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.     

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.     

Interruption of intrachromosomal looping by CCCTC binding factor decoy proteins abrogates genomic imprinting of human insulin-like growth factor II

Monoallelic expression of IGF2 is regulated by CCCTC binding factor (CTCF) binding to the imprinting control region (ICR) on the maternal allele, with subsequent formation of an intrachromosomal loop to the promoter region. The N-terminal domain of CTCF interacts with SUZ12, part of the polycomb repressive complex-2 (PRC2), to silence the maternal allele. We synthesized decoy CTCF proteins, fusing the CTCF deoxyribonucleic acid-binding zinc finger domain to CpG methyltransferase Sss1 or to enhanced green fluorescent protein. In normal human fibroblasts and breast cancer MCF7 cell lines, the CTCF decoy proteins bound to the unmethylated ICR and to the IGF2 promoter region but did not interact with SUZ12. EZH2, another part of PRC2, was unable to methylate histone H3-K27 in the IGF2 promoter region, resulting in reactivation of the imprinted allele. The intrachromosomal loop between the maternal ICR and the IGF2 promoters was not observed when IGF2 imprinting was lost. CTCF epigenetically governs allelic gene expression of IGF2 by orchestrating chromatin loop structures involving PRC2.     

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.     

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.     

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.     

Rasgrf1 imprinting is regulated by a CTCF-dependent methylation-sensitive enhancer blocker

Imprinted methylation of the paternal Rasgrf1 allele in mice occurs at a differentially methylated domain (DMD) 30 kbp 5' of the promoter. A repeated sequence 3' of the DMD regulates imprinted methylation, which is required for imprinted expression. Here we identify the mechanism by which methylation controls imprinting. The DMD is an enhancer blocker that binds CTCF in a methylation-sensitive manner. CTCF bound to the unmethylated maternal allele silences expression. CTCF binding to the paternal allele is prevented by repeat-mediated methylation, allowing expression. Optimal in vitro enhancer-blocking activity requires CTCF binding sites. The enhancer blocker can be bypassed in vivo and imprinting abolished by placing an extra enhancer proximal to the promoter. Together, the repeats and the DMD constitute a binary switch that regulates Rasgrf1 imprinting.     

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     

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     

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.     

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.