Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 3 295-305
© 2003 Oxford University Press

Epigenetic modifications in an imprinting cluster are controlled by a hierarchy of DMRs suggesting long-range chromatin interactions

Susana Lopes^1,, Annabelle Lewis^1,, Petra Hajkova², Wendy Dean¹, Joachim Oswald², Thierry Forné³, Adele Murrell¹, Miguel Constância¹, Marisa Bartolomei⁴, Jörn Walter² and Wolf Reik^1,*

¹Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme, The Babraham Institute, Cambridge CB2 4AT, UK, ²Universität des Saarlandes, Fr 8.2 Genetik, 66041 Saarbrücken, Germany, ³Institut de Génétique Moléculaire de Montpellier, Montpellier, France and ⁴Howard Hughes Medical Institute and Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA

Received September 20, 2002; Accepted November 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Imprinted genes and their control elements occur in clusters in the mammalian genome and carry epigenetic modifications. Observations from imprinting disorders suggest that epigenetic modifications throughout the clusters could be under regional control. However, neither the elements that are responsible for regional control, nor its developmental timing, particularly whether it occurs in the germline or postzygotically, are known. Here we examine regional control of DNA methylation in the imprinted Igf2-H19 region in the mouse. Paternal germline specific methylation was reprogrammed after fertilization in two differentially methylated regions (DMRs) in Igf2, and was reestablished after implantation. Using a number of knockout strains in the region, we found that the DMRs themselves are involved in regional coordination in a hierarchical fashion. Thus the H19 DMR was needed on the maternal allele to protect the Igf2 DMRs 1 and 2 from methylation, and Igf2 DMR1 was needed to protect DMR2 from methylation. This regional coordination occurred exclusively after fertilization during somatic development, and did not involve linear spreading of DNA methylation, suggesting a model in which long-range chromatin interactions are involved in regional epigenetic coordination. These observations are likely to be relevant to other gene clusters in which epigenetic regulation plays a role, and in pathological situations in which epigenetic regulation is disrupted.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Imprinted genes in mammals are those genes that are expressed from predominantly one of the parental alleles. These genes have important functions in mammalian development particularly in fetal growth, placental development and function, and in certain behaviours after birth (1–4). There are approximately 50 imprinted genes currently identified in the mouse genome, with over 100 expected, and most are conserved in the human genome (www.mgu.har.mrc.ac.uk/imprinting/imprin-viewdatagenes.html). One of the interesting features of organization of imprinted genes is that most of them occur in clusters in the genome. The imprinting cluster on distal chromosome 7 in mouse and proximal chromosome 11p15.5 in human, for example, contains 14 imprinted genes (2).

Imprinted genes are characterized by regions of parent-specific epigenetic modifications including DNA methylation, histone acetylation and histone methylation (1–6). Genetically, the maintenance of DNA methylation has been shown to be crucial for maintaining imprinting, with either biallelic expression or biallelic silencing of imprinted genes in a knockout of the Dnmt1 gene (7). Recently, a number of elements have been characterized whose parent-specific methylation regulates expression or repression of imprinted genes. These include promoters, promoters of antisense RNAs, chromatin boundaries, silencers and activators. These elements are often involved in regulating more than one gene in an imprinting cluster. Some of these differentially methylated regions (DMRs) receive their methylation imprints in the parental germ cells, and these are then maintained through all developmental stages, whereas others are significantly reprogrammed during development. The signals for inheritance or reprogramming of DNA methylation imprints are unknown.

The clustering of imprinted genes and their sharing of differentially methylated control sequences suggest that epigenetic modifications throughout the cluster might be under regional control. Indeed, earlier observations on patients with the classical imprinting disorders Prader–Willi/Angelman syndromes (PWS/AS) or Beckwith–Wiedemann syndrome had suggested that defects in putative imprinting centres (IC) in the clusters could lead to altered epigenetic modifications throughout the cluster, and consequently altered expression of imprinted genes (8–10). In the PWS/AS cluster it was proposed that this regional coordination of the epigenotype occurred in the germline, and was defective in PWS or AS patients with IC mutations (8). Subsequent work, however, showed that IC mutations can also alter the epigenotype in the cluster during somatic development (11). Thus, whether coordination occurs in the germline or during somatic development (or both) is not known.

Within the imprinting cluster on distal chromosome 7 in mouse there are likely to be two subdomains which are regulated separately, although a higher level of coordination between the two domains cannot be ruled out (2). One of these domains includes the Cdkn1c and Kcnq1 genes and others, and the antisense RNA gene Kcnq1ot1. The other domain contains the paternally expressed insulin 2, insulin-like growth factor 2, and maternally expressed untranslated RNA gene H19. Within this domain there are four DMRs. The paternally methylated H19 DMR contains a methylation sensitive chromatin boundary and silencer element, which restricts, when unmethylated, the access to Igf2 of enhancers located distal to H19 (12–17). DMR1 is located upstream of the fetal promoters of Igf2, is paternally hypermethylated, and contains a methylation-sensitive silencer (18,19). DMR2 is located in the last exon of Igf2 and contains a methylation-sensitive activator (20,21). The function of the maternally methylated DMR0 which is located in the promoter of the placenta specific transcript of Igf2 is currently unknown (22).

We have previously shown that maternal deletion of the H19 DMR and gene in the mouse leads to methylation of the maternal DMR1 and 2 regions, suggesting regional coordination of the epigenotype (23). Here we examine systematically methylation patterns in the DMRs during embryogenesis, and the control of these patterns in cis by using various knockouts in the cluster. We show that coordination requires a hierarchy of the DMRs themselves, occurs during somatic development, and may involve long-range chromatin interactions.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Reprogramming of germline methylation in Igf2 DMRs
The Igf2-H19 region has four DMRs, three of which (H19 DMR, Igf2 DMRs 1 and 2) are paternally methylated (Fig. 1, this figure also summarizes the regions in DMR1 and 2 analysed by Southern blotting and bisulphite sequencing, and the knockout alleles that were used). The H19 DMR is methylated in sperm but not oocytes and this differential methylation is maintained during all stages of development and all tissues, except in germ cells where the pattern is erased and re-established (24,25). Figure 2 summarizes the methylation pattern of the H19 DMR during preimplantation development (55). The methylation patterns of the Igf2 DMRs were established by bisulphite sequencing (Fig. 2A). The gametic pattern of DMR2 had previously been determined (26) (Fig. 2A). DMR1, just as DMR2, was highly methylated in sperm, and not methylated in oocytes. In striking contrast however to the H19 DMR, methylation was lost from the paternal copy already in the zygote (DMR2 more pronounced than DMR1) and essentially all differential methylation was erased at the morula stage (Fig. 2). The diagrams in Figure 2 show the methylation patterns of a representative selection of template sequences from each developmental stage. The corresponding graphs summarize the methylation levels from all analysed sequences. The total number of sequences analysed for each region and time point is listed in the legend to Figure 2.

View larger version (18K): [in this window] [in a new window]   Figure 1. Map of the Igf2-H19 locus and knockouts within the region. All exons of the Igf2 gene and promoters of both genes are represented. DMRs are shown underneath together with their allele-specific methylation status (filled, hypermethylated). DMR0 methylation pattern refers only to placenta. DMRs 1 and 2 are expanded to show maps of restriction enzymes used for methylation analysis by Southern blotting [R, B, D, E, N, F and h represent EcoRI, BamHI, DraI, EcoNI, FspI and HpaII, respectively; (m) or (s) indicate that the restriction enzyme is specific to M.m.domesticus or to M.spretus, respectively] and further expanded to show regions analysed by bisulphite sequencing. CpGs are represented by circles and numbered. s is a polymorphism specific to the M.spretus strain. Underneath is a map of the knockout strains used.

 

View larger version (44K): [in this window] [in a new window]   Figure 2. Methylation levels in the Igf2-H19 locus at early developmental stages. (A) Methylation levels of DMRs in gametes and preimplantation embryos determined by bisulphite sequencing. DNA samples were treated with sodium bisulphite, amplified by PCR (see Fig. 1 for amplified region), cloned and sequenced. Dot diagrams show the methylation profile of DMR1 in oocytes (n=12), sperm (n=13), one-cell embryos (n=20) and morulae (n=16). Each line represents a single DNA template molecule (where n = the total number of sequences analysed). A representative selection of sequences is shown. Filled circles represent methylated CpGs. A polymorphism was used to distinguish the parental origin of the sequences in one-cell embryos and morulae. The graphs underneath summarize the methylation levels (overall percentage of methylated CpGs out of total CpGs in analyzed region) in gametes, zygotes and morulae in Igf2 DMR1, DMR2 and H19 DMR. Note the reprogramming of Igf2 DMR2 and DMR1 gametic methylation differences, and the maintenance of the germline H19 DMR methylation levels. The Igf2 DMR2 and H19 DMR methylation levels are taken from the indicated references. (B) Methylation levels of Igf2 DMR1 and DMR2 in postimplantation embryos. Whole embryo E9 DNA and liver and kidney E15 DNA samples were treated with sodium bisulphite, amplified by PCR (see Fig. 1 for amplified region), cloned and sequenced. Dot diagrams show the methylation profile of E9 whole embryos at DMR1 (n=19) and DMR2 (n=18), E15 kidney at DMR1 (n=33) and DMR2 (n=20) and E15 liver at DMR1 (n=21) and DMR2 (n=20). Each line represents a single DNA template molecule (where n = the total number of sequences analysed). A representative selection of sequences is shown. Filled circles show methylated CpGs. Polymorphisms were used to distinguish between sequences of maternal and paternal origin. The graphs underneath summarize the methylation levels (overall percentage of methylated CpGs out of total CpGs in analysed region). Note that the adult methylation pattern of DMR1 and DMR2 is established by E15 except for DMR2 in the kidney, which is still hypomethylated.

 
Paternal methylation patterns became re-established at postimplantation stages (Fig. 2B). For both DMR1 and 2, there was some increase in methylation on the paternal allele in whole embryonic day 9 (E9) fetuses (to 43% in DMR1 and 20% in DMR2). Further increases occurred up to E15 and later in individual tissues. Interestingly, in largely mesodermal tissues such as the kidney, an increase in DMR1 methylation preceded the increase in DMR2 methylation temporally. Thus, while DMR1 was differentially methylated in E15 kidney, DMR2 differential methylation was not yet established on E15 (Fig. 2B), but was in place at late fetal stages (data not shown). A small increase in methylation of both DMRs was also observed on the maternal allele (Fig. 2B).

DMRs protect from regional methylation in a hierarchical fashion
In order to see whether we could identify cis acting elements within the cluster that were important either for coordinating methylation imprints in the germline (all three DMRs are methylated in sperm but not oocyte) or postzygotically when differential methylation is re-established, we tested three different types of sequence with a total of six knockouts (Fig. 1). The Igf2 and H19 genes themselves, the known endoderm enhancers, which are located 3' of H19 and are required for expression of both genes, and the three DMRs themselves were tested. We measured differential methylation in the three DMRs, Igf2 promoters 1 and 3, and three different locations between the Igf2 and H19 genes, following maternal or paternal transmission of the knockouts. The overall results are summarised in Figure 5.

View larger version (10K): [in this window] [in a new window]   Figure 5. Summary of methylation analysis in the Igf2-H19 region with maternal transmission of the H1913, Igf2DMR1-U2 and Igf2lacZDMR2 alleles. The regions analysed are shown; from left to right, DMR1, Igf2 promoter1, Igf2 promoter 3 (3 and 4 CpGs tested, respectively), DMR2, probes A4, A11 and AE31 (4,2 and 7 CpGs tested, respectively) (32) and H19 DMR. A ratio of 1 indicates there was no change. Altered methylation patterns are specific to DMR1 and 2.

 
Since both bisulphite sequencing and Southern blotting were used for the analysis of differential methylation (see below), we needed to establish whether the two techniques resulted in comparable results. Hence, an experiment was carried out in which liver DNA was examined for DMR1 and 2 methylation at a single restriction enzyme site by Southern blotting and at the same CpG by bisulphite sequencing (Fig. 3A). The quantitative results were remarkably similar, so we decided that we could compare results obtained by the two different methods.

View larger version (27K): [in this window] [in a new window]   Figure 3. Effect of maternal deletion of the H19 DMR and of Igf2 DMR1 on methylation levels in the Igf2 DMRs. (A) Comparison of methylation levels of CpG 5 in DMR1 and of CpG 21 in DMR2 in wild-type liver using Southern Blot analysis and bisulphite sequencing. M.m.domesticus females were mated with SD7 males and E15 offspring analysed. For Southern blot analysis of DMR1 genomic DNAs (n=4) were digested with XbaI, DraI and NaeI and probed with a XbaI–NaeI probe. The methylation-sensitive enzyme NaeI restriction site corresponds to CpG 5 analysed by bisulphite sequencing. For Southern blot analysis of DMR2 genomic DNAs (n=7) were digested with BamHI, FspI and EcoNI and probed with a FspI–BamHI probe. The methylation-sensitive enzyme FspI corresponds to CpG 21 analysed by bisulphite sequencing. Graphs show comparison of methylation levels in paternal and maternal allele in liver. Following Southern blot analysis methylation levels were calculated as the intensity of each allele-specific band normalized to the total amount of DNA loaded in the gel. Methylation levels for bisulphite sequencing are shown as percentage of methylated CpGs. (B) Maternal transmission of the H1913 knockout results in increased maternal methylation levels in the DMR1 and DMR2 regions of Igf2. Liver and kidney were collected from E15 H1913-/+ and H1913+/+ mice. DNA was extracted, treated with sodium bisulphite, amplified by PCR (see Fig. 1 for amplified region), cloned and sequenced. Maternal and paternal alleles were distinguished using polymorphisms (Fig. 1). The graphs summarize the methylation levels (overall percentage of methylated CpGs out of total CpGs in analysed region) of the paternally and maternally inherited alleles of wild-type and H1913maternal transmission samples (n17 for all samples). The levels of maternal methylation are strongly increased in both DMR1 and DMR2 following maternal transmission of the H1913allele. (C) Maternal transmission of H19DMD results in increased methylation in Igf2 DMR1 and DMR2. Southern blot analysis of methylation levels of DMR1 and DMR2 in kidney and liver of post-natal day 1 H19DMD-/+ (n=1) compared with H19DMD+/+ (n=1) mice. For DMR1 methylation analysis genomic DNAs were digested with EcoRI and HpaII and probed with an EcoRI–XbaI probe. For DMR2 methylation analysis genomic DNAs were digested with BamHI and HpaII and probed with a KpnI–BamHI probe. Graphs represent the methylation levels calculated as a ratio between density of higher molecular weight bands (for DMR1, h5, h6 and h7; for DMR2, h1, hm and h3) and lower molecular weight bands (for DMR1, h2, h3 and h4; for DMR2, h3 and h4). Note the increase in methylation in kidney for DMR1 and in kidney and liver for DMR2. (D) Maternal transmission of Igf2DMR1-U2 results in methylation changes in the Igf2 DMR2. Southern blot analysis of methylation levels of DMR2 in kidney and liver of E18 Igf2DMR1-U2-/+ (n=2) compared with Igf2DMR1-U2+/+ (n=2) mice. Genomic DNAs were digested with BamHI, FspI and EcoNI and probed with a KpnI–BamHI probe. Graphs represent the methylation levels of maternal and paternal alleles in wild-type and mutant offspring, calculated as the density of each allele-specific band normalized to the total amount of DNA loaded in the gel. Note the significant increase in methylation of the maternal allele in mutants, in both kidney and liver.

 
Deletion of the H19 DMR in the H19¹³ knockout (27) (Fig. 1) had a striking effect on methylation of both Igf2 DMRs in both endodermal and mesodermal tissues (Fig. 3B). Thus maternal transmission of the H19¹³ knockout resulted in a substantial increase in methylation of the maternal DMR1 and 2 regions [from 38 to 63% in kidney (P<0.01) and from 24 to 44% in liver (P<0.01) in DMR1, and from 8 to 35% in kidney (P<0.0001) and from 21 to 58% in liver (P<0.0001) in DMR2], whereas paternal transmission of the same allele had little effect on DMR1 and 2 (data not shown). Significantly, the maternal increase could be directly attributed to the H19 DMR through analysis of samples from the H19^DMD knockout (28), which qualitatively gave the same result as the H19¹³ allele (Fig. 3C).

The effect of the DMR1 knockout, Igf2^DMR1-U2 (19) (Fig. 1), on methylation of the other regions was similarly tested (Fig. 3D). Maternal transmission of this knockout resulted in a very substantial increase in methylation in DMR2. A representative CpG (29) showed an increase in both kidney (from 10 to 89%) and liver (from 18 to 65%) tissues, whereas paternal transmission again had little effect (data not shown). However, DMR1 deletion did not have any effect on the methylation of the H19 DMR (data not shown, Fig. 5). Finally, two different DMR2 deletions (Igf2^laczDMR2, Igf2^DMR2) (20) (Fig. 1) did not have any effect on either DMR1 or H19 DMR methylation, or methylation of the Igf2 promoters or the intergenic regions (data not shown, Fig. 5).

Disruption of the Igf2 gene (Igf2^lacz) (19) (Fig. 1) did not have any significant effect on methylation of the DMRs, the Igf2 promoters, or the intergenic regions (data not shown). Deletion of the endoderm enhancers (30) (Fig. 1) did not have any effect on methylation of all three DMRs (data not shown), suggesting that methylation and expression can be uncoupled. This conclusion is further supported by the observation mentioned above that the Igf2^DMR2 knockout, in which Igf2 transcription is repressed up to 5-fold (20), had no effect on DNA methylation.

Thus, the unmethylated maternal H19 DMR seems to protect DMRs 1 and 2 from methylation, whereas the maternal DMR1 protects DMR2 from methylation.

Regional control acts postzygotically
It was important to ask whether maternal methylation in DMRs 1 and 2 as a result of H19 DMR deletion arose in germ cells during oogenesis, or was a postzygotic event (or both). Thus, oocytes carrying the H19¹³ deletion were analysed for DMR1 and DMR2 methylation by bisulphite sequencing (Fig. 4A). The results clearly show that DMRs 1 and 2 were unmethylated in the knockout oocytes, just as in wild-type ones (compare with Fig. 2). In addition, morulae containing the maternally transmitted H19¹³ allele were unmethylated at DMR1 (summarized in Fig. 4B). However, DMR1 and in particular DMR2 show a significant increase in methylation of the maternal H19¹³allele by E9 (summarized in Fig. 4B). Maternal methylation of DMRs in the knockout is therefore a postzygotic event, which appears to parallel the de novo methylation events occurring on the wild-type paternal allele (Fig. 4B).

View larger version (22K): [in this window] [in a new window]   Figure 4. Developmental timing of methylation changes at Igf2 DMR1 and 2 following maternal transmission of the H1913 knockout. (A) Bisulphite analysis shows no change of methylation levels in oocytes from H1913-/- females at Igf2 DMR1 and DMR2. DNA was extracted from oocytes, treated with sodium bisulphite, amplified by PCR (see Fig. 1 for amplified region), cloned and sequenced. Note low levels of methylation in both samples not significantly different from wild-type ones (compare with Fig. 2). (B) Summary of the effect of maternal transmission of the H1913allele on methylation at Igf2 DMR1 and DMR2. Graphs show overall percentages of methylation in wild-type (solid lines) and H1913-/+ (dashed lines) liver samples shown in Figures 2 and 3. Methylation profiles in H1913-/+ embryos are shown only when clearly different from wild-type ones. (DMR2 methylation was not analysed in H1913-/+ embryos at the morulae stage but we have assumed from the DMR2 oocyte data and DMR1 morulae data that there is no change.) Both DMR1 and DMR2 show differential methylation at the E9 and E15 stages in wild-type samples. This differential methylation is lost in H1913-/+ embryos due to an increase in maternal levels by the E9 stage. This maternal increase occurs at the same time as the establishment of adult differential methylation patterns in wild-type embryos.

 
Does coordination occur by linear spreading?
The only mechanistic model for coordination of DNA methylation patterns proposes linear spreading along the DNA (31). We therefore analysed DNA methylation in the Igf2 promoters and in the intergenic regions between the two genes in all knockouts. The regions were chosen so that they contained both relatively methylated areas (Igf2-H19 intergenic region, probes A11 and AE31) (32) and relatively undermethylated areas (Igf2 promoter region and Igf2-H19 intergenic region, probe A4) (32) (Fig. 5). No changes were observed in any of the knockouts; in particular no changes were observed in the Igf2 promoters with maternal transmission of Igf2^DMR1-U2, which led to increased methylation in DMR2, nor in the Igf2 promoters or the intergenic regions with maternal transmission of H19¹³(data not shown). These results, summarized in Figure 5, show that the methylation changes observed in the absence of the maternal DMRs were highly specific to DMRs 1 and 2 and did therefore not arise from linear methylation spreading between the two genes, or between the two DMRs in Igf2 (Fig. 5).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We have carried out the first systematic analysis of coordination of epigenetic modifications in an imprinting cluster in the mouse. The most significant result of this analysis is that methylation in three DMRs in the Igf2-H19 region is coordinately regulated, with the three DMRs acting in a hierarchical fashion to achieve this coordination. Epigenetic coordination does not occur in the germ line, but happens during postimplantation development in a tissue-specific fashion and may thus be linked to cellular differentiation. Finally, the observation that coordinated methylation patterns are highly specific to DMRs, and do not involve linear spreading along the DNA, suggests that secondary structures in chromatin allow remote DMRs to interact with each other. We propose a model of how this might occur. These observations explain how in imprinting clusters the monoallelic expression of many genes can be regulated by local differential methylation, when only some genes in the cluster carry methylation imprints that are inherited from the gametes unchanged (2). Our observations may also apply to other epigenetically regulated gene clusters (e.g. olfactory receptor and interleukin genes) (33), and to pathological situations in which epigenetic regulation is disrupted by mutations or epimutations.

The three DMRs investigated (DMRs 1 and 2 in Igf2 and the H19 DMR) are fully methylated in sperm, and unmethylated in oocytes. We therefore considered the possibility that all three are germ line methylation imprints, which are maintained unchanged in the embryo after fertilization. While this is true for the H19 DMR, the DMRs in Igf2 lose paternal methylation soon after fertilization. Previous work on DMR2 has shown that there is massive loss of paternal methylation in the zygote which probably occurs by an active demethylation mechanism (26). Loss of methylation in DMR1 is less dramatic, but there are clearly some CpG sites that are fully methylated in sperm and completely demethylated in zygotes, again indicating active removal. In addition, there is more delayed loss as well over the subsequent cleavage divisions, indicating perhaps a role of passive demethylation. A small amount of de novo methylation occurs on the maternal allele after fertilization, as previously observed for DMR2 (26). Thus, by the morula and blastocyst stage all paternal methylation in the Igf2 DMRs is erased, consistent with the proposal that most paternal germ line methylation imprints are erased by reprogramming in the early embryo (34). How the H19 DMR is protected against demethylation remains unknown.

Following implantation and early postimplantation development, there is de novo methylation of the paternal alleles of DMR1 and 2. This de novo methylation occurs relatively slowly, and there are lineage specific differences, with earlier methylation of DMR1 than DMR2 in some tissues like the kidney. De novo methylation is likely to be caused by Dnmt3a and b, since DMR2 methylation in ES cells was shown to be dependent on the two de novo methylase enzymes (35). There is also a small but consistent amount of de novo methylation on the maternal allele, which we interpret as the same de novo methylation process acting on both parental alleles, but the maternal allele being relatively more protected against this methylation (see below). It is interesting to note that expression of Igf2 in blastocysts is from both parental alleles, but becomes increasingly monoallelically paternal after embryo implantation. This parallels a dramatic increase in levels of Igf2 mRNA (36). Thus, a progressive increase of Igf2 expression during early development is accompanied by increased DNA methylation in the paternal copies of DMR1 and 2, which is consistent with their functions as silencer and activator, respectively (18–20).

Maternal deletion of the H19 DMR and of DMR1 in Igf2, but of no other cis acting element in the cluster, leads to de novo methylation of the maternal DMR1 and 2, and of DMR2, respectively. This de novo methylation of the maternal allele occurs precisely at the times in development at which the paternal allele becomes methylated normally (summarized in Fig. 4B). Therefore, maternal deletion of the H19 DMR has removed the protection against de novo methylation of the maternal DMR1 and 2, and maternal deletion of DMR1 has removed protection against methylation of DMR2.

While the methylation state of Igf2 DMRs 1 and 2 is important for expression of the gene, as mentioned above, it is likely that the methylation changes observed here are not directly caused by changes in expression. As far as the paternal allele is concerned, transcriptional repression of Igf2 by enhancer deletion (30) or DMR2 deletion (20) leaves methylation in DMRs unchanged. On the maternal allele, Igf2^DMR1-U2 causes methylation in DMR2 in all tissues, but expression of the maternal Igf2 allele is limited to mesodermal tissues (19). Similarly, maternal transmission of the minute mutation leads to methylation of DMR1 in all tissues, but the maternal Igf2 allele is only expressed in endodermal organs (37).

Coordination of methylation in the cluster is highly specific to DMRs, and there are no changes anywhere else between or in the genes, ruling out that it occurs by linear spreading of methylation along the chromosome. How does coordination occur? It has been previously proposed that there are higher-order chromatin interactions between the H19 and Igf2 genes (38). Thus in our model (Fig. 6) the maternal H19 DMR together with its protein factors, including CTCF, may protect DMR1 (and secondarily DMR2) from de novo methylation. It is interesting to note that not only maternal deletion of the H19 DMR, but also its maternal methylation, leads to methylation of DMR1 (K.Davies, unpublished data). This model also suggests how the DMR of H19 (acting as a chromatin boundary) and the DMR1 in Igf2 might cooperate in silencing of the maternal Igf2 allele, and thus how both deletion of the DMR or deletion of DMR1 could disrupt this cooperation and lead to expression of the maternal Igf2 allele (Fig. 6). Deletion of DMR2, by contrast, does not reactivate the silent maternal Igf2 (20). The proposal of long-range chromatin interactions is supported by recent findings in the beta globin locus in which local interactions between the beta major gene and the LCR were directly demonstrated (39). Long-range chromatin rearrangements in the nucleus are generally thought to underlie changes in gene expression during differentiation, and are often tissue-specific (40–42), as observed here.

View larger version (12K): [in this window] [in a new window]   Figure 6. Model for methylation protection of the maternal allele of the Igf2 gene. In the wild-type situation a specific chromatin structure promotes physical proximity between the H19 DMR and the Igf2 DMR1. The two regions interact, potentially via a complex of proteins (orange oval) that may involve CTCF or GCF2 or both, and DMR1 is thus protected from de novo methylation. As a consequence the unmethylated DMR1 protects DMR2 from de novo methylation, also possibly via the binding of protein complexes. When the H19 DMR is deleted the protection is disrupted and DMR1 and consequently DMR2 become de novo methylated. When DMR1 is deleted the methylation protection over DMR2 is disrupted and this region becomes de novo methylated. The model also shows how the two knockouts can relieve repression of the maternal allele of Igf2.

 
Our results also show that regional coordination by protection from de novo methylation occurs exclusively during postzygotic embryonic development, and does not take place during oogenesis where deletion of DMRs did not have any effect on regional methylation. Thus the factors that protect from de novo methylation (if any) during oogenesis remain unknown. The recent observation that deletion of Dnmt3l in oocytes results in inability of a number of maternal methylation imprints to be established (43,44) may suggest that demethylation of DMRs in oocytes is a default state, rather than requiring specific protection. Similarly, the factors leading to methylation of the three DMRs during spermatogenesis remain unknown. That regional coordination does not operate during gametogenesis is significant in the light of the proposal that the imprinting centre in the Prader–Willi/Angelman syndrome imprinting cluster on chromosome 15q11–13 is required for regional coordination of methylation in the germline (8). However, more recently it has been shown that the IC may operate postzygotically (11), as shown for the Igf2-H19 region here. Thus whether the PWS/AS IC can operate in the germline as proposed is still unknown.

Our work appears to reveal a general principle which is applicable to other imprinting clusters. Thus, in the PWS/AS orthologous cluster in the mouse, the Snrpn DMR1 (part of the IC) carries a maternal germline imprint, and a number of linked imprinted genes are maternally methylated and paternally express (45,46). When the DMR1 region is deleted on the paternal chromosome, DMRs in the linked genes become methylated. One of these linked genes, Ndn, has a maternal methylation imprint in oocytes, which is reprogrammed after fertilization and re-established during postimplantation development (47). Interestingly, a higher-order chromatin structure involving matrix attachment regions (MARs) has been proposed to be involved in the regional coordination in this cluster (48). Although less well characterized, similar situations are likely to exist in other imprinting clusters (1,49,50) and perhaps also in other gene clusters that involve epigenetic regulation (33). The challenge now is to work out how long-range chromatin interactions are involved in these situations

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mouse crosses
Wild-type crosses.
M.m.domesticus females were mated to SD7 males (SD7 is a C57Bl/6 X CBA M.m.domesticus strain containing the distal portion of M.spretus chromosome 7). One-cell zygotes and morulae were collected from super-ovulated females (51). Whole embryos and tissue samples were collected on E9 and E15, respectively. Oocytes were collected from super-ovulated homozygous M.m.domesticus females and epididymal sperm was collected from homozygous M.m.domesticus males.

Maternal transmission of H19¹³.
Homozygous H19¹³ females were mated to SD7 males. Tissue samples were collected from embryos on E15. Oocytes were collected from super-ovulated homozygous H19¹³ females.

Maternal transmission of H19^DMD.
Heterozygous females were mated to M.m.domesticus males. Tissue samples were collected from neonates on the day of birth.

Maternal transmission of Igf2^DMR1-U2.
Heterozygous females were mated to SD7 males. Tissue samples were collected from embryos on E18.

Nuclease sensitivity assays.
Igf2^DMR1-U2 heterozygous were mated with SD7 males. Tissue samples were collected from mice on post-natal day 21.

For developmental staging purposes, day of conception is considered to be day 1 of pregnancy.

Bisulphite sequencing
Genomic DNA was isolated from E15 tissues, E9 whole embryos, 50–60 morulae and epididymal sperm. The samples were digested with Proteinase K (50 µg/ml) for 0.5–3 h. The DNA was then purified by extraction with phenol : chloroform : isoamyl alcohol, precipitated with 100% ethanol and resuspended in H₂O. Yeast tRNA (5 µg) carrier was used in the extraction of DNA from morulae. ß-Mercaptoethanol (70 mM) was added to the proteinase K solution to increase yield of sperm DNA. The DNA was then digested with EcoRI for 1–16 h and denatured at 100°C for 5–10 min. NaOH was added to a final concentration of 0.35 M.

Denatured DNA, 1–25 ng, or whole cells (50–100 oocytes or one-cell zygotes) were embedded in LMP agarose (2%), digested with EcoRI and treated with sodium bisulphite solution as previously described (52). An H₂O-only agarose bead was also treated in each reaction to control for contamination.

PCR amplification was carried out on single agarose beads with primers specific for bisulphite-treated DNA. Both DMR1 and DMR2 regions of Igf2 were amplified using a nested primer approach. DMR1 amplifications were performed in a 100 µl reaction using the primers 5'-GGTTAGGTGAAGGTTTTGTGGGTAGTTATA-3' and 5'-ATATTCCCCTTTCAAATTC CAATCTACATC-3' (94°C for 60 s, 50°C for 120 s, 72°C for 180 s for five cycles, then 94°C for 30 s, 50°C for 120 s, 72°C for 90 s (+5 s each cycle) for 25 cycles followed by 72°C for 6 min. Two microlitres of the first amplification products were used in a second reaction with the nested primers 5'-GGTGGTTTTTTAATGGATATTTTAAGGTGA-3' and 5'-CCAACCTCTATCCCTAACTTTTCTAACCTC using the conditions above. Amplification of Igf2 DMR2 was carried out using the primers and conditions previously described (52).

The resulting PCR products were gel-purified (Qiaquick, Qiagen) and ligated into pCR2.1 (Topo-TA cloning kit, Invitrogen). Individual clones were sequenced using an ABI sequencer. Clones were only accepted with 95% cytosine conversion. The non-converted cytosine residues were used to ensure each accepted clone originated from a different template DNA. Two separate sodium bisulphite treatments were carried out for the samples shown to verify the results.

Statistical analysis of bisulphite sequence data
Statistical analysis was carried out in collaboration with E. Walter, Cambridge. The methylation percentages were obtained for each individual clone within a sample (number of methylated CpGs per clone divided by the total number of CpGs per clone). These were then used to calculate the overall methylation level, standard deviation and standard error of the mean of each sample. The graphs show the overall methylation level of each sample. Where error bars are shown, these represent the standard error of the mean. A logistical regression test from the Genstat statistical package was used to test for differences between samples. The samples are considered significantly different when P<0.01.

Southern blot analysis
Genomic DNA was extracted from tissues as previously described (53) and phenol-chloroformed. Twenty micrograms of DNA were digested with 65 units of each restriction enzyme, loaded on a 1% agarose gel, blotted onto Nytran-plus membrane (Schleicher and Schuell) in alkaline conditions and hybridized with a specific probe. Hybridizations and washes were performed as described by Church and Gilbert (54).

Quantifications of allelic methylation were performed with a Phosphorimager (Fujifilm FLA-3000 and AIDA software).

	ACKNOWLEDGEMENTS

 
We thank S. Tilghman for making the endoderm enhancer and H19¹³ knockouts available to us. We thank G. Kelsey for comments on the manuscript, K. Davies for communicating results prior to publication, and E. Walters for help with statistical analysis. This work was supported by BBSRC, CRC and MRC. S.L. was in receipt of a Praxis XXI/FCT Scholarship (Portugal).

	FOOTNOTES

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

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors

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