CTCF elements direct allele-specific undermethylation at the imprinted H19 locus

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.     

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.     

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.     

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.     

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.     

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.     

Tissue-specific relationship of S-adenosylhomocysteine with allele-specific H19/Igf2 methylation and imprinting in mice with hyperhomocysteinemia

DNA methylation is linked to homocysteine metabolism through the generation of S-adenosylmethionine (AdoMet) and S-Adenosylhomocysteine (AdoHcy). The ratio of AdoMet/AdoHcy is often considered an indicator of tissue methylation capacity. The goal of this study is to determine the relationship of tissue AdoMet and AdoHcy concentrations to allele-specific methylation and expression of genomically imprinted H19/Igf2. Expression of H19/Igf2 is regulated by a differentially methylated domain (DMD), with H19 paternally imprinted and Igf2 maternally imprinted. F1 hybrid C57BL/6J x Castaneous/EiJ (Cast) mice with (+/−), and without (+/+), heterozygous disruption of cystathionine-β-synthase (Cbs) were fed a control diet or a diet (called HH) to induce hyperhomocysteinemia and changes in tissue AdoMet and AdoHcy. F1 Cast x Cbs+/− mice fed the HH diet had significantly higher plasma total homocysteine concentrations, higher liver AdoHcy, and lower AdoMet/AdoHcy ratios and this was accompanied by lower liver maternal H19 DMD allele methylation, lower liver Igf2 mRNA levels, and loss of Igf2 maternal imprinting. In contrast, we found no significant differences in AdoMet and AdoHcy in brain between the diet groups but F1 Cast x Cbs+/− mice fed the HH diet had higher maternal H19 DMD methylation and lower H19 mRNA levels in brain. A significant negative relationship between AdoHcy and maternal H19 DMD allele methylation was found in liver but not in brain. These findings suggest the relationship of AdoMet and AdoHcy to gene-specific DNA methylation is tissue-specific and that changes in DNA methylation can occur without changes in AdoMet and AdoHcy.     

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.     

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.     

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.     

Mechanisms of Igf2/H19 imprinting: DNA methylation, chromatin and long-distance gene regulation

The mouse Igf2 and H19 genes lie 70-kb apart on chromosome 7 and are reciprocally imprinted. Two regulatory regions are important for their parental allele-specific expression: a differentially methylated region (DMR) upstream of H19 and a set of tissue-specific enhancers downstream of H19. The enhancers specifically activate Igf2 on the paternal chromosome and H19 on the maternal chromosome. The interactions between the enhancers and the genes are regulated by the DMR, which works as a selector by exerting dual functions: a methylated DMR on the paternal chromosome inactivates adjacent H19 and an unmethylated DMR on the maternal chromosome insulates Igf2 from the enhancers. These processes appear to involve methyl-CpG-binding proteins, histone deacetylases and the formation of chromatin insulator complexes. The Igf2/H19 region provides a unique model in which to study the roles of DNA methylation and chromatin structure in the regulation of chromosome domains.     

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.