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Human Molecular Genetics, 2003, Vol. 12, No. 23 3123-3132
DOI: 10.1093/hmg/ddg338
© 2003 Oxford University Press

Paternal imprints can be established on the maternal Igf2-H19 locus without altering replication timing of DNA

Flavia Cerrato1,2,{dagger}, Wendy Dean1, Karen Davies1, Kazuhiro Kagotani3, Kohzoh Mitsuya1, Katsuzumi Okumura3, Andrea Riccio1,2,{dagger} and Wolf Reik1,*

1Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme, The Babraham Institute, Cambridge CB2 4AT, UK, 2Dipartimento di Scienze Ambientali, Seconda Università di Napoli, Caserta, Italy and 3Laboratory of Molecular and Cellular Biology, Faculty of Bioresources, Mie University, Japan

Received July 29, 2003; Revised September 23, 2003; Accepted September 30, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Genomic imprinting in mammals marks the parental alleles in gametes, resulting in differential gene expression in offspring. A number of epigenetic features are associated with imprinted genes. These include differential DNA methylation, histone acetylation and methylation, subnuclear localization and DNA replication timing. While DNA methylation has been shown to be necessary both for establishment and maintenance of imprinting, the connections with the other types of epigenetic marking systems are not clear. Specifically, it is not known whether the other marking systems, either on their own or in conjunction with DNA methylation, are required for imprinting. Here we show that in the mouse mutant Minute (Mnt) the Igf2-H19 locus acquires a paternal methylation imprint in the maternal germline. DNA methylation of the H19 DMR is established in oogenesis, maintained during postzygotic development on the maternal allele, and erased in primordial germ cells. The fact that a paternal type methylation imprint can also be established in the maternal germline indicates that trans-acting factors that target methylation to this imprinted region are likely to be the same in both germlines. Surprisingly, however, asynchrony of DNA replication of the locus is maintained despite the altered expression and methylation imprint of Igf2 and H19. These results show clearly that replication asynchrony of this region is neither the determinant factor for, nor a consequence of, epigenetic modifications that are critical for genomic imprinting. Replication asynchrony may thus be regulated differently from methylation imprints and have a separate function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Gene regulation in higher eukaryotes is increasingly thought to depend on epigenetic marks of DNA and chromatin (1–5). DNA and histone methylation (in certain residues) generally have repressive effects on transcription, whereas histone acetylation (and histone methylation in other residues) mark transcriptionally active regions. In some systems heritable repression is associated with specific intranuclear localization of gene loci near pericentromeric heterochromatin (6). The timing of DNA replication of a locus during S phase has also been associated with its transcriptional activity, with early replication of active genes (7). While mechanistic links between DNA methylation and histone modifications are beginning to be uncovered (8), the precise relationship between timing of DNA replication and gene silencing or expression remains unclear.

Imprinted genes provide an excellent model system to study the different facets of epigenetic gene control. Imprinted genes are epigenetically modified during gametogenesis so that their expression in somatic cells becomes dependent on the parent of origin (9,10). Many imprinted genes have sequences which are differentially methylated between the parental alleles (DMRs). DMRs are also differentially marked by histone acetylation and methylation (11–14). Some DMRs have their DNA methylation differences established in the germ cells and maintained throughout development (germline DMRs). Germline DMRs generally correspond to regulatory elements controlling the imprinted expression of multiple genes in a cluster (imprinting centres, ICs). DNA methylation of the imprinted genes is erased in the primordial germ cells (PGCs) and re-established later during gametogenesis according to the sex of the organism (9,5,15). The importance of DNA methylation in the establishment and maintenance of genomic imprinting has been demonstrated in mice in which DNA methyltransferase (Dnmt) genes have been knocked out. Dnmt3L cooperates with Dnmt3a and Dnmt3b to establish maternal methylation imprints during oocyte development (16,17). Dnmt1 and its variant Dnmt1o are required for maintenance of allele-specific methylation and expression of many imprinted genes in preimplantation and postimplantation embryos (18,19). In addition to DNA and histone modifications, the parental alleles of imprinted gene regions differ in their replication timing during S phase (7). Replication asynchrony fulfils the formal criteria for an imprinting mechanism in that asynchronous replication is established in the gametes, maintained throughout development and erased in PGCs (20). It has therefore been suggested that it may have a role in the acquisition and /or maintenance of other epigenetic marks, leading to allele-specific expression (20). Recently it has been shown that replication asynchrony (and subnuclear localization) of imprinted regions is maintained in the Dnmt1-mutant, at least in ES cells (21). This suggests that replication asynchrony can be maintained in the absence of methylation imprints. However, whether or not methylation and expression imprints can be established and maintained independently of replication asynchrony is unknown.

The paternally expressed Insulin like growth factor 2 (Igf2) and maternally expressed H19 genes are part of a large cluster of imprinted genes located on mouse distal chromosome 7 and human chromosome 11p15.5 (9,22). Igf2 and H19 are coordinately transcribed during embryonic development and both use the same set of tissue-specific enhancers located downstream of H19 (23–25). A paternally methylated germline DMR (H19 DMR) is present between Igf2 and H19 (26,27). This region (IC1) harbours a chromatin insulator (28–32). The protein CTCF binds the DMR/IC1 and this binding is prevented by CpG methylation (29–32). Thus, the reciprocal imprinting of the Igf2 and H19 genes is explained by the parent of origin-specific methylation of the DMR. The unmethylated DMR present on the maternal chromosome 7 limits the action of the enhancers to the H19 promoter, while methylation of the DMR inactivates the insulator on the paternal homologue, thereby allowing activation of the Igf2 promoter by the enhancers.

Mutation of the CTCF sites abolishes the enhancer blocking activity of the DMR and causes activation of the Igf2 gene on the maternal chromosome (33). It also leads to DNA methylation of the maternal DMR but this only happens postzygotically. Thus CTCF binding apparently protects the maternal allele from methylation during somatic development, but whether there is protection needed during oogenesis by other factors is not known. Alternatively the machinery that methylates the H19 DMR in the paternal germline may be absent in the maternal one.

We have recently identified the genetic defect in a mutant mouse line, minute (Mnt), which shows intrauterine growth retardation on paternal transmission and foetal overgrowth on maternal transmission (25). These animals carry a 3 Mb inversion on distal chromosome 7, whose first breakpoint (BP1) is near the imprinting cluster at 25 kb distal to the H19 gene. Upon paternal transmission of the Mnt mutation, Igf2 expression is suppressed in mesodermal tissues and placenta, due to disruption or removal of tissue-specific enhancers by the inversion. Maternal transmission leads to silencing of the H19 gene and methylation of its upstream DMR in all tissues. This aberrant methylation results in derepression of the maternal Igf2 allele in those tissues (endodermal) for which enhancers are intact and thus increases the overall expression of the growth factor.

Here we show that in the Mnt mutant the maternal Igf2-H19 locus acquires a methylation imprint which is identical to the paternal wild-type one. Methylation is acquired during oogenesis, maintained during development, and erased in PGCs. Most surprisingly, replication asynchrony of the Igf2-H19 locus is unaffected by the Mnt mutation, indicating that the methylation imprint of the Igf2-H19 locus can be established and maintained aberrantly on the maternal chromosome, without interfering with its normal replication timing in S phase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Paternal methylation pattern of the Igf2-H19 locus on the maternal Mnt allele
It was previously demonstrated that maternal transmission of the Mnt mutation resulted in methylation of the H19 DMR and promoter in E17 fetuses (25). This analysis was performed by Southern blotting after digestion with the methylation-sensitive restriction enzyme AatII, which determines the methylation status of a single CpG in the H19 DMR. In this study, we wished to determine whether the methylation pattern of the whole locus on the mutant maternal allele was similar to or different from that of the paternal wild-type one. We examined the methylation status of 25 CpGs located in the DMR (including two CTCF sites) by the bisulfite-genomic sequencing procedure. Progeny from maternal transmission of the Mnt mutation [F1(C57Bl /6JxCBA /Ca)/Mnt femalexSD7/SD7 (Mus spretus distal chrom. 7 in M. mus domesticus background) male] were collected on postnatal day 1 (P1) and DNA methylation was analysed in the liver. Sequence polymorphisms allowed us to distinguish maternal from paternal alleles. The results showed that all the CpGs of the H19 DMR analysed were hypermethylated on both the maternal and paternal alleles in the Mnt/SD7 mice, while most CpGs were methylated on the paternal allele only in the +/SD7 (wild-type) mice (Fig. 1). Thus, maternal transmission of the Mnt mutation resulted in extensive hypermethylation of the H19 DMR on the maternal chromosome, such that its methylation pattern resembled that of the normal paternal allele.



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  		Figure 1. DNA methylation analysis of the H19 DMR in neonatal liver. (A) Diagram showing the relative positions of the H19 DMR (filled box), the transcriptional start site and orientation of the H19 gene (arrow), the two endodermal enhancer elements (ovals) and the DNA sequence analysed in this experiment (GenBank accession no. AF049091). The region extends from position -4147 to -3611 relative to the H19 transcriptional start site and contains 25 CpGs of which six are polymorphic (asterisks) between M. mus domesticus and M. spretus. Only the sequence of the domesticus allele is shown. The shaded boxes on the sequence represent the AatII site whose methylation was previously analysed (25) and two binding sites for CTCF (30). (B) Methylation profiles of the H19 DMR in the liver of 1-day-old mice following maternal transmission of the Mnt mutation (Mnt/SD7) and corresponding wild-type (+/SD7). DNA samples were treated with sodium bisulfite, amplified by PCR, cloned and sequenced. Each line corresponds to a single template DNA molecule and each circle represents a CpG dinucleotide. Solid circles designate methylated cytosines and open circles correspond to non-methylated cytosines. Dashed lines indicate CpGs whose methylation could not be determined. Polymorphisms between M. mus domesticus and SD7 were used to distinguish the parental origin of the sequences. Polymorphic CpGs are indicated by asterisks. In this experiment, the domesticus allele was of maternal origin and the SD7 allele of paternal origin. Note that the H19 DMR shows the normal paternal methylation pattern on the maternal Mnt chromosome.

 
Two additional DMRs (DMR1 and DMR2) methylated on the paternal chromosome are present in the Igf2 gene (34). Maternal deletion of the H19 DMR leads to methylation of the maternal Igf2 DMR1 and 2 (34). We observed that with maternal transmission of Mnt there was an increase in methylation of DMR1 and DMR2 on the maternal allele (data not shown). This effect could be either direct or secondary to hypermethylation of the H19 DMR. Thus, based on this analysis of differential methylation, the epigenotype of the maternal allele has been converted to a paternal one by the Mnt mutation.

Expression of the Nctc1 gene and methylation of the H19 endodermal enhancers are unaffected by the Mnt mutation
The H19 DMR and Igf2 DMRs were hypermethylated, and the H19 gene silenced and the Igf2 gene activated on the maternal Mnt allele. We wanted to determine the effect of the Mnt mutation on other genes and regulatory elements located between H19 and the inversion breakpoint BP1 (Fig. 2A), to see whether the inversion resulted in spreading of methylation (similar to a position effect) or whether it specifically affected imprinting. For this reason, the expression of the Nctc1 gene and the methylation of the H19 endodermal enhancers were investigated in the Mnt mice. Nctc1 is a gene coding for a non-translated RNA, which is not imprinted and is expressed predominantly in the adult skeletal muscle (35). Expression from the maternal and paternal Nctc1 alleles was distinguished by an EcoRV RFLP present in the Nctc1 transcript (Fig. 2A). The results showed that, although the SD7 (spretus) allele was more efficiently expressed than the domesticus allele, the relative expressions of the domesticus wild-type allele and the domesticus Mnt allele were similar and were not affected by parental inheritance (Fig. 2B). Thus, the Mnt mutation has no effect on the expression of Nctc1. This contrasts with the complete silencing of the H19 gene in the Mnt/SD7 mice (25 and data not shown).



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  		Figure 2. Allele-specific expression analysis of the Nctc1 gene. (A) Schematic representation of the H19-Nctc1 locus. The transcriptional start sites and orientation of the H19 and Nctc1 genes are indicated by arrows. The primers used for the RT–PCR (located in the first and second Nctc1 exon, respectively) are shown as arrows underneath the exons. The vertical arrow represents the breakpoint 1 of the Mnt inversion (BP1) and the dashed line indicates the chromosomal region that is inverted in Mnt. An EcoR V site polymorphic between the domesticus and the spretus alleles is indicated by an asterisk. (B) RT–PCR analysis of Nctc1 in mice following maternal (Mnt/SD7) or paternal (SD7/Mnt) transmission of the Mnt mutation and corresponding +/SD7 and SD7/+ mice. The EcoR V RFLP was used to distinguish the parental origin of the alleles. RNAs extracted from the skeletal muscle of adult mice were RT–PCR amplified, digested with EcoR V and separated by electrophoresis on non-denaturing polyacrylamide gels. To test for contamination by genomic DNA, RNA samples were run in duplicate with (RT+) or without (RT-) addition of reverse transcriptase. The DNAs derived from SD7 and M. mus domesticus (+) mice were analysed as controls. The relative intensities of the bands corresponding to the domesticus (Mnt or wild-type) and SD7 (wild-type) alleles are shown in the lower part of the figure. Note that the expression of the Nctc1 gene is not affected by the Mnt mutation.

 
Two enhancer elements (end. enh. 1 and 2; Fig. 2A) required for the expression of the Igf2 and H19 genes in tissues of endodermal origin are located between H19 and Nctc1 (23). DNA methylation of nine CpGs in the sequence of enhancer 1 and six CpGs of enhancer 2 was determined in the liver of P1 mice (Fig. 3A). Enhancer 1 was heavily methylated on both parental alleles in +/SD7 (wild-type) mice and this pattern was not modified by the Mnt mutation (Fig. 3B). Enhancer 2 was largely unmethylated and the patterns of the Mnt/SD7 and +/ SD7 mice were very similar (Fig. 3C). No significant difference was evident in the methylation of the maternal and paternal alleles. Whether the function of the enhancers is affected by methylation is not known.



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  		Figure 3. DNA methylation analysis of the H19 endodermal enhancers. (A) Diagram representing the H19 locus and detail of the sequence analysed (GenBank accession no. AF049091). The regions analysed extend from position +7203 to +7492 and from +8731 to +9039 relative to the H19 transcriptional start site and contain nine CpGs in enhancer 1 and 6 CpGs in enhancer 2 of which two are polymorphic (asterisks) between M. mus domesticus and M. spretus. Only the sequence of the domesticus allele is shown. Methylation profiles of the H19 endodermal enhancer 1 (B) and enhancer 2 (C) in the liver of 1-day-old mice following maternal transmission of the Mnt mutation (Mnt/SD7) and corresponding wild-type (+/SD7). Methylation of individual CpG dinucleotides was determined and is shown as in Figure 1. The domesticus allele was of maternal origin and the SD7 allele of paternal origin. Note that methylation of the endodermal enhancer 1 and 2 is not affected by the Mnt mutation.

 
These results indicate that the Mnt mutation does not affect expression and methylation of genes and regulatory sequences located between BP1 and H19, suggesting that the Mnt inversion specifically alters the imprinting epigenotype of the Igf2-H19 locus on the maternal chromosome.

The Mnt mutation converts the maternal germline imprint of the H19 DMR into a paternal one
The maternal wild-type allele of the H19 DMR is maintained unmethylated throughout mouse development and therefore is protected from the extensive de novo methylation of the genome occurring post-implantation (26,27). This postzygotic protection from methylation requires CTCF (33). We wondered whether the maternal Mnt allele acquired its methylation during these early developmental stages. For this purpose, embryos from crosses between Mnt/SD7 females and SD7/SD7 males were collected at the morula stage (eight to 16 cells) and analysed for methylation of the H19 DMR. Since the amount of DNA was limiting, the bisulfite-treated DNA was amplified by nested PCR and 10–11 CpGs were analysed in this case (Fig. 4A). The results showed that six CpGs encompassing a CTCF site were highly methylated on the maternal Mnt allele. A pool of morulae was used for this analysis, and so two thirds of the SD7 (wild-type) chromosomes were of paternal origin and one-third of maternal origin. Accordingly, 11/16 template sequences derived from the SD7 allele were found to be methylated. As a control, the morulae derived from +/+x SD7/SD7 crosses were analysed and found to have the H19 DMR methylated only on the paternal allele (Fig. 4B). This clearly establishes that the maternal Mnt allele acquires methylation at the H19 DMR prior to the morula stage.



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  		Figure 4. DNA methylation analysis of the H19 DMR in preimplantation embryos. (A) Methylation profiles of the H19 DMR in morulae collected from Mnt/SD7 females mated with SD7/SD7 males. DNA extracted from pooled embryos was treated with sodium bisulfite, amplified by nested PCR, cloned and sequenced. Methylation of individual CpG dinucleotides (5–15 of Fig. 1) is shown as in Figure 1. In this experiment, the domesticus Mnt allele was of maternal origin, while one-third of the wild-type SD7 alleles were of maternal origin and two-thirds were of paternal origin. (B) Methylation profiles of the H19 DMR in +/SD7 morulae. Methylation of individual CpG dinucleotides was determined and is shown as in (A). In this experiment, the domesticus allele was of maternal origin and the SD7 allele of paternal origin. Note that the the H19 DMR is methylated on the maternal Mnt chromosome in preimplantation embryos, in contrast to the wild-type allele.

 
Methylation was then analysed in female gametes (Fig. 5). Unfertilized oocytes were collected from Mnt/SD7 and +/+ females. All eight CpGs analysed were methylated in more than 90% of the template sequences derived from the Mnt allele, showing clearly that the Mnt mutation leads to acquisition of H19 DMR methylation in the maternal germline. In contrast, the wild-type allele was consistently unmethylated in both the Mnt/SD7 and +/+ mice.



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  		Figure 5. DNA methylation analysis of the H19 DMR in unfertilized oocytes. (A) Methylation profiles of the H19 DMR in oocytes collected from adult females following maternal transmission of the Mnt mutation (Mnt/SD7). In this experiment, the domesticus allele is on the Mnt chromosome and the SD7 allele on the wildtype chromosome. (B) Methylation profiles of the H19 DMR in oocytes collected from +/+ mice. Methylation of individual CpG dinucleotides was determined and is shown as in Figure 4. Note that methylation of the H19 DMR on the maternal Mnt chromosome is already established in the oocyte.

 
In the morula and gamete DNAs we observed that some Cs followed by bases other than G were resistant to the reaction with sodium bisulfite, suggesting the occurrence of non-symmetrical methylation (data not shown). The most frequent of these corresponded to the C preceding CpG 10 and included in the CTCF site 1 (Fig. 1). This cytosine was unconverted exclusively on alleles that also had CpG methylation, with particularly high frequency in the sequences derived from the Mnt allele in oocytes (20/25 molecules), but less frequently in the sequences derived from the Mnt allele in sperm (1/10 molecules) or from the wild-type allele in sperm (6/18 molecules). Although it cannot be excluded that this finding represents an artefact of the bisulfite-sequencing procedure (36), its more frequent occurrence in oocytes than in sperm may indicate that it corresponds to a type of non-symmetrical methylation which is preferentially established in female gametes. This type of non-symmetrical methylation in oocytes has not been observed in other imprinted genes so far (37).

Overall these results demonstrate that the methylation of the H19 DMR caused by the maternal transmission of the Mnt mutation is already established in oocytes and is maintained throughout mouse development.

Methylation of the Mnt allele is erased in PGCs
The wild-type paternal allele of the H19 DMR is demethylated in primordial germ cells between E11.5 and E12.5, and is remethylated in spermatogonia at later fetal stages, but not in oocytes (38,39). We sought to determine whether or not methylation of the maternal Mnt allele was erased in PGCs or if it was fixed and escaped demethylation. For this purpose, Mnt/SD7 female embryos were collected at E14.5 and PGCs were isolated from the gonads. These embryos therefore had a methylated maternal H19 DMR (Mnt allele) and a methylated paternal one (SD7 wild-type allele). At this stage of PGC development, the wild-type H19 DMR is expected to be completely demethylated regardless of the parental origin (40). We found that the maternal Mnt allele was methylated in only 38% of template molecules in our sample, and the paternal SD7 allele was methylated in 55% (Fig. 6). Considering that PGCs were sorted by their morphological characteristics, we attribute most of the methylated sequences derived from the SD7 wild-type allele (and therefore also those from the Mnt allele) to contaminating somatic cells that are methylated in the Mnt/SD7 mice (see also Materials and Methods). Indeed, in other experiments in which immunoaffinity purification of PGCs was used (giving higher purity of PGCs), the paternal allele of H19 was methylated in ~30% of molecules (40). Because in our experiment the maternal Mnt allele was less methylated than the paternal SD7 wild-type allele, these results indicate that the methylation of the H19 DMR is as efficiently erased on the Mnt allele as on the wild-type allele in PGCs. This shows that the Mnt allele undergoes a normal life cycle of imprint establishment, maintenance and erasure, but does so in the inappropriate (maternal) germline.



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  		Figure 6. DNA methylation analysis of the H19 DMR in primordial germ cells (PGCs). Methylation profiles of the H19 DMR in PGCs collected from E14.5 female embryos following maternal transmission of the Mnt mutation (Mnt/SD7). In this experiment, the domesticus allele is on the Mnt chromosome and the SD7 allele on the wild-type chromosome. Methylation of individual CpG dinucleotides was determined and is shown as in Figure 4. Note that methylation of the H19 DMR is erased on the Mnt chromosome as well as on the wild-type (SD7) chromosome in PGCs.

 
Asynchronous replication of the Igf2-H19 locus is maintained in the Mnt mutant
Maternal and paternal alleles of the Igf2-H19 locus have been shown by different methods to replicate asynchronously during S phase (20,41). It has been proposed that asynchronous replication, which is set up in the germline, is an epigenetic mark that may be required for establishment and maintenance of methylation imprints (20). We have previously shown that replication asynchrony extends throughout the entire distal chromosome 7 imprinting cluster (42). We have now used the FISH technique to determine the replication timing of Igf2-H19 in Mnt mice. Spleen lymphocytes (which show the same H19 DMR methylation imprint as other somatic cells; data not shown) were collected from Mnt/+, +/Mnt and +/+ mice, stained with BrdU and analysed by DNA FISH. Among the BrdU-positive nuclei, those with a single spot and a double spot indicate that one allele has replicated and the other has not, which shows asynchrony (SD pattern). FISH probes to imprinted regions detect a higher percentage of SD patterns than probes to non-imprinted loci (41,42). Consistent with these observations, we found that a control probe hybridizing to a non-imprinted locus revealed a low percentage of SD patterns, while probes specific for the H19 and Igf2 genes detected significantly higher numbers of SD patterns in wild-type cells (Table 1). Strikingly, the percentage of SD patterns detected by the H19 and Igf2 probes remained the same in the Mnt/+ (and +/Mnt cells), indicating that the asynchronous replication of the Igf2-H19 locus was not affected by the Mnt mutation (Table 1).


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  		Table 1. Analysis of replication timing in the Mnt mouse
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
In this study, we investigated the molecular and developmental features of the Mnt inversion, which leads to methylation and silencing of the H19 locus and loss of Igf2 imprinting upon maternal transmission. The maternal Mnt allele shows a pattern of methylation that resembles that of the wild-type paternal allele at the H19 DMR and the Igf2 DMRs. Expression of a gene and methylation of two regulatory elements located between H19 and the nearest inversion breakpoint are not affected. Our results indicate that an abnormal paternal methylation imprint is established at the H19 DMR in oocytes and is maintained throughout development. This methylation imprint is erased in primordial germ cells, as is the normal paternal imprint. Contrary to expectation, our study also demonstrates that the normal replication asynchrony at this locus is maintained in Mnt mice. The persisting replication asynchrony clearly does not prevent establishment of a paternal methylation imprint on the maternal chromosome, nor does the paternal methylation imprint lead to an altered pattern of DNA replication timing.

Establishment of paternal epigenotype in the maternal germline
In our previous study we reported that the H19 locus was methylated on the maternal chromosome when this carried the Mnt inversion (25). This study demonstrates that de novo methylation is not caused by a position effect in which methylation spreads along the DNA from the inversion breakpoint, but rather represents a specific modification of the H19 DMR and Igf2 DMRs that resembles that of the paternal wild-type allele in its pattern and developmental life-cycle. Cranston et al. (43) demonstrated that transgenes extending from 5.7 kb upstream to 8 kb downstream of the H19 gene reproduce the endogenous H19 imprinting pattern, indicating that all cis-acting elements required for the germ line-specific epigenetic modification of the H19 DMR may reside relatively close to the DMR itself. However, these transgenes require higher copy numbers in order to be consistently imprinted, while larger transgenes are consistently imprinted even at low copy number. This suggests that more distantly located sequences also have a role, at least in imprinting stabilization (43–45). Indeed, our study shows that altering sequences located further than 30 kb away abolishes protection from methylation of the H19 DMR in the female germline, suggesting that remote cis-acting sequences may be required for this protection. This study also demonstrates for the first time that a paternal-type methylation imprint can also be established in oocytes, although the precise timing of de novo methylation during oogenesis needs to be established, in particular in relation to that of other imprinted genes (37). These observations suggest that the same factors that are needed for methylation of the H19 DMR are present in both the maternal and paternal germlines, and thus how the maternal allele is normally protected in oocytes from becoming methylated is an important question. The cis- and trans-acting factors required for protection will be further elucidated in the Mnt mutant. This may also shed light on mechanisms of imprinting alterations in human genetic disorders.

It has been proposed that certain DNA repeats may be important for the acquisition of differential methylation and that genomic imprinting has arisen as a side-effect of cellular defence mechanisms against exogenous and transposon DNA (46). It has also been observed that SINE but not LINE repeats tend to be excluded from imprinted loci (47,48). We have compared the distribution of repetitive elements located downstream of H19 in the wild-type and Mnt chromosomes (Supplementary Material, Fig. S1). Interestingly, the Mnt allele has substantially higher numbers of LTR and tandem repeats in 30 kb 3' of BP1 than the wild-type allele. In addition, these two types of repetitive sequence are not abundant on the wild-type chromosome within 100 kb surrounding the H19 DMR. This raises the possibility that the repeats brought into proximity by the Mnt inversion may attract methylation to the H19 DMR during oogenesis, thus perhaps overwhelming the protection system that normally operates in oocytes.

Dissociation of replication timing and imprint establishment and maintenance
It has been shown that the gamete-specific pattern of replication timing of imprinted genes is established prior to the acquisition of imprinted methylation in oocytes (20). Additional evidence indicates that asynchronous replication timing does not require parent-specific DNA methylation (21). Our study clearly shows that altered replication timing is not needed in order to establish altered methylation imprinting, and conversely that establishing methylation in the Igf2-H19 locus does not alter its replication timing. This is consistent with previous observations that alterations in imprinting status do not necessarily correspond to altered regional replication timing and that parental alleles of imprinted transgenes do not necessarily replicate asynchronously (49–52).

Thus we propose that the epigenetic control of replication timing is different from the epigenetic control of methylation imprinting. While replication timing is not altered by DNA hypomethylation (21) it clearly is by histone hyperacetylation (53,42). It is possible that asynchronous replication timing is important for maintaining imprinted expression of those genes whose imprinting status is not affected by hypomethylation (54). Nevertheless, DNA replication asynchrony is lost in parthenogenetic embryos and ES cells (20,21) and is altered in the H19 {Delta}13 mutation, which also results in loss of imprinting of Igf2 (55). This shows that both imprinting and replication asynchrony are dependent on a biparental origin and suggests that cis-acting sequences for imprinting control, and for control of replication asynchrony, may occur in close proximity. This would then explain why in general imprinted domains also tend to replicate asynchronously. Perhaps a more ancient and less stable mechanism of imprinting was based on replication asynchrony (and other epigenetic marks) and this was subsequently superseded by a more stable mechanism involving DNA methylation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mice
Mouse strains used for all the experiments have been previously described (25). The mice were typed for the presence of the Mnt inversion as described (25).

Collection of embryonic tissues and gametes
Unfertilized oocytes and morulae were collected from superovulated females. Primordial germ cells (PGCs) were collected from sexed individual E14.5 embryos, as described by Buehr and McLaren (56). Gonads were disaggregated and PGCs identified by their morphological characteristics and separated from somatic cells. Samples of germ cells released were fixed, air-dried, and stained with alkaline phosphatase. On average, preparations contained 70–80% germ cells as judged by positive alkaline phosphatase staining. Embryos were genotyped before methylation analysis of PGCs.

Nucleic acid isolation
DNA was extracted from neonatal and adult tissues, according to standard techniques. Oocytes (~200), PGCs (~500) and morulae (~60) were resuspended in 0.03% SDS, 10 µg glycogen (Roche), 10 µg proteinase K and 1xPBS to a final volume of 33 µl. Samples were incubated at 37°C for 90 min followed by 15 min at 95°C. Total RNA was extracted from tissues by using a Qiagen RNeasy kit according to the protocol of the manufacturer.

RT–PCR analysis
For detection of the Nctc1 expressed alleles, 1 µg total RNA was treated with RNase-free DNase (Promega) and first-strand cDNA was synthesized by using Superscript II Reverse Transcriptase (Invitrogen) and random hexamers as primers, according to the protocol of the manufacturer.

cDNA was amplified by hot-stop PCR by adding [{alpha}-32P] dCTP before the last cycle (57). The primers used were: forward, 5'-GAGATGTTCAAGGATGGGAC-3'; reverse, 5'-GTGGGTCTTGAGGTACAATG-3'. PCR conditions were: 2 min at 95°C followed by 94°C for 30 s, 60°C for 30 s and 72°C for 30 s for 30 cycles followed by 72°C for 5 min. PCR products were digested with EcoR V and separated by electrophoresis on non-denaturing 6% polyacrylamide gel. The intensity of the bands was measured by computer analysis of the image captured by Molecular Dynamics phosporimager.

Bisulfite sequencing
Sodium bisulfite treatment of DNA was performed as described in Arnaud et al. (58). DNA (1–2 µg) extracted from neonatal tissues was digested with BamH I for 16 h prior to bisulfite treatment.

For neonatal samples, PCRs were carried out in 25 µl reaction mixture containing 0.2 mM dNTP, 1.5 mM MgCl2 and 2.5 U Expand-High Fidelity Taq polymerase (Roche) and 100 ng of the following primers: for the H19 DMR, 5'-GATTAGATAGTATTGAGTTTGTTTGGAGTTTGAG-3' and 5'-AAAAACTAACATAAACCCCTAACCTCATAA-3' (95°C for 5 min followed by 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 30 cycles followed by 72°C for 5 min); for the H19 endodermal enhancer 1, 5'-GATAGATGAAAGAAGATTTTTGGAGGGATTATGG-3' and 5'-AACCACCACCCCCAAAACCCTAACAATAAACC-3' (95°C for 5 min followed by 94°C for 1 min, 57°C for 1 min, 72°C for 1 min for 35 cycles followed by 72°C for 5 min); for the H19 endodermal enhancer 2: 5'-GGTATAGAGGGAGATTGAGTTGATAAATTGGG-3' and 5'-AAACCAAAATTACACAAAACCCTACAACCTAACC-3' (95°C for 5 min followed by 94°C for 1 min, 53°C for 1 min, 72°C for 1 min for 35 cycles followed by 72°C for 5 min).

A semi-nested primer strategy was used to amplify the H19 DMR in bisulphite-treated morula, oocyte and PGC DNAs. The primers used were: 5'-GTAGGGTATTTATATTTAGGATTTAAAGGAATATG-3' (outside forward); 5'-AGAATTTTGTAAGGAGATTATGTTTTATTTTTGG-3' (inside forward); and 5'-CTAAAATACTAAACTTAAATAACCCACAACATTA-3' (reverse) (first and second rounds: 95°C for 5 min followed by 94°C for 1 min, 55°C for 1 min, 72°C for 1 min for 35 cycles followed by 72°C for 5 min). MgCl2 was 2.5 mM in the first round.

The resulting PCR products were gel-purified (gel extraction kit, Qiagen) and ligated into pCR 2.1 (topo-TA cloning kit, Invitrogen). Individual positive clones were sequenced using an ABI sequencer or DNA sequencing was obtained from Lark Technologies Inc. (UK). Sequence polymorphisms allowed to distinguish the M. mus domesticus from the SD7 alleles. To ensure that the sequences obtained were not biased toward methylated or non-methylated molecules, PCR products were digested prior to cloning with restriction enzymes whose recognition sites were created by the bisulfite conversion of methylated or non-methylated cytosines, as described (58). Residual unconverted non-CpG cytosines indicated that the majority of the sequences with similar CpG methylation pattern were not a clonal product.

FISH-based replication timing
Lymphocytes were isolated from mouse spleen, labelled with BrdU and analysed by FISH, as previously described (42). Igf2, H19 and control (random genomic clone) probes have been described before (42). A minimum of 200 BrdU-positive nuclei were counted for each genotype investigated.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are grateful to Bruce Cattanach (MRC Harwell) for the Mnt mice. We gratefully acknowledge Philippe Arnaud, Natasha Lane and Annabelle Lewis for helpful advice in the bisulfite technique. We thank Paul Smith and Emma Gordon for excellent technical assistance and Miguel Constancia, Gavin Kelsey and Hugh Morgan for comments on the manuscript. This work was supported by MRC and BBSRC and Associazione Italiana Ricerca sul Cancro. F.C. was partially supported by a fellowship from Associazione Leonardo Di Capua and A.R. was recipient of a Marie Curie Individual Fellowship Category 40 from the European Community Programme in Quality of Life (under contract number QLCA-CT-2000-52040).


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +44 1223496338; Fax: +44 1223496015; Email: wolf.reik{at}bbsrc.ac.uk Back

{dagger} The work was carried out while the authors were at The Babraham Institute on sabbatical leave. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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