IGF2 Imprinting Research Articles

Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome

The overgrowth- and tumor-associated Beckwith-Wiedemann syndrome results from dysregulation of imprinted genes on chromosome 11p15.5. Here we show that inherited microdeletions in the H19 differentially methylated region (DMR) that abolish two CTCF target sites cause this disease. Maternal transmission of the deletions results in hypermethylation of the H19 DMR, biallelic IGF2 expression, H19 silencing and Beckwith-Wiedemann syndrome, indicative of loss of function of the IGF2-H19 imprinting control element.

Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations.

Imprinted expression at the H19-Igf2 locus depends on a differentially methylated domain (DMD) that acts both as a maternal-specific, methylation-sensitive insulator and as a paternal-specific site of hypermethylation. Four repeats in the DMD bind CCCTC-binding factor (CTCF) on the maternal allele and have been proposed to attract methylation on the paternal allele. We introduced point mutations into the DMD to deplete the repeats of CpGs while retaining CTCF-binding and enhancer-blocking activity. Maternal inheritance of the mutations left H19 expression and Igf2 imprinting intact, consistent with the idea that the DMD acts as an insulator. Conversely, paternal inheritance of these mutations disrupted maintenance of DMD methylation, resulting in biallelic H19 expression. Furthermore, an insulator was established on the paternally inherited mutated allele in vivo, reducing Igf2 expression and resulting in a 40% reduction in size of newborn offspring. Thus, the nine CpG mutations in the DMD showed that the two parental-specific roles of the H19 DMD, methylation maintenance and insulator assembly, are antagonistic.

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.

Igf2 Gene imprinting in preimplantation mouse embryos

Background and Significance: Epigenetic effects on embryos produced from in vitro fertilization are unknown. Recent epidemiological studies show that individuals conceived through in vitro fertilization (IVF) are at twice the risk of birth defects which are associated with a loss of imprinting or altered imprinting of Igf2,and other genes, as compared with babies conceived naturally. These birth defects include diseases associated with aberrant imprinting, such as, Beckwith-Wiedemann Syndrome, Prader-Willi and Angelman’s Syndrome.Objective: In this study, we use a mouse model system to investigate Igf2 imprinting in embryos conceived in vitro.Materials and Methods: M. Musculus female mice (C57 Black, Jackson Laboratories, Maine), were superovulated with pregnant mare serum gonadotropin (10 IU of PMSG, Sigma, St. Louis MO). In vitro fertilization was performed using oocytes recovered from the superovulated mice, 36–40 hours after an injection of 7.5 IU of human chorionic gonadotropin (hCG). The oocytes were fertilized with sperm removed from the epididymus of Mus Spretus male mice. Embryos were placed in Quinn’s Advantage Cleavage Medium (Sage Biopharma, Inc.). The embryos were recovered and analyzed at the 2-cell, 4-cell, morula and blastocyst stages of development.Igf2 expression was evaluated by RT PCR (1Hu J.F. Vu T.H. Hoffman A.R. Promoter-specific modulation of insulin-like growth factor II genomic imprinting by inhibitors of DNA methylation.JBC. 1996; 271: 18253-18262Crossref PubMed Scopus (62) Google Scholar). Maternal and paternal alleles were differentiated by restriction endonuclease digestion specific for polymorphic sites between M. Musculus and Mus Spretus.Results: Our results show that Igf2 gene expression is detectable in morula and blastocyst of our mouse model system. Igf2 expression is biallelic in the morula stage, whereas, some blastocyst have only monallelic expression. Further studies will compare Igf2 imprinting in embryos conceived in vitro vs. in vivo, as well as, evaluate the imprinting patterns of other genes.Conclusion: We have developed a mouse model system for evaluating Igf2 imprinting for in vitro studies. This model enables us to discriminate maternal vs. paternal gene expression in morula and blastocyst. Background and Significance: Epigenetic effects on embryos produced from in vitro fertilization are unknown. Recent epidemiological studies show that individuals conceived through in vitro fertilization (IVF) are at twice the risk of birth defects which are associated with a loss of imprinting or altered imprinting of Igf2,and other genes, as compared with babies conceived naturally. These birth defects include diseases associated with aberrant imprinting, such as, Beckwith-Wiedemann Syndrome, Prader-Willi and Angelman’s Syndrome. Objective: In this study, we use a mouse model system to investigate Igf2 imprinting in embryos conceived in vitro. Materials and Methods: M. Musculus female mice (C57 Black, Jackson Laboratories, Maine), were superovulated with pregnant mare serum gonadotropin (10 IU of PMSG, Sigma, St. Louis MO). In vitro fertilization was performed using oocytes recovered from the superovulated mice, 36–40 hours after an injection of 7.5 IU of human chorionic gonadotropin (hCG). The oocytes were fertilized with sperm removed from the epididymus of Mus Spretus male mice. Embryos were placed in Quinn’s Advantage Cleavage Medium (Sage Biopharma, Inc.). The embryos were recovered and analyzed at the 2-cell, 4-cell, morula and blastocyst stages of development. Igf2 expression was evaluated by RT PCR (1Hu J.F. Vu T.H. Hoffman A.R. Promoter-specific modulation of insulin-like growth factor II genomic imprinting by inhibitors of DNA methylation.JBC. 1996; 271: 18253-18262Crossref PubMed Scopus (62) Google Scholar). Maternal and paternal alleles were differentiated by restriction endonuclease digestion specific for polymorphic sites between M. Musculus and Mus Spretus. Results: Our results show that Igf2 gene expression is detectable in morula and blastocyst of our mouse model system. Igf2 expression is biallelic in the morula stage, whereas, some blastocyst have only monallelic expression. Further studies will compare Igf2 imprinting in embryos conceived in vitro vs. in vivo, as well as, evaluate the imprinting patterns of other genes. Conclusion: We have developed a mouse model system for evaluating Igf2 imprinting for in vitro studies. This model enables us to discriminate maternal vs. paternal gene expression in morula and blastocyst.

Igf2 Gene imprinting in preimplantation mouse embryos

Background and Significance: Epigenetic effects on embryos produced from in vitro fertilization are unknown. Recent epidemiological studies show that individuals conceived through in vitro fertilization (IVF) are at twice the risk of birth defects which are associated with a loss of imprinting or altered imprinting of Igf2,and other genes, as compared with babies conceived naturally. These birth defects include diseases associated with aberrant imprinting, such as, Beckwith-Wiedemann Syndrome, Prader-Willi and Angelman’s Syndrome.

Epigenetic differences between Wilms' tumours in white and east-Asian children

Variations in the international incidence of Wilms' tumour might be due to genetic factors. The maternal insulin-like growth factor 2 gene (IGF2) is imprinted in normal tissues, whereas in some Wilms' tumours and other tumour types the imprint is lost, leading to biallelic transcription of IGF2. We investigated whether the difference in incidence of Wilms' tumour between children of east-Asian descent and white children is due to variations in proportion of tumours with loss of IGF2 imprinting (IGF2 LOI). We assessed IGF2 LOI by use of an ApaI polymorphism in IGF2 exon 9 or quantitative PCR measuring DNA methylation of the H19 and KvDMR1 alleles. The frequencies of perilobar nephrogenic rests associated with Wilms' tumour were assessed histologically in Japanese children and children of white and east-Asian descent. IGF2 LOI was present in Wilms' tumours from predominantly white children from New Zealand (13 of 41 tumours) but absent in tumours from Japanese children (0 of 21 tumours; difference in proportions 0.32, 95% CI 0.07-0.52). Frequency of perilobar nephrogenic rests accompanying tumours from white American children (1192 of 5002, 24%) was significantly higher than in Japanese (one of 56, 1%, difference in proportions 0.22, 95% CI 0.14-0.25) and east-Asian American children (seven of 92, 8%, 0.16, 0.09-0.21). Wilms' tumours in the east-Asian population rarely arise from the IGF2 LOI mechanism frequently noted in white patients. Our findings that IGF2 LOI and perilobar nephrogenic rests associated with this mechanism arise at low frequency in Japanese and east-Asian American children lend support to this conclusion. Variation in frequency of this epigenetic mechanism provides one explanation for the difference in incidence of Wilms' tumour between populations.

Role of H19 3′ sequences in controlling H19 and Igf2 imprinting and expression

The regulation of H19 and Igf2 imprinting and expression depends on common elements. Using comparative analysis between human and mouse, we identified conserved regions 3′ of the H19 transcription unit, including the H19/ Igf2 endodermal enhancers and elements within a 4.2-kb domain between the H19 transcription unit and the enhancers. Transgene experiments implicate these elements in imprinting regulation. To establish whether they are required at the endogenous locus, first we replaced the endodermal enhancers with the α -fetoprotein endodermal enhancers ( H19 Afp ). Second, we deleted the 4.2-kb region ( H19 Δ 4.2 ). Our analysis revealed that H19 and Igf2 imprinting and tissue-specific expression were maintained for both mutations, except for a slight reduction in paternal Igf2 expression from the H19 Afp allele in liver. These results demonstrate that the H19 insulator can interact with heterologous enhancers to imprint Igf2. Furthermore, for H19, chromatin context or additional sequences possibly compensate for loss of conserved 3′ elements.

Developmentally regulated functions of the H19 differentially methylated domain.

Igf2 and H19 are physically linked imprinted genes. In embryonic liver, their reciprocal expression (paternal for Igf2 and maternal for H19) is controlled by a paternally methylated region (H19 DMD) located 5' of H19. This region contains a methylation-sensitive insulator that prevents the Igf2 promoters being activated by downstream enhancers on the maternal chromosome. In adult liver, Igf2 is normally not expressed but is reactivated upon tumour formation. By analysing three deletions of the H19 locus, we investigated the mechanism regulating the imprinted expression of the Igf2 gene in the course of liver tumourigenesis. We observed that the role of the H19 DMD in the control of Igf2 expression changes during tumourigenesis. The H19 DMD is required on the paternal chromosome for Igf2 activation in the early stages while its maternal allele is necessary for maintaining Igf2 imprinting only in the late stages. A positive regulatory function of the paternal H19 DMD is also evident in normal neonatal liver, but its relevance for Igf2 expression becomes higher in the second post-natal week. Our results support a model in which both methylated and non-methylated parental copies of the H19 DMD have active roles in the regulation of Igf2 expression in the liver and these activities are under developmental control.

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites.

The mouse insulin-like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11-13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent-specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males x C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF-associated factors that may be needed to coordinate Igf2 imprinting.

Alterations in Promoter Usage and Expression Levels of Insulin-like Growth Factor-II and H19 Genes in Cervical and Endometrial Cancer.

The biallelic expression of insulin-like growth factor-II (IGF2) and H19 has been reported to be associated with the progression of several tumors. Here, the promoter usage and expression levels of IGF2 and H19 are reported to be altered in cervical and endometrial cancers showing loss of imprinting (LOI). The imprinting status of IGF2 and H19 was examined in 32 cervical carcinomas, their matched normal tissues, 13 endometrial cancer and 33 normal endometrial tissues. The LOI of IGF2 was observed in 7 of 18 (39%) and 1 of 13 (8.3%) informative cervical carcinomas and informative endometrial cancers, respectively. The LOI of the H19 gene was detected in 5 of 14 (36%) and in all 11 (100%) informative cervical carcinoma cases and informative endometrial cancer cases, respectively. The use of promoter P1 was observed in the LOI tumors of IGF2, but not in the tumors showing maintenance of IGF2 imprinting (MOI), or in cervical and endometrial cancers. Unlike MOI tumors, some LOI tumors revealed a lack of IGF2 transcription from the promoter P3. The LOI tumors of IGF2 showed increased expression of the IGF2 level, but a down-regulation of the H19, relative to normal tissues, whereas the MOI tumors revealed no significant alterations. These results suggest that the promoter P1 could be involved in the biallelic expression of IGF2, and that the altered expression of the IGF2 and H19 levels might be associated with the progression of cervical and endometrial cancers that exhibit biallelic IGF2 expression.

Loss of imprinting of IGF2 is an epigenetic marker independently associated with colorectal cancer and not related to tobacco or alcohol consumption

Loss of imprinting of IGF2 is an epigenetic marker independently associated with colorectal cancer and not related to tobacco or alcohol consumption

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site

Loss of imprinting of IGF2 and H19 in osteosarcoma is accompanied by reciprocal methylation changes of a CTCF-binding site

Altered imprinting, promoter usage, and expression of insulin-like growth factor-II gene in gestational trophoblastic diseases

Objective We aimed to understand the involvement of imprinted genes in the pathogenesis of gestational trophoblastic diseases (GTD) such as hydatidiform mole (H-mole) and gestational trophoblastic tumors (GTT). Methods An allelic-typing assay was performed using a PCR-RFLP-based method for identification of heterozygous informative cases. The usage of insulin-like growth factor-II (IGF2) promoters was examined by RT-PCR using promoter-specific primers. The mRNA expression of IGF2 and H19 was quantified using a densitometer. Results The imprinting of IGF2 and H19 was maintained in all normal placenta tissues ( n = 15) but relaxed in GTD ( n = 47). Loss of imprinting (LOI) of IGF2 was in the order of GTT (57%) > complete H-mole (43%) > partial H-mole (25%). Similarly, LOI of H19 was in the order of GTT (40%) > complete H-mole (18%) > partial H-mole (0%). Promoter usage pattern of IGF2 changed with gestation stage of normal placentae and GTD. In normal placentae, the usage of promoter P1 was higher than that of P4 in the first trimester but lowered in the full term. H-mole and GTT predominantly used promoter P1 with relative silencing of promoter P4. Although normal early placenta and various GTD tissues showed the similar usage of IGF2 promoter P1, GTT tissues revealed the higher expression levels of IGF2 but a down-regulation of H19 relative to the normal early placentae. Conclusion These results suggest that LOI, deregulation of IGF2 promoters, and the altered expression levels of IGF2 and H19 genes might be associated with the progression of GTD.

Loss of Igf2 Imprinting in Monoclonal Mouse Hepatic Tumor Cells Is Not Associated with Abnormal Methylation Patterns for the H19, Igf2, andKvlqt1 Differentially Methylated Regions

IGFII, the peptide encoded by the Igf2 gene, is a broad spectrum mitogen with important roles in prenatal growth as well as cancer progression. Igf2 is transcribed from the paternally inherited allele, whereas the linked H19 is transcribed from the maternal allele. Igf2 imprinting is thought to be maintained by differentially methylated regions (DMRs) located at multiple sites such as upstream of H19 and Igf2 and within Kvlqt1 loci. Biallelic expression (loss of imprinting (LOI)) of Igf2 is frequently observed in cancers, and a subset of Wilms' and intestinal tumors have been shown to exhibit abnormal methylation at H19DMR associated with loss of maternal H19 expression, but it is not known whether such changes are common in other neoplasms. Because cancers consist of diverse cell populations with and without Igf2 LOI, we established four independent monoclonal cell lines with Igf2 LOI from mouse hepatic tumors. We here demonstrate retention of normal differential methylation at H19, Igf2, or Kvlqt1 DMR by all of the cell lines. Furthermore, H19 was found to be expressed exclusively from the maternal allele, and levels of CTCF, a multifunctional nuclear factor that has an important role in the Igf2 imprinting, were comparable with those in normal hepatic tissues with no mutational changes detected. These data indicate that Igf2 LOI in tumor cells is not necessarily linked to abnormal methylation at H19, Igf2, or Kvlqt1 loci.

Different imprinting status of IGF-2 in epithelial ovarian tumors.

To explore whether the imprinting status of IGF-2 in the malignant epithelial ovarian tumors is different from that in benign tumors, the target sequences (DNA and RNA) which contain a polymorphism site for ApaI restriction endonuclease digestion were amplified with PCR and RT-PCR methods. Then the PCR/RT-PCR products were digested by ApaI. The IGF-2 transcriptional pattern came out from the results of endonucleases digestion. Among the 36 cases of benign epithelial ovarian tumors, 20 were heterozygous for ApaI locus and all showed genomic imprinting. While in the malignant group, 22 were heterozygous for ApaI locus but six were found to lose imprinting. Significant differences existed between the two groups (P < 0.05). Loss of imprinting of IGF-2 may serve as a marker for differentiating the malignant ovarian cancers from the benign ones. In a new field of molecular genetics, our research provides an experimental basis for genetic diagnosis and treatment of the ovarian cancers.

Chromosome arm 16q in Wilms tumors: unbalanced chromosomal translocations, loss of heterozygosity, and assessment of the CTCF gene.

Chromosome arm 16q is a common site of loss of heterozygosity (LOH) in Wilms tumors (WTs). The mechanism and consequences of 16q LOH are not known, but the CTCF gene, in band 16q22, is a candidate target gene. CTCF protein binds to DNA upstream of the H19 gene on chromosome band 11p15, and maintains normal imprinting of H19 and IGF2. Thus, its loss might predispose to de novo methylation of the maternal allele of H19 and loss of imprinting (LOI) of IGF2 in WTs. We mapped Chr16 LOH in WTs and correlated this with unbalanced chromosomal translocations and histopathology. We also analyzed the CTCF gene for its mRNA and protein expression and DNA sequence, and we investigated correlations with methylation of H19(mat). In our series, unbalanced t(1;16) chromosomal translocations were a major pathway for Chr16 loss of heterozygosity, and this LOH was correlated significantly with tumor anaplasia. CTCF mapped in the minimal region of Chr16 LOH. However, we found no correlation between Chr16 LOH, or loss of CTCF mRNA or protein, and methylation of H19(mat). Some WTs contained reduced amounts of CTCF protein, but among these were cases with and without H19(mat) methylation. Rare WTs with simultaneous 16q LOH and H19(mat) methylation lacked CTCF mutations. These data argue against the CTCF gene as a target of Chr16 LOH in WTs, but leave open the possibility that post-transcriptional loss of CTCF protein may account for some instances of LOI.

Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages.

Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.

Alterations in promoter usage and expression levels of insulin-like growth factor-II and H19 genes in cervical carcinoma exhibiting biallelic expression of IGF-II

Biallelic expression of insulin-like growth factor-II (IGF2) has been reported to be associated with progression of several tumors. Here, we report that the promoter usage and expression levels of IGF2 and H19 were altered in cervical carcinoma showing loss of imprinting. The imprinting status was examined in 32 cervical carcinomas and their matched normal tissues. Loss of imprinting (LOI) of IGF2 was observed in seven of 18 (39%) informative cases. LOI of H19 gene was detected in five of 14 informative cases. The usage of promoter P1 was observed in LOI tumors of IGF2, but not in the tumors showing maintenance of IGF2 imprinting (MOI) and in the normal cervical tissues. Unlike MOI tumors, some LOI tumors revealed the lack of IGF2 transcription from promoter P3. LOI tumors of IGF2 showed an increased expression level of IGF2 but a down-regulation of H19 relative to the normal tissues whereas MOI tumors did not reveal significant alterations. These results suggest that promoter P1 could be involved in the biallelic expression of IGF2 and that the altered expression levels of IGF2 and H19 gene might be associated with the progression of cervical carcinoma showing biallelic expression of IGF2.

A human H19 transgene exhibits impaired paternal-specific imprint acquisition and maintenance in mice.

Genomic imprinting, the differential expression of autosomal genes based on their parent of origin, is observed in all eutherian mammals that have been examined. In most instances the genes that are imprinted in one species are imprinted in others as well, suggesting that imprinting predated eutherian radiation. For example, the RNA-coding H19 gene is repressed upon paternal inheritance in all species examined to date. Thus, it is surprising that there is remarkably little sequence conservation among the cis-acting DNA regulatory elements that are required for imprinting of H19 and the tightly linked Igf2 gene. The most conserved characteristic in the imprinting control region (ICR) is the presence of multiple binding sites for the zinc finger protein CTCF, raising the possibility that CTCF binding might be sufficient for the reciprocal imprinting of H19 and Igf2. To investigate whether a human H19 transgene, harboring seven CTCF sites, is correctly recognized and imprinted in the mouse, a 100 kb transgene containing the human H19 gene was introduced into the mouse germline. The human transgene was specifically methylated after passage through the male germline in a copy number-dependent manner, but the methylation was unstable, undergoing progressive loss during development. Consequently, the transgene was highly expressed upon both maternal and paternal inheritance. These results argue that the signals for both the acquisition and maintenance of methylation imprinting are diverging rapidly.

Deletion of a direct repeat element has no effect on Igf2 and H19 imprinting.

Deletion of a direct repeat element has no effect on Igf2 and H19 imprinting.