enrichment of CpG dinucleotides and low (TpG+CpA)/(2·CpG) ratios indicate reduced CpG deamination. Interestingly, paternally and maternally expressed genes can be distinguished by differences in G+C and CpG contents that might be associated with unusual epigenetic features. Especially noncoding conserved elements of paternally expressed genes are exceptionally G+C and CpG rich. In addition, we confirmed a frequent occurrence of intronic CpG islands and observed a decelerated degeneration of ancient LINE-1 repeats. We also found a moderate enrichment of YY1 and CTCF binding sites in imprinted regions and identified several short sequence motifs in highly conserved elements that might act as additional regulatory elements.
Conclusions
We discovered several novel conserved DNA features that might be related to allele-specific DNA methylation. Our results hint at reduced CpG deamination rates in imprinted regions, which affects mostly noncoding conserved elements of paternally expressed genes. Pronounced differences between maternally and paternally expressed genes imply specific modes of evolution as a result of differences in epigenetic features and a special response to selective pressure. In addition, our data support the potential role of intronic CpG islands as epigenetic key regulatory elements and suggest that evolutionary conserved LINE-1 elements fulfill regulatory functions in imprinted regions.
doi:10.1186/1471-2164-11-649
PMCID: PMC3091771  PMID: 21092170
12.  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.
doi:10.1098/rstb.2012.0151
PMCID: PMC3539366  PMID: 23166401
marsupials; monotremes; eutherians; retrotransposons; placentation; lactation
13.  The Importance of Imprinting in the Human Placenta 
PLoS Genetics  2010;6(7):e1001015.
As a field of study, genomic imprinting has grown rapidly in the last 20 years, with a growing figure of around 100 imprinted genes known in the mouse and approximately 50 in the human. The imprinted expression of genes may be transient and highly tissue-specific, and there are potentially hundreds of other, as yet undiscovered, imprinted transcripts. The placenta is notable amongst mammalian organs for its high and prolific expression of imprinted genes. This review discusses the development of the human placenta and focuses on the function of imprinting in this organ. Imprinting is potentially a mechanism to balance parental resource allocation and it plays an important role in growth. The placenta, as the interface between mother and fetus, is central to prenatal growth control. The expression of genes subject to parental allelic expression bias has, over the years, been shown to be essential for the normal development and physiology of the placenta. In this review we also discuss the significance of genes that lack conservation of imprinting between mice and humans, genes whose imprinted expression is often placental-specific. Finally, we illustrate the importance of imprinting in the postnatal human in terms of several human imprinting disorders, with consideration of the brain as a key organ for imprinted gene expression after birth.
doi:10.1371/journal.pgen.1001015
PMCID: PMC2895656  PMID: 20617174
14.  Characterisation of marsupial PHLDA2 reveals eutherian specific acquisition of imprinting 
Background
Genomic imprinting causes parent-of-origin specific gene expression by differential epigenetic modifications between two parental genomes. We previously reported that there is no evidence of genomic imprinting of CDKN1C in the KCNQ1 domain in the placenta of an Australian marsupial, the tammar wallaby (Macropus eugenii) whereas tammar IGF2 and H19, located adjacent to the KCNQ1 domain in eutherian mammals, are imprinted. We have now identified and characterised the marsupial orthologue of PHLDA2, another gene in the KCNQ1 domain (also known as IPL or TSSC3) that is imprinted in eutherians. In mice, Phlda2 is a dose-sensitive negative regulator of placental growth, as Cdkn1c is for embryonic growth.
Results
Tammar PHLDA2 is highly expressed in the yolk sac placenta compared to other fetal tissues, confirming a similar expression pattern to that of mouse Phlda2. However, tammar PHLDA2 is biallelically expressed in both the fetus and yolk sac placenta, so it is not imprinted. The lack of imprinting in tammar PHLDA2 suggests that the acquisition of genomic imprinting of the KCNQ1 domain in eutherian mammals, accompanied with gene dosage reduction, occurred after the split of the therian mammals into the marsupials and eutherians.
Conclusions
Our results confirm the idea that acquisition of genomic imprinting in the KCNQ1 domain occurred specifically in the eutherian lineage after the divergence of marsupials, even though imprinting of the adjacent IGF2-H19 domain arose before the marsupial-eutherian split. These data are consistent with the hypothesis that genomic imprinting of the KCNQ1 domain may have contributed to the evolution of more complex placentation in the eutherian lineage by reduction of the gene dosage of negative regulators for both embryonic and placental growth.
doi:10.1186/1471-2148-11-244
PMCID: PMC3170258  PMID: 21854573
15.  Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution  
PLoS Genetics  2007;3(5):e65.
Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.
Author Summary
Imprinted genes are expressed in a parent-of-origin manner, where one of the two inherited copies of the imprinted gene is silenced. Aberrations in the expression of these genes, which generally regulate growth, are associated with various developmental disorders, emphasizing the importance of their discovery and analysis. In this study, we identify a novel imprinted gene, named KLF14, on human Chromosome 7. It is predicted to bind DNA and regulate transcription and was shown to be expressed from the maternally inherited chromosome in all human and mouse tissues examined. Surprisingly, we did not identify molecular signatures generally associated with imprinted regions, such as DNA methylation. Additionally, the identification of numerous DNA sequence variants led to an in-depth analysis of the gene's evolution. It was determined that there is greater variability in KLF14 in the human lineage, when compared to other primates, with a significantly increased number of polymorphisms encoding for changes at the protein level, suggesting human-specific accelerated evolution. As the first example of an imprinted transcript undergoing accelerated evolution in the human lineage, we propose that the accumulation of polymorphisms in KLF14 may be aided by the silencing of the inactive allele, allowing for stronger selection.
doi:10.1371/journal.pgen.0030065
PMCID: PMC1865561  PMID: 17480121
16.  Identification of the Imprinted KLF14 Transcription Factor Undergoing Human-Specific Accelerated Evolution  
PLoS Genetics  2007;3(5):e65.
Imprinted genes are expressed in a parent-of-origin manner and are located in clusters throughout the genome. Aberrations in the expression of imprinted genes on human Chromosome 7 have been suggested to play a role in the etiologies of Russell-Silver Syndrome and autism. We describe the imprinting of KLF14, an intronless member of the Krüppel-like family of transcription factors located at Chromosome 7q32. We show that it has monoallelic maternal expression in all embryonic and extra-embryonic tissues studied, in both human and mouse. We examine epigenetic modifications in the KLF14 CpG island in both species and find this region to be hypomethylated. In addition, we perform chromatin immunoprecipitation and find that the murine Klf14 CpG island lacks allele-specific histone modifications. Despite the absence of these defining features, our analysis of Klf14 in offspring from DNA methyltransferase 3a conditional knockout mice reveals that the gene's expression is dependent upon a maternally methylated region. Due to the intronless nature of Klf14 and its homology to Klf16, we suggest that the gene is an ancient retrotransposed copy of Klf16. By sequence analysis of numerous species, we place the timing of this event after the divergence of Marsupialia, yet prior to the divergence of the Xenarthra superclade. We identify a large number of sequence variants in KLF14 and, using several measures of diversity, we determine that there is greater variability in the human lineage with a significantly increased number of nonsynonymous changes, suggesting human-specific accelerated evolution. Thus, KLF14 may be the first example of an imprinted transcript undergoing accelerated evolution in the human lineage.
Author Summary
Imprinted genes are expressed in a parent-of-origin manner, where one of the two inherited copies of the imprinted gene is silenced. Aberrations in the expression of these genes, which generally regulate growth, are associated with various developmental disorders, emphasizing the importance of their discovery and analysis. In this study, we identify a novel imprinted gene, named KLF14, on human Chromosome 7. It is predicted to bind DNA and regulate transcription and was shown to be expressed from the maternally inherited chromosome in all human and mouse tissues examined. Surprisingly, we did not identify molecular signatures generally associated with imprinted regions, such as DNA methylation. Additionally, the identification of numerous DNA sequence variants led to an in-depth analysis of the gene's evolution. It was determined that there is greater variability in KLF14 in the human lineage, when compared to other primates, with a significantly increased number of polymorphisms encoding for changes at the protein level, suggesting human-specific accelerated evolution. As the first example of an imprinted transcript undergoing accelerated evolution in the human lineage, we propose that the accumulation of polymorphisms in KLF14 may be aided by the silencing of the inactive allele, allowing for stronger selection.
doi:10.1371/journal.pgen.0030065
PMCID: PMC1865561  PMID: 17480121
17.  At Least Ten Genes Define the Imprinted Dlk1-Dio3 Cluster on Mouse Chromosome 12qF1 
PLoS ONE  2009;4(2):e4352.
Background
Genomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.
Methodology/Principal Findings
To identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.
Conclusions/Significance
Ten imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/“Rian”, AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.
doi:10.1371/journal.pone.0004352
PMCID: PMC2632752  PMID: 19194500
18.  A Prevalence of Imprinted Genes within the Total Transcriptomes of Human Tissues and Cells 
Genomic imprinting is an epigenetic phenomenon that causes a differential expression of paternally and maternally inherited alleles of a subset of genes (the so-called imprinted genes). Imprinted genes are distributed throughout the genome and it is predicted that about 1% of the human genes may be imprinted. It is recognized that the allelic expression of imprinted genes varies between tissues and developmental stages. The current study represents the first attempt to estimate a prevalence of imprinted genes within the total human transcriptome. In silico analysis of the normalized expression profiles of a comprehensive panel of 173 established and candidate human imprinted genes was performed, in 492 publicly available SAGE libraries. The latter represent human cell and tissue samples in a variety of physiological and pathological conditions. Variations in the prevalence of imprinted genes within the total transcriptomes (ranging from 0.08% to 4.36%) and expression profiles of the individual imprinted genes are assessed. This paper thus provides a useful reference on the size of the imprinted transcriptome and expression of the individual imprinted genes.
doi:10.1155/2012/793506
PMCID: PMC3446743  PMID: 22997578
19.  Evolution of the CDKN1C-KCNQ1 imprinted domain 
Background
Genomic imprinting occurs in both marsupial and eutherian mammals. The CDKN1C and IGF2 genes are both imprinted and syntenic in the mouse and human, but in marsupials only IGF2 is imprinted. This study examines the evolution of features that, in eutherians, regulate CDKN1C imprinting.
Results
Despite the absence of imprinting, CDKN1C protein was present in the tammar wallaby placenta. Genomic analysis of the tammar region confirmed that CDKN1C is syntenic with IGF2. However, there are fewer LTR and DNA elements in the region and in intron 9 of KCNQ1. In addition there are fewer LINEs in the tammar compared with human and mouse. While the CpG island in intron 10 of KCNQ1 and promoter elements could not be detected, the antisense transcript KCNQ1OT1 that regulates CDKN1C imprinting in human and mouse is still expressed.
Conclusion
CDKN1C has a conserved function, likely antagonistic to IGF2, in the mammalian placenta that preceded its acquisition of imprinting. CDKN1C resides in synteny with IGF2, demonstrating that imprinting of the two genes did not occur concurrently to balance maternal and paternal influences on the growth of the placenta. The expression of KCNQ1OT1 in the absence of CDKN1C imprinting suggests that antisense transcription at this locus preceded imprinting of this domain. These findings demonstrate the stepwise accumulation of control mechanisms within imprinted domains and show that CDKN1C imprinting cannot be due to its synteny with IGF2 or with its placental expression in mammals.
doi:10.1186/1471-2148-8-163
PMCID: PMC2427030  PMID: 18510768
20.  Developmental Stage-Specific Imprinting of IPL in Domestic Pigs (Sus scrofa) 
Imprinted in placenta and liver (IPL) gene has been identified as an imprinted gene in the mouse and human. Its sequence and imprinting status, however, have not been determined in the domestic pigs. In the present study, a 259 base pair-specific sequence for IPL gene of the domestic pig was obtained and a novel SNP, a T/C transition, was identified in IPL exon 1. The C allele of this polymorphism was found to be the predominant allele in Landrace,Yorkshire, and Duroc. The frequency of CC genotype and C allele are different in Duroc as compared with Yorkshire (P = .038 and P = .005, resp.). Variable imprinting status of this gene was observed in different developmental stages. For example, it is imprinted in 1-dayold newborns (expressed from the maternal allele), but imprinting was lost in 180-day-old adult (expressed from both parental alleles). Real-time PCR analysis showed the porcine IPL gene is expressed in all tested eight organ/tissues. The expression level was significantly higher in spleen, duodenum, lung, and bladder of 180-day-old Lantang adult compared to that in 1-day-old newborns Lantang pigs (P < .05). In conclusion, the imprinting of the porcine IPL gene is developmental stage and tissue specific.
doi:10.1155/2010/527539
PMCID: PMC2879551  PMID: 20589073
21.  Convergent and divergent evolution of genomic imprinting in the marsupial Monodelphis domestica 
BMC Genomics  2012;13:394.
Background
Genomic imprinting is an epigenetic phenomenon resulting in parent-of-origin specific monoallelic gene expression. It is postulated to have evolved in placental mammals to modulate intrauterine resource allocation to the offspring. In this study, we determined the imprint status of metatherian orthologues of eutherian imprinted genes.
Results
L3MBTL and HTR2A were shown to be imprinted in Monodelphis domestica (the gray short-tailed opossum). MEST expressed a monoallelic and a biallelic transcript, as in eutherians. In contrast, IMPACT, COPG2, and PLAGL1 were not imprinted in the opossum. Differentially methylated regions (DMRs) involved in regulating imprinting in eutherians were not found at any of the new imprinted loci in the opossum. Interestingly, a novel DMR was identified in intron 11 of the imprinted IGF2R gene, but this was not conserved in eutherians. The promoter regions of the imprinted genes in the opossum were enriched for the activating histone modification H3 Lysine 4 dimethylation.
Conclusions
The phenomenon of genomic imprinting is conserved in Therians, but the marked difference in the number and location of imprinted genes and DMRs between metatherians and eutherians indicates that imprinting is not fully conserved between the two Therian infra-classes. The identification of a novel DMR at a non-conserved location as well as the first demonstration of histone modifications at imprinted loci in the opossum suggest that genomic imprinting may have evolved in a common ancestor of these two Therian infra-classes with subsequent divergence of regulatory mechanisms in the two lineages.
doi:10.1186/1471-2164-13-394
PMCID: PMC3507640  PMID: 22899817
Genomic imprinting; Marsupials; Eutherians
22.  Molecular Characterization of the Neuronatin Gene in the Porcine Placenta 
PLoS ONE  2012;7(8):e43325.
Imprinted genes play important roles in placental and embryonic development. Neuronatin (NNAT), first identified as an imprinted gene in human and mouse brains, played important roles in neuronal differentiation in the brain and in glucose-mediated insulin secretion in pancreatic β cells. In the pig, NNAT was reported to be imprinted in eleven tissues. Our previous microarray hybridization study showed that NNAT was differentially expressed in Yorkshire and Meishan pig placentas, but the imprinting status and function of NNAT in the placenta have not been investigated. We demonstrated for the first time that NNAT was monoallelically expressed in the placenta. Immunochemistry analysis showed that NNAT was located in the uterine luminal and glandular epithelium in placentas. We also confirmed the differential expression of NNAT in Meishan and Yorkshire pig placentas by qPCR. Using IPA software and the published literature, we created a model network of the possible relationships between NNAT and glucose transporter genes. A dual luciferase reporter assay demonstrated that the crucial promoter region of NNAT contained a CANNTG sequence in the +210 to +215 positions, which corresponded to the E-box. Our findings demonstrated important roles of NNAT in placenta function.
doi:10.1371/journal.pone.0043325
PMCID: PMC3427331  PMID: 22937033
23.  Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting 
PLoS Genetics  2007;3(4):e55.
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.
Author Summary
Genomic imprinting is a gene regulatory mechanism controlling parent-of-origin-dependent expression of genes. In eutherians, imprinting is essential for fetal and placental development and defects in this mechanism are the cause of several genetic disorders. In eutherian mammals, genomic imprinting is controlled by differential methylation of the DNA. However, no such methylation-dependent mechanism had been previously identified in association with marsupial imprinting. By comparing the genome of all three extant classes of mammals (eutherians, marsupials, and monotremes), we have investigated the evolution of PEG10 (paternally expressed 10), a retrotransposon-derived imprinted gene that is essential for the formation of the placenta in the mouse. PEG10 was present in a marsupial species, the tammar wallaby, but absent from an egg-laying monotreme species, the platypus. Therefore, PEG10 was inserted into the genome at the time when the placenta and viviparity were evolving in therian mammals. This study has shown that PEG10 is not only imprinted in a marsupial, but that its imprint is regulated by differential methylation, suggesting a common origin for methylation in the therian ancestor. These results provide direct evidence that retrotransposon insertion can drive the evolution of genomic imprinting in mammals.
doi:10.1371/journal.pgen.0030055
PMCID: PMC1851980  PMID: 17432937
24.  Retrotransposon Silencing by DNA Methylation Can Drive Mammalian Genomic Imprinting 
PLoS Genetics  2007;3(4):e55.
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.
Author Summary
Genomic imprinting is a gene regulatory mechanism controlling parent-of-origin-dependent expression of genes. In eutherians, imprinting is essential for fetal and placental development and defects in this mechanism are the cause of several genetic disorders. In eutherian mammals, genomic imprinting is controlled by differential methylation of the DNA. However, no such methylation-dependent mechanism had been previously identified in association with marsupial imprinting. By comparing the genome of all three extant classes of mammals (eutherians, marsupials, and monotremes), we have investigated the evolution of PEG10 (paternally expressed 10), a retrotransposon-derived imprinted gene that is essential for the formation of the placenta in the mouse. PEG10 was present in a marsupial species, the tammar wallaby, but absent from an egg-laying monotreme species, the platypus. Therefore, PEG10 was inserted into the genome at the time when the placenta and viviparity were evolving in therian mammals. This study has shown that PEG10 is not only imprinted in a marsupial, but that its imprint is regulated by differential methylation, suggesting a common origin for methylation in the therian ancestor. These results provide direct evidence that retrotransposon insertion can drive the evolution of genomic imprinting in mammals.
doi:10.1371/journal.pgen.0030055
PMCID: PMC1851980  PMID: 17432937
25.  Genomic imprinting mechanisms in embryonic and extraembryonic mouse tissues 
Heredity  2010;105(1):45-56.
Imprinted genes in mice and humans mainly occur in clusters that are associated with differential DNA methylation of an imprint control element (ICE) and at least one nonprotein-coding RNA (ncRNA). Imprinted gene silencing is achieved by parental-specific insulator activity of the unmethylated ICE mediated by CTCF (CCCTC-binding factor) binding, or by ncRNA expression from a promoter in the unmethylated ICE. In many imprinted clusters, some genes, particularly those located furthest away from the ICE, show imprinted expression only in extraembryonic tissues. Recent research indicates that genes showing imprinted expression only in extraembryonic tissues may be regulated by different epigenetic mechanisms compared with genes showing imprinted expression in extraembryonic tissues and in embryonic/adult tissues. The study of extraembryonic imprinted expression, thus, has the potential to illuminate novel epigenetic strategies, but is complicated by the need to collect tissue from early stages of mouse development, when extraembryonic tissues may be contaminated by maternal cells or be present in limited amounts. Research in this area would be advanced by the development of an in vitro model system in which genetic experiments could be conducted in less time and at a lower cost than with mouse models. Here, we summarize what is known about the mechanisms regulating imprinted expression in mouse extraembryonic tissues and explore the possibilities for developing an in vitro model.
doi:10.1038/hdy.2010.23
PMCID: PMC2887385  PMID: 20234385
genomic imprinting; placenta; yolk sac; noncoding RNA; insulator

Results 1-25 (970432)