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Human Molecular Genetics Advance Access originally published online on August 23, 2006
Human Molecular Genetics 2006 15(19):2945-2954; doi:10.1093/hmg/ddl237
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus

Nora Engel, Joanne L. Thorvaldsen and Marisa S. Bartolomei*

Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA

* To whom correspondence should be addressed at: 415 Curie Boulevard, CRB Room 363, Philadelphia, PA 19104, USA. Tel: +1 2158989063; Fax: +1 2155736434; Email: bartolom{at}mail.med.upenn.edu

Received June 28, 2006; Accepted August 15, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Imprinting at the H19/Igf2 locus depends on a differentially methylated domain (DMD) acting as a maternal-specific, methylation-sensitive insulator and a paternal-specific locus of hypermethylation. Four repeats in the DMD bind CTCF on the maternal allele and have been proposed to recruit methylation on the paternal allele. We deleted the four repeats and assayed the effects of the mutation at the endogenous locus. The H19DMD-{Delta}R allele can successfully acquire methylation during spermatogenesis and silence paternal H19, indicating that these paternal-specific functions are independent of the CTCF binding sites. Maternal inheritance of the mutations leads to biallelic Igf2 expression, consistent with the loss of a functional insulator. Additionally, we uncovered two previously undescribed roles for the CTCF binding sites. On the mutant allele, H19 RNA is barely detectable in 6.5 d.p.c. embryos and 9.5 d.p.c. placenta, for the first time identifying the repeats as the elements responsible for initiating H19 transcription. Furthermore, methylation is abruptly acquired on the mutant maternal allele after implantation, a time when the embryo is undergoing genome-wide de novo methylation. Together, these experiments show that in addition to being essential for a functional insulator, the CTCF repeats facilitate initiation of H19 expression in the early embryo and are required to maintain the hypomethylated state of the entire DMD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genomic imprinting regulates the expression of a group of developmentally crucial mammalian genes. Disruption of imprinting is implicated in several cancers (1) and in diseases such as Beckwith–Wiedemann syndrome, a congenital overgrowth condition (2). Transcriptional regulation of imprinted loci poses a unique challenge to cells for several reasons. First, two parental alleles are present in the same nuclear environment but only one is expressed. Second, the transcriptional status of each allele is stably maintained following mitosis. Furthermore, which allele is active depends on whether it was introduced via the oocyte or the sperm. Although many imprinted genes have been described (www.mgu.har.mrc.ac.uk/research/imprinting), few cis-acting elements governing tissue-specific expression and imprinting patterns have been identified.

Most imprinted genes lie within clusters that exhibit parental-specific epigenetic features, such as differential DNA methylation. The Beckwith–Wiedemann region on human chromosome 11p15.5 is one of the most intensively studied clusters and is orthologous to the imprinting region at the distal end of mouse chromosome 7. The mouse H19 and Igf2 genes lie within a subdomain of this region, and essential regulatory elements coordinating their imprinted expression have been well characterized. Shared enhancers that regulate H19 and Igf2 in endodermal (3) and mesodermal tissues (4) have been delimited. An imprinting control region (ICR) has been well defined and consists of a CG-rich differentially methylated domain (DMD), located –2 to –4 kb relative to the H19 transcription start site (5,6). This region is essential for establishing the pattern of imprinting by which H19, a non-coding RNA, is exclusively expressed from the maternal chromosome and Igf2, a fetal growth factor, is only active paternally. Targeted mutations have revealed that the DMD has at least two distinct regulatory roles on each of the parental chromosomes, but have also yielded results suggestive of additional functions (4,6). The activity of the DMD is determined by its methylation status. On the maternal chromosome, the DMD is hypomethylated and binds the protein CTCF via four highly conserved CG-rich repetitive sites (7–9). This association leads to the establishment of an insulator that blocks the interaction of the Igf2 promoter with the downstream enhancers, silencing the maternal Igf2 gene (10–12) (Fig. 1A). Maternal binding of CTCF to the four sites within the DMD has been further hypothesized to protect actively the region from becoming methylated during oogenesis and development (13). The DMD is also essential for optimal H19 and Igf2 expression and is required on the maternal allele for the initiation of H19 expression (14). Absence of the DMD on the maternal allele delays of H19 expression until later stages in development. Since CTCF has been shown to be a transcriptional regulator at several loci (15), we hypothesize that CTCF is involved in the initiation of H19 expression.


Figure 2371
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  		Figure 1. (A) Model for the imprinting regulation of H19 and Igf2. Maternal and paternal chromosomes are indicated. Thick black arrows denote active transcription, DMD is the differentially methylated domain, filled lollipops indicate CpG methylation and hatched ovals represent the downstream enhancers. (B) Distribution and sequence of the four repeats within the DMD, with the 21 bp repeats capitalized and denoted as R1 through R4. CpG dinucleotides are underlined. The sequence resulting from the absence of the repeats in the mutant DMD (H19DMD-{Delta}R) is shown below.

 
The paternal allele of the DMD is hypermethylated, a modification acquired during spermatogenesis and heritably maintained in somatic cells (5,16). There are two transcriptional consequences of the hypermethylation of the DMD. First, the DMD acts as a center for the spread of methylation that silences the H19 gene. Second, since CTCF cannot bind methylated DNA (8,9), the insulator is not assembled, thus allowing paternal Igf2 to access the downstream enhancers and be expressed. In addition to these well-established functions, it has been hypothesized that the DMD contains sufficient information to acquire the methylation imprint during spermatogenesis. Since it has been posited that repetitive sequences in the vicinity of imprinted genes could be the initial targets of methylation (17), the four 21 bp CG-rich repeats within the DMD are candidates for embodying the signal for germline methylation.

We sought to investigate the role of the 21 bp CG-rich repeats that bind CTCF in the different functions of the DMD, both established and hypothetical. Previous replacement alleles (10–12) had tested whether CTCF binding was necessary for insulator function by mutating the recognition sites. We deleted precisely the 21 bp core consensus sites at the endogenous locus. We show that deleting the repeats does not affect the acquisition of methylation of the DMD during spermatogenesis. There is a slight loss of methylation on the paternal allele during development, suggesting that the density of CpGs could be important for the persistence of the mark. When the mutant DMD is transmitted maternally, imprinting of Igf2 is perturbed, resulting in biallelic Igf2 expression. In addition, H19 expression levels are drastically reduced in 6.5 d.p.c. embryo and 9.5 d.p.c. placenta, for the first time directly implicating the CTCF recognition sites in the initiation of H19 transcription. Expression of H19 is present but reduced in a tissue-specific manner in neonatal liver and kidneys, arguing for an involvement of the repeats in the full transcriptional activation of H19 in these tissues. Although the repeats are not necessary to maintain the hypomethylated state of the DMD in oocytes and pre-implantation embryos, when de novo methylation is limited, methylation is acquired post-implantation on the mutant maternal allele, leading to levels similar to wild-type paternal methylation in neonatal tissues. Thus, although the repeats are not implicated in the paternal functions of the DMD, they affect the insulator assembly and initiation of H19 expression on the maternal allele. Moreover, the repeats are required on the maternal chromosome to maintain the allele in a hypomethylated state during the period of active de novo methylation that occurs after implantation. These three distinct maternal effects can be attributed to CTCF binding within the DMD, and two of these mutant phenotypes suggest novel functions of CTCF.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Deletion of CTCF sites in the DMD
The DMD is postulated to have independent roles on the maternal and paternal H19/Igf2 alleles. On the maternal chromosome, the DMD insulates Igf2 from enhancers lying downstream of H19 (Fig. 1A). On the paternal allele, it is hypermethylated and required for H19 silencing. To test whether the four CTCF binding sites mediate these functions, we deleted them by site-directed mutagenesis (Fig. 1B). In contrast to previously described CTCF mutations (10–12), these deletions specifically remove the core repeats. By computational analysis, the remaining sequences did not contain any cryptic CTCF binding sites. The mutant version of the DMD was targeted to the endogenous locus by homologous recombination in ES cells (Fig. 2A), and two correctly targeted clones were used to generate two independent founder mouse lines that transmitted the H19DMD-{Delta}R-neo allele through the germline (Fig. 2B). The neor cassette was removed by crossing male founders with females carrying a Cre recombinase gene under the control of the Zp3 promoter (18).


Figure 2372
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  		Figure 2. Strategy for the targeted replacement of the 5' H19 DMD and effects of paternal inheritance. (A) Illustrated from top to bottom are the targeting vector (pDMD-{Delta}R), the wild-type locus, the correctly targeted allele (H19DMD-{Delta}R-neo) and the targeted allele after excision of the neor selection marker (H19DMD-{Delta}R). The vector includes bacterial sequences (broken lines), 5' H19 sequence (hatched line), a neor selection cassette (open box), the flanking loxP sites (black triangles) and H19 exon sequences (open boxes). Fragments used as probes for screening (A and B) are shown as thick lines below the endogenous locus. Positions are indicated relative to the start of H19 transcription. (B) Southern blot analysis for detection of targeting events. Genomic DNA from the parental E14 (+/+) and targeted clones (DMD-{Delta}R, two independent recombinant clones, 46 and 64) was digested either with EcoRV and hybridized to probe A from the 5' region (left panel) or with StuI and hybridized to probe B from the 3' end of the locus (right panel). (C) Paternal inheritance of the H19DMD-{Delta}R allele. B6(CAST7) females were crossed to heterozygous mutant males (B6 background), and 3 µg of RNA from neonatal liver was analyzed by RNAse protection assay. Wild-type mice are designated +/+ and mutant littermates are designated –/+. B6 and Cast digestion products are designated. (D) Total levels of Igf2 expression in neonatal liver relative to the internal control rpL32. Ratios of Igf2 to rpL32 levels are indicated below each lane.

 
Paternal inheritance of the H19DMD-{Delta}R allele
We first assessed the effect of transmitting the H19DMD-{Delta}R allele paternally. To determine whether the deletions affected the ability of the DMD to silence H19 expression, mice harboring the mutation were crossed to B6(CAST7) mice, which have a Mus castaneus chromosome 7 on a C57BL/6 background (19). This cross allows the parental alleles to be distinguished in the F1 progeny. Imprinted expression of H19 was assayed on RNA from neonatal liver by an allele-specific RNAse protection assay (Fig. 2C). No loss of imprinting was observed, i.e. the mutant H19 allele remained silent. Furthermore, assay of Igf2 expression relative to rpL32 showed that Igf2 was at wild-type levels (Fig. 2D). Thus, the loss of the repeats did not affect the silencing capacity of the DMD. These results contrast with the loss of imprinted H19 expression and reduction of Igf2 RNA levels observed upon the deletion of the complete DMD (20).

The DMD acquires methylation during male gametogenesis, and the preferential paternal methylation is maintained throughout embryogenesis, resisting the genome-wide demethylation that occurs prior to implantation. To determine if methylation had been acquired normally during spermatogenesis, sperm DNA was examined by bisulfite mutagenesis and sequencing. The wild-type and mutant DMD were similarly hypermethylated, indicating that establishment of methylation was unaffected by the mutations (Fig. 3A). Analysis of neonatal liver DNA showed slightly lower than normal methylation levels on the paternally transmitted H19DMD-{Delta}R allele (Fig. 3B). This minor decrease in methylation did not affect imprinted H19 expression.


Figure 2373
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  		Figure 3. Methylation profile of the paternally transmitted H19DMD-{Delta}R allele. (A) Schematic of the region analyzed by bisulfite mutagenesis and sequencing, with the primers shown as arrows. (B) Methylation status of individual DNA strands of the paternally inherited wild-type and H19DMD-{Delta}R alleles in sperm of heterozygous males with the paternally inherited mutant alleles. (C) Methylation profile from neonatal liver of heterozygous animals. In the graph, summary is shown for the wild-type (light gray bars) and H19DMD-{Delta}R alleles (dark gray bars). Bars represent the fraction of methylated cytosines at each position. Note that sites 4, 5, 6 and 11,12,13 are deleted in H19DMD-{Delta}R. CpG dinucleotides are depicted as open circles when unmethylated and filled circles when methylated. Each line of circles represents an independent strand of DNA, with multiple strands showing the same pattern indicated to the left. The results are from two independent PCR reactions.

 
Effects of the maternal inheritance of the H19DMD-{Delta}R allele
Although paternal transmission of the H19DMD-{Delta}R allele revealed results similar to previously described CTCF mutations (10–12), maternal transmission uncovered significant new information regarding the imprinting and transcriptional regulation of this locus. As expected, deletion of the CTCF sites affected the imprinted expression of Igf2, as shown with an allele-specific RNAse protection assay (Fig. 4A). Igf2 was biallelically expressed in livers of mutant neonates, with reactivation of the normally silent maternal allele similar to that occurring in neonatal liver RNA from mice bearing a maternally inherited mutation in which the complete DMD has been removed (H19{Delta}3.8 kb-5'H19/+).


Figure 2374
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  		Figure 4. Imprinting of Igf2 in mice upon maternal inheritance of the H19DMD-{Delta}R allele. (A) Heterozygous mutant females (B6 background) were crossed to B6(CAST7) males, and 3 µg of liver RNA from neonates was analyzed by RNAse protection assay. Wild-type mice are designated +/+ and mutant littermates are designated –/+. Percent expression of maternal alleles relative to paternal alleles is shown below each lane. Activation of maternal Igf2 in mice inheriting the H19{Delta}3.8 kb-5'H19 allele maternally is shown for comparison. (B) Allelic analysis of Igf2 expression in 6.5 d.p.c. embryos inheriting the wild-type (+/+) or H19DMD-{Delta}R allele (–/+) from the mother. RNA was assayed by RT-PCR and restriction digestion, with Tsp509I for the exon 6 product and with BanI for the placental-specific transcript. Activation of maternal Igf2 in mice inheriting the H19{Delta}3.8 kb-5'H19 allele maternally is shown for comparison. Genotype of each sample is indicated above the lane, and non-digested (ND) and –RT controls are shown. Products derived from the C57BL/6 (B) and Mus castaneus (C) alleles are noted. Top panel, allelic expression of all Igf2 transcripts; bottom panel, allelic Igf2 expression from the placental-specific promoter.

 
We extended our analysis to determine if Igf2 imprinted expression was already disturbed on the H19DMD-{Delta}R allele in 6.5 d.p.c. embryos, the stage in which Igf2 starts to be fully expressed. We used an RT-PCR assay that amplifies a region in exon 6 of Igf2, which is common to all Igf2 transcripts. Restriction digestion of the PCR products reveals a polymorphism between the C57BL/6 and M. castaneus alleles. Igf2 was highly expressed from the maternally inherited mutant alleles. Since expression of Igf2 at this stage is expected to arise from the placental-specific promoter Po (21), we carried out an allele-specific assay to measure expression from this promoter (14) and found derepression of the maternal allele (Fig. 4B).

Transcriptional effects in the embryo upon maternal inheritance of the H19DMD-{Delta}R allele
Previous experiments on mice harboring a complete deletion of DMD sequence at the endogenous locus (H19{Delta}3.8 kb-5'H19) suggested that there is a positive transcriptional element influencing H19 expression initiation at the late blastocyst stage (14). To examine if the absence of the repeats in the DMD affected transcription of H19 in a similar way, total H19 RNA was determined relative to Gapdh levels in 6.5 d.p.c. embryos (Fig. 5B). Levels of H19 expression from the mutant allele were drastically reduced relative to the wild-type allele, suggesting that transcriptional initiation of H19 is developmentally delayed, similar to what was observed in the complete DMD deletion. We also assayed H19 RNA in 9.5 d.p.c. embryos. A striking reduction of H19 levels was observed in placenta and embryos (Fig. 5C), recapitulating the results previously determined with the H19{Delta}3.8 kb-5'H19 allele and thus identifying the repeats as the precise elements responsible for this effect. Our data strongly implicate a role for the repeats in the transcriptional modulation of H19.


Figure 2375
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  		Figure 5. H19 expression levels upon maternal inheritance of the H19DMD-{Delta}R allele. (A) Left panel, northern blot analysis of neonatal liver RNA. Gapdh was used as an internal control. Ratios of H19 to Gapdh are indicated beneath the lanes. Right panel, RNAse protection assay of kidney RNAs isolated from neonatal mice. Ribosomal protein rpL32 was used as a control. Position of H19 and rpL32 protected fragments are indicated, and the ratios of H19 to rpL32 are shown beneath the lanes. (B) H19 expression levels in 6.5 d.p.c. embryos. H19 RNA is measured relative to Gapdh RNA levels by RT-PCR. Wild-type mice (dark gray bars) are designated +/+ and mutant littermates (light gray bars) are designated –/+. Results are presented graphically, with the error bars reflecting the results from triplicate experiments. Results for the H19{Delta}3.8 kb-5'H19 allele are shown for comparison. An image of the H19 and Gapdh products is shown below. (C) H19 expression in 9.5 d.p.c. placenta and embryos.

 
A role for CTCF in tissue-specific expression was further demonstrated in neonatal tissues. Levels of H19 RNA in neonatal liver and kidney were found to be reduced by ~45% and 95%, respectively, indicating that deletion of the repeats removes the specific regulatory elements required for full H19 expression in liver and kidney (Fig. 5A).

Methylation profile of the maternally inherited H19DMD-{Delta}R allele
To assess whether the absence of the repeats affected the hypomethylated state of the maternal H19DMD-{Delta}R allele, we carried out bisulfite mutagenesis and sequencing on DNA from neonatal liver. The mutant allele was hypermethylated to levels similar to a wild-type paternal allele (Fig. 6A). To determine the stage when the methylation was acquired, the H19DMD-{Delta}R allele was examined in oocytes, blastocysts 6.5 and 9.5 d.p.c. embryos (Fig. 6B and data not shown). The mutant DMD was hypomethylated in oocytes and blastocysts, suggesting that maintenance of hypomethylation during these early stages does not depend on CTCF binding sites. Methylation was acquired by 6.5 d.p.c., a time when the embryo is undergoing de novo methylation, demonstrating that after implantation, the CTCF binding sites are important for maintaining the hypomethylated state of the entire DMD. Full methylation of the region as seen in neonatal liver was still not observed in 9.5 d.p.c. embryos (data not shown) suggesting that the methylation is acquired progressively during development.


Figure 2376
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  		Figure 6. Methylation profile of the maternally transmitted H19DMD-{Delta}R alleles. (A) Top, schematic of the region analyzed by bisulfite mutagenesis and sequencing, with the primers shown as arrows. Below, methylation status of individual DNA strands of the maternally inherited wild-type and H19DMD-{Delta}R alleles in neonatal liver of heterozygous animals. (B) Methylation profile of maternally inherited H19DMD-{Delta}R alleles from oocytes, blastocysts, 6.5 and 9.5 d.p.c. embryos. Details are given in Figure 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Disruption of genomic imprinting has been increasingly recognized as an important factor in many genetic diseases and cancers, either as a direct cause or as a contributing factor. Targeted mutations in the mouse have strongly impacted our understanding of the mechanisms involved in the regulation of imprinting, complementing and, at times, even anticipating clinical findings. Deletion mutants had shown that the H19 DMD had an essential role in the imprinting pattern at the Igf2/H19 locus, and a deletion in the H19 DMD was later reported in individuals with Beckwith–Wiedemann syndrome (22). Discrete deletions of specific regulatory sequences, analogous to microdeletions of CTCF target sites in the DMD recently described in a family with cases of Beckwith–Wiedemann syndrome (23), can give insight into the molecular mechanisms involved in imprinting control.

The Igf2/H19 locus DMD is a multifaceted sequence, with distinct roles on each chromosome that depend upon on sex-specific epigenetic differences inherited from the gametes. The DMD is crucial for at least two genetic functions implicated in maintaining imprinted expression at this locus. First, both loss-of-function (10–12) and gain-of-function mutations (24) have strongly supported the model whereby the DMD is a methylation-sensitive insulator assembled upon binding of CTCF to four recognition sites. The insulator is established on the hypomethylated maternal chromosome and precludes activation of Igf2 by blocking interaction between Igf2 and the downstream enhancers (8). Second, the DMD functions to silence the H19 gene in cis. This activity is exclusive to the paternal allele and depends on the spreading of methylation from the DMD to the H19 promoter, probably at late gestation stages (5). We generated an allele, H19DMD-{Delta}R, which completely removed the 21 bp repeats that are the core of the CTCF consensus binding sites. Our results show that the absence of the repeats leads to loss of enhancer blocking, confirming other reports that the CTCF binding sites are essential for this activity (10–12). Our H19DMD-{Delta}R allele can successfully substitute for the wild-type paternal DMD, indicating that acquisition of methylation during spermatogenesis and silencing of H19 is independent of the CTCF binding sites. This result is consistent with a previously reported partial deletion of the DMD, which left the two 5'-CTCF binding sites intact and in which there was loss of silencing of paternal H19 (25), and suggests that there are other as-yet-unknown cis-elements within the 3' region of the DMD that endow it with silencing activity. With regard to the acquisition of methylation in the male germline, it is intriguing that none of the mutations and deletions within and adjacent to the DMD of the H19/Igf2 locus to date has perturbed the establishment of the male-specific methylation mark, as defined by full methylation of the DMD in sperm (6,11,12,20,26–29). Thus, although it is possible that there are other unrevealed motifs within the DMD involved in recruiting methylation, it seems that imprint establishment is not an autonomous feature of the DMD and that the genomic context of the endogenous locus is essential.

Importantly, the H19DMD-{Delta}R allele has allowed us to identify two new properties of the DMD on the maternal chromosome and uncovered the role of CTCF in these novel functions. It has been hypothesized that CTCF binding on the maternal allele is essential to protect the region from becoming methylated (13). Our results show that even in the absence of CTCF binding sites, the DMD is refractory to methylation during both oogenesis and pre-implantation stages. However, the maternal H19DMD-{Delta}R allele is fully methylated in neonatal somatic tissues. We have determined that the precise stage at which methylation is acquired is immediately after implantation. These results indicate that CTCF has a direct role in maintaining an undermethylated state on the maternal allele during the wave of de novo methylation (30). The difference in the susceptibility of the DMD to methylation during oogenesis and after implantation could be the result of the distinct de novo methyltransferases acting at each stage: Dnmt 3a and 3l have been implicated in establishment of methylation imprints during oogenesis (31–33), whereas the genome-wide post-implantation methylation has been attributed to Dnmt 3a and 3b (34). Alternatively, specific nuclear conformation or localization could render the DMD less accessible to methylation events during oogenesis. What is clear is that the CTCF binding sites have a crucial role in protecting the DMD from the post-implantation genome-wide methylation event.

Earlier studies strongly suggested that the DMD had a transcriptional role in maintaining full levels of both H19 and Igf2 expression. Deletion of the DMD on the maternal allele caused a reduction in H19 RNA levels to different degrees depending on the tissue, whereas paternal inheritance of the deletion resulted in a decrease of Igf2 expression (20). Further studies in a model of experimental liver carcinogenesis showed that although Igf2 is activated during the induction phase, this does not occur in the absence of the DMD (35). Our results indicate that deletion of the CTCF binding sites on the maternal chromosome results in a decrease of H19 RNA levels, supporting their role in ensuring full expression of H19 in liver and kidney. Moreover, levels of paternal Igf2 were not affected by these deletions, indicating that other sequences in the DMD may be responsible for regulating Igf2 expression in liver.

It was recently postulated that the DMD had a crucial transcriptional role not only in maintaining the expression levels of H19 but also in the actual initiation of maternal H19 expression, since the absence of the DMD on the maternal chromosome led to a delay in the initiation of H19 expression (14). Our results show that upon deletion of the four CTCF binding sites, H19 expression is as drastically reduced in 6.5 and 9.5 d.p.c. embryos as it is when the complete DMD is absent, consistent with delay in expression initiation. Thus, a major finding of this work is that we have precisely identified for the first time the specific sequence elements responsible for this effect. Transcriptional activity of CTCF, both as an activator and as a repressor, has been reported at other loci (36). Our data argue for a transcriptional function of CTCF at this locus, which had not been described previously.

The transcriptional activity that we postulate for CTCF during embryogenesis coincides temporally with the requirement for the recognition sites to keep the region from being methylated, as described earlier; protection from methylating enzymes could simply be the consequence of the physical presence of CTCF in the region or could result from chromatin modifications that occur in conjunction with active transcription. Similarly, the enhancer-blocking activity could be a functional consequence of the transcriptional activity of CTCF at the DMD. Several insulators have promoter and, occasionally, enhancer activities (37–40). Moreover, some promoters and enhancers have been found to have boundary activity. Several insulator-binding proteins, in addition to CTCF, have multiple regulatory roles. This has led to the suggestion that an insulator, rather than a specific sequence with a single function, is a phenotype (41). At the H19/Igf2 locus, we suggest that the maternal enhancer-blocking phenotype of the DMD is a corollary of a primary transcriptional activity, mediated by CTCF. We propose a model in which the presence of CTCF engenders a tight engagement between the DMD, the H19 promoter and the downstream enhancers, thus excluding access of the Igf2 gene to the enhancers. This interaction could involve direct physical interactions and looping out of chromatin, thereby partitioning the region into either active or inactive domains, as suggested by chromosome conformation capture (3C) studies at this locus (42).

In conclusion, our studies confirm that on the maternal chromosome, binding of CTCF to the DMD repeats is essential for maintaining monoallelic Igf2 expression. Importantly, our results strongly suggest that CTCF has a positive transcriptional role in H19 expression, an activity that had not been previously described. In addition, our data show that the DMD repeats are required to maintain the methylation-free status of the region specifically after implantation when de novo methylation is occurring. Future studies will be directed at connecting this observation with the molecular mechanism responsible for maintaining a methylation-free region at the DMD. Furthermore, the challenge remains to unravel the mechanism by which the DMD is targeted for methylation in the germline and how paternal methylation later spreads and silences H19.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Targeting vector
H19 129/Sv genomic DNA sequence spanning the DMD contained in a pBluescript II KS vector (Stratagene) was modified by site-directed mutagenesis to delete the KpnI site at –3.7 kb. This plasmid was further mutagenized by successive deletions of each of the four CpG-rich repeats with site-directed mutagenesis (QuickChange site-directed mutagenesis kit, Stratagene). After digestion, the mutant DMD was ligated to a 6 kb fragment from the 5' region of H19 and cloned into a pBKS containing a Pgk1 promoter-driven neomycin-resistance cassette (neor) flanked with loxP sites and a HindIII/NotI fragment of H19 to serve as the 3' arm of the final targeting vector (Fig. 2A). The vector was linearized at a unique NotI site prior to electroporation.

Generation of mutant mice
Day 14 ES cells were electroporated as previously described (6). For screening, DNA prepared from the colonies was digested with StuI or EcoRV and run on 1% agarose gels. A BamHI/StuI probe (probe B, Fig. 2A) was used for the StuI-digested DNA for the 3' end, and an EcoRV/EcoRI probe (probe A, Fig. 2A) was used on EcoRV-digested DNA for the 5' end.

Two correctly targeted independent clones with the neor cassette (lines 46 and 64, Fig. 2B) were injected into C57BL/6 (B6) blastocysts and were then transferred to pseudopregnant female mice. The resulting chimeras were bred with B6 mice and DNA was isolated from tail biopsies of the progeny to determine germline transmission of the mutation. Mice were genotyped by PCR, using primers G2 and G5 (20). The mutant DMD was sequenced to confirm the presence of the mutations. The neor gene was removed by breeding to ZP3-Cre transgenic mice (18). Female progeny positive for the mutation and the Cre recombinase gene were crossed to B6 males, and the correct excision of neor in the following generation was ascertained by PCR. For methylation and allelic expression assays, heterozygous mutant mice were bred to the B6(CAST 7) strain of mice (19).

Methylation and expression analysis
DNA was isolated from tissues, and DNA bisulfite modification and sequencing analysis were carried out as described previously (6,20). The primers used were B1, B2, B3 and B4 (20), with the following modification in primer B3: 5'-CTAACCTCATAAAATCCCATAACTAT-3'. Total RNA was prepared from neonatal liver and kidney by the lithium chloride method (43). RNAse protection assays to detect H19, Igf2 and rpL32 expressions were performed as described previously (6). Total RNA was purified from 6.5 and 9.5 d.p.c. embryos, using the High Pure RNA kit (Roche). The RNA was reverse-transcribed as previously described and used in expression assays (14). H19 RNA levels relative to Gapdh were measured as described previously (14). Gels were exposed to phosphor screens and scanned on a Typhoon Trio Phosphorimager (Amersham Biosciences). Band intensities were calculated as described previously (6). For northern analyses, RNAs were fractionated on 0.9% agarose gels with 1x MOPS and 0.6% formaldehyde, transferred to Hybond-N+membranes (Amersham Pharmacia) in 20x SSC. H19, Gapdh and Igf2 exon 6 DNAs were labeled using Ready-To-Go DNA Labelling Beads (Amersham Pharmacia). Membranes were hybridized using Denhardt's buffer, and washed and stripped as recommended by the manufacturer.


   ACKNOWLEDGEMENTS
 
We thank Jean Richa at the University of Pennsylvania Transgenic Core Facility for the generation of chimeric mice, Christopher Krapp and Melanie Hullings for technical assistance and Raluca Verona for helpful comments. This work was supported by NIH grant GM51279, by the Howard Hughes Medical Institute and by NCI grant CA115906 (N.E.). N.E. was supported by NRSA post-doctoral fellowship HD41345.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Feinberg A.P., Oshimura M., Barrett J.C. (2002) Epigenetic mechanisms in human disease. Cancer Res. 62:6784–6787.[Free Full Text]

  2. Weksberg R., Smith A.C., Squire J., Sadowski P. (2003) Beckwith–Wiedemann syndrome demonstrates a role for epigenetic control of normal development. Hum. Mol. Genet. 12:R61–R68.[Abstract/Free Full Text]

  3. Leighton P.A., Saam J.R., Ingram R.S., Stewart C.L., Tilghman S.M. (1995) An enhancer deletion affects both H19 and Igf2 expression. Genes Dev. 9:2079–2089.[Abstract/Free Full Text]

  4. Kaffer C.R., Srivastava M., Park K.-Y., Ives E., Hsieh S., Batlle J., Grinberg A., Huang S.-P., Pfeifer K. (2000) A transcriptional insulator at the imprinted H19/Igf2 locus. Genes Dev. 14:1908–1919.[Abstract/Free Full Text]

  5. Tremblay K.D., Duran K.L., Bartolomei M.S. (1997) A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development. Mol. Cell. Biol. 17:4322–4329.[Abstract]

  6. Thorvaldsen J.L., Duran K.L., Bartolomei M.S. (1998) Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev. 12:3693–3702.[Abstract/Free Full Text]

  7. Stadnick M.P., Pieracci F.M., Cranston M.J., Taksel E., Thorvaldsen J.L., Bartolomei M.S. (1999) Role of a 461 bp G-rich repetitive element in H19 transgene imprinting. Dev. Genes Evol. 209:239–248.[CrossRef][Web of Science][Medline]

  8. Hark A.T., Schoenherr C.J., Katz D.J., Ingram R.S., Levorse J.M., Tilghman S.M. (2000) CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405:486–489.[CrossRef][Medline]

  9. Bell A.C. and Felsenfeld G. (2000) Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405:482–485.[CrossRef][Medline]

  10. Schoenherr C.J., Levorse J.M., Tilghman S.M. (2003) CTCF maintains differential methylation at the Igf2/H19 locus. Nat. Genet. 33:66–69.[CrossRef][Web of Science][Medline]

  11. Szabo P.E., Tang S.H., Silva F.J., Tsark W.M., Mann J.R. (2004) Role of CTCF binding sites in the Igf2/H19 imprinting control region. Mol. Cell. Biol. 24:4791–4800.[Abstract/Free Full Text]

  12. Pant V., Mariano P., Kanduri C., Mattsson A., Lobanenkov V., Heuchel R., Ohlsson R. (2003) The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. Genes Dev. 17:586–590.[Abstract/Free Full Text]

  13. Fedoriw A.M., Stein P., Svoboda P., Schultz R.M., Bartolomei M.S. (2004) Transgenic RNAi reveals essential function for CTCF in H19 gene imprinting. Science 303:238–240.[Abstract/Free Full Text]

  14. Thorvaldsen J.L., Fedoriw A.M., Nguyen S., Bartolomei M.S. (2006) Developmental profile of H19 differentially methylated domain (DMD) deletion alleles reveals multiple roles of the DMD in regulating allelic expression and DNA methylation at the imprinted H19/Igf2 locus. Mol. Cell. Biol. 26:1245–1258.[Abstract/Free Full Text]

  15. Ohlsson R., Renkawitz R., Lobanenkov V. (2001) CTCF is a uniquely versatile transcription regulator linked to epigenetics and disease. Trends Genet. 17:520–527.[CrossRef][Web of Science][Medline]

  16. Davis T.L., Yang G.J., McCarrey J.R., Bartolomei M.S. (2000) The H19 methylation imprint is erased and re-established differentially on the parental alleles during male germ cell development. Hum. Mol. Genet. 9:2885–2894.[Abstract/Free Full Text]

  17. Neumann B., Kubicka P., Barlow D.P. (1995) Characteristics of imprinted genes. Nat. Genet 9:12–13.[CrossRef][Web of Science][Medline]

  18. Lewandoski M., Wassarman K.M., Martin G.R. (1997) Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line. Curr. Biol. 7:148–151.[CrossRef][Web of Science][Medline]

  19. Mann M.R., Chung Y.G., Nolen L.D., Verona R.I., Latham K.E., Bartolomei M.S. (2003) Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol. Reprod. 69:902–914.[Abstract/Free Full Text]

  20. Thorvaldsen J.L., Mann M.R., Nwoko O., Duran K.L., Bartolomei M.S. (2002) Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting. Mol. Cell. Biol. 22:2450–2462.[Abstract/Free Full Text]

  21. Moore T., Constancia M., Zubair M., Bailleul B., Feil R., Sasaki H., Reik W. (1997) Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2. Proc. Natl Acad. Sci. USA 94:12509–12514.[Abstract/Free Full Text]

  22. Sparago A., Cerrato F., Vernucci M., Ferrero G.B., Silengo M.C., Riccio A. (2004) Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith–Wiedemann syndrome. Nat. Genet. 36:958–960.[CrossRef][Web of Science][Medline]

  23. Prawitt D., Enklaar T., Gartner-Rupprecht B., Spangenberg C., Oswald M., Lausch E., Schmidtke P., Reutzel D., Fees S., Lucito R., et al. (2005) Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith–Wiedemann syndrome and Wilms’ tumor. Proc. Natl Acad. Sci. USA 102:4085–4090.[Abstract/Free Full Text]

  24. Engel N., West A.G., Felsenfeld G., Bartolomei M.S. (2004) Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations. Nat. Genet. 36:883–888.[CrossRef][Web of Science][Medline]

  25. Drewell R.A., Brenton J.D., Ainscough J.F., Barton S.C., Hilton K.J., Arney K.L., Dandolo L., Surani M.A. (2000) Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic switch. Development 127:3419–3428.[Abstract]

  26. Szabo P.E., Pfeifer G.P., Mann J.R. (2004) Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region. Mol. Cell. Biol. 24:4858–4868.[Abstract/Free Full Text]

  27. Ripoche M.-A., Chantal K., Poirier F., Dandolo L. (1997) Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element. Genes Dev. 11:1596–1604.[Abstract/Free Full Text]

  28. Reed M.R., Riggs A.D., Mann J.R. (2001) Deletion of a direct repeat element has no effect on Igf2 and H19 imprinting. Mamm. Genome 12:873–876.[CrossRef][Web of Science][Medline]

  29. Lewis A., Mitsuya K., Constancia M., Reik W. (2004) Tandem repeat hypothesis in imprinting: deletion of a conserved direct repeat element upstream of h19 has no effect on imprinting in the igf2-h19 region. Mol. Cell. Biol. 24:5650–5656.[Abstract/Free Full Text]

  30. Ratnam S., Mertineit C., Ding F., Howell C.Y., Clarke H.J., Bestor T.H., Chaillet J.R., Trasler J.M. (2002) Dynamics of Dnmt1 methyltransferase expression and intracellular localization during oogenesis and preimplantation development. Dev. Biol. 245:304–314.[CrossRef][Web of Science][Medline]

  31. Bourc'his D., Xu G.L., Lin C.S., Bollman B., Bestor T.H. (2001) Dnmt3L and the establishment of maternal genomic imprints. Science 294:2536–2539.[Abstract/Free Full Text]

  32. Kaneda M., Okano M., Hata K., Sado T., Tsujimoto N., Li E., Sasaki H. (2004) Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting. Nature 429:900–903.[CrossRef][Medline]

  33. Webster K.E., O'Bryan M.K., Fletcher S., Crewther P.E., Aapola U., Craig J., Harrison D.K., Aung H., Phutikanit N., Lyle R., et al. (2005) Meiotic and epigenetic defects in Dnmt3L-knockout mouse spermatogenesis. Proc. Natl Acad. Sci. USA 102:4068–4073.[Abstract/Free Full Text]

  34. Okano M., Bell D.W., Haber D.A., Li E. (1999) DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257.[CrossRef][Web of Science][Medline]

  35. Vernucci M., Cerrato F., Pedone P.V., Dandolo L., Bruni C.B., Riccio A. (2004) Developmentally regulated functions of the H19 differentially methylated domain. Hum. Mol. Genet. 13:353–361.[Abstract/Free Full Text]

  36. Dunn K.L. and Davie J.R. (2003) The many roles of the transcriptional regulator CTCF. Biochem. Cell. Biol. 81:161–167.[CrossRef][Web of Science][Medline]

  37. Engel N. and Bartolomei M.S. (2003) Mechanisms of insulator function in gene regulation and genomic imprinting. Int. Rev. Cytol. 232:89–127.[Medline]

  38. Avramova Z. and Tikhonov A. (1999) Are scs and scs' ‘neutral’ chromatin domain boundaries of the locus? Trends Genet. 15:138–139.[CrossRef][Web of Science][Medline]

  39. Smith P.A. and Corces V.G. (1995) The suppressor of Hairy-wing protein regulates the tissue-specific expression of the Drosophila gypsy retrotransposon. Genetics 139:215–228.[Abstract]

  40. Ohtsuki S. and Levine M. (1998) GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo. Genes Dev. 12:3325–3330.[Abstract/Free Full Text]

  41. West A.G., Gaszner M., Felsenfeld G. (2002) Insulators: many functions, many mechanisms. Genes Dev. 16:271–288.[Free Full Text]

  42. Murrell A., Heeson S., Reik W. (2004) Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops. Nat. Genet. 36:889–893.[CrossRef][Web of Science][Medline]

  43. Auffray C. and Rougeon F. (1980) Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Biochem. 107:303–314.[Web of Science][Medline]


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