Genomic imprinting of IGF2, p57(KIP2) and PEG1/MEST in a marsupial, the tammar wallaby

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

A novel imprinted gene, KCNQ1DN, within the WT2 critical region of human chromosome 11p15.5 and its reduced expression in Wilms' tumors

WT2 is defined by a maternal-specific loss of heterozygosity on human chromosome 11p15.5 in Wilms' and other embryonal tumors. Therefore, the imprinted genes in this region are candidates for involvement in Wilms' tumorigenesis. We now report a novel imprinted gene, KCNQ1DN (KCNQ1 downstream neighbor). This gene is located between p57(KIP2) and KvLQT1 (KCNQ1) of 11p15.5 within the WT2 critical region. KCNQ1DN is imprinted and expressed from the maternal allele. We examined the expression of KCNQ1DN in Wilms' tumors. Seven of eighteen (39%) samples showed no expression. In contrast, other maternal imprinted genes in this region, including p57(KIP2), IMPT1, and IPL exhibited almost normal expression in these samples, although some samples expressed IGF2 biallelically. These results suggest that KCNQ1DN existing far from the H19/IGF2 region may play some role in Wilms' tumorigenesis along with IGF2.     

Methylation status of putative differentially methylated regions of porcine IGF2 and H19

This study was designed to identify the putative differentially methylated regions (DMRs) of the porcine imprinted genes insulin-like growth factor 2 and H19 (IGF2-H19), and to assess the genomic imprinting status of IGF2-H19 by identifying the methylation patterns of these regions in germ cells, and in tissues from porcine fetuses, an adult pig, as well as cloned offspring produced by somatic cell nuclear transfer (SCNT). Porcine IGF2-H19 DMRs exhibit a normal monoallelic methylation pattern (i.e., either the paternally- or the maternally derived allele is methylated) similar to the pattern observed for the same genes in the human and mice genomes. Examination of the methylation patterns of the IGF2-H19 DMRs revealed that the zinc finger protein binding sites CTCF1 and 2 did not exhibit differential methylation in both control and cloned offspring. In contrast, the CTCF3 and DMR2 loci of the IGF2 gene showed abnormal methylation in cloned offspring, but a normal differential or moderate methylation pattern in tissues from control offspring and an adult pig. Our data thus suggest that regulation of genomic imprinting at the porcine IGF2-H19 loci is conserved among species, and that the abnormal methylation pattern in the regulatory elements of imprinted genes may lead to an alteration in the coordinated expression of genes required for successful reprogramming, which, in consequence, may contribute to the low efficiency of porcine genome reprogramming induced by nuclear transfer.     

Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming

Loss of genomic imprinting of insulin-like growth factor II (IGF2) is a hallmark of many human neoplasms. We attempted to correct this aberrant epigenotype by transferring nuclei from human tumor cells that showed loss of IGF2 imprinting into enucleated mouse and human fibroblasts that had maintained normal IGF2 imprinting. After nuclear transfer, the abnormal biallelic expression of IGF2 in tumor nuclei transiently converted to normal monoallelic imprinted expression in the reconstructed diploid cells. In tetraploid hybrid cells, however, normal IGF2 imprinting was permanently restored in the tumor genome. Inhibition of the synthesis of putative trans imprinting factors with cycloheximide led to loss of IGF2 imprinting in normal cultured fibroblasts, suggesting that normal cells produce proteins that act in trans to induce or maintain genomic imprinting. These data demonstrate that an abnormal tumor epigenotype can be corrected by in vitro reprogramming, and suggest that loss of imprinting is associated with the loss of activity of non-CTCF trans imprinting factor(s) that are either inactivated or mutated in tumors.     

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.     

Bovine DNA methylation imprints are established in an oocyte size-specific manner, which are coordinated with the expression of the DNMT3 family proteins

A subset of genes, known as imprinted genes, is present in the mammalian genome. Genomic imprinting governs the monoallelic expression of these genes, depending on whether the gene was inherited from the sperm or the egg. This parent-of-origin specific gene expression is generally dependent on the epigenetic modification, DNA methylation, and the DNA methylation status of CpG dinucleotides residing in loci known as differentially methylated regions (DMRs). The enzymatic machinery responsible for the addition of methyl (-CH(3)) groups to the cytosine residue in the CpG dinucleotides are known as DNA methyltransferases (DNMTs). Correct establishment and maintenance of methylation patterns at imprinted genes has been associated with placental function and regulation of embryonic/fetal development. Much work has been carried out on imprinted genes in mouse and human; however, little is known about the methylation dynamics in the bovine oocyte. The primary objective of the present study was to characterize the establishment of methylation at maternally imprinted genes in bovine growing oocytes and to determine if the expression of the bovine DNMTs-DNMT3A, DNMT3B, and DNMT3L-was coordinated with DNA methylation during oocyte development. To this end, a panel of maternally imprinted genes was selected (SNRPN, MEST, IGF2R, PEG10, and PLAGL1) and putative DMRs for MEST, IGF2R, PEG10, and PLAGL1 were identified within the 5' regions for each gene; the SNRPN DMR has been reported previously. Conventional bisulfite sequencing revealed that methylation marks were acquired at all five DMRs investigated in an oocyte size-dependent fashion. This was confirmed for a selection of genes using pyrosequencing analysis. Furthermore, mRNA expression and protein analysis revealed that DNMT3A, DNMT3B, and DNMT3L are also present in the bovine oocyte during its growth phase. This study demonstrates for the first time that an increase in bovine imprinted gene DMR methylation occurs during oocyte growth, as is observed in mouse.     

Gene expression pattern of IGF2, PHLDA2, PEG10 and CDKN1C imprinted genes in spontaneous miscarriages or fetal deaths

Genomic imprinting is defined as an epigenetic modification that leads to parent-of-origin specific monoallelic expression. Some current research on the fetal control growth has been focused on the study of genes that display imprinted expression in utero. Four imprinted genes, two paternally expressed (IGF2 and PEG10) and two maternally expressed (PHLDA2 and CDKN1C), are well known to play a role in fetal growth and placental development. Pregnancy loss in the general reproductive population is a very common occurrence and other genetic causes beyond chromosomal abnormalities could be involved in spontaneous miscarriages or fetal deaths, such as alteration of expression in imprinted genes particularly those related to fetal or placental growth. Quantitative Real Time PCR was performed to evaluate gene expressions patterns of the four mentioned genes in spontaneous miscarriages or fetal deaths from 38 women. Expression levels of PHLDA2 gene were upregulated in the first trimester pregnancy cases and all four imprinted genes studied were upregulated in the second trimester of pregnancy cases comparing with controls. In third trimester PEG10 was downregulated in fetal samples group. This is the first study presenting data from human imprinted genes expression in spontaneous miscarriages or fetal deaths cases from the three trimesters of pregnancy.     

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.     

Allelic expression of IGF2 in marsupials and birds

Genomic imprinting, the parent-of-origin- specific expression of genes, has been observed in a variety of eutherian mammals. One gene that has been shown to be imprinted in all eutherians examined is the IGF2 gene. This gene encodes a potent fetal-specific growth factor that is expressed almost exclusively from the paternal chromosome. Several other imprinted genes in the IGF2 pathway are imprinted as well, suggesting that IGF2 is a focal point for the selective pressure leading to imprinted gene expression. This observation is in keeping with a proposal that imprinting arose as the result of a genetic conflict between parents over the allocation of maternal resources to the embryo. One prediction of this model is that imprinting exists in species in which there is at least some contribution of maternal resources to the embryo, and in which polyandry is observed. To test this prediction the allelic expression of the IGF2 gene was examined in two noneutherian species. The IGF2 gene was shown to be expressed in a paternal-specific manner identical to that in eutherians in Monodelphis domestica, a placental South American opossum. In contrast, the IGF2 gene is biallelic in expression in chickens, which are oviparous, and make no postfertilization contribution of maternal resources to the offspring. Received: 24 June 1999 / Accepted: 28 July 1999     

Monotreme IGF2 expression and ancestral origin of genomic imprinting

IGF2 (insulin-like growth factor 2) and M6P/IGF2R (mannose 6-phosphate/insulin-like growth factor 2 receptor) are imprinted in marsupials and eutherians but not in birds. These results along with the absence of M6P/IGF2R imprinting in the egg-laying monotremes indicate that the parental imprinting of fetal growth-regulatory genes may be unique to viviparous mammals. In this investigation, we have cloned IGF2 from two monotreme mammals, the platypus and echidna, to further investigate the origin of imprinting. We report herein that like M6P/IGF2R, IGF2 is not imprinted in monotremes. Thus, although IGF2 encodes for a highly conserved growth factor in chordates, it is only imprinted in therian mammals. These findings support a concurrent origin of IGF2 and M6P/IGF2R imprinting in the late Jurassic/early Cretaceous period. The absence of imprinting in monotremes, despite apparent interparental conflicts over maternal-offspring exchange, argues that a fortuitous congruency of genetic and epigenetic events may have limited the phylogenetic breadth of genomic imprinting to therian mammals. J. Exp. Zool. (Mol. Dev. Evol.) 291:205-212, 2001.     

Genomic imprinting of the insulin-like growth factor 2 gene in sheep

A number of genes in the human and mouse genomes are subject to genomic imprinting, with selective inactivation of one allele of a gene in a parent-of-origin specific manner. One of the first imprinted genes identified was the Insulin-like Growth Factor 2 gene (IGF2), which promotes growth of the fetus and is expressed from only the paternal allele in most tissues in both the mouse and human. The aim of this study was to establish the imprinting status of IGF2 in sheep (Ovis aries). Sheep provide an interesting model to study imprinting, owing to differences in their placental development and the fact that they have been subject to strong artificial selection for various production traits. We report the identification of a length polymorphism in the transcribed 3′-untranslated region of the ovine IGF2 gene. This polymorphism was used to map IGF2 to sheep Chromosome (Chr) 21 and demonstrate that IGF2 is indeed imprinted in sheep, being expressed from the paternal allele. We also report that the developmental switch from imprinted IGF2 expression in the fetal liver to biallelic IGF2 expression in the adult liver, which occurs in the human but not mouse, also occurs in sheep. Differences in male- and female-specific recombination values reported around the IGF2 locus in the human were also observed around the ovine IGF2 locus. The techniques developed in this study will enable the imprinting status of IGF2 to be assessed in a variety of tissues and stages of development in normal sheep. Received: 3 October 1998 / Accepted 29 January 1999     

Identification of four highly conserved genes between breakpoint hotspots BP1 and BP2 of the Prader-Willi/Angelman syndromes deletion region that have undergone evolutionary transposition mediated by flanking duplicons

Prader-Willi and Angelman syndromes (PWS and AS) typically result from an approximately 4-Mb deletion of human chromosome 15q11-q13, with clustered breakpoints (BP) at either of two proximal sites (BP1 and BP2) and one distal site (BP3). HERC2 and other duplicons map to these BP regions, with the 2-Mb PWS/AS imprinted domain just distal of BP2. Previously, the presence of genes and their imprinted status have not been examined between BP1 and BP2. Here, we identify two known (CYFIP1 and GCP5) and two novel (NIPA1 and NIPA2) genes in this region in human and their orthologs in mouse chromosome 7C. These genes are expressed from a broad range of tissues and are nonimprinted, as they are expressed in cells derived from normal individuals, patients with PWS or AS, and the corresponding mouse models. However, replication-timing studies in the mouse reveal that they are located in a genomic domain showing asynchronous replication, a feature typically ascribed to monoallelically expressed loci. The novel genes NIPA1 and NIPA2 each encode putative polypeptides with nine transmembrane domains, suggesting function as receptors or as transporters. Phylogenetic analyses show that NIPA1 and NIPA2 are highly conserved in vertebrate species, with ancestral members in invertebrates and plants. Intriguingly, evolutionary studies show conservation of the four-gene cassette between BP1 and BP2 in human, including NIPA1/2, CYFIP1, and GCP5, and proximity to the Herc2 gene in both mouse and Fugu. These observations support a model in which duplications of the HERC2 gene at BP3 in primates first flanked the four-gene cassette, with subsequent transposition of these four unique genes by a HERC2 duplicon-mediated process to form the BP1-BP2 region. Duplicons therefore appear to mediate genomic fluidity in both disease and evolutionary processes.     

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.     

M6P/IGF2R imprinting evolution in mammals

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.