CpG methylation regulates the Igf2/H19 insulator

The differentially methylated 5'-flank of the mouse H19 gene unidirectionally regulates the communication between enhancer elements and gene promoters and presumably represses maternal Igf2 expression in vivo [1-6]. The specific activation of the paternally inherited Igf2 allele has been proposed to involve methylation-mediated inactivation of the H19 insulator function during male germline development [1-4, 6]. Here, we addressed the role of methylation by inserting a methylated fragment of the H19-imprinting control region (ICR) into a nonmethylated episomal H19 minigene construct, followed by the transfection of ligation mixture into Hep3B cells. Individual clones were expanded and analyzed for genotype, methylation status, chromatin conformation, and insulator function. The results show that the methylated status of the H19 ICR could be propagated for several passages without spreading into the episomal vector. Moreover, the nuclease hypersensitive sites, which are typical for the maternally inherited H19 ICR allele [1], were absent on the methylated ICR, underscoring the suggestion that the methylation mark dictates parent of origin-specific chromatin conformations [1] that involve CTCF [2]. Finally, the insulator function was strongly attenuated in stably maintained episomes. Collectively, these results provide the first experimental support that the H19 insulator function is regulated by CpG methylation.     

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.     

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.     

The chicken beta-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain

Imprinting of the mouse insulin-like growth factor 2 (Igf2) and H19 genes is regulated by an imprinting control region (ICR). The hypomethylated maternal copy functions as a chromatin insulator through the binding of CTCF and prevents Igf2 activation in cis, while hypermethylation of the paternal copy inactivates insulator function and leads to inactivation of H19 in cis. The specificity of the ICR sequence for mediating imprinting and chromatin insulation was investigated by substituting it for two copies of the chicken beta-globin insulator element, (Ch beta GI)(2), in mice. This introduced sequence resembles the ICR in size, and in containing CTCF-binding sites and CpGs, but otherwise lacks homology. On maternal inheritance, the (Ch beta GI)(2) was hypomethylated and displayed full chromatin insulator activity. Monoallelic expression of Igf2 and H19 was retained and mice were of normal size. These results suggest that the ICR sequence, aside from CTCF-binding sites, is not uniquely specialized for chromatin insulation at the Igf2/H19 region. On paternal inheritance, the (Ch beta GI)(2) was also hypomethylated and displayed strong insulator activity--fetuses possessed very low levels of Igf2 RNA and were greatly reduced in size, being as small as Igf2-null mutants. Furthermore, the paternal H19 allele was active. These results suggest that differential ICR methylation in the female and male germ lines is not acquired through differential binding of CTCF. Rather, it is likely to be acquired through a separate or downstream process.     

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.     

A dyad oct-binding sequence functions as a maintenance sequence for the unmethylated state within the H19/Igf2-imprinted control region

DNA methylation of an imprinted control region (ICR) directs the allele-specific and reciprocal expression of the mouse H19 and the insulin-like growth factor 2 (Igf2) genes, mediated by controlling enhancer access. The ICR shows enhancer blocking activity through CTCF binding to an unmethylated sequence. The unmethylated state of the maternal ICR is maintained throughout development after establishment in the germ line; however, little is known of the molecular mechanisms that regulate DNA methylation. Hence, in this study we show that a dyad Oct-binding sequence (DOS) in the ICR mediates the demethylation of low-density methylation but not hypermethylation and is required to maintain the unmethylated state against the tendency for de novo methylation within the ICR in the embryonic carcinoma cell line P19. Furthermore, we also reveal that the unmethylated state of at least one CTCF-binding site within the ICR is under the control of DOS. Our results suggest that the ICR, as a CTCF-dependent insulator, requires DOS as well as CTCF-binding sites and that DOS maintains the maternal specific unmethylated state of the ICR at postimplantation stages.     

Expression of H19 does not influence the timing of replication of the Igf2/H19 imprinted region

Allele specific timing of replication is believed to be a hallmark of imprinted genes, however recent evidence suggests that this might not be the case for the insulin-like growth factor 2 (Igf2) and H19 locus. In this report, we assayed the timing of replication of Igf2 and H19 in two mouse embryonic cell lines expressing both H19 and Igf2, and one cell line maternally disomic for the Igf2/H19 mouse locus which expresses H19 but not Igf2. In all cell lines, Igf2 and H19 were replicated early in the S phase of the cell cycle, and both alleles replicated at the same time. This indicates that any differences in the timing of replication at the Igf2/H19 locus are of a lesser magnitude than those found in other imprinted regions. Dev Genet 20:29–35, 1997. © 1997 Wiley-Liss, Inc.     

Role of CTCF binding sites in the Igf2/H19 imprinting control region

A approximately 2.4-kb imprinting control region (ICR) regulates somatic monoallelic expression of the Igf2 and H19 genes. This is achieved through DNA methylation-dependent chromatin insulator and promoter silencing activities on the maternal and paternal chromosomes, respectively. In somatic cells, the hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites and blocks activity of the proximal Igf2 promoter by insulating it from its distal enhancers. CTCF binding is thought to play a direct role in inhibiting methylation of the ICR in female germ cells and in somatic cells and, therefore, in establishing and maintaining imprinting of the Igf2/H19 region. Here, we report on the effects of eliminating ICR CTCF binding by severely mutating all four sites in mice. We found that in the female and male germ lines, the mutant ICR remained hypomethylated and hypermethylated, respectively, showing that the CTCF binding sites are dispensable for imprinting establishment. Postfertilization, the maternal mutant ICR acquired methylation, which could be explained by loss of methylation inhibition, which is normally provided by CTCF binding. Adjacent regions in cis-the H19 promoter and gene-also acquired methylation, accompanied by downregulation of H19. This could be the result of a silencing effect of the methylated maternal ICR.     

Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region

The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.     

Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region

The imprinted expression of the H19 and Igf2 genes in the mouse is controlled by an imprinting control center (ICR) whose activity is regulated by parent-of-origin differences in methylation. The only protein that has been implicated in ICR function is the zinc-finger protein CTCF, which binds at multiple sites within the maternally inherited ICR and is required to form a chromatin boundary that inhibits Igf2 expression. To identify other proteins that play a role in imprinting, we employed electrophoresis mobility shift assays to identify two novel binding sites within the ICR. The DNA binding activity was identified as the heterodimer Ku70/80, which binds nonspecifically to free DNA ends. The sites within the ICR bind Ku70/80 in a sequence-specific manner and with higher affinity than previously reported binding sites. The binding required the presence of Mg(2+), implying that the sequence is a pause site for Ku70/80 translocation from a free end. Chromatin immunoprecipitation assays were unable to confirm that Ku70/80 binds to the ICR in vivo. In addition, mutation of these binding sites in the mouse did not result in any imprinting defects. A genome scan revealed that the binding site is found in LINE-1 retrotransposons, suggesting a possible role for Ku70/80 in transposition.     

Relationship between DNA methylation, histone H4 acetylation and gene expression in the mouse imprinted Igf2-H19 domain

DNA methylation and histone H4 acetylation play a role in gene regulation by modulating the structure of the chromatin. Recently, these two epigenetic modifications have dynamically and physically been linked. Evidence suggests that both modifications are involved in regulating imprinted genes - a subset of genes whose expression depends on their parental origin. Using immunoprecipitation assays, we investigate the relationship between DNA methylation, histone H4 acetylation and gene expression in the well-characterised imprinted Igf2-H19 domain on mouse chromosome 7. A systematic regional analysis of the acetylation status of the domain shows that parental-specific differences in acetylation of the core histone H4 are present in the promoter regions of both Igf2 and H19 genes, with the expressed alleles being more acetylated than the silent alleles. A correlation between DNA methylation, histone hypoacetylation and gene repression is evident only at the promoter region of the H19 gene. Treatment with trichostatin A, a specific inhibitor of histone deacetylase, reduces the expression of the active maternal H19 allele and this can be correlated with regional changes in acetylation within the upstream regulatory domain. The data suggest that histone H4 acetylation and DNA methylation have distinct functions on the maternal and paternal Igf2-H19 domains.     

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.     

Mutagenesis in mice of nuclear hormone receptor binding sites in the Igf2/H19 imprinting control region

The H19/Igf2 imprinting control region (ICR) is a DNA methylation-dependent chromatin insulator in somatic cells. The hypomethylated maternally inherited ICR binds the insulator protein CTCF at four sites, and blocks activity of the proximal Igf2 promoter by insulating it from the shared distal enhancers. The hypermethylated paternally inherited ICR lacks CTCF binding and insulator activity, but induces methylation-silencing of the paternal H19 promoter. The paternal-specific methylation of the ICR is established in the male germ cells, while the ICR emerges from the female germ line in an unmethylated form. Despite several attempts to find cis-regulatory elements, it is still unknown what determines these male and female germ cell-specific epigenetic modifications. We recently proposed that five in vivo footprints spanning fifteen half nuclear hormone receptor (NHR) binding sites within the ICR might be involved, and here we report on the effects of mutagenizing all of these half sites in mice. No effect was obtained--in the female and male germ lines the mutant ICR remained hypomethylated and hypermethylated, respectively. The ICR imprinting mechanism remains undefined.