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.     

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.     

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.     

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.     

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.     

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 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.     

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.     

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.     

Nucleosome positioning signals in the DNA sequence of the human and mouse H19 imprinting control regions

We have investigated the sequences of the mouse and human H19 imprinting control regions (ICRs) to see whether they contain nucleosome positioning information pertinent to their function as a methylation-regulated chromatin boundary. Positioning signals were identified by an in vitro approach that employs reconstituted chromatin to comprehensively describe the contribution of the DNA to the most basic, underlying level of chromatin structure. Signals in the DNA sequence of both ICRs directed nucleosomes to flank and encompass the short conserved sequences that constitute the binding sites for the zinc finger protein CTCF, an essential mediator of insulator activity. The repeat structure of the human ICR presented a conserved array of strong positioning signals that would preferentially flank these CTCF binding sites with positioned nucleosomes, a chromatin structure that would tend to maintain their accessibility. Conversely, all four CTCF binding sites in the mouse sequence were located close to the centre of positioning signals that were stronger than those in their flanks; these binding sites might therefore be expected to be more readily incorporated into positioned nucleosomes. We found that CpG methylation did not effect widespread repositioning of nucleosomes on either ICR, indicating that allelic methylation patterns were unlikely to establish allele-specific chromatin structures for H19 by operating directly upon the underlying DNA-histone interactions; instead, epigenetic modulation of ICR chromatin structure is likely to be mediated principally at higher levels of control. DNA methylation did, however, both promote and inhibit nucleosome positioning at several sites in both ICRs and substantially negated one of the strongest nucleosome positioning signals in the human sequence, observations that underline the fact that this epigenetic modification can, nevertheless, directly and decisively modulate core histone-DNA interactions within the nucleosome.     

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.     

Characterization of differentially methylated regions in 3 bovine imprinted genes: a model for studying human germ-cell and embryo development

Correct imprinting is crucial for normal fetal and placental development in mammals. Experimental evidence in animal models and epidemiological studies in humans suggest that assisted reproductive technologies (ARTs) can interfere with imprinted gene regulation in gametogenesis and early embryogenesis. Bos taurus is an agriculturally important species in which ARTs are commonly employed. Because this species exhibits a similar preimplantation development and gestation length as humans, it is increasingly being used as a model for human germ-cell and embryo development. However, in contrast to humans and mice, there is relatively little information on bovine imprinted genes. Here, we characterized the bovine intergenic IGF2-H19 imprinting control region (ICR) spanning approximately 3 kb. We identified a 300-bp differentially methylated region (DMR) approximately 6 kb upstream of the H19 promoter, containing a CpG island with CTCF-binding site and high sequence similarity with the human intergenic ICR. Additional differentially methylated CpG islands lie -6 kb to -3 kb upstream of the promoter, however these are less conserved. Both classical bisulfite sequencing and bisulfite pyrosequencing demonstrated complete methylation of the IGF2-H19 ICR in sperm, complete demethylation in parthenogenetic embryos having only the female genome, and differential methylation in placental and somatic tissues. In addition, we established pyrosequencing assays for the previously reported bovine SNRPN and PEG3 DMRs. The observed methylation patterns were consistent with genomic imprinting in all analyzed tissues/cell types. The identified IGF2-H19 ICR and the developed quantitative methylation assays may prove useful for further studies on the relationship between ARTs and imprinting defects in the bovine model.     

Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight

IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ~25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.     

Altered DNA Methylation Patterns of the H19 Differentially Methylated Region and the DAZL Gene Promoter Are Associated with Defective Human Sperm

DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ) and asthenozoospermia (AZ) usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control), with only complete methylation and mild hypomethylation(<50% unmethylated CpGs) identified, and there was no significant difference in the occurrence of these two methylation patterns between AZ and NZ (P>0.05). However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs ) and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05%) and AZ (0.58±1.77%) (P<0.001).Additional, our data indicated the occurrence of >20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05). In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.     

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.     

Epigenetic and genetic variation at the IGF2/H19 imprinting control region on 11p15.5 is associated with cerebellum weight

IGF2 is a paternally expressed imprinted gene with an important role in development and brain function. Allele-specific expression of IGF2 is regulated by DNA methylation at three differentially methylated regions (DMRs) spanning the IGF2/H19 domain on human 11p15.5. We have comprehensively assessed DNA methylation and genotype across the three DMRs and the H19 promoter using tissue from a unique collection of well-characterized and neuropathologically-dissected post-mortem human cerebellum samples (n = 106) and frontal cortex samples (n = 51). We show that DNA methylation, particularly in the vicinity of a key CTCF-binding site (CTCF3) in the imprinting control region (ICR) upstream of H19, is strongly correlated with cerebellum weight. DNA methylation at CTCF3 uniquely explains ∼25% of the variance in cerebellum weight. In addition, we report that genetic variation in this ICR is strongly associated with cerebellum weight in a parental-origin specific manner, with maternally-inherited alleles associated with a 16% increase in cerebellum weight compared with paternally-inherited alleles. Given the link between structural brain abnormalities and neuropsychiatric disease, an understanding of the epigenetic and parent-of-origin specific genetic factors associated with brain morphology provides important clues about the etiology of disorders such as schizophrenia and autism.Key words: epigenetic, DNA methylation, genomic imprinting, cerebellum, IGF2, H19, brain, expression, frontal cortex, genetic, single nucleotide polymorphism     

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.