Epigenetic regulation of the Igf2/H19 gene cluster

Igf2 (insulin‐like growth factor 2) and H19 genes are imprinted in mammals; they are expressed unevenly from the two parental alleles. Igf2 is a growth factor expressed in most normal tissues, solely from the paternal allele. H19 gene is transcribed (but not translated to a protein) from the maternal allele. Igf2 protein is a growth factor particularly important during pregnancy, where it promotes both foetal and placental growth and also nutrient transfer from mother to offspring via the placenta. This article reviews epigenetic regulation of the Igf2/H19 gene‐cluster that leads to parent‐specific expression, with current models including parental allele‐specific DNA methylation and chromatin modifications, DNA‐binding of insulator proteins (CTCFs) and three‐dimensional partitioning of DNA in the nucleus. It is emphasized that key genomic features are conserved among mammals and have been functionally tested in mouse. ‘The enhancer competition model’, ‘the boundary model’ and ‘the chromatin‐loop model’ are three models based on differential methylation as the epigenetic mark responsible for the imprinted expression pattern. Pathways are discussed that can account for allelic methylation differences; there is a recent study that contradicts the previously accepted fact that biallelic expression is accompanied with loss of differential methylation pattern.     

Insulin Like Growth Factor 2 Expression in the Rat Brain Both in Basal Condition and following Learning Predominantly Derives from the Maternal Allele

Insulin like growth factor 2 (Igf2) is known as a maternally imprinted gene involved in growth and development. Recently, Igf2 was found to also be regulated and required in the adult rat hippocampus for long-term memory formation, raising the question of its allelic regulation in adult brain regions following experience and in cognitive processes. We show that, in adult rats, Igf2 is abundantly expressed in brain regions involved in cognitive functions, like hippocampus and prefrontal cortex, compared to the peripheral tissues. In contrast to its maternal imprinting in peripheral tissues, Igf2 is mainly expressed from the maternal allele in these brain regions. The training-dependent increase in Igf2 expression derives proportionally from both parental alleles, and, hence, is mostly maternal. Thus, Igf2 parental expression in the adult rat brain does not follow the imprinting rules found in peripheral tissues, suggesting differential expression regulation and functions of imprinted genes in the brain.     

Aberrant expression of imprinted genes in post-implantation rat embryos

AimImprinted genes are known regulators of embryo growth. Studies from our laboratory have demonstrated that treatment of adult male rats with tamoxifen increased post-implantation loss at around midgestation. Expression of insulin like growth factor 2 (Igf2), a paternally expressed imprinted gene was down-regulated in the resorbing embryos obtained at embryonic day 13. Hypomethylation of Igf2-H19 imprint control region was observed in the resorbing embryo sires and spermatozoa obtained from tamoxifen-treated rats thereby suggesting that errors in imprint acquisition during spermatogenesis can result in embryo loss. The present study aims at studying the expression of other imprinted genes, besides Igf2 in the embryos sired by tamoxifen-treated males.Main methodsGene expression profiles of resorbing versus normal embryos were assessed by microarrays. Real time quantitative RT-PCR for six imprinted genes and four genes involved in cell cycle was done to validate gene expression data. The affected pathways and functions were identified in the resorbing embryos and effect on cell cycle was confirmed by flow cytometry.Key findingsAberrant expression of a number of imprinted genes was observed in the resorbing embryos when compared to the normal embryos at embryonic days 11 and 13. Down-regulation of Notch signaling, Wnt signaling and cell cycle pathway was observed in the resorbing embryos.SignificanceThe study suggests that exposure of male germ cells to tamoxifen during adulthood results in aberrant expression of imprinted genes and down-regulation of development associated pathways in the F₁ progeny thereby causing embryo loss.     

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.     

Unregulated expression of the imprinted genes H19 and Igf2r in mouse uniparental fetuses.

The present study shows that the H19 and Igf2r genes, which are imprinted and expressed solely from maternal alleles, are expressed in an unregulatable manner in mouse uniparental, androgenetic, and parthenogenetic fetuses at day 9.5 of gestation. In the androgenetic fetuses, the H19 and Igf2r genes were respectively expressed at 12 and 40% of the levels in biparental fetuses. In addition, the expression of both genes was excessive (1259 and 482%, respectively) in the parthenotes. These expressions of the imprinted genes were not regulated by methylation in the regulatory regions. Moreover, the expression of the antisense Igf2r RNA (Air) was also excessive and was not correlated with Igf2r gene expression in the uniparental fetuses. Taken together, these results indicate that the parental specific expression of imprinted genes is not maintained in particular genes in uniparental embryos, which in turn suggests that both parental genomes are required to establish maternal specific expression of the H19 and Igf2r genes by trans-acting mechanisms.     

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.     

Ubiquitous expression and imprinting of Snrpn in the mouse

Snrpn is known to be abundantly expressed in rodent brain and heart, and in two separate studies with neonatal mouse brain it has been shown to be maternally imprinted, that is, the maternal allele is normally repressed. We now provide evidence on the expression profile and imprinting status of Snrpn throughout development. Using RT-PCR, we have established that Snrpn is further expressed at low levels in lung, liver, spleen, kidney, skeletal muscle, and gonads. Moreover, using mice with only maternal copies of Snrpn (maternal duplication for the chromosome region involved and parthenogenotes), we have shown that the gene is imprinted in all of these tissues and, generally, from the time the gene is first expressed at 7.5 days gestation. In contrast to the findings made with the imprinted genes, Igf2, Ins1, and Ins2, there is no evidence of tissue-specific imprinting in the embryo with Snrpn. Nor, as found with Igf2 and Igf2r, is there evidence of a window of biallelic expression between the germ line imprint and the time of gene repression. The absence of Snrpn expression in early embryos contrasts with the findings in ES cells.     

Epigenotype switching of imprintable loci in embryonic germ cells

 Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar. Received: 1 September 1997 / Accepted: 15 October 1997     

TGFα Reactivates Imprinted <Emphasis Type="Italic">Igf2</Emphasis> in the Parthenogenetic Mice Embryos and Placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE), there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here, we studied effect of transforming growth factor alpha (TGFα) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5 days of gestation, 25 somites) and their placentas (PP). Using RT-PCR, we showed that TGFα reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfα expression in the preimplantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments, it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFα treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP, both Igf2 and H19 were expressed. It seems that TGFα can play an important role as modulator of the Igf2/H19 locus.     

Towards unravelling the Igf2/H19 imprinted domain

Genomic imprinting is an epigenetic marking process that confers parent-of-origin-dependent expression on certain genes. These imprinted genes are sometimes found in clusters, suggesting a possible involvement of higher order regulatory elements controlling expression and imprinting of genes organised in such clusters. In the distal chromosome 7 there are at least four imprinted genes: Mash2, Ins2, Igf2 and H19. Recent evidence⁽¹⁾ suggests that imprinting and expression of at least Igf2 and H19 may be mechanistically linked.     

TGFalpha reactivates imprinted Igf2 in the parthenogenetic mice embryos and placenta

Imprinted genes play important roles in the mammalian development. In the parthenogenetic embryos (PE) there is only expression of maternally expressed genes. Therefore, PEs are appropriate experimental models to study genomic imprinting controlling mechanisms. The maternally expressed H19 and paternally expressed Igf2 are reciprocally imprinted genes in normal embryos. Here we studied effect of transforming growth factor alpha (TGFalpha) treatment in vitro (10 ng/ml at the morula stage) on the expression of Igf2/H19 locus in mice PE (9.5-days of gestation, 25 somites) and their placentas (PP). Using RT-PCR we showed that TGFalpha reactivated maternally imprinted Igf2 gene in parthenogenetic embryos and placentas. In spite of similar Tgfalpha expression in the pre-implantation stages, its expression in the 9.5-day parthenogenetic embryos is significantly less than in normal embryos (NE). In our experiments it was shown that reactivation of Igf2 gene occurred independently of H19 gene. In vitro TGFalpha treatment of mouse PE reactivated paternally expressed Igf2 gene in the PE and PP. In the PE and PP both Igf2 and H19 were expressed. It seems that TGFalpha can play an important role as modulator of the Igf2/H19 locus.     

Expression of imprinted <Emphasis Type="Italic">Igf2</Emphasis> and <Emphasis Type="Italic">Peg1/Mest</Emphasis> genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor α in vitro

The effect of transforming growth factor α (TGFα) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA × C57BL/6)F₁ mice during the postimplantation period of embryogenesis. The PEs were treated with TGFα in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf₂and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf₂ before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFα) PEs, neither gene was expressed at the same 21-to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30-to 35-somite stages, while the Peg1/Mes t expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30-to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFα-treated PEs at the 40-to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40–45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFα can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.     

Expression of imprinted Igf2 and Peg1/Mest genes in postimplantation parthenogenetic mouse embryos treated with transforming growth factor alpha in vitro

The effect of transforming growth factor alpha (TGFt) on the expression of imprinted Igf2 and Peg1/Mest genes was studied in diploid parthenogenetic embryos (PEs) of (CBA x C57BL/6)F1 mice during the postimplantation period of embryogenesis. The PEs were treated with TGFalpha in vitro at the morula stage and, after they developed to the blastocyst stage, were implanted into the uterus of false-pregnant females. On the tenth day of pregnancy, the PEs were explanted for subsequent in vitro culturing for 24 or 48 h. The expression of the imprinted Igf2 and Peg1/Mest genes was studied by means of whole mount in situ hybridization using digoxigenin-labeled antisense RNAs. The expression of the imprinted Igf2 and Peg1/Mest genes was studied in embryos on the tenth day of in utero development before culturing and after 24 and 48 h of culturing in vitro. The expression of Igf2 before culturing was detected only in the brain of 60% of PEs on the tents day of pregnancy (the 21-to 25-somite stages); while the Peg1/Mest expression was not detected at all. In control (not treated with TGFalpha) PEs, neither gene was expressed at the same 21- to 25-somite stages. After 24 h of culturing, the Igf2 expression was detected in the brain of 71% of PEs at the 30- to 35-somite stages, while the Peg1/Mest expression was not detected. In control (untreated) PEs, neither imprinted gene was expressed at the 30- to 35-somite stage. After 48 h of culturing, Igf2 was expressed in the regions of the brain, developing jaws, heart, liver, and somites of all TGFalpha-treated PEs at the 40- to 45-somite stages; and Peg1/Mest was expressed in the brain, heart, and liver of these embryos. In control (untreated) PEs, neither Igf2 nor Peg1/Mest was expressed at these stages The expression patterns of the imprinted Igf2 and Peg1/Mest genes in PEs at the most advanced developmental stages (40-45 somites) and in normal (fertilized) embryos at the same stages were similar; however, their expression rate in PEs was substantially lower than in normal embryos. These data indicate that exogenous TGFalpha can reactivate the expression of the two imprinted genes, modulating the effects of genomic imprinting in such a way that the PE development is improved and substantially prolonged.     

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

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

Novel conserved elements upstream of the H19 gene are transcribed and act as mesodermal enhancers

The reciprocally imprinted H19 and Igf2 genes form a co-ordinately regulated 130 kb unit in the mouse controlled by widely dispersed enhancers, epigenetically modified silencers and an imprinting control region (ICR). Comparative human and mouse genomic sequencing between H19 and Igf2 revealed two novel regions of strong homology upstream of the ICR termed H19 upstream conserved regions (HUCs). Mouse HUC1 and HUC2 act as potent enhancers capable of driving expression of an H19 reporter gene in a range of mesodermal tissues. Intriguingly, the HUC sequences are also transcribed bi-allelically in mouse and human, but their expression pattern in neural and endodermal tissues in day 13.5 embryos is distinct from their enhancer function. The location of the HUC mesodermal enhancers upstream of the ICR and H19, and their capacity for interaction with both H19 and Igf2 requires critical re-evaluation of the cis-regulation of imprinted gene expression of H19 and Igf2 in a range of mesodermal tissues. We propose that these novel sequences interact with the ICR at H19 and the epigenetically regulated silencer at differentially methylated region 1 (DMR1) of Igf2.