Human Molecular Genetics, 2001, Vol. 10, No. 15 1601-1609
© 2001 Oxford University Press
Distant cis-elements regulate imprinted expression of the mouse p57 Kip2 (Cdkn1c) gene: implications for the human disorder, Beckwith–Wiedemann syndrome
Wellcome/CRC Institute of Cancer and Developmental Biology, Tennis Court Road, Cambridge CB2 1QR, UK
Received April 20, 2001; Revised and Accepted May 30, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Complex phenotypes and genotypes characterize the human disease, Beckwith–Wiedemann syndrome (BWS). Genetic and epigenetic mutations are found in five different genes which all lie within a 1 Mb imprinted domain on human chromosome 11p15. Only two of these genes, p57KIP2 (CDKN1C) and IGF2, are likely to be functionally involved in this disease. The presence of the additional mutations therefore suggests a role for the regulation of these two genes by distant cis-elements. The mouse Igf2 gene is regulated by enhancers and imprinting elements which lie >120 kb downstream of its promoter. Here we show that key elements for expression of the mouse p57Kip2 (Cdkn1c) gene also lie at a distance. Enhancers for expression within skeletal muscle and cartilage lie >25 kb downstream of the gene. In addition, we find no evidence for allele-specific expression of p57Kip2 (Cdkn1c) from our bacterial artificial chromosome transgenes that span 315 kb around the locus. This suggests that a key imprinting element for p57Kip2 (Cdkn1c) also lies at a distance. Therefore, BWS in humans may result from disruption of appropriate expression of the p57KIP2 (CDKN1C) gene through mutations that occur at a substantial distance from the gene.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Beckwith–Wiedemann syndrome (BWS) is a complex disorder associated with numerous growth abnormalities (1–3). The phenotypic expression of BWS can vary between patients but it generally includes exomphalos, macroglossia, giantism and organomegaly. Other features that can be present are hypoglycaemia, hemihypertrophy, genitourinary abnormalities, cleft palate and a susceptibility to embryonal tumours.
In BWS, genetic and epigenetic mutations disrupt the expression of at least five different imprinted genes, all of which map to human chromosome 11p15. Nearly half of the patients with familial BWS have germline mutations in the coding sequence of the maternally-expressed p57KIP2 (CDKN1C) gene (4–7). However, BWS is predominantly seen as a sporadic occurrence. In this group of patients, p57KIP2 (CDKN1C) mutations are relatively infrequent (<5%). Instead, loss of imprinting (LOI) is commonly observed for the paternally-expressed IGF2 gene which lies 1 Mb away from p57KIP2 (CDKN1C) (8,9). This is sometimes, but not invariably, associated with LOI of the maternally-expressed H19 gene (7). A small number of patients (>1%) have germline balanced chromosomal translocations which disrupt a fourth imprinted gene within the region, KvLQT1 (KCNQ1) (10,11). The fifth, and most predominant, mutation seen in BWS patients is LOI of an unspliced and untranslated RNA, LIT1 (KCNQ1OT1/KvLQT1-AS) (12–17). The 5' end of LIT1 (KCNQ1OT1/KvLQT1-AS) originates at a CpG island, which is methylated on the maternal allele both in somatic cells and in the female germline. The 56 kb transcript is entirely contained within the KvLQT1 (KCNQ1) gene and would be disrupted by some, but not all, of the translocation events seen in BWS patients in this region (12,13).
The genetic complexity of BWS is currently explained by the functional involvement of more than one imprinted gene. This is supported by recent studies which suggest that the phenotypic differences between BWS patients may define subgroups of mutations (7). Thus, exomphalos is frequently seen in patients with p57KIP2 (CDKN1C) mutations but not in patients with biallelic IGF2. Patients with p57KIP2 (CDKN1C) mutations do not, however, present with childhood tumours. These are more commonly associated with LOI of IGF2 and/or H19. In addition, patients with LOI of LIT1 (KCNQ1OT1/KvLQT1-AS) are generally associated with exomphalos rather than embryonal tumours. Indirectly, this suggests a link between LOI of LIT1 (KCNQ1OT1/KvLQT1-AS) and loss of expression of p57KIP2 (CDKN1C). Studies in the mouse suggest that both IGF2 and p57KIP2 (CDKN1C) play a role in BWS. Maternal inheritance of a targeted deletion of the mouse p57Kip2 (Cdkn1c) gene results in developmental abnormalities including exomphalos and cleft palate (18,19). Over-expression of Igf2, either by means of transgenes or by targeted deletion of the nearby H19 gene, results in an overgrowth phenotype similar to that seen in BWS patients (20–23). Individually, neither mutation fully duplicates the human disease in mice. However, a combination of over-expression of Igf2 and loss of expression of p57Kip2 (Cdkn1c) does fulfil many of the criteria as a mouse BWS model (24).
In mice, the homologous cluster to the human 11p15 region is located on distal chromosome 7. Comparative sequencing of the human and mouse regions has demonstrated a high degree of conservation in gene order and imprinting of at least nine genes within the domain (15,16). Previous work suggested that the region could be subdivided into at least three domains: one encompassing H19, Igf2 and Ins2; one central domain comprising biallelically expressed genes; and a second imprinted domain containing Kvlqt1 (Kcnq1), p57Kip2 (Cdkn1c) and other maternally-expressed genes (25–27). The existence of H19 and Igf2 within an independent domain is supported, in part, by experiments in the mouse (28,29). A region upstream of H19 has been described as an imprinting control region (ICR). ICRs are primarily defined by the existence of epigenetic marks that are present in the germline. They are functionally defined by LOI of the nearby genes which results when these regions are deleted, either from the endogenous loci or from transgenes (30,31). Evidence for an ICR at the LIT1 (KCNQ1OT1/KvLQT1-AS) locus, within the KvLQT1 (KCNQ1)/p57KIP2 (CDKN1C) domain, is suggested primarily by its resemblance to these ICRs. Also, deletion of this putative ICR in a somatic cell line results in LOI of KvLQT1 (KCNQ1) and SMS4 without affecting imprinting of H19 (32). Currently, most of the available data is consistent with mutations in the two separate domains leading to BWS. The shared phenotypes in BWS patients may result from a functional interaction in a growth control pathway independent of their physical association (33). However, some studies show that LOI of LIT1 (KCNQ1OT1/KvLQT1-AS) is often associated with LOI of IGF2 (7). This data would suggest that these two putative domains are not, in fact, independent. In addition, the imprinted Mash2 lies between the two proposed domains and has not been shown to be affected by deletion of either ICR.
Clearly, complex mechanisms regulate imprinting in the 11p15 domain. We have therefore been applying a transgenic approach in mice to establish how genes within this locus are regulated (28,34,35). A 130 kb transgene spanning the Igf2/H19 locus was found to maintain imprinted expression independent of the distal 7 domain. However, we observed no imprinting with a 38 kb human p57KIP2 (CDKN1C) transgene. We have now extended this analysis using bacterial artificial chromosome (BAC) transgenes spanning 315 kb around the mouse p57Kip2 (Cdkn1c) locus. Our work demonstrates that key regulatory factors for tissue- and allele-specific expression of p57Kip2 (Cdkn1c) lie at a distance from the gene. This has important implications both for studies of imprinting mechanisms and for BWS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene content mapping p57Kip2 (Cdkn1c) BAC contig
To assess a molecular basis for the involvement of the human p57KIP2 (CDKN1C) gene in BWS, we wished to establish the expression and imprinting capabilities of the genomic region surrounding the mouse p57Kip2 (Cdkn1c) gene. We had previously isolated an 85 kb BAC, 144D14, spanning the p57Kip2 (Cdkn1c) locus (35). Subsequently, the publication of a physical map of the region allowed us to identify two additional clones for our analysis (36). The size and degree of overlap of these BAC clones was initially determined by pulse-field gel analysis and hybridization studies. BAC 144D14 covered 60 kb of sequence upstream of the p57Kip2 (Cdkn1c) gene and 25 kb of sequence downstream. BAC 35G23 extended 10 kb upstream and 250 kb downstream of the gene and BAC 96P6 did not contain the gene. The position of the BAC clones within the mouse distal 7 domain was further defined by PCR-based gene content analysis to generate a contig of 315 kb. PCR was performed for exons 1 and 15 of the Nap1l4 gene, for exons 1 and 2 of the p57Kip2 (Cdkn1c) gene, for the 3'-UTR, exon 10 and exon 1
of the Kvlqt1 (Kcnq1) gene and for the mouse Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island (Fig. 1A). In addition to p57Kip2 (Cdkn1c), the 85 kb BAC clone, 144D14, was found to contain exon 15 of the Nap1l4 gene but not the 3'-UTR of Kvlqt1 (Kcnq1) which lies 
30 kb downstream of p57Kip2 (Fig 1B). The 260 kb BAC, 35G23, encompassed more that half of the Kvlqt1 (Kcnq1C gene including the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island and exon 10 of Kvlqt1 (Kcnq1). None of the BAC clones extended to the Kvlqt1 (Kcnq1) promoter (data not shown).
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Expression of p57Kip2 (Cdkn1c) from a modified 85 kb BAC transgene
We first examined expression from the 85 kb BAC. In order to distinguish between expression of p57Kip2 (Cdkn1c) from the mouse BAC transgene and that of the endogenous locus, a lacZ-alkP reporter was inserted into the 3'-UTR of the gene by homologous recombination (Fig. 2A and B; Materials and Methods). At the same time, a SalI-loxP site was inserted into the 5'-UTR. This construct was designed to leave the locus intact but left the p57Kip2 (Cdkn1c) gene non-functional.
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The linearized transgene was injected into fertilized eggs and embryos were recovered and stained for lacZ at embryonic day (E) 11.5 and E13.5. At E11.5, five out of the 21 embryos we recovered carried the transgene as determined by the presence of the SalI polymorphism (Fig. 2C). Three embryos showed an identical pattern of lacZ activity (Fig. 2D) and two embryos showed no staining. Strikingly, expression from the transgene was restricted to a subset of the tissues that normally express p57Kip2 (Cdkn1c; Fig. 2E). Careful examination of transverse sections of one of the lacZ-stained embryos revealed expression in the ependymal layer of the neural tube (Fig. 2F), Rathke’s pouch (Fig. 2G) and the lens epithelium of the eye (Fig. 2H).
At E13.5, seven out of the 37 embryos were transgenic. Six of these showed an identical pattern of lacZ staining (Fig. 3I). Again, this was restricted compared with the endogenous expression pattern at E13.5 (Fig. 2J). In addition to expression within neural tissues and Rathke’s pouch (Fig. 2K), expression was also apparent in the epithelia of the lung (not shown) and in the podocytes of the kidney glomerulus and the adrenal cortex (Fig. 2L). However, there was a striking absence of lacZ expression in the cranial ganglia, the skeletal muscle within the limbs, body and tongue, the cardiac muscle and the mesenchymal cells of the palate.
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Expression of p57Kip2 (Cdkn1c) from the unmodified 85 kb BAC transgene
To exclude the possibility that our modification disrupted appropriate expression of the lacZ reporter under the p57Kip2 (Cdkn1c) promoter, expression from the unmodified BAC was examined. Embryonic stem cell lines were generated carrying 1–20 copies of the 144D14 BAC (Materials and Methods). Transgenic mice were established for three of these lines (1–2 copies). Expression was examined at E13.5 by in situ hybridization (Fig. 3A). To distinguish between expression from the endogenous locus and expression from the transgene, it was necessary to perform this analysis on embryos which had inherited a maternal deletion of p57Kip2 (Cdkn1c) gene (18). These embryos do not express any endogenous p57Kip2 (Cdkn1c) since the paternal allele is completely silent in mice (Fig. 3B). Consistent but restricted expression of p57Kip2 (Cdkn1c) was observed which exactly correlated with that observed for the modified transgene. The analysis was extended to extra-embryonic tissues. In non-transgenic embryos, there is marked expression of p57Kip2 (Cdkn1c) in the labyrinth zone of the placenta (Fig. 3C). This was not seen with either the unmodified 85 kb BAC transgene (Fig. 3D) or with the modified version (data not shown).
This work confirmed that enhancers for expression of p57Kip2 (Cdkn1c) in some neural tissues, Rathke’s pouch, the lung epithelium, the kidney, the adrenal cortex and the lens epithelium were present within the 85 kb transgene. Enhancers for expression in all muscle cell types, cartilage, cranial ganglia, the palate and the labyrinth layer of the placenta lie outside this region, at least 60 kb upstream of the p57Kip2 (Cdkn1c) gene or >25 kb downstream (Table 1).
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Expression of p57Kip2 (Cdkn1c) from a 260 kb BAC
To identify additional p57Kip2 (Cdkn1c) enhancers, we modified the 260 kb BAC clone, 35G23, which extends 10 kb upstream and 250 kb downstream of the gene (Fig. 1B). We used this modified BAC to generate three transgenic lines by pronuclear injection, lines 24-1 (three copies), 24-4 (eight copies) and 24-10 (12 copies). Line 24-4 showed no expression of the lacZ reporter. In lines 24-1 and 24-10, lacZ expression was observed in all the regions seen with the smaller BAC transgene. In addition, expression was seen in skeletal muscle and cartilage, in the cardiac muscle and the muscle of the tongue, the intestinal epithelium, the nasal epithelium and the palate (Fig. 4A). Importantly, in both cases no ectopic expression of the lacZ reporter was observed. The endogenous p57Kip2 (Cdkn1c) gene is not expressed in the liver at this stage. This tissue was negative for lacZ staining with both the expressing lines. External examination of an embryo at E13.5 revealed expression in the ear pinnae and the vibrissal follicles (Fig. 4B). All the enhancers for expression of p57Kip2 (Cdkn1c) in the embryo appeared to be present on the 260 kb transgene. However, no lacZ staining was observed in the placenta (Fig. 4C). The enhancer for expression of p57Kip2 (Cdkn1c) in the placenta must lie either more than 60 kb upstream or more than 250 kb downstream of the gene (Fig. 4D).
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p57Kip2 (Cdkn1c) transgenes do not show parent-of-origin expression or methylation differences
Finally, the imprinting capabilities of the p57Kip2 (Cdkn1c) transgenic lines were tested. The endogenous p57Kip2 (Cdkn1c) gene is only active after maternal transmission. In mice, no expression is seen after paternal transmission. Initially, paternal expression of the 85 kb unmodified BAC transgene was tested on the p57Kip2 (Cdkn1c) null background by in situ hybridization. The pattern of expression was identical to that seen for maternal transmission of the transgene (Fig. 3A and Table 1). All three lines carrying the 85 kb unmodified BAC (1–2 copies) showed a similar pattern of expression after paternal inheritance. The in situ hybridization signal from the transgene (transgene on a null background) was compared with the signal from the endogenous gene (non-transgenic embryo). The levels of expression from the transgenes were comparable with wild-type levels. Subsequently we also generated one transgenic line carrying three copies of the modified 85 kb BAC, line 10-15. The lacZ expression pattern in this line was identical to that seen for the pre-germline embryos (data not shown). Again, the pattern and level of expression after maternal and paternal inheritance was indistinguishable in all tissues and at all stages examined (Table 1). None of the four lines we tested for the 85 kb BAC showed any indication of paternal silencing.
The three BAC transgenic lines carrying the 260 kb modified transgene were also tested for their ability to imprint. In lines 24-1 and 24-10, the pattern and degree of staining for the lacZ reporter gene after maternal and paternal transmission were indistinguishable. The reporter remained silent in line 24-4 (Table 1).
We also examined the methylation status of the p57Kip2 (Cdkn1c) gene in our transgenic lines. No difference was detected in the methylation status of this locus after maternal and paternal inheritance (data not shown) consistent with the comparable levels of lacZ expression we observed. A second imprinted locus, the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island, was also present on the 260 kb transgene. We therefore examined the methylation status of this island in all three lines after maternal and paternal inheritance of the transgene. Again, no parent-of-origin difference in the methylation was observed (data not shown). We found no evidence for imprinting of either the p57Kip2 (Cdkn1c) gene or the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island in any of the transgenic lines.
Our transgenic analysis of the mouse p57Kip2 (Cdkn1c) locus demonstrates that key cis-regulatory elements probably lie at a distance from the gene. All the p57Kip2 (Cdkn1c) embryonic enhancers appear to lie downstream of the p57Kip2 (Cdkn1c) promoter and some are >25 kb downstream, probably within a second imprinted gene, Kvlqt1 (Kcnq1). In addition, we found no evidence for imprinting of p57Kip2 (Cdkn1c) from our transgenes, suggesting that important imprinting elements are also not closely associated with the gene.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent studies on BWS patients suggest a major role for p57KIP2 (CDKN1C) in the pathogenesis of this disease (7,12); but paradoxically, few patients present with mutations within the coding sequence of this gene (4–7). We previously suggested that BWS might occur through a physical or epigenetic separation of p57KIP2 (CDKN1C) from key regulatory elements, located at a distance from the gene (35). Our characterization of a 315 kb region spanning the mouse p57Kip2 (Cdkn1c) locus supports this conclusion.
We observed restricted expression of p57Kip2 (Cdkn1c) from the 85 kb transgene which contains 60 kb of sequence from upstream of the gene and 25 kb of sequence from downstream. In tissues where expression was observed, the levels were comparable to the endogenous gene. In contrast, a human transgene with 20 kb of upstream sequence and 15 kb of sequence downstream to the gene showed expression at low levels (35). This previous study led us to suggest that the human transgene either lacked enhancers or a key imprinting regulatory element required for activation after maternal inheritance. We can now conclude that the reduced expression from the human transgene was most likely due to the absence of enhancers, many of which seem to lie at a distance from the homologous mouse gene.
Since we observed the full pattern of expression of p57Kip2 (Cdkn1c) in the embryo from a 260 kb transgene, the remaining enhancers for skeletal muscle, cardiac muscle, cartilage, cranial ganglia, intestine and the palate must lie within a 225 kb region downstream of the gene, the majority of which is encompassed by the Kvlqt1 (Kcnq1) gene (Fig. 4D). Enhancers for expression in neural tissues, Rathke’s pouch, the lens epithelium, the lung epithelium, the kidney and the adrenal cortex must lie within the minimal overlap between the two transgenes, a region of 35 kb (Fig. 4D). Given the high degree of conservation between human and mouse sequences at the p57Kip2 (Cdkn1c) locus (15,16) it is likely that, in both species, all these enhancers lie 3' to the gene. As yet, we have been unable to identify the enhancer for expression in the placenta which must lie outside the 315 kb region we have tested.
None of the transgenes we examined showed imprinting through parental-specific differences in expression or methylation. Transgenes of this large size range show efficient imprinting in other instances (28,30,37,38). In the case of Igf2r and Igf2/H19, known ICRs are present within the transgene regions tested. The Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island, which is present on our 260 kb transgene, resembles an ICR (12–14,32), therefore a lack of imprinting of p57Kip2 (Cdkn1c) is somewhat surprising. However, there is no direct evidence that Lit1 (Kcnq1ot1/Kvlqt1-AS) is an ICR for p57Kip2 (Cdkn1c). First, although the Lit1 (Kcnq1ot1/Kvlqt1-AS) transcript does resemble Igf2r-AS and Tsix in that it is antisense to an oppositely imprinted gene, originates at a differentially methylated CpG island and does not appear to be spliced or to encode any protein, Igf2r-AS and Tsix are antisense and overlap with their target imprinted genes, Igf2r and Xist (31,39). In contrast, Lit1 (Kcnq1ot1/Kvlqt1-AS) and p57Kip2 (Cdkn1c) are transcribed in the same orientation and they are 160 kb apart. Secondly, deletion of the human LIT1 (KCNQ1OT1/KvLQT1-AS) island in a somatic cell line resulted in LOI of KvLQT1 (KCNQ1) and SM34 but it caused hyperactivation of p57KIP2 (CDKN1C) (32). It is possible that the activation of p57KIP2 (CDKN1C) resulted from changes in transcriptional regulation rather than LOI or that this island is required only for maintenance of the imprint in somatic cells. Finally, it has been suggested that imprinted regulation of p57Kip2 (Cdkn1c) may resemble that seen at the H19/Igf2 locus, with the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island acting as a boundary element between p57Kip2 (Cdkn1c) and its enhancers (12,40). This study directly demonstrates that there are enhancers for p57Kip2 (Cdkn1c) that lie between the gene and the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island, which excludes this model.
Two explanations remain for our data. The unspliced Kvlqt1 (Kcnq1) transcript and Lit1 (Kcnq1ot1/Kvlqt1-AS) transcript may establish the imprint by antisense interference and subsequent spreading of imprint to p57Kip2 (Cdkn1c). If this is the case, we do not observe imprinting of p57Kip2 (Cdkn1c) from our 260 kb transgene because it lacks the Kvlqt1 (Kcnq1) promoter. However, our preferred explanation is that Lit1 (Kcnq1ot1/Kvlqt1-AS) is not the major ICR for p57Kip2 (Cdkn1c). Since we do not see imprinting of either p57Kip2 (Cdkn1c) or Lit1 (Kcnq1ot1/Kvlqt1-AS) in the transgene, other sequences outside the 260 kb region are required for the imprinting of these genes.
Our work has major implications for the study of BWS. The only genetic mutations in the BWS region consist of point mutations within the p57KIP2 (CDKN1C) gene and translocations within KvLQT1 (KCNQ1). The point mutations disrupt p57KIP2 (CDKN1C) activity directly. This study demonstrates that the translocations would result in a physical separation of p57Kip2 (Cdkn1c) from key regulatory elements, inactivating p57KIP2 (CDKN1C) indirectly. The simplest explanation is that BWS results from a physical separation of p57KIP2 (CDKN1C) from some of its enhancers (Fig. 5A). Alternatively, BWS may result from the physical separation of the gene from an activating ICR, which may be established by an interaction between LIT1 (KCNQ1OT1/KvLQT1-AS) and KvLQT1 (KCNQ1, Fig. 5B).
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The vast majority of BWS patients show epigenetic changes at a number of imprinted loci. In the Prader–Willi/Angelmann syndromes, epigenetic changes over several megabases are found associated with deletions of a small region near the SNRPN locus (41). Thus regions proposed to act as imprinting centres (ICs) regulate the imprinting of genes many megabases away. It remains possible that the key IC region within the 11p15/distal 7 domain is not LIT1 (KCNQ1OT1/KvLQT1-AS) or the region upstream of H19, but an element lying elsewhere. This would explain why none of the targeted deletions in mice of the known ICRs result in LOI of both Igf2 and Lit1 (Kcnq1ot1/Kvlqt1-AS). A single mutation at this hypothetical IC would account for those BWS patients with LOI of both IGF2 and LIT1 (KCNQ1OT1/KvLQT1-AS) (7) (Fig. 5C).
While we have not yet determined how the p57Kip2 (Cdkn1c) imprint is established, the presence of enhancers between p57Kip2 (Cdkn1c) and the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island excludes a simple boundary model. In addition, our analysis demonstrates that, similar to the Igf2 gene, crucial enhancers and ICRs for the mouse p57Kip2 (Cdkn1c) gene lie at a distance from the gene. Finally, in BWS cases where there are translocations within the 11p15 region, the disease may simply result from a physical separation of p57KIP2 (CDKN1C) from its regulatory sequences. Phenotypic analysis of p57Kip2 (Cdkn1c) function in the mouse and indirect evidence from human studies suggest that changes in p57KIP2 (CDKN1C) expression could be responsible for a large number of BWS patients. Our studies in the mouse reveal how these changes in expression may come about.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of p57Kip2 (Cdkn1c) BACs
One clone, 144D14, was isolated by screening a Genome Systems gridded library as described previously (35). Two additional clones were ordered directly from Genome Systems (36). BAC DNA digested with NotI was resolved on pulse-field gels and the position of p57Kip2 (Cdkn1c) determined by Southern blot analysis with the p57Kip2 (Cdkn1c) cDNA. Gel electrophoresis of HindIII and EcoRI digests was used to determine the degree of overlap between the clones. A PCR-based gene content analysis was performed using primers for exon 1 (R93, 5'-AGGGGACAAAGGCGTTCAGATG-3' and R94, 5'-GAACTTCCTCGTAGAACTTAGC-3') and exon 15 (R95, 5'-AGTTGGTGGTTGGTGTGTGTGG-3' and R96, 5'-GAGCACACTCTACAATGGCTGG-3') of Nap1l4 and the Kvlqt1 (Kcnq1) gene as described by Paulsen et al. (42). Note that the 5' and 3' primers for Kvlqt1 (Kcnq1) are reversed in this paper. Primers R27, 5'-ACTGAGAGCAAGCGAACA-3' and R28, 5'AAGCGTTCCATCGCTGTTCTG-3', which span intron 1 of the mouse p57Kip2 (Cdkn1c) gene, were used to detect a 120 bp product as described previously (35). Primer sc34.1 and sc34.2 were used to amplify the Lit1 (Kcnq1ot1/Kvlqt1-AS) CpG island as described previously by Smilinich et al. (13).
Modification of the BAC transgenes
To generate the targeting construct, a 3 kb genomic BamHI clone containing the p57Kip2 (Cdkn1c) gene was cloned into pKan. A SalI-LoxP linker (5'-CGTGGATCCATAACTTCGTATAGCATACATTATACGAAGTTAT-3') was inserted into a unique AflIII site in the 5'-UTR. A 5' 2 kb SacI fragment was cloned into a unique SacI site to extend the 5' sequence. This was excised with HindIII and inserted into a NotI site in a IRESßgeoloxPalkP cassette (38). A 2.8 kb HindIII genomic fragment was cloned into the unique SrfI site in the vector linker to provide homology on the 3' side of the modification. The p57Kip2-IRESßgeoloxPalkP construct was cloned into the SalI site of pSV1.RecA. This was transformed into chemical competent bacteria containing the target BAC clone (43). Co-integrant (BACs containing the original locus and the modified locus) and resolved (BACs which have lost either the original locus or the modified locus by homologous recombination) clones were detected by southern analysis of EcoRI/SalI digested DNA obtained by alkaline lysis from 2 ml cultures.
Analysis of transgenic animals
Transgenic founders were generated by pronuclear injection of SrfI-linearized BAC DNA (28). Genomic DNA was extracted as described by Hogan et al. (44) from yolk sacs, tail tips or whole embryos. Transgenic embryos and lines were characterized by hybridization of Southern blots of EcoRI/SalI-digested genomic DNA with a 0.5 kb XhoI-EagI fragment from the p57Kip2 (Cdkn1c) cDNA. Copy number was determined by comparing the intensity of hybridization of this fragment with the endogenous locus using a PhosphoImager. A comparison of hybridization signal of transgene end probes derived from the BAC vector was made to confirm the integrity of the transgene insertion events.
In situ hybridization and histology
In situ hybridization was performed using a 1.4 kb mouse p57Kip2 (Cdkn1c) cDNA probe. Sense and antisense RNA probes were prepared by in vitro transcription using the DIG RNA labelling kit (Roche Molecular Biochemicals). Ten micrometre sagittal and transverse sections from transgenic mouse embryos at E11.5 and E13.5 was used for in situ hybridization (35). For whole-mount lacZ staining, embryos were dissected free of extra-embryonic tissues and fixed for 1–3 h in formaldehyde (2%), glutaraldehyde (0.2%), NP-40 (0.02%), MgCl2 (1 mM) and sodium deoxychlolate (20 mM) at 4°C and washed three times in PBS before staining for 16 h at room temperature in ß-galactosidase (0.4 mg/ml), potassium ferricyanide (4 mM), potassium ferrocyanide (4 mM), MgCl2 (2 mM) in PBS. At E13.5, a mid-sagittal incision was made midway through fixation to facilitate penetration of the stain. Embryos were washed extensively in PBS post-staining, fixed overnight in 4% formaldehyde and cleared in 70% ethanol. LacZ-stained embryos were dehydrated through ascending alcohol series, cleared in xylene and embedded in fibrowax (BDH). Sagittal and transverse sections were cut at 8 µm, mounted on slides, dewaxed and rehydrated in water through descending alcohol series and counterstained with 4% eosin. Mounted sections were photographed under dark field where the lacZ signal is pink.
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ACKNOWLEDGEMENTS |
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Our thanks go to Dr Paul Schofield for his insightful comments on this manuscript, to Kathy Hilton for technical assistance and to members of the Surani and McLaren laboratories for their support. This work was funded by the Wellcome Trust Grant 036481.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1223 334138; Fax: +44 1223 334089; Email: rmj22@cus.cam.ac.uk
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Beckwith, J.B. (1969) Macroglossia, omphalocele, adrenal cytomegaly, giantism, and hyperplastic visceromegaly. Birth Defects, 5, 188–196.
2 Wiedemann, H.R. (1983) Tongues and hemihypertrophy associated with Wiedemann–Beckwith syndrome. Eur. J. Paediatr., 141, 129.[Web of Science]
3 Elliott, M. and Maher, E.R. (1994) Beckwith–Wiedemann syndrome. J. Med. Genet., 31, 560–556.
4 Hatada, I., Ohashi, H., Fukushima, Y., Kaneko, Y., Inoue, M., Komoto, Y., Okada, A., Ohishi, S., Nabetani, A., Morisaki, H. et al. (1996) An imprinted gene p57(KIP2) is mutated in Beckwith–Wiedemann syndrome. Nat. Genet., 14, 171–173.[Web of Science][Medline]
5 O’Keefe, D., Dao, D., Zhao, L., Sanderson, R., Warburton, D., Weiss, L., Anyane-Yeboa, K. and Tycko, B. (1997) Coding mutations in p57KIP2 are present in some cases of Beckwith–Wiedemann syndrome but are rare or absent in Wilms tumors. Am. J. Hum. Genet., 61, 295–303.[Web of Science][Medline]
6 Lee, M.P., DeBaun, M., Randhawa, G., Reichard, B.A., Elledge, S.J. and Feinberg, A.P. (1997) Low frequency of p57KIP2 mutation in Beckwith–Wiedemann syndrome. Am. J. Hum. Genet., 61, 304–349.[Web of Science][Medline]
7 Engel, J.R., Smallwood, A., Harper, A., Higgins, M.J., Oshimura, M., Reik, W., Schofield, P.N. and Maher, E.R. (2000) Epigenotype-phenotype correlations in Beckwith–Wiedemann syndrome. J. Med. Genet., 37, 921–926.
8 Weksberg, R., Shen, D.R., Fei, Y.L., Song, Q.L. and Squire, J. (1993) Disruption of insulin-like growth factor 2 imprinting in Beckwith–Wiedemann syndrome. Nat. Genet., 5, 143–150.[Web of Science][Medline]
9 Joyce, J.A., Lam, W.K., Catchpoole, D.J., Jenks, P., Reik, W., Maher, E.R. and Schofield, P.N. (1997) Imprinting of IGF2 and H19: lack of reciprocity in sporadic Beckwith–Wiedemann syndrome. Hum. Mol. Genet., 6, 1543–1548.
10 Hoovers, J.M.N., Kalikin, L.M., Johnson, L.A., Alders, M., Redeker, B., Law, D.J., Bliek, J., Steenman, M., Benedict, M., Wiegant, J. et al. (1995) Multiple genetic loci within 11p15 defined by Beckwith–Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments. Proc. Natl Acad. Sci. USA, 92, 12456–12460.
11 Lee, M.P., Hu, R.J., Johnson, L.A. and Feinberg, A.P. (1997) Human KvLQT1 gene shows tissue-specific imprinting and encompasses Beckwith–Wiedemann syndrome chromosomal rearrangements. Nat. Genet., 15, 181–185.[Web of Science][Medline]
12 Lee, M.P., DeBaun, M.R., Mitsuya, K., Galonek, H.L., Brandenburg, S., Oshimura, M. and Feinberg, A.P. (1999) Loss of imprinting of a paternally expressed transcript, with antisense orientation to KvLQT1, occurs frequently in Beckwith–Wiedemann syndrome and is independant of insulin-like growth factor II imprinting. Proc. Natl Acad. Sci. USA, 96, 5203–5208.
13 Smilinich, N.J., Day, C.D., Fitzpatrick, G.V., Caldwell, G.M., Lossie, A.C., Cooper, P.R., Smallwood, A.C., Joyce, J.A., Schofield, P.N., Reik, W. et al. (1999) A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith–Wiedemann syndrome. Proc. Natl Acad. Sci. USA, 96, 8064–8069.
14 Mitsuya, K., Meguro, M., Lee, M.P., Katoh, M., Schulz, T.C., Kugoh, H., Yoshida, M.A., Niikawa, N., Feinberg, A.P. and Oshimura, M. (1999) LIT, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. Hum. Mol. Genet., 8, 1209–1217.
15 Onyango, P., Miller, W., Lehoczky, J., Leung, C.T., Birren, B., Wheelan, S., Dewar, K. and Feinberg, A.P. (2000) Sequence and comparative analysis of the mouse 1-megabase region orthologous to the human 11p15 imprinted domain. Genome Res., 10, 1697–1710.
16 Engemann, S., Strodicke, M., Paulsen, M., Franck, O., Reinhardt, R., Lane, N., Reik, W. and Walter, J. (2000) Sequence and functional comparison in the Beckwith–Wiedemann region: implications for a novel imprinting centre and extended imprinting. Hum. Mol. Genet., 9, 2691–2706.
17 Bliek, J., Maas, S.M., Ruijter, J.M., Hennekam, R.C., Alders, M., Westerveld, A. and Mannens, M.M.A.M. (2001) Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1QT1 methylation: occurence of KCNQ1QT1 hypomethylation in familial cases of BWS. Hum. Mol. Genet., 10, 467–476.
18 Zhang, P., Liegeois, N.J., Wong, C., Finegold, M., Hou, H., Thompson, J.C., Silverman, A., Harper, J.W., DePinho, R.A. and Elledge, S.J. (1997) Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith–Wiedemann syndrome. Nature, 387, 151–158.[Medline]
19 Yan, Y., Frisen, J., Lee, M.H., Massague, J. and Barbacid, M. (1997) Ablation of the CDK inhibitor p57Kip2 results in increased apoptosis and delayed differentiation during mouse development. Genes Dev., 11, 973–983.
20 Ward, A., Bates, P., Fisher, R., Richardson, L. and Graham, C.F. (1994) Disproportionate growth in mice with Igf-2 transgenes. Proc. Natl Acad. Sci. USA, 91, 10365–10369.
21 Wolf, E., Kramer, R., Blum, W.F., Foll, J. and Brem, G. (1994) Consequences of postnatally elevated insulin-like growth factor-II in transgenic mice: endocrine changes and effects on body and organ growth. Endocrinology, 135, 1877–1886.[Abstract]
22 Eggenschwiler, J., Ludwig, T., Fisher, P., Leighton, P.A., Tilghman, S.M. and Efstratiadis, A. (1997) Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes. Genes Dev., 11, 3128–3142.
23 Sun, F.-l., Dean, W., Kelsey, G., Allen, N.D. and Reik, W. (1997) Transactivation of Igf2 in a mouse model of Beckwith–Wiedemann syndrome. Nature, 389, 809–815.[Medline]
24 Caspary, T., Cleary, M.A., Perlman, E.J., Zhang, P., Elledge, S.J. and Tilghman, S.M. (1999) Oppositely imprinted genes p57(Kip2) and Igf2 interact in a mouse model for Beckwith–Wiedemann syndrome. Genes Dev., 13, 3115–3124.
25 Caspary, T., Cleary, M.A., Baker, C.C., Guan, X.J. and Tilghman, S.M. (1998) Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster. Mol. Cell Biol., 18, 3466–3474.
26 Lee, M.P., Brandenburg, S., Landes, G.M., Adams, M., Miller, G. and Feinberg, A.P. (1999) Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting. Hum. Mol. Genet., 8, 683–690.
27 Paulsen, M., El-Maarri, O., Engemann, S., Strodicke, M., Franck, O., Davies, K., Reinhardt, R., Reik, W. and Walter, J. (2000) Sequence conservation and variability of imprinting in the Beckwith–Wiedemann syndrome gene cluster in human and mouse. Hum. Mol. Genet., 9, 1829–1841.
28 Ainscough, J.F.-X., Koide, T., Tada, M., Barton, S. and Surani, M.A. (1997) Imprinting of Igf2 and H19 from a 130kb YAC transgene. Development, 124, 3621–3632.[Abstract]
29 Thorvaldsen, J.L., Duran, K.L. and Bartolomei, M.S. (1998) Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev., 12, 3693–3702.
30 Wutz, A., Smrzka, O.W., Schweifer, N., Schellander, K., Wagner, E.F. and Barlow, D.P. (1997) Imprinted expression of the Igf2r gene depends on an intronic CpG island. Nature, 389, 745–749.[Medline]
31 Lyle, R., Watanabe, D., te Vruchte, D., Lerchner, W., Smrzka, O.W., Wutz, A., Schageman, J., Hahner, L., Davies, C. and Barlow, D.P. (2000) The imprinted antisense RNA at the Igf2r locus overlaps but does not imprint Mas1. Nat. Genet., 25, 19–21.[Web of Science][Medline]
32 Horike, S., Mitsuya, K., Meguro, M., Kotobuki, N., Kashiwagi, A., Notsu, T., Schulz, T.C., Shirayoshi, Y. and Oshimura, M. (2000) Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith–Wiedemann syndrome. Hum. Mol. Genet., 9, 2075–2083.
33 Grandjean, V., Smith, J., Schofield, P.N. and Ferguson-Smith, A.C. (2000) Increased IGF-II protein affects p57Kip2 expression in vivo and in vitro: implications for Beckwith–Wiedemann syndrome. Proc. Natl Acad. Sci. USA, 97, 5279–5284.
34 Brenton, J.D., Drewell, R.A., Viville, S., Hilton, K.J., Barton, S.C., Ainscough, J.F. and Surani, M.A. (1999) A silencer element identified in Drosophila is required for imprinting of H19 reporter transgenes in mice. Proc. Natl Acad. Sci. USA, 96, 9242–9247.
35 John, R.M., Hodges, M., Little, P., Barton, S.C. and Surani, M.A. (1999) A human p57KIP2 transgene is not activated by passage through the maternal mouse germline. Hum. Mol. Genet., 8, 2211–2219.
36 Gould, T.D. and Pfeifer, K. (1998) Imprinting of mouse Kvlqt1 is developmentally regulated. Hum. Mol. Genet., 7, 483–487.
37 Shemer, R., Hershko, A.Y., Perk, J., Mostoslavsky, R., Tsuberi, B., Cedar, H., Buiting, K. and Razin, A. (2000) The imprinting box of the Prader–Willi/Angelman syndrome domain. Nat. Genet., 26, 440–443.[Web of Science][Medline]
38 John, R.M., Aparicio, S.A.J.R., Ainscough, J.F.-X., Arney, K.L., Khosla, S., Hilton, K.J., Barton, S.C. and Surani, M.A. (2001) Imprinted expression of neuronatin from modified BAC transgenes reveals regulation by distinct and distant enhancers. Dev. Biol., in press.
39 Lee, J.T. (2000) Disruption of imprinted X inactivation by parent-of-origin effects at Tsix. Cell, 103, 17–27.[Web of Science][Medline]
40 Reik, W. and. Walter, J. (2001) Genomic Imprinting: Parental influence on the genome. Nat. Rev. Genet. 2, 21–32.
41 Buiting, K., Saitoh, S., Gross, S., Dittrich, B., Schwartz, S., Nicholls, R.D. and Horsthemke, B. (1995) Inherited microdeletions in the Angelman and Prader–Willi syndromes define an imprinting centre on human chromosome 15. Nat. Genet., 9, 395–400.[Web of Science][Medline]
42 Paulsen, M., Davies, K.R., Bowden, L.M., Villar, A.J., Franck, O., Fuermann, M., Dean, W.L., Moore, T.F., Rodrigues, N., Davies, K.E. et al. (1998) Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith–Wiedemann syndrome region in chromosome 11p15.5. Hum. Mol. Genet., 7, 1149–1159.
43 Yang, X.W., Model, P. and Heintz, N. (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat. Biotechnol., 15, 859–865.[Web of Science][Medline]
44 Hogan, B., Beddington, R., Constantini., F. and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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