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Human Molecular Genetics Pages 1149-1159  

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5
Introduction
Results
   Genetic mapping and establishment of a physical contig from Nap1l4 to H19
   Structure and imprinting of Kcnq1
   Mouse Nap1l4 is not imprinted
Discussion
Materials And Methods
   Libraries and clones
   DNA and RNA preparations
   Field inversion gel electrophoresis (FIGE) and pulse-field gel electrophoresis (PFGE)
   Hybridization probes
   Hybridization of immobilized DNA and RNA
   Genotyping by allele-specific PCR
   Quantification of allele-specific RT-PCR products
   Single nucleotide primer extension (SNuPE)
   5[prime] Rapid amplification of cDNA ends (RACE)
   Sequencing
Acknowledgements
References

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5

Martina Paulsen1,2, Karen R. Davies1, Lucy M. Bowden1, Angela J. Villar1,3, Olivia Franck2, Martina Fuermann2, Wendy L. Dean1, Tom F. Moore1, Nanda Rodrigues4, Kay E. Davies4, Ren-J. Hu5, Andrew P. Feinberg5, Eamonn R. Maher6, Wolf Reik1,*, Jörn Walter2

1Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB2 4AT, UK, 2Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany, 3Department of Pediatrics, University of California, San Francisco, CA 94143-0546, USA, 4Genetics Laboratory, Biochemistry Department, University of Oxford, South Parks Road, Oxford OX1 3QU, UK, 5Department of Medicine and Departments of Molecular Biology & Genetics and Oncology, John Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA and 6Division of Medical and Molecular Genetics, Department of Paediatrics and Child Health, University of Birmingham, Medical School, Edgbaston, Birmingham B15 2TG, UK

Received February 17, 1998; Revised and Accepted April 24, 1998

In human and mouse, most imprinted genes are arranged in chromosomal clusters. Their linked organization suggests co-ordinated mechanisms controlling imprinting and gene expression. The identification of local and regional elements responsible for the epigenetic control of imprinted gene expression will be important in understanding the molecular basis of diseases associated with imprinting such as Beckwith-Wiedemann syndrome. We have established a complete contig of clones along the murine imprinting cluster on distal chromosome 7 syntenic with the human imprinting region at 11p15.5 associated with Beckwith-Wiedemann syndrome. The cluster comprises ~1 Mb of DNA, contains at least eight imprinted genes and is demarcated by the two maternally expressed genes Tssc3 (Ipl) and H19 which are directly flanked by the non-imprinted genes Nap1l4 (Nap2) and Rpl23l (L23mrp), respectively. We also localized Kcnq1 (Kvlqt1) and Cd81 (Tapa-1) between Cdkn1c (p57Kip2) and Mash2. The mouse Kcnq1 gene is maternally expressed in most fetal but biallelically transcribed in most neonatal tissues, suggesting relaxation of imprinting during development. Our findings indicate conserved control mechanisms between mouse and human, but also reveal some structural and functional differences. Our study opens the way for a systematic analysis of the cluster by genetic manipulation in the mouse which will lead to animal models of Beckwith-Wiedemann syndrome and childhood tumours.

INTRODUCTION

Imprinted genes are those genes in the genome that are expressed predominantly from only one of the parental chromosomes (1-7). The imprinting mechanism involves epigenetic modifications of DNA such as DNA methylation. Imprinted genes play important roles in development, in particular in fetal growth, placental development, cell proliferation and behaviour after birth. Deregulation of imprinting is implicated in a number of human cancers and genetic diseases.

A significant number of imprinted genes are organized in clusters in the genome. Imprinting clusters seem to be embedded in chromosomal domains characterized by epigenetic features such as differences in DNA replication and meiotic recombination frequencies between the two parental copies (8-11). This suggests that aspects of regulation of imprinting could occur at the level of the whole cluster. Indeed in the PWS/AS cluster (see below), an `imprinting centre' exists that controls imprinting of genes in the cluster (5,12). Whether other clusters have `imprinting centres' is not known. Clustering could also implicate more local relationships between individual genes in terms of co-ordinated regulation and perhaps also of functional interaction. Mechanistic interactions of clustered imprinted genes may occur, for example by enhancer competition or cis-acting RNAs (1,3,6). In addition, individual imprinted genes have gene-specific control elements that are likely to be involved in tissue- and developmental-specific regulation of imprinting (1,6).

In humans, two major imprinting clusters are associated with two of the classical imprinting diseases, Prader-Willi/Angelman syndromes (PWS/AS) and Beckwith-Wiedemann syndrome (BWS), respectively. The PWS/AS cluster on chromosome 15 contains at least six imprinted genes (4,5,13,14). An `imprinting centre' has been identified for this cluster: a proportion of PWS and AS patients show deregulated imprinting probably throughout the cluster, and mutations (imprinting mutations) have been identified in SNRPN exon 1 (PWS) and upstream RNA transcripts of the SNRPN gene (AS) (5,12). It has been postulated that the upstream transcript itself interacts in cis with an imprinting initiation site (or imprinting initiation centre) in SNRPN exon 1 and thereby promotes the switch from the paternal to the maternal imprint in the maternal germline (12).

The BWS-associated imprinting cluster is on chromosome 11p15.5 and currently contains the paternally expressed insulin-like growth factor 2 (IGF2) (15,16) and insulin (INS) (17) genes, and the maternally expressed H19 (18), ASCL2 (19), CDKN1C (p57KIP2) (20), TSSC3 (IPL) (21) and KCNQ1 (KVLQT1) (22) genes. Clone contigs of the human cluster (800-1000 kb long) have been established (23-25), and flanking non-imprinted genes [NAP1L4 (NAP2) on the centromeric side (26) and RPL23L (L23MRP) on the telomeric side (27)] have been identified.

In the fetal overgrowth and tumour syndrome, BWS, the most common molecular abnormality in patients is biallelic (instead of monoallelic) expression of IGF2 [loss of imprinting (LOI)] (28-30). This can be associated with hypermethylation of the maternal alleles of H19 and IGF2 (31). LOI might be caused by translocations (32) or other mutations in BWS patients. One translocation breakpoint cluster has been mapped to the KCNQ1 gene centromeric to IGF2 (22-24,33,34). The other translocation breakpoint cluster is several megabases further centromeric (23,33). The translocations might disrupt an important imprinting element (such as an imprinting centre or maintenance element), or a gene whose expression is important for the control of regional imprinting. Translocations could also separate parts of the imprinting cluster from necessary cis-acting elements. A minority of patients have mutations in CDKN1C (35-37), but whether these lead to LOI or otherwise increased IGF2 expression is not known. The mutations and molecular pathology of BWS are therefore complex, and may involve several genes in the cluster (with possibly an ultimate effect on IGF2 action) (30).

Mechanistic aspects of regulation and deregulation of imprinted genes in the cluster together with their phenotypic consequences can be best addressed in mouse transgenic and knockout models. In the mouse, the BWS-associated imprinting cluster is located on distal chromosome 7. Several knockouts and overexpression transgenics have provided important insights into the phenotypes and regulation of genes in the cluster, and have resulted in valuable mouse models of BWS (38-41). However, detailed information about the physical organization of the equivalent region in the mouse is missing. The region from Rpl23l to Cdkn1c has been shown to be physically linked in the mouse (42-44) and to contain the Kcnq1 gene as does the human BWS cluster (44). We have established a contig of phage P1-derived (P1), bacterial (BAC) and yeast artificial chromosome (YAC) clones for the entire cluster and show that the syntenic region in mouse and human also covers the Nap1l4 gene. We present a detailed physical map of the imprinting cluster in the mouse and an imprinting analysis of the Nap1l4 and Kcnq1 genes.

RESULTS

Genetic mapping and establishment of a physical contig from Nap1l4 to H19

A genetic map of the region was generated using a backcross of SD7/+ males against +/+ females (SD7 is a congenic mouse strain which carries a defined segment of distal chromosome 7 of Mus spretus on a Mus musculus domesticus background). A total of 133 progeny from the cross were typed by PCR followed by restriction enzyme digestion which allowed us to distinguish between M.m.domesticus and M.spretus alleles for Nap1l4, Cdkn1c, Kcnq1, H19, Rpl23l and Nttp1 (45). One recombination event was observed between Kcnq1 and H19, and two recombination events were observed between Rpl23l and Nttp1. This allowed us to localize Nttp1 to the downstream region of H19 (Fig. 1). The order of these genes on distal chromosome 7 is (Nap1l4/Cdkn1c/Kcnq1)-(0.75 cM)-(H19/Rpl23l)-(1.5 cM)-Nttp1.In order to see whether imprinted genes on distal chromosome 7 of the mouse are indeed confined to the imprinting cluster, we examined the imprinting status of Nttp1. Our RT-PCR analysis revealed biallelic expression of Nttp1 in all tissues analysed (data not shown).


Figure 1. The imprinting cluster on distal chromosome 7. (A) Schematic physical map of the imprinting cluster on distal chromosome 7. The relative positions of Mash2, Ins2, Igf2, H19 and Rpl23l (42,43), respectively, are based on previously published data. The map is completed by our data on the positions of Nap1l4, Tssc3, Cdkn1c, Kcnq1, Cd81 and Nttp1, and the position of Th in mouse has not been mapped so far. Th is present in the human imprinting cluster on chromosome 11p15.5 (24,25) and is therefore shown in parentheses. The gap in the bar between Rpl23l and Nttp1 indicates that the physical distance between Nttp1 and the imprinting cluster is so far unknown. Filled boxes, maternally expressed genes; hatched boxes, paternally expressed genes; open boxes, biallelically expressed genes; grey boxes, the imprinting status in the mouse has not been determined. YACs are drawn as thin black bars according to their position. The orientation of transcription of the genes is indicated by arrows. (B) Detailed map of the Nap1l4 to Mash2 region. Recognition sites of restriction enzymes are depicted as vertical lines: B, BssHII; M, MluI; No, NotI; S, SalI. The restriction map is based on the analysis of at least two independent clones. Genes are shown as grey boxes. P1 clones and BAC clones are shown underneath. The exons of Kcnq1 are numbered in accordance with the designation of exons of the human KCNQ1 gene (22). Exons are drawn as black bars if their positions have been determined. Grey exons have not been mapped so far but are expected in the shown positions according to the synteny with human KCNQ1 (22).

In order to show that the genes between Nap1l4/Cdkn1c and H19/Rpl23l are not only genetically linked but localized in the same cluster syntenic with the human BWS cluster, we established a contig of BAC, P1 and YAC clones for this region. These clones were isolated using genomic and cDNA probes from Igf2, Ins2, Mash2, Cdkn1c, Nap1l4 and Kcnq1 which were presumed to map to the region (Fig. 1). The BAC, P1 and YAC inserts were linked by Southern blot hybridization using (i) the aforementioned genomic and cDNA probes; (ii) intact inserts of the clones; and (iii) probes derived from the ends of the cloned DNA fragments (see Materials and Methods). A physical map of these genes was constructed and a basic restriction map was established for the region from Nap1l4 to Mash2. The whole cluster from Nap1l4 to H19 is ~1 Mb in length. The positions of Nap1l4, Cdkn1c, Tssc3, Cd81 (Tapa-1) (46) and Kcnq1 were established on the map along with their transcriptional orientation. All these genes are transcribed in the same direction, with the exception of Kcnq1 and Cd81 that are transcribed in the opposite direction. These results demonstrate complete synteny of this segment of distal mouse chromosome 7 with the human 11p15.5 region (24,25).

Structure and imprinting of Kcnq1

Although there is considerable information concerning human KCNQ1 (which encodes a potassium channel protein), little is known about the genomic structure of murine Kcnq1.Using the mouse cDNA sequence of Kcnq1 (accession no. U70068), we were able to localize its position between Cd81 and Cdkn1c and to place several exons on the physical map (Fig. 1B). Human KCNQ1 has at least two different 5[prime] leader exons, with additional splice variants that can give rise to untranslated RNA isoforms (22). To establish whether this is also the case in mouse, we carried out 5[prime] RACE using primers in exons 2 and 3 to identify all upstream exons. Our results showed that, in addition to the published mouse 5[prime] leader exon (47) (henceforth referred to as 1[alpha]), a novel sequence was identified which did not show homology to any known sequences. Subsequent RT-PCR using primers specific for the 5[prime] end of the new sequence and exon 2 confirmed the existence of the original 5[prime] RACE product and resulted in three additional RT-PCR products. Sequencing and Southern blot analysis revealed the existence of four new exons (1[beta]-1[epsis]) (accession nos AJ002199, AJ002200, AJ002201, AJ002202) downstream of exon 1[alpha]. It should be noted that sequences corresponding to exons 1b, 1c and 2a described in the human (22) were not identified in the mouse. The exons 1[alpha]-1[epsis] are differentially spliced onto exon 1 so that, in all, there are at least five different splice variants of the Kcnq1 RNAinthe mouse. We expect the two promoters of these transcripts to be upstream of 1[alpha] and of 1[beta], respectively. The splice variants I and II contain an `in-frame' ATG and therefore have the potential to be translated (Fig. 2B). Assuming that splice variant II is translated, it would result in an altered form of the predicted first transmembrane domain of the functional protein (S1) (47). This isoform could function to inhibit the formation of functional Kcnq1 channels in a similar fashion to the truncated isoform 2 identified in humans (48). We predict that splice variants III and IV are untranslated as they contain stop codons upstream of exon 1 `in-frame' with the rest of the Kcnq1 transcript. While they do contain start codons in alternative reading frames, these are unlikely to be translated because they either would produce very small peptides (<20 amino acids) or do not meet the criteria for functional ATG start codons (49). Splice variant V does not contain any `in-frame' start or stop codons upstream of exon 1 so we are unable to predict if this splice variant is translated. It is possible that the transcribed sequence may start further 5[prime].


Figure 2. Imprinting of the Kcnq1 gene. (A)Representation of the different regions identified by 5[prime] RACE and RT-PCR. M denotes in-frame start codons. The relative positions of the exons 1[delta] and 1[epsis] are not known. Splice variant I is identical to the published mouse sequence (accession no. U70068). Splice variant II contains an in-frame start codon, and this leads to the alteration of the predicted first transmembrane S1 domain. (B) Comparison of the published amino acid sequence [splice variant I in (A)] with that of the predicted amino acid sequence for the novel translated splice variant II. The shaded sequence corresponds to the beginning of exon 1 and the position of the S1 domain of splice variant I is shown. (C)Stage- and tissue-specific allelic expression patterns of murine Kcnq1, determined using RT-PCR to analyse tissues from C57BL/6×SD7F1 mice. A 10 bp length polymorphism was identified between M.spretus and M.m.domesticus. Base pairs 2287-2296 of the published sequence (M.musculus) are directly duplicated in M.spretus. The results obtained were confirmed through the analysis of the reciprocal cross (SD7 × C57BL/6, data not shown). The only exception was the spleen, where a genotypic effect was observed, with the M.domesticus allele being more highly expressed regardless of parental origin.

We also performed RT-PCR in order to try and establish if there was a difference in the spatial and/or temporal expression of splice variants I and II, as both are potentially translated. PCR primers were designed to amplify from exon 1[alpha] to exon 2, and from exon 1[delta] to exon 2. Both splice variants are transcribed in all tissues analysed, and no significant differences in site or level of expression were found (data not shown). This does not allow any conclusions to be drawn about the imprinting status of these splice variants or their biological roles. It should be noted that nothing is known about the expression patterns, relative abundance or imprinting status of the other splice variants.

It has been shown that Kcnq1 is maternally expressed during the early stages of development and that the paternal allele progressively becomes active so that in late juvenile and adult mice Kcnq1 is expressed biallelically (44). Our results on Kcnq1 imprinting confirm and extend these observations. We found that Kcnq1 is expressed in all embryonic and neonatal tissues analysed using RT-PCR (Fig. 2C). Using a length polymorphism in the 3[prime]-untranslated region (UTR) of the gene, we observed predominantly maternal expression in all fetal tissues derived from (M.m.domesticus × SD7) F1 embryos and from reciprocal crosses at day 15 of gestation, with the exception of the brain, which showed biallelic expression (Fig. 2C). In neonates, both parental alleles were expressed in all tissues. However, there remained a slight bias towards the maternal allele, which was particularly prominent in the tongue (Fig. 2C). These results contrast with those obtained for human KCNQ1 for which maternal expression was observed in all expressing fetal tissues except in the heart where biallelic expression was seen (22).

Mouse Nap1l4 is not imprinted

The human NAP1L4 gene encodes a nucleosome assembly protein (NAP) which is part of a small protein family and is located next to TSSC3 (IPL)and CDKN1C (25,26). The human NAP1L4 gene was found to be expressed biallelically in fetal tissues (26). This suggested a boundary between imprinted and non-imprinted genes at the centromeric end of the 11p15.5 cluster between NAP1L4 and TSSC3 (IPL).

Hybridization of the human NAP1L4 cDNA (26) to the contig confirmed the map location of mouse Nap1l4 proximal of Cdkn1c. A mouse cDNA clone for Nap1l4 was isolated using a murine PCR probe which corresponded to exon 11 of the human cDNA. To confirm that we had isolated a full-length cDNA, 5[prime] RACE experiments were performed which extended the length of the cDNA sequence by 28 nucleotides. The coding sequence of the mouse cDNA (accession no. AJ002198) shows 86% identity to the coding sequence of the human cDNA (accession no. U77456) leading to 95% identity for the peptide sequences.

Sequencing of RT-PCR products from fetal RNA revealed two additional splice variants (Fig. 3A). In the peptide sequences of the variants II and III, the last valine and stop codon are replaced by KEPSQPAECKQQ (Fig. 3A). This ECKQ motif is highly conserved in all known NAPs, suggesting that it may be functionally significant. Additionally, in isoform III, the C-terminal acidic domain is shortened by 10 amino acid residues. This truncation might affect the function of the protein since the acidic domain has been shown to be essential for the function of the human NAP1L4 (50). Although the relative abundance of the splice variants has not been quantified exactly, splice variant II appears to be the predominant form in all tissues analysed by RT-PCR.


Figure 3. Expression of Nap1l4. (A) Alternative splicing in the coding region of Nap1l4. The three different splice variants were detected by RT-PCR in total RNA from embryo (15 d.p.c.). Splice variant I corresponds to the published sequence (accession no. AJ002198). The affected exons 11-15 are numbered in the same way as the homologous exons of the splice variant I of human NAP1L4 (49). The exons are located at the following positions in the mouse Nap1l4 cDNA sequence: exon 11, nucleotides 982-1101; exon12, 1102-1131; exon 13, 1132-1188; exon 14, 1189-1223; exon 15, 1224-2301. Stop codons in the splice variants are shown. The correspondingdeduced C-terminal peptide sequences of the splice variants I-III start at nucleotide 1078 in exon 11. The C-terminal acidic domains are shown as grey boxes. (B) Northern analysis of maternal disomy distal 7 fetuses and placenta shows an ~1.5-fold increase in Nap1l4 RNA compared with controls. The maternally expressed genes Cdkn1c and H19, and the biallelically expressed Gapdh gene are shown for comparison. (C) Ratio of transcripts (maternal disomy distal 7 RNA/control RNA) derived from the northern blot shown in (B). Amounts of transcripts were quantified based on normalization against Gapdh expression. (D)RT-PCR analysis using a length polymorphism which distinguishes between the parental alleles shows equal expression from both alleles in tissues from (C57BL/6 × SD7) and (SD7 × C57BL/6) embryos (15 d.p.c.).

Northern blot analysis using the Nap1l4 cDNA as a probe revealed widespread expression of a 2.3 kb transcript in all neonatal tissues (data not shown). The expression pattern is similar to that observed for human NAP1L4 (26) and is in contrast to the expression patterns observed for imprinted genes in the cluster such as H19 and Cdkn1c, which show marked tissue- and developmental-specific expression patterns.

As an initial assay for the imprinting status, the Nap1l4 cDNA was also hybridized to northern blots of maternal disomy distal 7 and wild-type control RNAs (Fig. 3B). The scanned images revealed a slight excess of Nap1l4 RNA in the disomy compared with the wild-type controls in both the fetus and placenta (Fig. 3C). In order to investigate this further, RT-PCR experiments were performed on selected tissues from (M.m.domesticus × SD7)F1 fetuses (15 d.p.c.) and neonates (1 day after birth), and also from reciprocal crosses. The allele-specific products could be distinguished using a length polymorphism between the M.spretus and M.m.domesticus Nap1l4 alleles. This revealed biallelic expression in both fetal and neonatal tissues (Fig. 3D), and these results were confirmed by single nucleotide primer extension (SNuPE) analysis (results not shown). Hence the analysis in these crosses strongly suggests that the gene is not imprinted, although embryos with a maternal disomy of distal chromosome 7 express Nap1l4 at higher levels than normal embryos (see Discussion).

DISCUSSION

We established a contig for the imprinting cluster on distal chromosome 7 in the mouse. The imprinting region from H19 to Tssc3 is genetically and physically linked. Upstream of Tssc3 we localized Nap1l4 which appears to be non-imprinted. Kcnq1 was mapped in the centre of the cluster between Cdkn1c and Cd81. The detailed mapping analysis of the region between Mash2 and Nap1l4 together with previously published data (42) revealed a size of ~1 Mb for the murine cluster including Tssc3, Cdkn1c, Kcnq1, Cd81, Mash2, Ins2, Igf2 and H19. The size is similar to that of the human cluster (24,25). These results confirm the predicted synteny of the murine imprinting cluster on distal chromosome 7 to the human BWS imprinting cluster. Although the relative transcriptional orientation of all genes appears to be conserved between mouse and human, their precise structures and relative distances still remain to be defined. In light of the functional similarities, the knowledge of the precise structures of the genes in both organisms will enable comparative analyses to identify local and regional imprinting control elements.

The mouse imprinting cluster on distal chromosome 7 is flanked on either side by non-imprinted genes, Nap1l4 and Rpl23l (43), and further downstream Nttp1 (Fig. 1A). However, it cannot be excluded that (i) the imprinting cluster contains genes which are not imprinted [i.e. previous analysis of Cd81 and Th knockouts did not provide any indications for imprinting (51,52)] and (ii) that additional imprinted genes are present further upstream of Nap1l4 or upstream of Rpl23l in the vicinity of the cluster. It is possible that the whole cluster is organized into an epigenetic domain which may, for example, lead to differences in allele-specific replication timing. A recent report (53) suggests the existence of a replication control `boundary' that separates the asynchronously replicating Igf2-H19 from the synchronously replicating Rpl23l region. In contrast, our preliminary results on replication timing analysis show asynchronous replication with P1 and cosmid probes from outside (Nap1l4, Rpl23l and Nttp1 genes) and inside (Igf2, H19, Cdkn1c and Kcnq1) the imprinting cluster (K. Okumura et al., unpublished data). This suggests that either the imprinted domain is larger, or that the asynchronous replication domain is larger than the region of imprinted genes.

In humans, the region between NAP1L4 and TSSC3 contains a repeat element consisting of a large block of tandemly repeated GTGTGAATA motifs (21) (human 155 kb contig from chromosome 11p15.5, accession no. U90582). Direct repeats frequently are found associated with imprinted genes and are thought to play a role in the establishment or maintenance of the imprint (54). The direct repeats associated with imprinted genes so far characterized are CG rich, whereas the repeat between TSSC3 and NAPIL4 has a low CG content. Apart from a possible function as a regulatory element for TSSC3 imprinting, the repeat could act as a structural boundary element between NAP1L4 and the imprinting cluster. So far, it is unknown if there is a similar element present between mouse Tssc3 and Nap1l4.

The Nap1l4 gene encodes a protein with homology to NAPs. The NAP family has been implicated in nucleosome assembly in vitro (50,55,56), and in cell cycle control through the binding to cyclin-dependent kinase complexes (57,58). It is therefore intriguing to find Nap1l4 linked to a region which is epigenetically controlled by chromatin factors and DNA methylation, and which contains genes that are positive and negative regulators of cell proliferation. Although in normal embryos Nap1l4 expression was shown to be biallelic in all tissues and at all stages analysed, Nap1l4 transcript levels were reproducibly higher in maternal disomy distal 7 embryos compared with the normal biparental embryos. The same holds for H19 transcript levels, which were higher in maternal disomies than the expected 2-fold increase. In contrast, Nttp1 (non-imprinted, data not shown) and Cdkn1c (imprinted) transcript levels were as expected. It has also been reported that Igf2 levels were higher than expected in the paternal disomy of distal chromosome 7 (59). The reasons for the expression changes of imprinted and non-imprinted genes in disomic embryos could include developmental differences between disomy and normal embryos, the presence of imprinted regulatory genes that affect transcription of other genes in the region, or the requirement for a biparental constitution for the appropriate establishment of some epigenetic modifications that control expression of individual genes. The differentially methylated region 1 in the Igf2 gene, for example, shows clear differences between paternal and maternal alleles in normal embryos (60) whereas the differences are considerably reduced between maternal and paternal disomies (J. Oswald, personal communication). Some of the phenotypes observed in disomic embryos in human and mouse could therefore be a consequence of deregulation of gene expression in addition to simple dosage effects.

The murine Kcnq1 gene is located in the centre of the imprinting cluster covering a region of 300 kb. The gene is maternally expressed in most embryonic tissues as is the human homologue. The murine Kcnq1 gene is expressed biallelically in neonatal tissues but retains a bias towards the maternal allele. This bias is strongest in the tongue, which is interesting with regard to macroglossia as a BWS symptom.

Together with the data obtained by Gould and Pfeifer (44), our analysis shows that the imprinted expression during early embryonic development is converted gradually into biallelic expression during later stages of fetal and early post-natal development. This gradual loss of imprinted expression raises a number of questions about possible mechanisms controlling Kcnq1 expression. The simplest scenario would be that relaxation of imprinted expression is a consequence of a gradual activation of the silenced paternal allele. A second possibility might be that our [and probably also Gould and Pfeifer's (44)] RT-PCR products are mixtures of biallelic and monoallelic transcripts which might be transcribed from different promoters. Relaxation of imprinting could be caused by the appearance during development of biallelic transcripts, with or without repression of the monoallelic transcripts. Our present knowledge about the structure of the Kcnq1 transcripts and promoters, however, does not allow us to distinguish between these possibilities.

Point mutations in the coding region of human KCNQ1 cause long QT syndromes (LQTS), characterized by cardiac arrhythmias (61). LQTS patients do not show symptoms implicated in BWS. The human KCNQ1 spans translocation breakpoints on the maternal allele in a number of BWS patients, but these patients do not show cardiac arrhythmias (22). The transcript encoding the intact potassium channel protein seems to be expressed biallelically in the heart (22). This indicates that different mutations are involved in LQTS and BWS syndromes. The BWS-associated translocations could remove an `imprinting centre', could directly disrupt an imprinting element in the cluster, possibly the KCNQ1 RNA, or could simply alter the allele-specific chromatin structure by the interruption of the cluster. This could lead to the observed biallelic expression of IGF2 in KCNQ1 translocation carrier BWS patients. However, changes in regional imprinting or disruption of the allele-specific chromatin structure could also cause biallelic expression of unknown paternally expressed genes with growth-enhancing properties in the vicinity of KCNQ1.

Overall, our study establishes a sound molecular genetic basis for functional experiments in this important region. This will include searching for the possibility of an overall `imprinting centre'; currently H19 seems to constitute a local imprinting control element that affects imprinting of Igf2, Igf2 antisense transcripts (62) and Ins2, but probably not other genes further away in the cluster (1,63). Other work should address the intriguing possibility (suggested from analysis of BWS patients and mouse knockouts) that various genes in this cluster may interact in physiological pathways in the control of cell proliferation and growth.

MATERIALS AND METHODS

Libraries and clones

Clones were isolated from the following libraries from the Resource Centre (Berlin, Germany): YAC library no. 902 (C57BL/6) (64), P1 library no. 703 (C57BL/6) and the cDNA library no. 522 (9 d.p.c. mouse embryo, C57BL/6). A BAC library (C57BL/6) was kindly provided by H. Himmelbauer. The YAC and P1 clones and the Nap1l4 cDNA clone are distributed by the Resource Centre using the following database names: YAC1, ICRFy902H0522; YAC17, ICRFy902H06119DD2; P1-2, ICRFP703C21274; P1-4, ICRFP703D23243; P1-15, ICRFP703C03218; P1-62, ICRFP703I1043Q2; P1-64, ICRFP703G19266QD2; P1-68, ICRFP703F07327QD2; Nap1l4 cDNA clone, ICRFp522I2410Q2. YAC22 (well position 288 C 3) was obtained from the Whitehead Mouse YAC I library based on the database information that the microsatellite marker D7Mit46 is present on this clone (Mouse Genome Database, Jackson Laboratory: www.informatics.jax.org).

DNA and RNA preparations

P1, BAC and plasmid DNA were isolated from liquid cultures using the QIAGEN Midi kit (Qiagen). High molecular weight DNA from yeast was prepared from liquid cultures (0.67% yeast nitrogen base, 1% casamino acid hydrolysate, 0.01% adenine, 2% glucose, pH 5.8). The DNA preparation was carried out in low melting point agarose (65). Genomic DNA from mice was prepared according to a standard protocol (66).

RNA preparations were performed either by a standard method (67) or using a RNeasy Mini Kit (Qiagen). Randomly primed first strand cDNA synthesis of total RNA was performed using Gibco BRL Superscript II Reverse Transcriptase according to the manufacturer's protocol.

Field inversion gel electrophoresis (FIGE) and pulse-field gel electrophoresis (PFGE)

Restriction fragments of P1 and BAC DNA (5-100 kb long) were separated in 1% agarose gels (0.5× TBE) on a FIGE-Gel Mapper Electrophoresis System (Bio-Rad) according to the manufacturer's protocol.

For the size estimation of YACs, PFGE of yeast DNA was performed in 1% low melting point agarose in 0.5× TBE on a CHEF PFGE System (Bio-Rad) (6 V/cm, 13 h switch time intervals of 50 s, 9 h switch time intervals of 90 s, electrophoresis buffer chilled at 14°C).

Hybridization probes

The hybridization probe for the isolation of a mouse Nap1l4 cDNA clone was amplified by PCR from clone P1-15 (primers Pr 1, Pr 2, Table 1).

P1, BAC and YAC clones were isolated and mapped using the following murine probes: (i) cDNA probes of Mash2 (kindly provided by F. Guillemot) and Nap1l4; (ii) genomic probes of Igf2 and Ins2; and (iii) RT-PCR probes from Cdkn1c (primers Pr 3, Pr 4, Table 1) and from the exons 4-11 of Kcnq1 (primers Pr 5, Pr 6, Table 1) (22).

The following probes were used to characterize the clone contig by Southern blot hybridization: RT-PCR probes of Kcnq1, exon 1[alpha]-2 (primers Pr 7, Pr 8, Table 1), exon 10 (primers Pr 9, Pr 10, Table 1) and 3[prime] UTR (primers Pr 11, Pr 12, Table 1). A 5[prime] RACE product (see below) including the exons 1[beta], 1[gamma], 1[epsis] and 1 was used for the localization of these exons on the clone contig. A probe for Tssc3 (21) was amplified from clone P1-15 using the primer pair Pr 13 and Pr 14 (Table 1). For Cd81 (46), two probes were amplified for exon 1 (primers Pr 15, Pr 16, Table 1) and exon 2 (primers Pr 17, Pr 18, Table 1), respectively.

EcoRI-restricted vector DNA of P1 and BAC clones was used for the characterization of regions of overlap. Probes specific for the flanks of the P1 clone were generated by the following method: P1 DNA was restricted either with ApoI or with Csp6I and religated according to standard protocols (68). The end-specific probes were amplified from the religated DNA using either the primer pair Pr 19, Pr 20 (Table 1) for DNA cut with ApoI or the primer pair Pr 21, Pr 22 (Table 1) for DNA cut with Csp6I.

Table 1. Sequences of primers used for PCR amplifications, reverse transcription and single nucleotide primer extension
Primer Primer sequence MgCl2concentration (mM) Annealing temperature (°C)
Pr 1 5[prime]-TCTCAGGATGAAGATTCCGAG-3[prime] 1.5 53
Pr 2 5[prime]-TTGTCATCATCCTCTATGGCC-3[prime] 1.5 53
Pr 3 5[prime]-GTCCATCACCAATCAGCCAGC-3[prime] 3.5 62
Pr 4 5[prime]-ACCTTGGGACCAGCGTACTCC-3[prime] 3.5 62
Pr 5 5[prime]-GGGGTATCCGCTTCCTTCAGA-3[prime] 2.5 59
Pr 6 5[prime]-CGTCGTAGGGCTTCCGGCTT-3[prime] 2.5 59
Pr 7 5[prime]-GCCCCACCTCCCTCTCAACAG-3[prime] 2.5 66
Pr 8 5[prime]-GTGGCGTGAAAGAGGCTGGAG-3[prime] 2.5 66
Pr 9 5[prime]-AAGGAAGAGCCCTACACTGCTG-3[prime] 2.5 54
Pr 10 5[prime]-ACATGGGTGATGGGGGTCAG-3[prime] 2.5 54
Pr 11 5[prime]-GGTGAGCCTTGACCCGCGGGTCTCCAT-3[prime] 2.5 58
Pr 12 5[prime]-ATGGAAATGGGCTTCCGGGCAAAACGT-3[prime] 2.5 58
Pr 13 5[prime]-TCCACTCCATCCTCAAGGTG-3[prime] 1.5 54
Pr 14 5[prime]-CGTTCCAGCAGCTCTCCA-3[prime] 1.5 54
Pr 15 5[prime]-AACCAGCTGCACCAAGTTCC-3[prime] 1.5 60
Pr 16 5[prime]-AGAAGACGAAATTGAAGACGAAGA-3[prime] 1.5 60
Pr 17 5[prime]-CTGGCTGGAGGCGTGATCCTAG-3[prime] 1.5 57
Pr 18 5[prime]-TAGAAGGTGTTGGGTGCCGGTT-3[prime] 1.5 57
Pr 19 5[prime]-GCCGTCGACATTTAGGTGACACT-3[prime] 1.5 52
Pr 20 5[prime]-TCCCTCGACTACGTCGTTAAGGC-3[prime] 1.5 52
Pr 21 5[prime]-GCATGACCCAGTCACGTAGCGAT-3[prime] 1.5 52
Pr 22 5[prime]-AGCTGACTGGGTTGAAGGCTCTC-3[prime] 1.5 52
Pr 23 5[prime]-GAAGAATTAATGCCCTGAAGC-3[prime] 2.5 55
Pr 24 5[prime]-AACTTGTCCTCCTCCTCATTC-3[prime] 2.5 55
Pr 25 5[prime]-GTGAAGCTGAAAGAACAGATGGTG-3[prime] 2.5 56.5
Pr 26 5[prime]-GTAGGGCATGTTGAACACTTTATG-3[prime] 2.5 56.5
Pr 27 5[prime]-CTATGACAGCAGGATTTGGAC-3[prime] 2.5 58
Pr 28 5[prime]-GGTATGTGTGCTGTGAATGTG-3[prime] 2.5 58
Pr 29a 5[prime]-GCAGGAAAGCTGGGCAGCT-3[prime] 2.5 60
Pr 30a 5[prime]-CACGTGGCGAGGAGCTGG-3[prime] 2.5 60
Pr 31 5[prime]-TGGGTTGTGGCACACACCTCTG-3[prime] 1.5 60
Pr 32 5[prime]-GCATGGCACAGCACAGCACAG-3[prime] 1.5 60
Pr 33 5[prime]-AGAGGGAAGCCCATCCAGGTA-3[prime] 2.5 58
Pr 34 5[prime]-CTGCACGTTCCCTGATGGTCT-3[prime] 2.5 58
Pr 35 5[prime]-TGTGGAAGCTGCTAAAAATGC-3[prime] 1.5 57
Pr 36 5[prime]-CAAAGTCAGAGGCTAAGGTGAAC-3[prime] 1.5 57
Pr 37 5[prime]-AGATTCTCCATCTCCAGAGGCTTT-3[prime]    
Pr 38 5[prime]-AGGCTACAACCACGAT-3[prime]    
Pr 39 5[prime]-AGACCAGAGGCGGACCACATA-3[prime] 1.5 55
Pr 40 5[prime]-TCCCAAAGAACACCACAAGGA-3[prime] 1.5 55
Pr 41a 5[prime]-CGGAAGAAAGCCTGATGGAAC-3[prime] 3.5 57
Pr 42 5[prime]-TCTGATCCCCAAGAACATGCC-3[prime] 2.5 57
Pr 43 5[prime]-CCGGAATTCTGCATCACCTGGTCTG-3[prime] 1.5 55
For primers that were used for PCR, the chosen MgCl2 concentrations and annealing temperatures are shown. If not described differently in the text, amplifications by PCR were carried out under standard conditions.
aPrimers that were used in the presence of 10% DMSO.

Hybridization of immobilized DNA and RNA

DNA and RNA were transferred on to positively charged membranes (Hybond N+; Amersham) according to standard protocols (68). Library filters were hybridized with 32P-labelled probes in the presence of 35S-labelled vector or yeast DNA. DNAs of P1 and BAC clones of the imprinting cluster were used as hybridization probes in the presence of 5 µg/ml of denatured unlabelled cot1-DNA (Gibco). The probe labelling was performed by the random priming method (69). Pre-hybridizations and hybridizations were performed in hybridization buffer (0.5 M sodium phosphate, pH 7.2, 1 mM EDTA, 7% SDS) at 65°C (murine probes) or 55°C (human probes). The filters were washed in 25 mM sodium phosphate, pH 7.2, 1% SDS at 65°C (murine probes) or in 2× SSC, 0.1% SDS at 55°C (human NAP1L4 probe).

Genotyping by allele-specific PCR

Recombination events were analysed by PCR on genomic DNA from the progeny of C57BL/6 × (C57BL/6 × SD7) crosses (SD7 is a C57BL/6 × CBA M.m.domesticus strain which carries a defined segment of distal chromosome 7 of M.spretus SEG origin). The PCR products obtained contain a polymorphic restriction site. The amplified products were cut with the denoted restriction enzymes and separated by gel electrophoresis. PCR was performed for Nap1l4 (primers Pr 23, Pr 24, Table 1, M.spretus-specific HphI site), H19 [primers Pr 25, Pr 26, Table 1, M.spretus-specific BglI site (70)], Rpl23l (primers Pr 27, Pr 28, Table 1, M.m.domesticus-specific NcoI site), Nttp1 (primers Pr 29, Pr 30, Table 1, M.spretus-specific HinfI site) and Kcnq1 (primers Pr 8, Pr 9, M.spretus-specific EcoRV site).

Quantification of allele-specific RT-PCR products

For the analysis of the allele-specific expression of Kcnq1 and Nap1l4, C57BL/6 females were crossed with SD7 males and vice versa. Tissues were collected from embryos and newborn mice obtained from these crosses. Nap1l4 RT-PCR products (Pr 31, Pr 32, Table 1) were restricted with BbvI which generates an allele-specific fragment of either 46 bp (M.m.domesticus) or 41 bp (M.spretus). This length polymorphism results from a 5 bp deletion of nucleotides 1936-1941 (accession no. AJ002198) in the M.spretus sequence.

A 10 bp length polymorphism resulting from a tandem duplication of the sequence motif nucleotides 2287-2297 (accession no. U70068) in the M.spretus sequence allows the M.m.domesticus and M.spretus alleles to be distinguished in Kcnq1 RT-PCR products (primers Pr 33, Pr 34).

Kcnq1 RT-PCR products and Nap1l4 restriction fragments were resolved on a 10% non-denaturing polyacrylamide gel. The intensities of the different alleles were quantified using the Gel Documentation System.

Single nucleotide primer extension (SNuPE)

SNuPE was performed for quantification of allele-specific RT-PCR products of Nap1l4. Mus m.domesticus and M.spretus alleles show a G/A base exchange polymorphism at nucleotide 980 (accession no. AJ002198) which is suitable for SNuPE assays. RT-PCR products (primers Pr 35, Pr 36, Table 1) were purified from 1% agarose gels (1× TBE) using the QIAEX II Kit, (Qiagen). SNuPE was carried out in a 25 µl reaction on 50-100 ng of template in the presence of 0.5 U of PwoI polymerase (Boehringer Mannheim) and 100 pmol of primer Pr 37 (Table 1). For each PCR product, two primer extension reactions were carried out: one in the presence of 0.3 nmol (0.037 MBq) of [[alpha]-32P]dCTP (M.m.domesticus-specific extension products) the other in the presence of 0.3 nmol (0.037 MBq) of [[alpha]-32P]dTTP (M.spretus-specific extension products) (five cycles: 30 s, 94°C; 30 s, 55°C; 30 s, 68°C). The extension products were separated on a denaturing 12% polyacrylamide gel. The signal intensities were scanned using the Phosphoimager System (Molecular Dynamics).

5[prime] Rapid amplification of cDNA ends (RACE)

5[prime] RACE was carried out using the Gibco BRL system, version 2, according to the protocol of the manufacturer. First strand cDNA from murine adult heart RNA was synthesized by reverse transcription using the Kcnq1-specific primer Pr 38 (Table 1). The PCR product was generated by nested PCR: 35 amplification cycles using the anchor-primer provided and the Kcnq-specific primer Pr 39 (Table 1) (1.5 mM MgCl2, annealing temperature: 55°C), 30 cycles on 1:1000 diluted PCR product using the anchor primer and a second gene-specific primer Pr 40 (Table 1) (same conditions as the first amplification).

The 5[prime] RACE product was verified by RT-PCR using Pr 39 and Pr 41 which is specific for the 5[prime] end (exon 1[beta]) of the 5[prime] RACE product. The tissue-specific expression of splice variants I and II was analysed by RT-PCR on randomly primed cDNA using either the primers Pr 7 and Pr 8 (splice variant I) or Pr 39 and Pr 42 (splice variant II).

The amplification of the 5[prime] end of the Nap1l4 transcript was performed using randomly primed cDNA synthesized from total RNA of a mouse embryo (C57BL/6, 16 d.p.c.). For the amplification, the anchor primer provided and the Nap1l4- specific primer Pr 43 (Table 1) were used (1.5 mM MgCl2, four cycles at 55°C annealing temperature, 30 cycles at 60°C annealing temperature).

Sequencing

Sequencing was performed from cloned PCR products (TA cloning kit; Invitrogen) and cloned restriction fragments (68) using the ABI DNA Sequencing System (PE Applied Biosystems).

ACKNOWLEDGEMENTS

We thank T. Tang for technical assistance, and F. Francis and H. Himmelbauer for helpful discussions. We thank H. Himmelbauer, S. Meier-Ewert and the Resource Centre Berlin, Germany, for providing the mouse libraries, and K. Okumura and J. Oswald for allowing us to quote their unpublished findings. The Mash2 cDNA probe was a generous gift from F. Guillemot. This work was supported by the Deutsche Forschungsgemeinschaft (WA1029), BBSRC, Action Research, the EC (BMH4-CT96- 0050) and the NIH (grant CA54358 to A.P.F.).

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*To whom correspondence should be addressed. Tel: +44 1223 832312; Fax: +44 1223 836481; Email: wolf.reik@bbsrc.ac.uk

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