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.     

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.     

Complete Biallelic Insulation at the H19/Igf2 Imprinting Control Region Position Results in Fetal Growth Retardation and Perinatal Lethality

Background

The H19/Igf2 imprinting control region (ICR) functions as an insulator exclusively in the unmethylated maternal allele, where enhancer-blocking by CTCF protein prevents the interaction between the Igf2 promoter and the distant enhancers. DNA methylation inhibits CTCF binding in the paternal ICR allele. Two copies of the chicken β-globin insulator (ChβGI)₂ are capable of substituting for the enhancer blocking function of the ICR. Insulation, however, now also occurs upon paternal inheritance, because unlike the H19 ICR, the (ChβGI)₂ does not become methylated in fetal male germ cells. The (ChβGI)₂ is a composite insulator, exhibiting enhancer blocking by CTCF and chromatin barrier functions by USF1 and VEZF1. We asked the question whether these barrier proteins protected the (ChβGI)₂ sequences from methylation in the male germ line.

Methodology/Principal Findings

We genetically dissected the ChβGI in the mouse by deleting the binding sites USF1 and VEZF1. The methylation of the mutant versus normal (ChβGI)₂ significantly increased from 11% to 32% in perinatal male germ cells, suggesting that the barrier proteins did have a role in protecting the (ChβGI)₂ from methylation in the male germ line. Contrary to the H19 ICR, however, the mutant (mChβGI)₂ lacked the potential to attain full de novo methylation in the germ line and to maintain methylation in the paternal allele in the soma, where it consequently functioned as a biallelic insulator. Unexpectedly, a stricter enhancer blocking was achieved by CTCF alone than by a combination of the CTCF, USF1 and VEZF1 sites, illustrated by undetectable Igf2 expression upon paternal transmission.

Conclusions/Significance

In this in vivo model, hypomethylation at the ICR position together with fetal growth retardation mimicked the human Silver-Russell syndrome. Importantly, late fetal/perinatal death occurred arguing that strict biallelic insulation at the H19/Igf2 ICR position is not tolerated in development.     

The 3' portion of the mouse H19 Imprinting-Control Region is required for proper tissue-specific expression of the Igf2 gene

Genomic imprinting at the H19/Igf2 locus is governed by a cis-acting Imprinting-Control Region (ICR), located 2 kb upstream of the H19 gene. This region possesses an insulator function which is activated on the unmethylated maternal allele through the binding of the CTCF factor. It has been previously reported that paternal transmission of the H19(SilK) deletion, which removes the 3' portion of H19 ICR, leads to the loss of H19 imprinting. Here we show that, in the liver, this reactivation of the paternal H19 gene is concomitant to a dramatic decrease in Igf2 mRNA levels. This deletion alters higher-order chromatin architecture, Igf2 promoter usage and tissue-specific expression. Therefore, when methylated, the 3' portion of the H19 ICR is a bi-functional regulatory element involved not only in H19 imprinting but also in 'formatting' the higher-order chromatin structure for proper tissue-specific expression of both H19 and Igf2 genes.     

Maternal-specific footprints at putative CTCF sites in the H19 imprinting control region give evidence for insulator function

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 (Igf2) gene and the closely linked H19 gene are regulated by an intervening 2 kb imprinting control region (ICR), which displays parentspecific differential DNA methylation [1] [2]. Four 21 bp repeats are embedded within the ICR and are conserved in the putative ICR of human and rat Igf2 and H19, suggesting that the repeats have a function [3] [4]. Here, we report that prominent DNA footprints were found in vivo on the unmethylated maternal ICR at all four 21 bp repeats, demonstrating the presence of protein binding. The methylated paternal ICR displayed no footprints. Significantly, the maternal-specific footprints were localized to putative binding sites for CTCF, a highly conserved zinc-finger DNA-binding protein with multiple roles in gene regulation including that of chromatin insulator function [5] [6]. These results strongly suggest that the maternal ICR functions as an insulator element in regulating mutually exclusive expression of Igf2 and H19 in cis.     

Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance

The differentially methylated imprinting control region (ICR) region upstream of the H19 gene regulates allelic Igf2 expression by means of a methylation-sensitive chromatin insulator function. We have previously shown that maternal inheritance of mutated (three of the four) target sites for the 11-zinc finger protein CTCF leads to loss of Igf2 imprinting. Here we show that a mutation in only CTCF site 4 also leads to robust activation of the maternal Igf2 allele despite a noticeably weaker interaction in vitro of site 4 DNA with CTCF compared to other ICR sites, sites 1 and 3. Moreover, maternally inherited sites 1 to 3 become de novo methylated in complex patterns in subpopulations of liver and heart cells with a mutated site 4, suggesting that the methylation privilege status of the maternal H19 ICR allele requires an interdependence between all four CTCF sites. In support of this conclusion, we show that CTCF molecules bind to each other both in vivo and in vitro, and we demonstrate strong interaction between two CTCF-DNA complexes, preassembled in vitro with sites 3 and 4. We propose that the CTCF sites may cooperate to jointly maintain both methylation-free status and insulator properties of the maternal H19 ICR allele. Considering many other CTCF targets, we propose that site-specific interactions between various DNA-bound CTCF molecules may provide general focal points in the organization of looped chromatin domains involved in gene regulation.     

Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting

Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5' of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3' to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (Delta D MD Delta G) that removes the DMD and G-rich repeat and a 1.3-kb deletion (Delta G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb Delta DMD allele, we analyzed a 3.8-kb targeted allele (Delta 3.8kb-5'H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5' deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the Delta DMD and Delta DMD Delta G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5' CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.     

Multiple nucleosome positioning sites regulate the CTCF-mediated insulator function of the H19 imprinting control region

The 5' region of the H19 gene harbors a methylation-sensitive chromatin insulator within an imprinting control region (ICR). Insertional mutagenesis in combination with episomal assays identified nucleosome positioning sequences (NPSs) that set the stage for the remarkably precise distribution of the four target sites for the chromatin insulator protein CTCF to nucleosome linker sequences in the H19 ICR. Changing positions of the NPSs resulted in loss of both CTCF target site occupancy and insulator function, suggesting that the NPSs optimize the fidelity of the insulator function. We propose that the NPSs ensure the fidelity of the repressed status of the maternal Igf2 allele during development by constitutively maintaining availability of the CTCF target sites.     

A complex duplication created by gene targeting at the imprinted H19 locus results in two classes of methylation and correlated Igf2 expression phenotypes

Imprinting of the mouse H19 and Igf2 genes is dependent on the presence of an intervening imprinting control region (ICR) situated 2 kb upstream of H19 and approximately 70 kb downstream of Igf2. Several recent studies have provided substantial evidence that the unmethylated maternal ICR acts as an insulator that prevents activation of Igf2 by a suite of enhancers downstream of the H19 gene. The methylated paternal ICR and H19 promoter have no activity, allowing sole activation of Igf2 expression. We have produced mice in which a duplication of the H19/Igf2 ICR produces, in each generation, two classes of methylation levels that correlated with two Igf2 imprinting phenotypes. One hypermethylated class also shows activation of the normally silent Igf2 gene, whereas the other hypomethylated class shows only slight activation of Igf2, in agreement with methylation's role in ICR function. This study describes a rare, possibly unique type of mutation that induces two distinct phenotypes in each generation.     

CTCF-binding sites within the H19 ICR differentially regulate local chromatin structures and cis-acting functions

It is generally assumed that CTCF-binding sites are synonymous with the demarcation of expression domains by promoting the formation of chromatin loops. We have proposed earlier, however, that such features may be context-dependent. In support of this notion, we show here that chromatin loop structures, impinging on CTCF-binding sites 1/2 and 3/4 at the 5′ and 3′-ends, respectively, within the maternal allele of the H19 imprinting control region (ICR), differ significantly. Although abrogation of CTCF binding to the maternal H19 ICR allele results in loss of chromatin loops in the 3′-region, there is a dramatic gain of long-range chromatin loops impinging on the 5′-region. As the degree of occupancy of its four CTCF-binding sites discriminates between the chromatin insulator and replication timing functions, we submit that the CTCF-binding sites within the H19 ICR are functionally diverse and organize context-dependent higher order chromatin conformations.     

A differentially methylated imprinting control region within the Kcnq1 locus harbors a methylation-sensitive chromatin insulator

The mechanisms underlying the phenomenon of genomic imprinting remain poorly understood. In one instance, a differentially methylated imprinting control region (ICR) at the H19 locus has been shown to involve a methylation-sensitive chromatin insulator function that apparently partitions the neighboring Igf2 and H19 genes in different expression domains in a parent of origin-dependent manner. It is not known, however, if this mechanism is unique to the Igf2/H19 locus or if insulator function is a common feature in the regulation of imprinted genes. To address this question, we have studied an ICR in the Kcnq1 locus that regulates long range repression on the paternally derived p57Kip2 and Kcnq1 alleles in an imprinting domain that includes Igf2 and H19. We show that this ICR appears to possess a unidirectional chromatin insulator function in somatic cells of both mesodermal and endodermal origins. Moreover, we document that CpG methylation regulates this insulator function suggesting that a methylation-sensitive chromatin insulator is a common theme in the phenomenon of genomic imprinting.     

Novel cis-regulatory function in ICR-mediated imprinted repression of H19

Expression of coregulated imprinted genes, H19 and Igf2, is monoallelic and parent-of-origin-dependent. Like most imprinted genes, H19 and Igf2 are regulated by a differentially methylated imprinting control region (ICR). CTCF binding sites and DNA methylation at the ICR have previously been identified as key cis-acting elements required for proper H19/Igf2 imprinting. Here, we use mouse models to elucidate further the mechanism of ICR-mediated gene regulation. We specifically address the question of whether sequences outside of CTCF sites at the ICR are required for paternal H19 repression. To this end, we generated two types of mutant ICRs in the mouse: (i) deletion of intervening sequence between CTCF sites (H19^ICR?IVS), which changes size and CpG content at the ICR; and (ii) CpG depletion outside of CTCF sites (H19^ICR-8nrCG), which only changes CpG content at the ICR. Individually, both mutant alleles (H19^ICR?IVS and H19^ICR-8nrCG) show loss of imprinted repression of paternal H19. Interestingly, this loss of repression does not coincide with a detectable change in methylation at the H19 ICR or promoter. Thus, neither intact CTCF sites nor hypermethylation at the ICR is sufficient for maintaining the fully repressed state of the paternal H19 allele. Our findings demonstrate, for the first time in vivo, that sequence outside of CTCF sites at the ICR is required in cis for ICR-mediated imprinted repression at the H19/Igf2 locus. In addition, these results strongly implicate a novel role of ICR size and CpG density in paternal H19 repression.     

Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region

Parent-of-origin-specific expression of the mouse insulin-like growth factor 2 gene (Igf2) and the closely linked H19 gene located on distal chromosome 7 is regulated by a 2.4-kb imprinting control region (ICR) located upstream of the H19 gene. In somatic cells, the maternally and paternally derived ICRs are hypo- and hypermethylated, respectively, with the former binding the insulator protein CCCTC-binding factor (CTCF) and acting to block access of enhancers to the Igf2 promoter. Here we report on a detailed in vivo footprinting analysis-using ligation-mediated PCR combined with in vivo dimethyl sulfate, DNase I, or UV treatment-of ICR sequences located outside of the CTCF binding domains. In mouse primary embryo fibroblasts carrying only maternal or paternal copies of distal chromosome 7, we have identified five prominent footprints specific to the maternal ICR. Each of the five footprinted areas contains at least two nuclear hormone receptor hexad binding sites arranged with irregular spacing. When combined with fibroblast nuclear extracts, these sequences interact with complexes containing retinoic X receptor alpha and estrogen receptor beta. More significantly, the footprint sequences bind nuclear hormone receptor complexes in male, but not female, germ cell extracts purified from fetuses at a developmental stage corresponding to the time of establishment of differential ICR methylation. These data are consistent with the possibility that nuclear hormone receptor complexes participate in the establishment of differential ICR methylation imprinting in the germ line.     

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.     

CTCF-dependent chromatin bias constitutes transient epigenetic memory of the mother at the H19-Igf2 imprinting control region in prospermatogonia

Genomic imprints-parental allele-specific DNA methylation marks at the differentially methylated regions (DMRs) of imprinted genes-are erased and reestablished in germ cells according to the individual's sex. Imprint establishment at paternally methylated germ line DMRs occurs in fetal male germ cells. In prospermatogonia, the two unmethylated alleles exhibit different rates of de novo methylation at the H19/Igf2 imprinting control region (ICR) depending on parental origin. We investigated the nature of this epigenetic memory using bisulfite sequencing and allele-specific ChIP-SNuPE assays. We found that the chromatin composition in fetal germ cells was biased at the ICR between the two alleles with the maternally inherited allele exhibiting more H3K4me3 and less H3K9me3 than the paternally inherited allele. We determined genetically that the chromatin bias, and also the delayed methylation establishment in the maternal allele, depended on functional CTCF insulator binding sites in the ICR. Our data suggest that, in primordial germ cells, maternally inherited allele-specific CTCF binding sets up allele-specific chromatin differences at the ICR. The erasure of these allele-specific chromatin marks is not complete before the process of de novo methylation imprint establishment begins. CTCF-dependent allele-specific chromatin composition imposes a maternal allele-specific delay on de novo methylation imprint establishment at the H19/Igf2 ICR in prospermatogonia.     

The H19 Imprinting Control Region Mediates Preimplantation Imprinted Methylation of Nearby Sequences in Yeast Artificial Chromosome Transgenic Mice

In the mouse Igf2/H19 imprinted locus, differential methylation of the imprinting control region (H19 ICR) is established during spermatogenesis and is maintained in offspring throughout development. Previously, however, we observed that the paternal H19 ICR, when analyzed in yeast artificial chromosome transgenic mice (YAC-TgM), was preferentially methylated only after fertilization. To identify the DNA sequences that confer methylation imprinting, we divided the H19 ICR into two fragments (1.7 and 1.2 kb), ligated them to both ends of a λ DNA fragment into which CTCF binding sites had been inserted, and analyzed this in YAC-TgM. The maternally inherited λ sequence, normally methylated after implantation in the absence of H19 ICR sequences, became hypomethylated, demonstrating protective activity against methylation within the ICR. Meanwhile, the paternally inherited λ sequence was hypermethylated before implantation only when a 1.7-kb fragment was ligated. Consistently, when two subfragments of the H19 ICR were individually investigated for their activities in YAC-TgM, only the 1.7-kb fragment was capable of introducing paternal allele-specific DNA methylation. These results show that postfertilization methylation imprinting is conferred by a paternal allele-specific methylation activity present in a 1.7-kb DNA fragment of the H19 ICR, while maternal allele-specific activities protect the allele from de novo DNA methylation.     

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.