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Human Molecular Genetics Pages 1543-1547

Imprinting of IGF2 and H19: lack of reciprocity in sporadic Beckwith-Wiedemann syndrome
Introduction
Results
   Expression of IGF2 promoter-specific transcripts in BWS fibroblast cell lines
   Expression of H19 allele-specific transcripts in BWS fibroblast cell lines
Discussion
Materials And Methods
   Fibroblast cell lines
   IGF2 allele-specific expression
   H19 allele-specific expression
Acknowledgements
References

Imprinting of IGF2 and H19: lack of reciprocity in sporadic Beckwith-Wiedemann syndrome

Imprinting of IGF2 and H19 : lack of reciprocity in sporadic Beckwith-Wiedemann syndrome Johanna A. Joyce, Wayne K. Lam1,2, Daniel J. Catchpoole2, Paul Jenks3, Wolf Reik4, Eamonn R. Maher1,2 and Paul N. Schofield*

Laboratory of Stem Cell Biology, Department of Anatomy, and 2Department of Pathology, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK, 1Division of Medical Genetics, Department of Paediatrics and Child Health, University of Birmingham, Birmingham Womens' Hospital, Edgbaston, Birmingham B15 2TG, UK, 3Department of Cytogenetics, Addenbrookes NHS Trust, Cambridge CB2 2QQ, UK and 4Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB2 4AT, UK

Received April 29, 1997; Revised and Accepted June 19, 1997

Genomic imprinting is a novel form of control of gene expression in which the transcription of each allele of an imprinted gene is dependent on the sex of the gamete from which it was derived; to date >15 genes have been demonstrated to show imprinting. The maintenance of a normal imprinting pattern in many loci has been shown to be essential for normal development and adult life. Many tumours, and some developmental disorders, exhibit loss of imprinting (LOI) in key genes such as insulin-like growth factor 2 (IGF2) which often results in hyperplasia and is associated with cancer. The mechanism by which the genomic imprint is first established, then maintained, is not understood. However, in the case of IGF2, the expression of a neighbouring gene, H19, has been suggested to influence its transcription by competition for a common enhancer, thereby generating a mutually exclusive and allele-specific pattern of gene expression. Associated changes in CpG methylation in discrete areas of both genes have been implicated in maintenance of the imprint. We have examined the allele-specific expression of IGF2 and H19 in fibroblasts derived from patients with sporadic Beckwith-Wiedemann syndrome (BWS), a fetal overgrowth syndrome associated with an imprinted locus on 11p15.5. We report that the majority of karyotypically normal patients show LOI of IGF2 with biallelic expression. In a proportion of these patients,loss of IGF2 imprinting was associated with complete suppression of H19 expression, as predicted by the enhancer competition model. However, in a significant number of cases, IGF2 showed biallelic expression even though H19 expression and methylation status were normal. This indicates that there must be an alternative H19-independent pathway by which allele-specific IGF2 expression is established or maintained.

INTRODUCTION

Genomic imprinting is an epigenetic mechanism controlling gene activity in which the expression of each allele depends on its parent of origin. More than 15 genes have now been shown to be subject to this mode of regulation, many of which have profound effects on the growth of the embryo, which has led to the proposal that imprinting has evolved to moderate the conflicting demands of each parental genome on the reproductive process.

Genes subject to genomic imprinting appear to be clustered within both the mouse and human genomes, suggesting that part of the mechanism responsible for allele-specific expression/ suppression depends on cis-acting sequences (1 ,2 ).In the cluster of genes associated with Prader-Willi and Angelman syndromes, such cis-acting sequences may have been identified recently (3 ), yet the mechanism by which they work is still unknown. Insulin-like growth factor II (IGF2) lies within a cluster of imprinted genes; H19, p57 KIP2, KvLQT1 and insulin (INS) at 11p15.5 in the human, and distal chromosome 7 in the mouse. In the mouse, Igf2 and Ins2 are paternally expressed; p57KIP2and H19 are expressed from the maternal chromosome. The pattern in humans is similar for IGF2, H19 and p57KIP2. Whilst KvLQT1 shows a tissue-specific pattern of imprinting in humans it has not been shown to be imprinted in the mouse. As yet,INS has not been shown definitively to be imprinted in humans (4 ).

Transgenic experiments have demonstrated that deletion of the H19 gene, or removal of downstream enhancer sequences, causes loss of imprinting (LOI) of the nearby IGF2 gene, implying that the reciprocity of imprinting is determined by either the H19 gene product or possibly cis-acting sequences outside the gene itself (5 -7 ). This has led to the development of the `enhancer competition' model of reciprocal imprinting (7 ,8 ) in which it is envisaged that there is mutually exclusive competition between H19 and IGF2 for a single enhancer. However, a functional role for the H19 RNA in expression of its own gene has been suggested following the recent observation that deletion of sequences in exon 1 of the murine gene results in LOI in H19 itself (9 ).

Many primary tumours and a discrete set of fetal overgrowth syndromes are subject to LOI of IGF2 (10 -14 ). The subsequent increased levels of IGF2 mRNA, resulting from biallelic expression, may predispose certain tissues to the developmental abnormalities found in Beckwith-Wiedemann syndrome (BWS), and the hyperplasia associated with increased tumour risk (15 ,16 ). Consequently it might be expected that understanding of the mechanism by which the imprint is established and maintained might shed light on the aetiopathology of these abnormalities.


Figure 1. PCR amplification of genomic sequences and allelic cDNAs from H19 and IGF2. (A) H19 gene. The solid boxes represent exons 1-5; the primers used to PCR across the RsaI polymorphism are marked. The size of the product spanning exons 4 and 5 is 756 bp from genomic DNA and 676 bp from cDNA. (B) IGF2 gene. Coding exons are represented by solid boxes and non-coding exons by open boxes. Primers used to generate promoter-specific cDNAs and nested PCRs from these are indicated, as are expected product lengths. Primer designations are all as described in Materials and Methods.

The nature of the primary mechanism of imprinting is unknown; however, DNA methylation of specific sites within and around H19 and IGF2 has been shown to correlate with expression status in normal individuals, developmental tumours and cancer predisposing syndromes such as BWS (17 -20 ). In BWS, ~25% of cases show abnormal IGF2 and H19 methylation; of these most have isodisomy associated with a paternal pattern of methylation. The remaining cases, accounting for ~5% of all patients, show no karyotypic abnormalities of 11p but display a paternal methylation pattern on the maternal IGF2 and H19 alleles, accompanied by biallelic IGF2 expression and extinction of H19 expression (21 ,22 ). This combination is consistent with the presence of imprinting centre mutations.

The methylation status of IGF2 may be dissociated from its expression, at least as the result of gross chromosomal changes. We have previously reported one BWS family in which a chromosome 11 inversion was associated with biallelic expression of IGF2, normal maternal H19 expression and a normal IGF2 and H19 methylation pattern (23 ). This suggests the existence of a H19-independent mechanism of loss of IGF2 imprinting in the presence of chromosomal rearrangement.

In this study, we report the allele-specific expression pattern of H19 and IGF2 in fibroblasts from patients suffering from sporadic BWS previously shown to be karyotypically normal and normally methylated at each allele of each gene. The majority (9/11) of the cell lines show biallelic IGF2 expression; three of these show the predicted biallelic suppression of H19. Unexpectedly, four of the remaining lines exhibit normally imprinted, monoallelic H19 transcription, indicating that, in addition to competition for a putative single enhancer, there must be H19 enhancer-independent mechanisms by which biallelic IGF2 expression may be achieved.

RESULTS

We have investigated the mechanisms of H19 and IGF2 imprinting in dermal fibroblasts from BWS patients with a normal karyotype and normal IGF2 and H19 methylation patterns. Expression of IGF2 and H19 was investigated in fibroblast cell lines derived from 11 cytogenetically normal sporadic BWS patients. None of these showed isoallelism or aberrant methylation of IGF2 or H19 in a previous study (21 ,22 ).


Figure 2. Allele-specific expression of IGF2 in BWS patients. (a) Biallelic expression of IGF2 from promoters P1-P4 in BWS fibroblast cell line BWS49. cDNA derived from reverse transcribed total fibroblast RNA using an IGF2-specific reverse primer was amplified to generate full-length promoter templates, specific for each promoter (P1-P4). Exon 9 was amplified from each individual template and digested with ApaI. (+) exon 9 amplified from promoter template generated from cDNA set up with reverse transcriptase; (A) promoter-specific exon 9 digested with ApaI; (-) minus reverse transcriptase control. (b) Leader exons amplified from promoters P1-P4 in BWS fibroblast cell line BWS49. Leader-specific exons (exons 3, 4, 5 or 6) were amplified from individual promoter templates to confirm the identity of promoter-specific templates. (+) Leader exon amplified from promoter template generated from cDNA set up with reverse transcriptase; (-) minus reverse transcriptase control.

Expression of IGF2 promoter-specific transcripts in BWS fibroblast cell lines

Full-length cDNAs were generated from cultured fibroblast RNA using primers specific for each promoter (P1-P4) at the 5' end and co-terminous at the 3' end within exon 9 (common to all classes of transcript, Fig. 1 B). All 11 cell lines were informative for IGF2 expression; nine showed biallelic expression and two normal monoallelic paternal expression. This demonstrates that the majority of non-disomic BWS patients with normal IGF2 and H19 methylation have relaxation of IGF2 imprinting. In most normal adult tissues, IGF2 expression is monoallelic from promoters P2-P4, but biallelic from the P1 promoter (24 ). To determine if relaxation of IGF2 imprinting in BWS patients was caused by true LOI of P2-P4 or a switch in promoter usage to P1, the allele-specific expression status of each of the four promoters was examined in the nine patients. Biallelic IGF2 expression was detected at P1-P4 in all nine patients (Fig. 2 a), indicating that a simple promoter switch to the biallelically expressed P1 promoter did not account for the overall biallelic IGF2 expression observed in these patients. Under the conditions of the PCR used here, overall expression from P2-P4 appeared to be equivalent (Fig. 2 b). However, it was notable that the relative intensities of the two bands associated with transcripts from each allele were unequal for products from the P3 and P4 promoters (Fig. 2 a). This may reflect an allele-specific difference in the degree of relaxation of imprinting for different promoters, but as the PCR conditions used were non-quantitative, this interpretation is speculative.

Expression of H19 allele-specific transcripts in BWS fibroblast cell lines

To investigate the relationship between IGF2 and H19 imprinting, H19 cDNA was reverse transcribed from RNA harvested from subconfluent cultures. The positions of primers generating allele-specific RT-PCR products are shown in Figure 1 A. Cell lines from 11 patients were screened for H19 expression (Fig. 3 a) using primers spanning exons 4 and 5. Expression of H19 was demonstrated in eight out of 11 cell lines, and no trace of amplified product was found in the remainder. Co-amplification of cDNA for GAPDH was uniformly successful in all cell lines, indicating that failure to generate a H19-specific product in the remaining three lines reflected the absence of detectable amounts of H19 RNA in the cells (Fig. 3 b). Of the cell lines expressing IGF2 biallelically together with H19, four out of six were informative at the RsaI polymorphic site in exon 5. In all four cases, H19 expression was found to be monoallelic.


Figure 3. Allele-specific expression of H19 from BWS patients. (a) Monoallelic expression of H19 in BWS fibroblast cell line BWS3 (heterozygous at the H19 RsaI site). (g): 756 bp H19 product amplified from genomic DNA; (RsaI) 756 bp genomic DNA product digested with RsaI; (c) 676 bp product amplified from cDNA; (RsaI) 676 bp cDNA product digested with RsaI, i.e. monoallelic; (-RT) minus reverse transcriptase control. M; markers. (b) Examples (a-f) of BWS fibroblast cell lines showing either expression (a, b and d) of H19 or lack of H19 expression (c, e and f) from cDNAs from which GAPDH was amplified simultaneously. (+) RNAs set up for cDNA synthesis with reverse transcriptase. (-) minus reverse transcriptase control.

Previous studies carried out by ourselves (21 ) and others (13 ,17 ) indicate that normal cultured fibroblasts show stable retention of IGF2 imprints over the passage numbers used here. A significant number of the BWS cell lines in the current study show retention of H19 imprinting (4/4 informative lines) and IGF2 imprinting (2/11 informative lines), indicating that the heterogeneity we report is unlikely to result from sporadic LOI as a consequence of culture. Assays for allele-specific expression were carried out at several passage numbers between three and 10 on the same set of cell lines and showed retention of the expression pattern seen at the earliest passage.

Table 1 IGF2 and H19 expression status
Patient H19 expression IGF2 expression
  mono null NI (exp) mono bi
BWS3 +       +
BWS49 +       +
BWS67 +       +
BWS95 +       +
BWS12     +   +
BWS123     +   +
BWS47   +     +
BWS93   +     +
BWS98   +     +
BWS94     + +  
BWS110     + +  
Summary of expression data for patients informative at H19 and IGF2. mono: monoallelic expression of IGF2/H19, bi: biallelic expression of IGF2/H19,null: no H19 expression, NI (exp): not informative for H19 RsaI polymorphism (but expressing H19).

DISCUSSION

In the data presented here (summarized in Table 1 ), we describe a new group of karyotypically normal patients showing several novel combinations of IGF2 and H19 expression with normal H19 methylation. We report instances where IGF2 imprinting is normal (2/11) and abnormal (9/11), showing that LOI at IGF2, although predominantly associated, is not necessary for BWS. Furthermore, we have shown that overall biallelic expression of IGF2 results from true LOI and not from a shift in promoter usage. Several patients (4/11) showed loss of IGF2 imprinting with normally imprinted H19, a combination excluded by the `enhancer competition' model. In addition, we have identified a number of individuals with biallelic IGF2 expression, extinction of H19 transcription, but normal H19 methylation. In the patients presented in the current study, we previously have shown H19 methylation to be normal (22 ), but have not investigated allele-specific expression of IGF2/H19. This observation of biallelic IGF2 expression in BWS confirms that of Weksberg and co-workers, but this earlier study (17 ) did not investigate methylation or H19 expression. The current study suggests that mechanisms other than methylation in the region assayed are capable of affecting H19 expression. There are no clear differences between the phenotypes of each of these classes of patient to indicate that the different epigenotypes are associated with particular aspects of BWS.

The enhancer competition model (8 ) predicts that IGF2 may only be expressed by using the H19 enhancer in cis, and only when H19 is repressed (usually associated with methylation) on the same chromosome. We have demonstrated that, although some patients conform to this model in that H19 expression is suppressed with IGF2 LOI, a significant number show normal H19 imprinting with IGF2 LOI. This implies that either the enhancer competition model does not adequately describe the mechanism of IGF2 imprinting, or that it should be modified to take into account the potential for mutations to override the exclusive use of the H19 enhancer by one or the other gene (25 ). Although we previously have reported biallelic expression of IGF2 in a BWS patient with a chromosomal rearrangement in whom the normal pattern of methylation was retained at H19 and IGF2 (23 ), H19 imprinting being unaffected, the data presented here indicate that IGF2 LOI can occur in the absence of changes in IGF2 methylation pattern and in the presence of normal H19 imprinting with no gross chromosomal aberrations.

For karyotypically normal BWScases with normal H19 expression and imprinting, it must be considered that the H19 molecule itself may be important in establishing the expression status of IGF2, and that in a subset of the BWS population this, or perhaps co-operating factors may be defective. In addition, quantitative changes in H19 expression may underlie its action if a critical threshold of transcripts is necessary to repress IGF2 expression fully. Depending on the level of IGF2 promoter activity, this threshold may change from tissue to tissue and, therefore, such changes may not be immediately apparent. Thus monoallelic expression of H19 may be retained but loss of H19 function or reduction in H19 expression may fail to suppress IGF2 transcription on the same chromosome. A cis-acting role for a transcribed RNA has precedents in Xist and is discussed in reference (8 ). In mice, a BWS-like phenotype can be generated by an IGF2 transgene, providing compelling evidence that many aspects of the BWS phenotype result from enhanced IGF-II expression (unpublished data).

Other imprinted genes at 11p15 have been shown to be mutated in BWS. KvLQT1 frequently is interrupted in the chromosomal breakpoints at BWSCR1 (26 ), and the recent description of two BWS patients with germline mutations in the p57KIP2 gene, an inhibitor of G1 cyclin-Cdk complexes (27 ), may indicate that additional pathways can result in a similar phenotype. Given the nature of the gene products, it is difficult at present to reconcile the potential mode of action of KvLQT1 or p57KIP2 with what is known about the control of imprinting of IGF2 and H19. However, as the frequency of mutations in p57KIP2 is similar to those BWS cases with monoallelic IGF2 expression, they may represent a further class of lesion able to give rise to aspects of the BWS phenotype either independently of those described to date, or possibly by factors acting epistatically on the same pathway of growth control as IGF2/H19. Both cases showing monoallelic expression of IGF2 have been investigated recently for mutation in p57KIP2 (Lam et al., submitted), and, at least within the gene and its immediate environment, no mutations have been detected.

MATERIALS AND METHODS

Fibroblast cell lines

Fibroblast cell lines were derived from dermal punch biopsies as described (22 ). Cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, penicillin and streptomycin according to a 3T3 regime. All cells were used at passages between three and 10. Where replicates were carried out on the same line at different passage numbers no difference in allele-specific patterns of gene expression was detected.

IGF2 allele-specific expression

DNA was extracted from peripheral blood, and primers spanning the ApaI polymorphism in the 3' untranslated region in the ninth exon of IGF2 were used to screen genomic DNAs (28 ). RNA was extracted from informative fibroblast cell lines, using TriReagent (Gibco-BRL Life Technologies), and RNA samples were treated with DNase I to remove contaminating genomic DNA. One [mu]g of total RNA was then reverse transcribed into cDNA, with and without reverse transcriptase, using an IGF2-specific reverse primer; IGF2 Ra: 5'-ggtcgtgccaattacatttca (Gibco-BRL Life Technologies Superscript kit). The IGF2 exon 9 PCR was repeated for each cDNA sample and PCR products were digested with ApaI. Digestion was performed using 10 U of ApaI, 2 [mu]l of appropriate buffer and 7.0 [mu]l of PCR product for 3 h at 30oC. Complete digestion of exon 9 products was controlled for by setting up each digestion in parallel with a sample known to be homozygous for the presence of the ApaI site. Digested fragments were then resolved on 2% agarose and stained with ethidium bromide.

Full-length promoter transcripts were generated for each of the P1-P4 promoters using an IGF2-specific reverse primer: IGF2Rb, 5'-gggttgttgctattttcggat-3' and promoter-specific forward primers: P1f, 5'-gggactgcgcagggactaga-3'; P2f, 5'-gacggggtaaccattatcca-3'; P3f, 5'-cccgctctgccccgtcgcacattc-3'; and P4f, 5'-tcctgccccagcgagccttctgctg-3'. Promoter-specific leader exons and exon 9 were amplified from each of these templates. Leader exons were amplified using a common reverse primer: IGF2 7R, 5'-CAGCAATGCAGCACGAGGCGAAGGC-3' and P1f (exon 3, 106 bp); 4F, 5'-gtgtccaggaaagcgaccg-3' (exon 4, 200 bp); 5F, 5'-cgtcgcacattcggcccccgcgact-3' (exon 5, 211 bp); and 6F, 5'-tcctcctcctcctgccccagcg-3' (exon 6, 121 bp).

PCR conditions for the IGF2 ApaI polymorphism were according to Tadakoro et al. (28 ). Conditions for all four full-length promoters were: 94oC for 5 min, followed by 35 cycles of 94oC for 2 min, 50oC for 2 min and 72oC for 2.5 min. PCR products were then diluted 5-fold and nested PCRs for each of the leader exons and exon 9 were performed. PCR conditions for leader exons 3, 4, 5 and 6 were 94oC initial denaturation for 5 min, then 30 cycles of 94oC for 1 min, 55oC for 1 min and 72oC for 1 min.

H19 allele-specific expression

Using primers spanning a reported RsaI polymorphism in exon 5 of the H19 gene (29 ), genomic DNA samples were tested for informativity at the RsaI locus and RNA from informative samples was then analysed for allele-specific expression. As for IGF2, RNA samples were treated with DNase I and set up with minus reverse transcriptase controls, and the use of primers spanning the fourth intron allowed for the detection of any contaminating genomic DNA. H19 products were digested with RsaI using 10 U of enzyme, 2.0 [mu]l of appropriate buffer and 7.0 [mu]l of PCR product for 3 h at 37oC.

PCR conditions for the H19 primers were: initial denaturation at 94oC for 5 min, followed by 30 cycles of 94oC for 1 min, 55oC for 1 min and 72oC for 1 min for both DNA and RNA. Primer sequences for the H19 primers were: H19 4F, 5'-agattcaaaggctccac-3' and H19 5R, 5'-ctggactcatcatcaataaacact-3'. PCR conditions for the GAPDH primers were: initial denaturation at 94oC for 5 min, followed by 30 cycles of 94oC for 1 min, 58oC for 1 min and 72oC for 1 min. Primer sequences for the GAPDH primers were: GAPDH F, 5'-GACCCCTTCATTGACCTCAACTACA-3' and GAPDH R, 5'-CTAAGCAGTTGGTGGAGGA-3'.

ACKNOWLEDGEMENTS

The authors would like to express their thanks to the parents and children involved in this study. We are grateful to Dr Anne Ferguson-Smith for helpful comments on the manuscript. The work was funded by the Wellcome Trust. W.L. is funded by an Anglia/Oxford regional Research Award, J.A.J. and W.R. acknowledge the support of the BBSRC and W.R. Action Research.

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