Imprint Control Element-mediated Secondary Methylation Imprints at the Igf2/H19 Locus*

Madhulika SrivastavaDagger, Ella Frolova, Brian Rottinghaus, Steven P. Boe, Alexander Grinberg, Eric Lee, Paul E. Love, and Karl Pfeifer

From the Laboratory of Mammalian Genes and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, August 19, 2002, and in revised form, September 12, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Understanding the molecular basis of monoallelic expression as observed at imprinted loci is helpful in understanding the mechanisms underlying epigenetic regulation. Genomic imprinting begins during gametogenesis with the establishment of epigenetic marks on the chromosomes such that paternal and maternal chromosomes are rendered distinct. During embryonic development, the primary imprint can lead to generation of secondary epigenetic modifications (secondary imprints) of the chromosomes. Eventually, either the primary imprints or the secondary imprints interfere with transcription, leading to parent-of-origin-dependent silencing of one of the two alleles. Here we investigated several aspects pertaining to the generation and functional necessity of secondary methylation imprints at the Igf2/H19 locus. At the H19 locus, these secondary imprints are, in fact, the signals mediating paternal chromosome-specific silencing of that gene. We first demonstrated that the H19 secondary methylation imprints are entirely stable through multiple cell divisions, even in the absence of the primary imprint. Second, we generated mouse mutations to determine which DNA sequences are important in mediating establishment and maintenance of the silent state of the paternal H19 allele. Finally, we analyzed the dependence of the methylation of Igf2DMR1 region on the primary methylation imprint about 90 kilobases away.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mammals inherit two complete sets of chromosomes, one from the mother and one from the father. Most autosomal genes are expressed equivalently from the maternal and the paternal alleles. Imprinted genes, however, are expressed preferentially from only one chromosome in a parent-of-origin-dependent manner (1). Because the active and the inactive promoters of an imprinted gene are present in a single nucleus, the differences in their activity cannot be explained by differences in transcription factor abundance. Rather, the transcription of imprinted genes represents a clear situation in which epigenetic mechanisms restrict gene expression and therefore offers a model for understanding the role of heritable DNA modifications such as cytosine methylation in maintaining appropriate patterns of expression.

The imprinted Igf2-H19 gene pair is part of a large cluster of imprinted genes on the distal end of mouse chromosome 7. H19 and Igf2 share enhancers and therefore share developmentally complex patterns of gene expression (2). However, they are reciprocally imprinted. H19 is expressed exclusively from the maternal chromosome, whereas Igf2 expression is almost entirely paternal in origin (3, 4). The syntenic region in humans on chromosome 11p15.5 is highly conserved in genomic organization and in monoallelic gene expression patterns (5, 6). Loss-of-imprinting mutations at chromosome 11p15.5 are associated with Beckwith-Wiedemann syndrome and with several types of cancer (7-9).

Maternal chromosome-specific expression of H19 and paternal chromosome-specific expression of Igf2 are each dependent upon a cis acting imprinting control element (ICE),1 a 2-kb region spanning -2 kb to -4 kb upstream of the H19 promoter (10, 11) (Fig. 1a) and located about 90 kb downstream of Igf2 (distance based on GenBankTM sequence NW_000336). However, the mechanisms by which monoallelic expression is maintained at the two loci are distinct (12). Igf2 is regulated by a methylation-sensitive insulator element that also maps with the ICE. This insulator element must be continually present in its unmethylated state to maintain maternal Igf2 silencing. Repression of paternal H19 is at least a two-step process. A paternally inherited ICE is required in the developing embryo. The primary paternal imprint at the ICE somehow induces further epigenetic changes at the locus that silence the paternal H19 promoter such that the presence of the ICE is not subsequently required for maternally restricted transcription in H19 expressing cells. Thus, at H19, the primary imprint established during gametogenesis leads to a secondary imprint responsible for silencing the paternal H19 (12).


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Fig. 1.   a, schematic diagram of the Igf2/H19 locus showing the relative positions of the H19 and Igf2 genes (rectangles). Transcription start sites are shown as arrows with promoters for H19 (P) and Igf2 (P0, P1, P2, and P3) indicated. Exons are shown as blackened areas. Gray rectangles represent differentially methylated regions H19DMR (D), Igf2DMR0 (D0), Igf2DMR1 (D1), and Igf2DMR2 (D2). The H19DMR is coincident with the imprint control element, ICE (gray oval), for H19 and Igf2. Shared enhancers (black ovals) for expression of H19 and Igf2 in endoderm (E1) and skeletal muscle (E2) have been defined and are all located downstream of H19 (2, 32). b, strategy for the deletion of a part of H19 exon I by gene targeting. (i), wild type allele; (ii), targeting vector; (iii), targeted allele, H19exIflox-neo; (iv), targeted allele after the excision of the neomycin gene, H19exIflox, with loxP sites flanking the first 710 bp of H19; (v), targeted allele, H19Delta exI, after deletion of loxP-flanked region in vivo using EIIa-cre transgenic mice; (vi), correctly targeted clones and cre recombinase-mediated excision of neomycin gene were confirmed by Southern hybridization. Genomic DNA from the manipulated embryonic stem cell clones was digested with KpnI and hybridized to a 1.5-kb KpnI-BglII fragment from the region upstream of H19. Correctly targeted clones give a 5-kb band from the targeted H19exIflox-neo allele and a 7.7-kb band from the wild type allele (lane 1). Subsequent to cre recombinase-mediated excision of neo-tk, the targeted allele, H19exIflox, hybridizes only to the 7.7-kb DNA fragment (lane 2) like the wild type (lane 3); (vii), EcoRI-digested genomic DNA hybridized to a 1-kb BglII-EcoRI fragment downstream of H19. Correctly targeted clones show two bands of 12.7 and 10.7 kb from H19exIflox-neo and the wild type allele, respectively (lane 1). Subsequent to the excision of neo-tk, the H19exIflox and the wild type alleles are indistinguishable (lane 2) and resemble the wild type (lane 3). DTA, diphtheria toxin gene; neo-tk, neomycin resistance and thymidine kinase genes; solid arrowheads, loxP sites; thick lines, regions for homologous recombination; white rectangles, regions used as 5' and 3' probes for hybridization; RI, EcoRI; B, BsmI; Bg, BglII; D, DraIII; K, KpnI; S, SalI.

Developmental regulation of cytosine methylation at the H19 locus is consistent with, and most likely explains, the mechanisms of this two part silencing process. The ICE is so far indistinguishable from the H19DMR (or H19 differentially methylated region). Cytosine residues within this 2-kb region are methylated in sperm but not in oocytes (10, 13). This differential methylation survives the global changes in DNA methylation that occur during early mammalian development (14, 15). Post-implantation, the domain of paternal chromosome-specific methylation spreads to include the H19 promoter and exonic sequences (13, 16, 17). The mechanistic significance of these changes in DNA methylation is evidenced by the correlation between loss of biallelism and gain-of-methylation in the normally developing embryo (18, 19) and also by the loss of H19 monoallelism in mice carrying a deletion of the DNA methyltransferase gene (20). Mechanisms for this methylation spread are of great interest because of analogies with changes in DNA structure and expression of a number of tumor suppressor genes, as seen in many types of tumor cells.

In this investigation, we examined the silencing of the paternal H19 promoter and several aspects pertaining to the spread of cytosine methylation. We show first that the cytosine methylation of the H19 promoter and exonic sequences is stable in the absence of the originating differentially methylated ICE even through mitosis. Second, we have characterized the sequences required to establish and hold this secondary imprint. Finally, we show that the spread of the methylation can occur over long distances, as the paternally methylated ICE is responsible for secondary methylation changes that occur about 90 kilobases upstream at the Igf2 promoter.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of hCD2-cre Transgenic Mice-- The plasmid phCD2-cre, placing cre recombinase under the control of hCD2 promoter (21), was microinjected into mouse oocytes to obtain the transgenic mice.

Generation of H19Delta exI Mice-- We used cre-loxP-based deletion strategy for generating the mutants (22). As shown in Fig. 1b, the targeting vector carried an 11.7-kb BglII fragment with H19 sequences from between -2 and +9.7 kb (all base pairs are described relative to the H19 transcriptional start site). A loxP-flanked cassette with neomycin resistance and thymidine kinase genes (neo-tk) was inserted at DraIII (+3 bp), and an additional loxP site was inserted at BsmI (+710 bp). The diphtheria toxin-A gene was inserted for negative selection. Linearized vector was electroporated into mouse RI embryonic stem cells. Correct clones were identified by Southern hybridization. A correctly targeted clone was then re-electroporated with pBS185 (Invitrogen) to direct excision of neo-tk, and the excision was detected by Southern hybridization (Fig. 1b). Correct clones were injected into C57/BL6-J blastocysts to generate chimeric founder mice that were mated with EIIa-cre transgenic females (23) to generate strains deleted for exon I (H19Delta exI). The exon I excision was detected by PCR using primers Madhu25 (5'-GAA TTC TGG GCG GAG CCA C-3') and Madhu20 (5'-TGG GAT GTT GTG GCG GCT GG-3') upstream and downstream of the deletion, respectively. The 180-bp PCR product was confirmed by sequencing.

Isolation, Purification, and Induced Proliferation of T Cells-- Cells were isolated from lymph nodes of +/DMRflox and +/DMRflox,hCD2-cre mice, suspended in complete RPMI medium, and enumerated. Mature T cells were enriched by depletion of non-T cells with magnetic beads essentially as described (24). The purity of the resulting T cell populations was >= 90%. T cells were then labeled with the membrane-permeable intracellular covalent coupling fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) as described previously (24). For in vivo experiments, labeled cells were resuspended in phosphate-buffered saline (1 × 107/ml) and adoptively transferred into irradiated (T-depleted) hosts by tail vein injection. For in vitro experiments, labeled cells were incubated at 37 °C in 12-well plates that had previously been coated with stimulating antibodies (anti-CD3 and anti-CD28). The proliferation of viable cells was assessed by analyzing fluorescence at various time points on a FACSCalibur flow cytometer (BD Pharmingen).

Bisulfite-based DNA Methylation Analysis-- Genomic DNA was digested with restriction enzymes outside the sequence of interest. More specifically, DNA from T cells of +/DMRflox,hCD2-cre (carrying the deletion of H19DMR, DMRD) and skeletal muscle of +/+ and +/H19Delta exI mutants was digested with BamHI for analysis of H19 promoter methylation. DNA from hearts of +/DMRDelta G and DMRDelta G/+ mutants was digested with EcoRI for analysis of Igf2DMR1 methylation. The DNA was then treated with sodium bisulfite in agarose beads (25). Subsequently the H19 promoter region or the Igf2DMR1 region was amplified using a nested PCR strategy such that the primers recognized the bisulfite-converted DNA (Tables I and II). To ensure that the methylation information is derived for several chromosomes from each sample, PCR products from at least three separate PCR reactions were cloned and sequenced for each DNA sample. The parental origin of the sequences obtained was assigned by taking advantage of the polymorphic bases (+167 in the H19 relative to the transcription start site and -3776 in the Igf2 relative to the first nucleotide of exon I on the transcript originating from promoter P1) between domesticus and castaneus parental alleles.

                              
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Table I
Primers for the amplification of bisulfite converted DNA

                              
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Table II
Sequences of the primers used for methylation analysis

Nuclear Run-on Analysis-- Nuclei were isolated from the livers of p8 neonates and prepared as described (26). Briefly, the liver was homogenized in chilled buffer containing 2.1 M sucrose, 10 mM HEPES, pH 7.6, 2 mM EDTA, 15 mM KCl, 10% glycerol, 150 mM spermine, 500 mM dithiothreitol, 500 mM phenylmethylsulfonyl fluoride, and 7 µg/ml aprotinin. Nuclei were pelleted by ultracentrifugation through the same buffer, rinsed, resuspended in buffer containing 40% glycerol, 50 mM Tris-Cl, pH 8.3, 5 mM MgCl2, 0.1 mM and EDTA and kept frozen at -70 °C. Typically, nuclei isolated from one liver were resuspended in 450 µl of buffer. The run-on reaction was carried out with 150 µl of nuclei mixed with a buffer containing 10 mM Tris-Cl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 1 mg/ml heparin, 1 mM ATP, 1 mM GTP, 1 mM CTP, and 300 µCi of UTP (800 Ci/mmol) at 30 °C for 30 min with shaking. After DNase and proteinase K treatment, radiolabeled RNA was isolated using Trizol (Invitrogen). Separately, 5 µg of linearized plasmid DNA containing the target probes was immobilized on nitrocellulose. RNA equivalent to 2.5 × 107 cpm was hybridized to the DNA blot for 40-48 h at 43 °C. The probes for the run-on assay were parts of the H19 (1.9-kb PstI-SalI fragment of H19), Igf2 (first 640 nucleotides of the transcript initiated at P1), and actin (region encompassing nucleotides 91-709 of the actin cDNA) cloned in pBluescript.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Stability of the ICE-mediated Epigenetic Modifications-- Earlier analyses have shown that during embryogenesis, the primary imprint at H19DMR directs secondary epigenetic modifications that silence the paternal H19 promoter (12). We wanted to test whether the acquired epigenetic changes are developmental in nature and stable through mitosis or are lost with cell division and need to be re-established during each cycle of mitosis. Therefore, we deleted the H19DMR element in proliferating cells and assayed for the stability of the secondary methylation imprint of the H19 promoter.

The stability of the secondary imprint was analyzed in T cells, because they retain their proliferative ability even after terminal differentiation. Although T cells do not normally express H19, when they are fused with embryonal carcinoma cells, the maternal H19 locus of the T cells is activated, whereas the paternal H19 remains silent (27). Also, the H19DMR and the H19 promoter regions are hypermethylated on the paternal chromosome as shown by Southern blot analysis (27). Thus using these two criteria, we concluded that the H19 locus in the T cells is imprinted.

We generated a transgenic mouse in which the expression of cre recombinase was under the control of the human CD2 (hCD2) promoter so that cre recombinase was expressed in T cells at an early stage of development (i.e. cells that are double negative for cell surface markers CD4 and CD8). Females hemizygous for the hCD2-cre transgene were mated with males homozygous for the DMRflox allele. At the DMRflox allele, loxP sites flank the H19DMR and hence H19DMR can be deleted dependent upon cre expression (12). Thus, the paternal H19DMR was expected to be deleted in the T cells of the +/DMRflox,hCD2-cre progeny.

Mature T cells were isolated from the lymph nodes of +/DMRflox,hCD2-cre mice and their +/DMRflox nontransgenic littermates. To assay the mitotic stability of the secondary imprint at the H19 promoter, we induced the isolated T cells to proliferate in vitro. We assayed proliferation after 6 days in culture both by counting viable cells and by monitoring changes in CFSE-mediated fluorescence during the in vitro culture. CFSE is a nonspecific cell stain. Once a cell is labeled with CFSE, the intensity of fluorescence will depend upon dilution of the dye due to cell division (28). This is clearly evident in adoptive transfer experiments (Fig. 2a). CFSE-labeled cells, when transferred to T-depleted hosts, proliferate and have a reduction in fluorescence with each successive division, whereas similar cells transferred to T replete hosts, which do not allow proliferation, exhibit unaltered CFSE fluorescence even after 30 days. By monitoring CFSE fluorescence during in vitro culture (Fig. 2b) and by counting cells, we projected that the vast majority of T cells had divided at least six times.


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Fig. 2.   CFSE labeling as a measure of cell proliferation. a, purified lymph node T cells from C57BL/6 mice were labeled with the fluorescent dye CFSE (left panel) and injected intravenously into sublethally irradiated (T depleted) or nonirradiated (T replete) hosts. 30 days after transfer, experimental animals were sacrificed, and lymph node cells were harvested and analyzed by flow cytometry. The right panels show CFSE fluorescence on gated T cell populations. b, analysis of proliferation of in vitro stimulated T cells by CFSE fluorescence. Purified T cells from +/DMRflox mice (upper panels) or +/DMRflox, hCD2-cre mice (lower panels) were labeled with CFSE and cultured on plates coated with stimulating antibodies. Cells were analyzed for CFSE fluorescence on day 0 (left panels), day 2 (middle panels), and day 6 (right panels) by flow cytometry. The numbers above the graphs represent the percentage of cells in the defined CFSE fluorescence range.

As expected, in +/DMRflox, hCD2-cre transgenic mice, loxP-flanked H19DMR (Fig. 3a) on the paternal chromosome was excised efficiently in the T cells as evidenced by the appearance of a 340-bp deletion-specific product concomitant with a decrease in the DMRflox-specific 580-bp product (Fig. 3b) in the DNA isolated from T cells.


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Fig. 3.   Characterization of the stability of the secondary imprint during mitosis in the absence of H19DMR in T cells. a, map of the DMRflox allele showing the relative positions of H19, H19DMR (oval), and the primers used for a PCR-based strategy to detect deletion of the H19DMR. Primers A and B (PrA/PrB) amplify the region around the -7.0-kb HindIII site to give the 387-bp product from the wild type allele and a 520-bp fragment from the DMRflox allele. Primers A and D (PrA/PrD) generate a 340-bp fragment from the DMRD allele subsequent to deletion of the loxP-flanked region spanning the -7.0-kb HindIII site (H) to the -0.7-kb XbaI site (X) (12). Solid triangles, loxP sites. b, PCR-based amplification to detect deletion of the DMR in T cells of +/DMRflox,hCD2-cre mice (lanes 1 and 2) and +/DMRflox mice (lanes 3 and 4). DNA from mouse carrying an H19DMR deletion in the germ line (12) was included as a positive control for deletion (lane 5). M, DNA size marker; c, methylation analysis of the +/DMRflox,hCD2-cre T cells in the promoter region of H19 before and after proliferation in vitro. Status of methylation at each CpG dinucleotide was analyzed in the -170 to +167-bp region of the H19 gene. Bars represent the percentage of clones that were found methylated at the specific cytosine residue. The cytosine positions from left to right are -170, -164, -147, -143, -139, -131, -106, -97, -94, -58, -45, -20, -6, +3, +44, +82, +91, +102, and +167 relative to the transcription start site of H19 (+1). 6 and 15 clones were analyzed from the DNA of cells before and after proliferation, respectively.

Methylation of the H19 promoter region was analyzed in the T cells of +/DMRflox,hCD2-cre mice before and after in vitro proliferation (Fig. 3c). We used bisulfite sequencing to determine the methylation status of individual cytosine residues of the DNA in the region. Primers were designed to ensure that the information was acquired only from the chromosomes where the ICE had been deleted. Like the wild type paternal H19 promoter (10), the paternal H19 promoter carrying the ICE deletion was hypermethylated. These cells had undergone at least some proliferation in vivo as they differentiated into mature single positive T cells (CD4+ or CD8+). Thus, H19 secondary imprint survived mitosis in vivo despite the absence of ICE. The cells that were induced to proliferate extensively in vitro also exhibited hypermethylation of the H19 promoter region comparable with cells before proliferation. This demonstrated conclusively that the absence of ICE did not lead to a loss of methylation during mitosis.

Because, subsequent to the ICE deletion, there is no loss in methylation despite extensive proliferation in vivo or in vitro, we concluded that the secondary imprint at the H19 promoter is stable through mitosis and does not require continuous input from the primary imprint at the ICE.

Requirement of the H19 Structural Gene for the ICE Mediated Epigenetic Silencing-- We next wished to determine which regions of the H19 gene are essential for acquiring and/or maintaining the secondary imprint that actually represses paternal H19 transcription. On the wild type paternal chromosome, both the H19 promoter region and the H19 RNA coding sequences are hypermethylated (10). In fact, the first exon of H19 is the most consistently hypermethylated region outside the H19DMR. Previous transgenic and knock-in experiments have provided contradictory data regarding the relative importance of these two regions. Deletion of exon I from transgenic constructs results in the loss of transgene imprinting (29). Likewise, replacement of the whole coding region with firefly luciferase also results in biallelic expression of the unmethylated transgene (29). However, replacement of the whole H19 coding region at the endogenous locus with luciferase results in only sporadic activation upon paternal inheritance (30). Interpreting the luciferase constructions is complicated, and it is not clear whether the presence of luciferase or the absence of the test sequence is the cause for the loss of imprinting. We decided, therefore, to test directly the requirement of H19 exon I for silencing paternal H19 promoter. The endogenous H19 locus was manipulated by gene targeting methods to flank most of exon I with loxP sites (Fig. 1b). This region was deleted in the germ line using an EIIa-cre transgenic line and passed through the maternal and paternal germ lines to analyze the effect of the deletion on methylation of the promoter and on H19 gene expression.

We analyzed the status of methylation of H19 promoter by bisulfite sequencing (Fig. 4). The paternal promoters of H19Delta exI mutants were consistently hypermethylated like the wild type paternal chromosomes at all of the CpG dinucleotides. Our results clearly demonstrate that although highly methylated itself, exon I of H19 does not carry any information for the acquisition of methylation on the paternal H19 promoter.


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Fig. 4.   Methylation analysis of paternal H19 promoter region in +/H19Delta exI mutants. The methylation status at each CpG dinucleotide between -170 and +3 was analyzed by bisulfite sequencing of DNA isolated from skeletal muscle. The vertical bars represent the percentage of clones found methylated at the given cytosine position. The cytosine positions from left to right are -170, -164, -147, -143, -139, -131, -106, -97, -94, -58, -45, -20, -6, and +3 relative to the transcription start site of H19 (+1). 8 and 22 clones were analyzed for +/+ and +/H19Delta exI, respectively.

We wanted to determine whether exon I and its methylation are required to maintain repression of paternal H19 transcription. We initially attempted to quantitate expression via Northern analysis (Fig. 5b). The expression of H19 in +/H19Delta exI mutants was similar to the +/+ control littermates, initially suggesting that paternal H19 was not appreciably activated because of the deletion. However, maternal deletion mutants (H19Delta exI/+) also did not exhibit any H19 expression, suggesting that either the initiation of H19 transcript or its stability is abolished as a result of the deletion. Keeping this in view, it was clear that Northern analysis was not informative for discerning the effect of the deletion on H19 imprinting. We performed, therefore, nuclear run-on assays to derive information about the H19 imprinting in the H19Delta exI mutants, tentatively assuming that the deletion affects the stability of the transcript rather than its initiation.


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Fig. 5.   Expression analysis of H19 from H19Delta exI mutants. a, regions of H19 used as probes for Northern analysis (B/B, 1-kb BamHI fragment) and for the nuclear run-on analysis (P/S, 1.9-kb PstI-SalI fragment). b, Northern analysis of H19 RNA from the liver of +/+, +/H19Delta exI, and H19Delta exI/+ neonates. Elongation factor (EF) was detected on stripped blots. c, nuclear run-on analysis showing transcriptional initiation of H19, Igf2, and actin genes in the nuclei of +/H19Delta 13 (lane 1), H19Delta 13/H19Delta 13 (lane 2), H19Delta exI/H19Delta 13 (lane 3), and H19Delta 13/H19Delta exI (lane 4).

To directly test the role of exon I in initiation of transcription, we attempted nuclear run-on experiments, first using nuclei isolated from the livers of +/H19Delta 13 and H19Delta exI/H19Delta 13 mice. (The H19Delta 13 mutant removes the entire H19 promoter and the coding region (31) and thus allowed us to be certain of the parental origin of any transcripts that we measured.) The run-on analyses (Fig. 5c, lanes 1 and 3) showed that the mutated H19Delta exI allele does initiate significant, although reduced, levels of transcription when maternally inherited. Signal strength in a run-on analysis is affected both by alteration in the rate of transcript initiation and by alteration in the stability of the transcript. The reduced H19 signal in H19Delta exI/H19Delta 13 mutants could be the result of either one or both of these factors. However, given that transcription initiation was not entirely abrogated due to exon I deletion and that significant levels of H19 RNA were observed, it was clear that the run-on analysis could be used to determine the functional necessity of exon I for silencing of paternal H19.

We next looked at transcription from the H19Delta exI allele when it was paternally inherited (Fig. 5c, lane 4). Like a wild type paternal allele, the H19Delta exI chromosome remains silent when paternally inherited. In fact, the levels of transcription could not be distinguished from those noted in nuclei entirely lacking the H19 gene (Fig. 5c, lane 2).

Thus the absence of exon I sequences does not interfere with the ICE-mediated silencing of the H19 promoter. Even though exon I acquires ICE-mediated methylation, it is required neither for ICE-mediated establishment of the secondary imprint at H19 promoter nor for its maintenance.

Because H19Delta 13 mutation removes the ICE in addition to the H19 gene, use of these mutants allowed us to investigate the effect of the ICE on Igf2 transcription initiation. ICE deletion on the maternal chromosome activates normally silent maternal Igf2 significantly in liver although not to the same levels as the paternal allele (31). However, paternal deletion also reduces Igf2 expression to some degree (31, 32). In complete accordance with this steady state mRNA data, we observed significant initiation of Igf2 transcription in H19Delta 13/H19Delta exI mutants (Fig. 5c, lane 4) where the maternal Igf2 allele is expected to be active and the paternal H19Delta exI allele to be fully active. The signals are also intense for H19Delta 13/H19Delta 13 (Fig. 5c, lane 2) where both alleles are likely to express but at reduced levels. The initiation is least in +/H19Delta 13 and H19Delta exI/H19Delta 13 mutants (Fig. 5c, lanes 1 and 3) where the paternal allele has a reduced expression and the maternal allele is expected to be silent. In other words, the gain of maternal Igf2 expression seen in maternal ICE deletion mutants (like H19Delta 13) is attributed directly to gain of transcriptional initiation on this chromosome.

Role of the ICE in Methylation of DMR1 at the Igf2 Locus-- Methylation of Igf2DMR1 located 90 kb upstream of the H19DMR has been postulated to play a role in maintaining the expression of Igf2 (33). Deletion of Igf2DMR1 indicates that its role is specific to muscle, and it is especially important in maintaining appropriate expression in cardiac muscle (34). We therefore investigated the effect of H19DMR on methylation of Igf2DMR1 in cardiac tissue. We used bisulfite sequencing to derive information about methylation of the Igf2DMR1 region in DNA isolated from the hearts of wild type +/+ mice and of mice with a paternally inherited deletion of H19DMR,+/DMRDelta G (12). Examining the 12 CpG residues, we found that the mutant paternal chromosome (Fig. 6b) although still methylated was less so than the wild type paternal chromosome (Fig. 6a). The extent of methylation, in fact, resembled the wild type maternal chromosome (Fig. 6, d and e). This clearly indicated that the ICE is required for the paternal hypermethylation of Igf2DMR1 in cis. Comparing the methylation status of Igf2DMR1 in wild type chromosomes from +/DMRDelta G and DMRDelta G/+ mutants, it appears that the ICE deletion also has an effect on Igf2DMR1 methylation in trans, although the effect of paternal ICE deletion on Igf2DMR1 in cis is most pronounced.


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Fig. 6.   Methylation analysis of Igf2DMR1 region as affected by the deletion of H19DMR in cis or in trans. a and d, wild type (+/+); b and e, deletion on the paternal chromosome (+/DMRDelta G); c and f, deletion on the maternal chromosome (DMRDelta G/+). The methylation status of 12 CpG dyads of the Igf2DMR1 region was analyzed by bisulfite sequencing of DNA isolated from heart. Several clones were analyzed for each type of allele under investigation and are presented as a string of circles. Filled circles, methylated CpG; open circles, nonmethylated CpG; hatched circles, noninformative CpG dyad. The numbers with % represent the percentage of CpG residues methylated among those that were analyzed from all of the clones for a specific allele. A vertical arrow represents the CpG dyad that is important for methylation-sensitive GCF2 binding.

Given the enormous difference in the expression of Igf2 from the wild type paternal and the maternal chromosomes, we were surprised to note that the maternal Igf2DMR1 was also heavily methylated. The methylation was seen at 70 and 50% of the total CpG residues on the paternal and maternal chromosomes, respectively, in the wild type mice (Fig. 6, a and d). Additionally, no single residue could be identified that was specifically methylated on the paternal chromosome. Both the wild type paternal and wild type maternal chromosome populations had individual chromosomes that were heavily methylated and others that were practically devoid of any methylation. Deletion of ICE on the maternal chromosome activates Igf2 expression to high levels in the heart (32), and if Igf2 expression requires Igf2DMR1 hypermethylation, we would expect hypermethylation of Igf2DMR1 on the mutant maternal chromosome. Hence, we analyzed the effect of ICE deletion on maternal Igf2DMR1 methylation. However, the extent of methylation on the expressing mutant maternal chromosomes (Fig. 6f) is low, comparable with wild type chromosomes (Fig. 6, d and e). Thus, comparing maternal and paternal wild type and mutant chromosomes, we see a correlation of methylation of Igf2DMR1 with a methylated H19DMR but not with Igf2 expression.

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Repression of the paternal H19 allele is a two-step process. Molecular evidence strongly supports the idea that a paternal imprint at the ICE between -2 and -4 kb upstream of the H19 promoter marks the parental origin of the chromosome (10, 11). Molecular and genetic studies demonstrate that during development this primary H19DMR imprint directs further epigenetic changes that actually silence the paternal H19 promoter (12). These changes certainly involve DNA methylation. Here we demonstrate that the secondary methylation imprint is developmental. That is, it does not require continued signaling from the primary imprint at the ICE but is stable even during multiple mitoses.

The ICE acts epigenetically to modify the H19 promoter region and also part of the structural gene. Both of these regions are hypermethylated post-fertilization, and the paternal H19 promoter is silenced. In fact, the exon I region is most completely and consistently methylated. However, our results here indicate that the region is not required at all for acquiring the secondary imprint. Thus, CpG methylation does not always connote function but in some cases may occur only coincidentally with other changes.

Our results concerning the role of exon I in maintaining H19 imprinting are not really consistent with previous transgenic experiments (29). Our personal observation is that transgenic constructs are particularly sensitive to disruptions in imprinting. For example, many transgenes show a copy number dependence in imprinted expression and DNA methylation that is obviously not applicable to the endogenous locus. These experiments perhaps suggest that the imprinting signals may include large DNA structures and elements in a way that is really not yet appreciated. Until recently, most transgenic constructs extended only 4 kb on the 5' flank and therefore included neither the entire H19DMR nor all of the CTCF binding sites. Recent experiments from the Bartolomei laboratory (35) indicate that additional flanking DNA may provide copy number and position independence and may provide a better substrate for further mutagenesis of transgene constructs.

Interestingly, the loxP sites left downstream to the promoter after our manipulation of the locus were also methylated (data not shown). Thus it appears that any CpG residue at that locus may be modified during the spread of methylation irrespective of its sequence context. This result is consistent with appropriate imprinting of the NeoR gene inserted at the H19 locus (36). Thus, even the sequence of the H19 promoter may not actually play an important role for methylation spreading but may simply be a CpG-rich element in the right place. We are currently investigating the ability of the ICE to modify promoters other than H19 when they are placed in adjacent position.

Our experiments have also demonstrated that the ICE controls the imprinting of Igf2 at the transcript initiation level despite being 90 kb downstream of the promoter. This is in complete accordance with the presence of a transcriptional insulator at the ICE (32, 37, 38). Insulator elements, when present between the enhancer and promoter, prevent the expression of genes. The mechanistic details of the process seem to be diverse and not well understood (39). Our nuclear run-on analysis suggests that the insulator in the ICE truly prevents promoter activation and transcript initiation by the enhancer.

Finally, we investigated the role of H19DMR in methylation of the Igf2DMR1 element. Igf2DMR1 has been identified as methylated in a parent-of-origin manner (17, 33, 40). Subsequently, its crucial role in maintaining appropriate expression patterns of Igf2 in mesodermal tissues was demonstrated in vivo using embryonic stem cell-generated mutational analyses (34). Further, an Igf2DMR1-specific binding protein, GCF2, has been identified in which affinity is dependent upon the levels of CpG methylation (41). Our bisulfite sequencing results are puzzling in this regard because although we see a correlation between Igf2DMR1 methylation and the presence of a methylated H19DMR in cis, we do not note a correlation between Igf2 expression and the methylation of the 12 CpG dyads we examined. Further, investigations are clearly warranted.

Although our experiments do not support a crucial functional role for Igf2DMR1 methylation, they do confirm and extend previous DNA sequencing experiments indicating that the degree of Igf2DMR2 methylation was altered in trans upon deletion of the H19DMR (via the H19Delta 13 mutation) (42). These results are intriguing in that they suggest a communication between the maternal and paternal chromosomes sometime after implantation, when the methylation of the Igf2DMR1 is first established. The idea that imprinted alleles interact and that such interactions are crucial to maintaining monoallelic expression has always been an attractive hypothesis but one with limited experimental support. Paternal and maternal human 15q11-q13 domains interact specifically during S phase (43) although no functional role for the interaction has been subsequently supported. To date no such physical association of the chromosomes has been noted at the Igf2/H19 locus. We have never found any evidence for transvection (i.e. communication between enhancer and promoter elements in trans at the H19 locus) despite specific attempts to record such interactions (44). Finally, H19 transgenes do not require a partner to exhibit imprinting. Rather single copy hemizygous transgenes exhibit monoallelic expression even in an H19Delta 13 genetic background (32). Nonetheless, it is interesting to note that the ICE can alter methylation patterns not only 90 kb away but also across other chromosomes.

    ACKNOWLEDGEMENTS

We thank Shirley Tilghman for the H19Delta 13 mice and Heiner Westphal for the EIIa-cre mice.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: National Institute of Immunology, Aruna Asaf Ali Rd., New Delhi 110067, India. Tel.: 91-11-6162281 or 91-11-6167623 (ext. 482); Fax: 91-11-6162125 or 91-11-6177626; E-mail: madhus@nii.res.in.

Published, JBC Papers in Press, September 20, 2002, DOI 10.1074/jbc.M208437200

    ABBREVIATIONS

The abbreviations used are: ICE, imprinting control element; DMR, differentially methylated region; CFSE, carboxyfluorescein diacetate succinimidyl ester.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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