Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression.The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O’Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.     

Imprinting defects in mouse embryos: stochastic errors or polymorphic phenotype?

Summary: Defects in expression of imprinted genes are believed to cause developmental abnormalities and play a role in carcinogenesis. To determine whether spontaneous imprinting defects may occur in mouse embryos, we studied the expression of two imprinted genes H19 and Igf2 in individual postimplantation 7.5 d.p.c. and 8.5 d.p.c. embryos. Biallelic expression of H19 was found in 1.6% of the embryos, whereas biallelic expression of Igf2 was found in 0.5% of the embryos. The loss of H19 imprinting (LOI) observed in a small fraction of early postimplantation embryos may be purely stochastic. Alternatively, since we never observed it in an inbred background, it may depend on genetic factors acting in trans. Either mechanism could explain the occurrence of polymorphic imprinting as well as the genesis of sporadic imprinting defects, including cancer. The frequency of LOI of H19 was higher than the incidence of sporadic imprinting disorders in humans (about 1 in 20,000). This contradiction may be explained by different incidence of imprinting errors in different imprinted regions of the genome, in different species, or by loss of the majority of nonmosaic embryos with imprinting defects before birth. genesis 31:11–16, 2001. © 2001 Wiley‐Liss, Inc.     

Genomic imprinting in mammals

A review of the data on the mechanisms and effects of genomic imprinting, an epigenetic phenomenon regulating the development in placentate mammals, is presented. In contrast to the majority of gene loci with biallelic expression, the expression of imprinted loci is monoallelic. In humans and mice, more than 300 imprinted loci have been identified, in which maternal or paternal alleles may either be expressed or be found in a repressed state during ontogeny. Imprinting is established during gametogenesis, and the repression of an allele of the imprinted locus is determined by methylation of the key regulatory element of this allele. Both the maternal and paternal chromosome sets are required for normal development in mammals. This is why parthenogenesis and androgenesis in these animals are impossible in nature. As a result of differential gene expression of many imprinted loci, the balance of gene activity is established, which is necessary for normal proliferation and differentiation of various cell clones in embryogenesis. Many human developmental abnormalities and syndromes are determined by defective genomic imprinting. In particular, the loss of imprints, which is followed by the occurrence of biallelic expression of some imprinted loci, may cause malignant tumors.     

Genomic Imprinting in Mammals

A review of the data on the mechanisms and effects of genomic imprinting, an epigenetic phenomenon regulating the development in placentate mammals, is presented. In contrast to the majority of gene loci with biallelic expression, the expression of imprinted loci is monoallelic. In humans and mice, more than 30 imprinted loci have been identified, in which maternal or paternal alleles may either be expressed or be found in a repressed state during ontogeny. Imprinting is established during gametogenesis, and the repression of an allele of the imprinted locus is determined by methylation of the key regulatory element of this allele. Both the maternal and paternal chromosome sets are required for normal development in mammals. This is why parthenogenesis and androgenesis in these animals are impossible in nature. As a result of differential gene expression of many imprinted loci, the balance of gene activity is established, which is necessary for normal proliferation and differentiation of various cell clones in embryogenesis. Many human developmental abnormalities and syndromes are determined by defective genomic imprinting. In particular, the loss of imprints, which is followed by the occurrence of biallelic expression of some imprinted loci, may cause malignant tumors.     

Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice

The completely embryonic stem (ES) cell-derived mice (ES mice) produced by tetraploid embryo complementation provide us with a rapid and powerful approach for functional genome analysis. However, inbred ES cell lines often fail to generate ES mice. The genome of mouse ES cells is extremely unstable during in vitro culture and passage, and the expression of the imprinted genes is most likely to be affected. Whether the ES mice retain or repair the abnormalities of the donor ES cells has still to be determined. Here we report that the inbred ES mice were efficiently produced with the inbred ES cell line (SCR012). The ES fetuses grew more slowly before day 17.5 after mating, but had an excessive growth from day 17.5 to birth. Five imprinted genes examined (H19, Igf2, Igf2r, Peg1, Peg3) were expressed abnormally in ES fetuses. Most remarkably, the expression of H19 was dramatically repressed in the ES fetuses through the embryo developmental stage, and this repression was associated with abnormal biallelic methylation of the H19 upstream region. The altered methylation pattern of H19 was further demonstrated to have arisen in the donor ES cells and persisted on in vivo differentiation to the fetal stage. These results indicate that the ES fetuses did retain the epigenetic alterations in imprinted genes from the donor ES cells.     

Selective loss of imprinting in the placenta following preimplantation development in culture

Preimplantation development is a period of dynamic epigenetic change that begins with remodeling of egg and sperm genomes, and ends with implantation. During this time, parental-specific imprinting marks are maintained to direct appropriate imprinted gene expression. We previously demonstrated that H19 imprinting could be lost during preimplantation development under certain culture conditions. To define the lability of genomic imprints during this dynamic period and to determine whether loss of imprinting continues at later stages of development, imprinted gene expression and methylation were examined after in vitro preimplantation culture. Following culture in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed and undermethylated. However, only a subset of individual cultured blastocysts (approximately 65%) exhibited biallelic expression, while others maintained imprinted H19 expression. Loss of H19 imprinting persisted in mid-gestation conceptuses. Placental tissues displayed activation of the normally silent allele for H19, Ascl2, Snrpn, Peg3 and Xist while in the embryo proper imprinted expression for the most part was preserved. Loss of imprinted expression was associated with a decrease in methylation at the H19 and Snrpn imprinting control regions. These results indicate that tissues of trophectoderm origin are unable to restore genomic imprints and suggest that mechanisms that safeguard imprinting might be more robust in the embryo than in the placenta.     

Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo

The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression.     

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.     

Allelic switching of the imprinted IGF2R gene in cloned bovine fetuses and calves

Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3’UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied.     

Loss of methylation at H19 DMD is associated with biallelic expression and reduced development in cattle derived by somatic cell nuclear transfer

Although cloning of mammals has been achieved successfully, the percentage of live offspring is very low because of reduced fetal size and fewer implantation sites. Recent studies have attributed such pathological conditions to abnormal reprogramming of the donor cell used for cloning. The inability of the oocyte to fully restore the differentiated status of a somatic cell to its pluripotent and undifferentiated state is normally evidenced by aberrant DNA methylation patterns established throughout the genome during development to blastocyst. These aberrant methylation patterns are associated with abnormal expression of imprinted genes, which among other genes are essential for normal embryo development and gestation. We hypothesized that embryo loss and low implantation rates in cattle derived by somatic cell nuclear transfer (SCNT) are caused by abnormal epigenetic reprogramming of imprinted genes. To verify our hypothesis, we analyzed the parental expression and the differentially methylated domain (DMD) methylation status of the H19 gene. Using a parental-specific analysis, we confirmed for the first time that H19 biallelic expression is tightly associated with a severe demethylation of the paternal H19 DMD in SCNT embryos, suggesting that these epigenetic anomalies to the H19 locus could be directly responsible for the reduced size and low implantation rates of cloned embryos in cattle.     

Alterations of H19 Imprinting and IGF2 Replication Timing Are Infrequent in Beckwith–Wiedemann Syndrome

Beckwith–Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.     

Differential differences in methylation status of putative imprinted genes among cloned swine genomes

DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR) of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2) and two paternally imprinted loci (H19 and IGF2R). Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.     

Imprinting evolution and the price of silence

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