Human Molecular Genetics, 2000, Vol. 9, No. 18 2691-2706
© 2000 Oxford University Press
Sequence and functional comparison in the Beckwith–Wiedemann region: implications for a novel imprinting centre and extended imprinting
1Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin, Germany, 2University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK and 3Laboratory of Developmental Genetics and Imprinting, The Babraham Institute, Cambridge CB2 4AT, UK
Received 18 July 2000; Revised and Accepted 1 September 2000 .
DDBJ/EMBL/GenBank accession nos AJ276505,AJ271885, AJ278263–AJ278265, AJ276796, AJ404609, AJ40461.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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The clustered organization of most imprinted genes in mammals suggests coordinated genetic and epigenetic control mechanisms. Comparisons between human and mouse will help in elucidating these mechanisms by identifying structural and functional similarities. Previously we reported on such a comparison in the central part of the mouse imprinting cluster on distal chromosome 7 with the homologous Beckwith–Wiedemann syndrome (BWS) gene cluster on human chromosome 11p15.5. Here we focus on the adjacent sequences of 0.5 Mb including the KCNQ1/Kcnq1 and CDKN1C/Cdkn1c genes, which are implicated in BWS, and on one of the proposed boundary regions of the imprinting cluster. As in the previously analysed central region, this part of the cluster exhibits a highly conserved arrangement and structure of genes. The most striking similarity is found in the 3' part of the KCNQ1/Kcnq1 genes in large stretches of mostly non-coding sequences. The conserved region includes the recently identified KCNQ1OT1/Kcnq1ot1 antisense transcripts, flanked by a strikingly conserved cluster of LINE/Line elements and a CpG island which we show to carry a maternal germline methylation imprint. This region is likely to be the proposed second imprinting centre (IC2) in the BWS cluster. We also identified several novel genes inside and outside the previously proposed boundaries of the imprinting cluster. One of the genes outside the cluster, Obph1, is imprinted in mouse placenta indicating that at least in extra-embryonic tissues the imprinting cluster extends into a larger domain.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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In the last decade
40 genes have been described which are exclusively or predominantly expressed from only one of the parental alleles (1–5). This epigenetic phenomenon has been termed ‘genomic’ or ‘parental imprinting’. Imprinted genes play an important role in development, and deregulation of imprinting is associated with several human cancers and genetic diseases, such as Prader–Willi/Angelman syndrome (PWS) (6) and Beckwith–Wiedemann syndrome (BWS), which causes fetal overgrowth and predisposition to childhood tumours (7). The mechanism of imprinting is still poorly understood. It involves epigenetic modifications such as DNA methylation and altered chromatin structures (2). Interestingly, most imprinted genes are located in clusters that show specific epigenetic characteristics, such as sex-specific meiotic recombination frequencies, allele-specific DNA replication and pairing of homologous alleles (8–11). The clustering of imprinted genes suggests a coordinated regulation of imprinted genes which are located close to each other.
In the human, one of the two main clusters of imprinted genes is the BWS region on chromosome 11p15.5; in the mouse the homologous cluster is located on distal chromosome 7. These regions each span 1 Mb. Recent publications indicate that the BWS region can be subdivided into three subdomains that differ in the regulation of imprinting: two outer imprinted domains are separated by a central domain characterized by relaxation of imprinting (12–14). The genes in this central domain between LTRPC5/Ltrpc5 and TSSC6/Tssc6 are predominantly biallelically expressed. It should be noted that this relaxation of imprinting is not strictly conserved in human and mouse: human LTRPC5 is imprinted in hybrid cell lines (15), whereas mouse Ltrpc5 is clearly biallelically expressed in somatic tissues (14); mouse Tssc4 is tissue-specifically imprinted in placenta (14), whereas human TSSC4 is biallelically expressed (13).
One of the two imprinted subdomains includes the IGF2/Igf2 and H19 genes, the other the TSSC3/Tssc3–KCNQ1/Kcnq1 region. For the IGF2/Igf2–H19 subdomain regionally coordinated control of imprinting has been demonstrated in the mouse (16) and is most likely for the human (17–19). The regional imprinting control of H19 and Igf2 seems to be restricted to this subdomain as it does not affect imprinting of Kcnq1 and Cdknc1 (12). An imprinting centre (IC) has been identified upstream of mouse H19 which constitutes an epigenetically controlled boundary (20,21). A separate IC has so far not been identified for the TSSC3/Tssc3–KCNQ1/Kcnq1 region; however, a maternally methylated CpG island associated with KCNQ1OT1/Kcnq1ot1 repression has been proposed as a candidate region (7,22,23). Common control mechanisms for the entire imprinting cluster might nevertheless exist since loss of imprinting of the KCNQ1 antisense transcript (KCNQ1OT1) is accompanied by loss of imprinting (LOI) of IGF2 in a number of BWS patients (22,23). Additionally, some patients with translocation breakpoints in the KCNQ1 gene showed biallelic IGF2 expression (22,24).
To establish a better molecular basis for further analyses of imprinting control in the BWS region we have begun a detailed comparison of the human and mouse sequences in this region. Based on a contig of bacterial artificial chromosome (BAC) clones (25) we have already sequenced and described a 250 kb BAC insert covering the central part of the BWS region in the mouse between the genes Kcnq1 and Mash2. This central subdomain is characterized by relaxation of imprinting and species-specific variation of imprinting (14). Here we describe the sequence analysis of a further 520 kb adjacent to the already published sequence. Hence together the sequenced region comprises 770 kb of the imprinting cluster. The new sequence encompasses 300 kb of the 3' portion of Kcnq1 including Kcnq1ot, the adjacent Tssc3–Cdkn1c domain and a further 215 kb upstream of Tssc3 including the assumed telomeric boundary of the mouse cluster between Tssc3 and Napl14. In total the analysed sequence comprises 10 genes, four of them (Obph1, Tnfrh1, Tnfrh2, Cars) being newly identified in the mouse. In addition, we identified in the human a novel transcript, Beckwith–Wiedemann region transcript (BWRT), located between CDKN1C and KCNQ1. We also combined the comparative sequence analysis with some functional studies concerning the imprinting status of the identified genes. Together these results provide novel insights in the structural organization of the BWS region by identifying candidate elements for imprinting control.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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Sequencing of BAC clones and identification of genes
Based on a contig of BAC clones (25) covering
1 Mb of the imprinting cluster on distal chromosome 7 in the mouse, we chose the BAC clones 153G18, 182N20, 245N5 and 482D4 (library CITB; see Materials and Methods) for sequencing, covering 0.5 Mb, starting in the Kcnq1 gene between exons 9 and 10 and ending 200 kb upstream of Cars. The BAC clones were used to establish pUC18 libraries with an average insert size of 2.0–2.5 kb. A total of 9939 readings were assembled; in addition high throughput genomic sequence (htgs) database sequence AC015800, consisting of several unordered pieces of genomic mouse sequence, was used to fill some sequence gaps within the Kcnq1 gene (for details see Materials and Methods). Due to a last sequence gap of <1 kb, as determined by PCR analysis, the finished high quality sequence contig consists of two sequences with lengths of 278 848 and 241 523 bp (GenBank accession nos AJ276505 and AJ271885), respectively. The latter sequence has an overlap of exactly 1000 bp with the already published sequence comprising the central part of the BWS imprinting cluster between Kcnq1 and Mash2 (14) (GenBank accession no. AJ251835).
Genes were identified either by homology searches against the databases or by using a series of gene prediction programs (see Materials and Methods). The genomic locations and structures of 10 genes were identified, including 230 kb of the 3' part of the Kcnq1 gene (including exon 10). The genes Nap1l4 (25), Tssc3 (26), Tssc5 (27), Cdkn1c (28), Kcnq1 (25) and Kcnq1ot1 (22) have been described previously, the position of Cars upstream of the Nap1l4 gene had been only roughly determined before (29). Upstream of Cars we identified three novel genes in the order 5'-Obph1–Tnfrh1–Tnfrh2-3'. In addition, we identified the human BWRT gene between CDKN1C and KCNQ1. The precise placement of genes and individual exons is shown in Figure 1. The exact coordinates of the exon/intron structures are deposited with the BAC sequences.
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Oxysterol binding protein homologue 1 (Obph1/OBPH1)
The gene Obph1 was identified by a combination of exon prediction programs and homology searches in the database for non-redundant sequences and expressed sequence tags (ESTs). The first eight exons were suggested by exon prediction programs, exons 9–17 were indicated by two human EST clones (GenBank accession nos AA776467 and AI432623) showing a strong homology to the genomic mouse sequence and exons 17–21 were identified by perfect matches of numerous mouse EST clones (see Materials and Methods). RT–PCR analysis revealed that all exons belong to a single gene.
The mRNA (GenBank accession no. AJ278263) with a length of 3613 bp encodes a protein of 837 amino acids. Homology searches with the predicted protein revealed a strong homology of 84.72% similarity in the aligned part (Fig. 2) to the human protein KIAA1534 (GenBank accession no. AB040967) (30). Both proteins share a common motif stretch with several other proteins, namely oxysterol-binding proteins (data not shown), therefore we called the novel mouse gene Obph1 and, in analogy, the human gene OBPH1. Obph1 is transcribed in all organs analysed [placentae and embryos 12.5 days post-conception (d.p.c.), placentae and various organs from embryos 16.5 d.p.c. and newborn], indicating a ubiquitous expression.
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Although for the human OBPH1 region a continuous genomic sequence is not available so far, we identified an htgs sequence containing the whole region between CARS and OBPH1 in several unordered pieces (GenBank accession no. AC016765) and were thus able to deduce largely the intron/exon structure of the human OBPH1 gene. It consists of 21 exons as does the mouse one, and the exon distribution is similar, with the exception of an elongation of the sixth intron in the human. Both in the human and in the mouse, the open reading frame (ORF) reaches the 5' end. Therefore, we assume that the translational start points lie even more upstream.
Since the novel Obph1 gene is located beyond the assumed border of the BWS imprinting cluster, we were interested to analyse its imprinting status. We identified strain-specific polymorphisms between the inbred mouse strains C57BL/6 and SD7 in exon 6, which allowed us to determine the parental origin of the transcripts by RT–PCR analysis. The results (Fig. 3A) show predominant expression of the maternal allele in placenta, whereas all other organs from different developmental stages (embryos 12.5 and 16.5 d.p.c. and newborn) showed biallelic expression of Obph1. Our analysis indicates that Obph1 is maternally expressed in placenta.
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Tumour necrosis factor receptor p60 homologues 1 and 2 (Tnfrh1 and Tnfrh2)
Homology searches in the EST database indicated the existence of two independent genes with similarity to tumour necrosis factor receptor p60 (31), mainly in the extracellular domain (32). The gene Tnfrh1 was identified by EST clone AI747041 and confirmed by RT–PCR. Interestingly, the same EST clone matched to a second region in the genomic mouse sequence and revealed the existence of Tnfrh2 which is closely related to Tnfrh1.
Tnfrh1 consists of six exons. On the basis of database searches and RT–PCR analysis there are two Tnfrh1 transcripts, 569 and 544 bp long (GenBank accession nos AJ278264 and AI747041), respectively. The 569 bp transcript includes all six exons whereas in the 544 bp transcript the fourth exon is missing. The deduced proteins of 176 and 131 amino acids, respectively, share only the first 98 amino acids, due to a frameshift caused by the absence of the fourth exon in the shorter transcript.
For the Tnfrh2 gene we amplified a 704 bp long mRNA (GenBank accession no. AJ278265) which consists of seven exons and encodes a protein of 180 amino acids. The intron/exon distributions of Tnfrh1 and Tnfrh2 are quite similar, with the exception of the enlargement of the third intron and the absence of exon 6 in Tnfrh1. Exons 1–5 and the last exons of the Tnfrh1 and Tnfrh2 transcripts correspond to each other. Interestingly, the Tnfrh2 exon 6, although having no corresponding exon in Tnfrh1, shows similarity to an intronic sequence from Tnfrh1. This suggests that this intronic sequence might form a further exon in a so far unidentified alternative Tnfrh1 transcript. The identities on the protein and DNA levels between Tnfrh1 and Tnfrh2 (long transcript) are 79 and 74%, respectively (Fig. 4).
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Both genes are transcribed in all organs and developmental stages analysed (placentae and embryos 12.5 d.p.c., placentae and various organs from embryos 16.5 d.p.c. and from newborn), revealing similar ubiquitous expression patterns. We could not perform an imprinting analysis for these genes since for both genes no polymorphisms between different inbred mouse strains could be detected.
Cars/CARS
Cars encodes a cysteinyl-tRNA synthetase and lies 50 kb upstream of the Nap1l4 gene (29,33). Database searches revealed a partial mRNA sequence and numerous EST clones (see Materials and Methods) matching perfectly to the genomic sequence.
For the mouse only one 2719 bp transcript could be identified and confirmed by RT–PCR (GenBank accession no. AJ276796). It consists of 23 exons and encodes a protein of 831 amino acids. In contrast, in the human numerous EST clones indicate the existence of at least seven different transcripts of the homologous gene CARS. Based on these EST clones, a transcript similar to the mouse can be assembled. Both transcripts show high homology to each other (84.7% identity within the aligned parts) and the identity between the deduced proteins reaches 90% (Fig. 5).
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Since Cars is located next to the Nap1l4 gene which is supposed to be at the border of the BWS imprinting cluster, we were interested to know whether or not Cars is imprinted. Therefore, we carried out an imprinting analysis, using a polymorphism between the inbred mouse strains C57Bl/6 and SD7 in exon 4 for the determination of the parental origin of the amplified transcripts. All different organs and developmental stages analysed (placentae and embryos 12.5 d.p.c., placentae and various organs from embryos 16.5 d.p.c. and newborn), indicated a ubiquitous and biallelic expression of Cars in the mouse (Fig. 3B). Since we did not analyse earlier stages, we cannot exclude that Cars is imprinted at these stages.
BWRT
Based on five EST sequences (GenBank accession nos AJ404617, AB039920, AC0012228, AC005950 and AC005231) originating from three different EST clones we identified a novel human gene in the region between CDKCN1 and KCNQ1. BWRT is located 21 kb downstream of the last exon of KCNQ1 and transcribed in the same orientation as this gene. The transcript has a length of 1067 bp (GenBank accession no. AJ404617) and consists of two exons (Fig. 1). The transcript contains a translation start signal which matches perfectly to the degenerate Kozak consensus sequence (RNNATGG) (34). The ATG is followed by an ORF encoding a protein of 68 amino acids. Database searches revealed no significant homology of BWRT to known protein sequences. Furthermore, there are neither significant homologies to the corresponding mouse sequence nor matching mouse EST clones detectable. Therefore, we assume that the mouse homologue of BWRT does not exist.
TSSC3/Tssc3, CDKN1C/Cdkn1c and KCNQ1OT1/Kcnq1ot1
For some genes the published mRNA sequences were extended by the integration of EST clone sequences. In the case of the previously described TSSC3/Tssc3 genes both the human and the mouse mRNA sequences were extended at their 5' end (GenBank accession no. AF035444 for the mouse). The same applies to the mRNA of Cdknc1 which was extended by 20 nucleotides at its previously described 5' end and 376 nucleotides at its 3' end. The exact positions of the extended mRNA sequences are deposited with the BAC sequences.
The human KCNQ1OT1 has a length of 58 kb, as indicated by northern blot analysis and several ESTs (23,35). In addition, we identified several other EST clones, confirming these data (see Materials and Methods). For the mouse so far a single EST clone has been described as part of the mouse homologue Kcnq1ot1; the corresponding transcript could not be verified by northern blot analysis (22). We identified a cluster of EST clones, indicating that the mouse gene also covers 54 kb (Fig. 1; for details see Materials and Methods).
We also identified four and five EST clones, respectively, 5' of the KCNQ1OT1/Kcnq1ot1 CpG island in human (GenBank accession nos AA310701, AA584165, AA601658, AI863519) and mouse [GenBank accession nos AI504599, AU316783, BB016223, BE106937 (rat), AU024004], respectively, indicating a so far unidentified transcript in this region. All EST sequences identified match the intronic KCNQ1/Kcnq1 sequences and are co-linear with the genomic sequence.
Comparison of the genes from CARS/Cars to KCNQ1/Kcnq1
The sequence data presented in this work cover the mouse distal chromosome 7 region between the genes Obph1 and Kcnq1. Together with the already published genomic sequence between Kcnq1 (exons 1–9) and Mash2 (GenBank accession no. AJ251835) (14), a contiguous sequence was established which covers major parts of the BWS region and is disrupted only by a single gap of <1 kb, located between Cdkn1c and Kcnq1. Previously we compared the regions KCNQ1/Kcnq1 (exons 1–9) to ASCL2/Mash2; here we present the comparison of the adjacent regions. Since a contiguous assembly of human genomic sequences corresponding to the mouse region between Obph1 and Cars is not yet available, we could only compare the region CARS/Cars–KCNQ1/Kcnq1 in the human using the overlapping GenBank sequences AC001228 and AJ006345.
This region exhibits a strong conservation of gene order in human and mouse, just like the previously analysed region between KCNQ1/Kcnq1 and ASCL2/Mash2 in the central part of the imprinting cluster (Fig. 1). Also the transcriptional orientation and in most cases the size and distribution of exons are highly conserved, confirming a long-range homologous gene organization within the BWS gene cluster.
Although there is a general high level of conservation, a few orthologous genes show some minor but remarkable differences. First, NAP1l4/Nap1l4, TSSC3/Tssc3 and CDKNC1/Cdknc1 differ in the number of exons. In all three cases the number of exons is higher in mouse compared with human: (i) NAP1l4 consists of 14 exons (36) compared with the 19 exons of Nap1l4 (25); (ii) TSSC3 is composed of two exons compared with the three exons in Tssc3 (26); and (iii) CDKNC1 is composed of three exons (37) compared with the five exons of Cdknc1 (38). This higher number of exons, however, contrasts with a smaller size of introns. Hence, the NAP1l4 gene (14 exons in 46 kb) is longer compared with the mouse Nap1l4 (19 exons in 35 kb) and the entire human sequence is extended compared with the mouse. Second, some genes within the sequences compared in this work are apparently present only in the human. This concerns the recently described ORCTL2S gene (27) and the newly identified BWRT transcript. For both, neither significant sequence homologies nor EST clones were identified which would indicate the existence of their mouse homologues. Furthermore, several attempts to obtain RT–PCR products from mouse RNA (from several embryonic stages and newborn) with primers selected against the hypothetical Orctl2s region (using exon prediction programs) failed, revealing only an alternative first exon of Tssc5, which has already been described (39); its sequence was deposited in the GenBank database (accession no. AJ404609). Conversely, we have been unable so far to detect the homologues of Tnfrh1 and Tnfrh2 in the discontinous human genomic sequence available which most probably corresponds to the homologous region.
An unusually high degree of conservation for an intronic region was found in the 3' end (exons 10–15) of the KCNQ1/Kcnq1 genes (Figs 1 and 6). Except for a cluster of repetitive elements separating exons 9 and 10, the entire 3' regions of the KCNQ1/Kcnq1 genes of 265 kb in the human and 235 kb in the mouse are highly homologous. This homology is in striking contrast to the dissimilarities and prominent size differences in the previously analysed 5' region (5' of exon 9) of both genes (14). Within the 3' region of the KCNQ1/Kcnq1 genes not only the number, but also the length of highly conserved sequence stretches (identity >70%; Fig. 1) are significantly above the average. In several cases the homologous sequence stretches are up to 300 bp long and share >90% identity. The more detailed analysis shows that the highly conserved sequence stretches are not only located within the KCNQ1/Kcnq1 antisense transcripts (KCNQ1OT1/Kcnq1ot1), but also in the intronic sequences of the KCNQ1/Kcnq1 genes upstream of KCNQ1OT1/Kcnq1ot1 for which so far no transcripts orientated in opposite direction to KCNQ1/Kcnq1 have been described.
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Strikingly, the high degree of sequence homology does not extend into the CpG islands which may be associated with the promoter regions of KCNQ1OT1/Kcnq1ot1. For the human this prominent CpG island has been shown to be methylated on the maternal but not on the paternal allele (22,23,35). So far the only germline-derived methylation imprint documented in the whole cluster is in the H19 differentially methylated region (DMR) (40,41). However, if the CDKN1C/Cdkn1c–KCNQ1/Kcnq1 region has imprinting controls which are separate from those regulating H19, this region may itself contain a germline-derived methylation imprint. In order to test this hypothesis, bisulphite methylation analysis was carried out on the Kcnq1ot1 CpG island in germ cells and early embryos (Fig. 6). The CpG island was found to be highly methylated in oocytes and unmethylated in sperm. These differences persisted unchanged in zygotes and embryonic stem (ES) cells. The CpG island therefore has a germline methylation imprint and constitutes a prominent element of the proposed second IC.
Comparison of non-coding sequences
Similar to the central part of the BWS region previously analysed (14) the human and mouse sequences between CARS/Cars and KCNQ1/Kcnq1 are highly homologous but clearly differ in size (35%) with 473 kb of human sequence corresponding to 409 kb in the mouse. Cluster-wide sequence comparisons (Figs 1 and 7) and an evaluation of the RepeatMasker analyses (Table 1) show that the absolute size differences in the BWS cluster are mainly due to the overall higher amount of repetitive sequences in the human. Whereas the enlargement of the chromosomes in the central part mostly results from the integration of two prominent clusters of A/T-rich repetitive elements (Fig. 7), the size differences are less prominent (16%) and more dispersed along the entire CARS/Cars–KCNQ1/Kcnq1 region.
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It has been previously described that the human X chromosome is enriched in LINE elements compared with the average concentration of such retro-elements on autosomes. This finding supports the idea that LINE elements serve as signals propagating X inactivation along the chromosome (42–44). To investigate whether retro-elements like LINEs/Lines and SINEs/Sines may also be enriched in an imprinted region and play a role in epigenetic control in the imprinting cluster we analysed the relative distribution of these elements in the sequenced regions in mouse and human.
Our first observation was that the overall amount of sequences identified by the RepeatMasker program as interspersed repetitive elements (IREs) amount to 25.8% of the total sequence in the human (region from CARS to ASCL2) and to 22.6% in the mouse (region from Cars to Mash2). These values are significantly below the average of the corresponding X chromosomes [51.5% in human (43); 33% in mouse (our own data); see Materials and Methods] or even autosomes [40.0% in human (43), 33.6% in mouse (our own data)]. Hence IREs appear to be relatively under-represented in this cluster. However, a more detailed examination of the distribution of these elements revealed a remarkably uneven distribution, in that specific classes of IREs are quite concentrated in some but strikingly under-represented in other regions. LINE/Line elements are mainly found between the Tnfrh1 and Tnfrh2 genes in the mouse (for which a contiguous human sequence is not available) and along the entire KCNQ1/Kcnq1 region. The most prominent and extensive LINE/Line cluster is located directly adjacent to the proposed IC region in the 3' part of the KCNQ1/Kcnq1 gene. In contrast prominent clusters of SINE/Sine elements and LTRs are concentrated more towards the boundary of the imprinting cluster between CARS/Cars and CDKN1C/Cdkn1c and absent around the IC. Remarkably, this distinct pattern of the distribution of IREs within the clusters is highly conserved between human and mouse. Further comparative analysis in other imprinted and non-imprinted gene-rich regions will elucidate whether the observed over-representation of LINEs/Lines close to the proposed IC and the absence of SINEs/Sines are sequence features characteristic for imprinted domains.
It has been shown that several imprinted genes are associated with direct repeats of unique sequences suggesting that such repeats are hallmarks of imprinted regions (45). When searching for such repeats in the human and mouse sequences neither their sequences nor their positions were found to be conserved in the two species. A very pronounced repeat in the human consisting of 70 repetitions of the sequence TATTCACACYRAGCR, located between the genes NAP1L4 and TSSC3 (Fig. 1), is absent in the mouse. The opposite is true for an extensive repeat structure between exons 9 and 10 of Kcnq1 in the mouse. This repeat with numerous repetitions of poly(A) stretches is absent in the human. Hence our analyses strongly argue against a conserved function of such direct repeats in the process of imprinting in human and mouse.
Similarly, potential matrix attachment sites (MARs), which were previously proposed to be important elements of imprinting control (46) and which were found to be partially conserved in the central part of the BWS cluster (14), are apparently not conserved in the newly analysed part of the imprinting cluster (Fig. 1). However, this observation has to be treated with caution, as it is solely based on the use of prediction programs and thus will have to await experimental proof.
Imprinted genes are associated with CpG islands
Previously we suggested that CpG islands play an important role in genomic imprinting, based on the observation that in the region KCNQ1/Kcnq1–ASCL2/Mash2 imprinting of parental alleles and also the stringency of imprinting can be correlated to the presence and strength of CpG islands (14). The sequence comparison presented here confirms this association: (i) the CpG island:gene ratio (percentage) in the entire sequenced region (CARS/Cars–ASCL2/Mash2) in human and mouse is 86.6 and 75.0%, respectively, and thus clearly above the average of autosomes (56% in human and 47% in mouse) (47) or ratios of 50% found in two non-imprinted gene-rich regions (48,49); and (ii) the previously postulated relationship between the quality (‘strength’) of CpG islands and genomic imprinting can be substantiated. The relative ‘strength’ of the CpG island (Fig. 1) was determined by the program ‘CpG island’ as a ratio between extent and CpG density (see Materials and Methods). Genes with a more relaxed imprinting, like TSSC5/Tssc5 (27,45,50), had less strong CpG islands compared with those of genes with a robust imprinting, like TSSC3/Tssc3 (26) or KCNQ1OT1/Kcnq1ot1 (22,23,35) and CDKN1C/Cdkn1c (38,51,52). The only exceptions appear to be the NAP1L4/Nap1l4 genes, which also possess a strong CpG island, while we and others previously found the Nap1l4 gene not to be imprinted as judged by RT–PCR analyses. When reanalysing the allele-specific expression of Nap1l4 in embryos and placentae of different embryonic stages, however, we observed a variable, but clearly preferential expression of the maternal allele, mainly in placenta (Fig. 8). This finding is in accordance with our previous observation that mice with maternal disomies of distal chromosome 7 show a slightly biased expression towards the maternal allele particularly in placentae (25). In conclusion, we find that the Nap1l4 gene also exhibits some degree of imprinting in the placenta. Hence its strong CpG island is partially in agreement with the proposed connection between CpG island structure and genomic imprinting.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSNOTE ADDED IN PROOFREFERENCES |
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The BWS syndrome is genetically linked to human chromosome 11p15.5. The mechanisms responsible for the control of this cluster of imprinted genes are so far only partially understood. To obtain new insights into these mechanisms we undertook a large-scale comparison of the BWS imprinting cluster in human and the corresponding region on distal chromosome 7 in mouse. We previously determined the sequence of 250 kb of the central BWS region in mouse. Here we present the adjacent sequence which covers the current boundary of the imprinting cluster. Taken together, the sequence covers 770 kb and allows the most extensive comparison of contiguous sequences between human and mouse reported to date.
Conservation of the BWS clusters in human and mouse
A key finding of our analysis is that the majority of genes in the human and mouse imprinting clusters are highly conserved. Among the 15 genes identified in the mouse genomic sequence 13 have orthologues on human chromosome 11p15.5. These orthologues have the same relative order, conserved structure, transcriptional orientation and with a few exceptions also the same imprinted expression. In addition general structural features like the location of CpG islands and the specific distribution of IREs are remarkably similar in both organisms. Together this high structural and functional similarity clearly demonstrates that the mouse is an excellent model organism to study the role of genes in this cluster in human BWS and certain cancers.
Besides the high conservation, however, some differences were detected. First, as previously shown for the central region, the size expansion of the human chromosome compared with the mouse continues and is mainly attributable to enlarged clusters of IREs in the human chromosome. Remarkably, this higher IRE content (35.3% in human compared with 22.4% in mouse) is parallelled by an extended length of the CpG islands in human (2.7% of the total human sequence compared with 2.0% in the mouse; all values refer to the region CARS/Cars–ASCL2/Mash2). It might be possible that this simultaneous expansion of IREs and CpG islands reflects the necessity for a balanced composition of such structural elements to maintain a chromosomal architecture appropriate for gene expression. However, such scenarios may be not only restricted to imprinted regions but a more common difference between mouse and human. The second remarkable difference concerns the conservation of genes in the human and mouse imprinting cluster. For some genes no orthologues seem to exist in the homologous region in either the human or mouse chromosome. Examples are the RPL26 (14), ORCTL2S (27) and BWRT genes, for which we were unable to detect any mouse counterpart. On the contrary the Tnfrh1 and Tnfrh2 genes identified in the mouse apparently do not have orthologues in the corresponding human chromosomal region. Both genes exhibit an extraordinary sequence similarity indicating a recent gene duplication event as proposed for the TSSC6/Tssc6 and CD81/Cd81 genes (14). Since Tnfrh1 and Tnfrh2 are embedded in a region with a high IRE content these genes may have been integrated through a recent recombination event triggered by IREs. A similar scenario most likely led to the insertion of the RPL26 gene into an LTR cluster in human (14). In the case of the ORCTL2S and BWRT genes the situation is apparently more complex, reflecting a rapid evolution of sequence variation in mouse and human. The absence of these genes in the imprinting cluster in mouse may explain some of the phenotypic differences in BWS-related symptoms between the two species which were observed in several mouse models (53–55).
Conservation of a putative IC carrying a germline imprint
The most remarkable sequence conservation is observed in the 3' regions of the KCNQ1/Kcnq1 genes. Intriguingly, the BWS chromosomal breakpoint region 1 (BWSCR1) associated with balanced germline chromosomal rearrangements (56) disrupts the KCNQ1 gene (57) within this conserved region. The impaired function of the KCNQ1/Kcnq1 gene itself caused by the translocations is thought not to be involved in the pathogenesis of BWS (57,58). Rather it has been proposed that regulatory elements located within this 3' region of KCNQ1/Kcnq1 control the imprinting status of other genes involved in BWS, like CDKN1C/Cdkn1c (7). The paternally expressed ‘antisense’ transcript KCNQ1OT1/Kcnq1ot1 (22,35) and its prominent CpG island (23) were suggested to be candidates for such a control function. LOI of this antisense transcript is observed in a substantial portion of BWS patients (22,23). The hypothesis that this region forms a second IC in the imprinting cluster is now substantiated by our finding that the Kcnq1ot1 CpG island harbours a germline-derived methylation imprint in the mouse and that both regions in mouse and human exhibit a high structural and sequence homology. The extent of the IC and the precise location of control elements remains to be elucidated. However, it is noteworthy that not only the KCNQ1OT1/Kcnq1ot1 transcripts themselves but also sequences 5' of their proposed transcriptional start sites and a large cluster of LINE/Line elements 3' of the transcripts are highly conserved. The homology of the KCNQ1OT1/Kcnq1ot1 transcripts which are co-linear with the DNA covers 60 kb. It is possible that the high conservation within the KCNQ1OT1/Kcnq1ot1 transcript reflects either a structural conservation of the RNA as postulated for the XIST/Xist genes (59,60) or a selective pressure to maintain structural features on the DNA level. Knockout experiments as performed for the Xist and H19 genes in the mouse will clarify this question. In conclusion our comparative analysis together with previous functional analyses (12,14) provide evidence for a bipartite structure of the Beckwith–Wiedemann imprinting cluster in human and mouse controlled by two functional ICs. IC1 is the DMR upstream of H19 (influencing imprinting of H19, IGF2/Igf2 and INS2/Ins2); IC2 is located within the highly conserved 3' region of KCNQ1/Kcnq1 and possibly the conserved germline imprint in Kcnq1ot1 identified here. The functional difference of both ICs is also supported by the fact that methylation imprints at IC1 and IC2 are established in different germlines. The IC1 (H19) imprint is established in sperm, the IC2 (Kcnq1ot1) imprint in oocytes. Both regional ICs on the other hand are not operating completely independently since alterations in the higher order structure of the domain are apparently affecting the correct setting of both imprints as witnessed by BWS patients with translocations or LOI at KCNQ1OT1 (22–24).
Size and structural organization of the imprinting cluster
In previous studies we and others had assumed that the TSSC3/Tssc3 gene marks the centromeric (human) and telomeric (mouse) border of the BWS imprinting cluster, as the neighbouring NAP1L4/Nap1l4 genes were found to exhibit no or only a very weak imprinted expression (25,61). To confirm this assumption we analysed the imprinting status of genes further telomeric to the Nap1l4 gene in the mouse. Surprisingly, we found one of them (Obph1) to be imprinted in the placenta. This finding and the fact that re-examination of the Nap1l4 expression confirmed a maternal (although variable) placental imprinting strongly suggest that the location of a domain border of the imprinting cluster telomeric of Tssc3 has to be reconsidered. In addition, the fact that genes in this part of the gene cluster and 5' of KCNQ1/Kcnq1 show no or variable imprinting gives some important new insights into the structural and functional organization of this imprinting cluster. The only genes that are robustly imprinted in all embryonic and extra-embryonic lineages are CDKN1C/Cdkn1c (38,51,52), KCNQ1/Kcnq1 (12,25,57,62) and KCNQ1OT1/Kcnq1ot1 (22,23,35) which are located around the proposed IC. In contrast, genes that are further away from this centre either are not imprinted at all, like Cars, Ltrpc5 (14), TSSC6/Tssc6 (13,14), Tnfrh1 and Tnfrh2, or exhibit a very relaxed form of imprinting, like CD81 (63), Tssc4 (14), LTRPC5 (15), Tssc3 (26), Nap1l4 and Obph1. Moreover, in agreement with our previous analysis we found that CpG islands are hallmarks of imprinted genes as they are significantly more frequent in the imprinting cluster than in other regions (14). Furthermore, the strength of CpG islands (regarding both the length and the overall CpG density) is apparently correlated with the quality of imprinting during development. Together these observations suggest that a combination of factors, for example the vicinity to the proposed IC in KCNQ1/Kcnq1 together with gene-specific structural control elements like the gene-specific CpG islands, influences the imprinting of particular genes along the cluster. In addition imprinting effects are more widespread in extra-embryonic tissues. This may reflect either a biological selection or the fact that the setting/maintenance of imprints differs between embryonic and extra-embryonic tissues.
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MATERIALS AND METHODS |
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DNA sequencing
DNA of the BAC clones 153G18, 182N20, 245N5 and 482D4 [library CITB; Research Genetics (http://resgen.com )] was prepared according to Sambrook et al. (64) including two CsCl gradient ultra-centrifugations. Ten micrograms of DNA of each BAC clone were sonicated for 15 s using a cell disruptor B-30 (setting: output control: 5; Branson Sonic Power, Danbury, CT). After treatment with Klenow and T4 DNA polymerases (MBI Fermentas, Vilnius, Lithuania) 2.0–2.5 kb long DNA fragments of the individual BAC clones were purified from 1% agarose gels (1x TBE) using the QIAEXII kit (Qiagen, Studio City, CA), ligated into SmaI-digested pUC19 (MBI Fermentas) and introduced into Escherichia coli SURE cells (Stratagene, La Jolla, CA) according to standard protocols. In order to obtain on average a 5-fold sequence coverage, inserts of 7201 clones were unidirectionally sequenced on ABI 377 automatic sequencers using standard protocols (PE Applied Biosystems, Foster City, CA). The sequences were assembled using the Gap4 software (Sanger Centre, http://sanger.ac.uk/Software ). Sequence gaps were filled by reverse sequencing of individual clones or by sequencing PCR products obtained by amplification with oligonucleotides situated on both sides of the gaps. Finally
10 000 sequences were assembled; per nucleotide on average 5 independent readings were used. In addition some remaining gaps could be filled by sequences from the htgs database; these gaps are indicated in our sequences deposited in GenBank.
Sequence analysis
The human and the mouse sequences were compared by dot plot analysis using the software package MacMolly (Institute of Technology, http://CCR-081.mit.edu/GENSCAN.html ) and Genefinder (Sanger Centre, http://genomic.sanger.ac.uk/gf/gf.shtml ) software. Searches for homologous sequences were performed in the GenBank databases for non-redundant sequences and ESTs (National Centre for Biotechnology Information, http://www.ncbi.nlm.nih.gov ) and in the TIGR database for mouse EST clusters (Institute for Genomic Research, http://www.tigr.org ). Repetitive elements were detected and masked using the Censor and RepeatMasker software (http://charon.girinst.org/server/censor.html ; GCG package, Genetics Computer Group, Madison, WI) (65). CpG and G+C distribution were obtained using the ‘window’ function of the GCG package [window size 500, shift increment 3]. Additionally CpG islands were detected with the ‘CpG islands’ search (input score threshold 20; http://www2.ebi.ac.uk/cpg/ ). The base content of sequences was determined using ‘composition’ of the GCG package, tandem repeats were detected with the ‘compare’ function of the same software package. The searches for MARs were performed using MarFinder (http://www.ncgr.org ).
RNA preparations
Total RNAs of various mouse tissues and developmental stages were prepared according to a standard protocol (66). Total RNA was randomly primed and reverse transcribed using reverse transcriptase (Promega, Madison, WI) according to the manufacturer’s protocol.
RT–PCR analysis
RT–PCRs were carried out on randomly primed cDNA on Perkin Elmer Thermocyclers (Perkin Elmer Cetus, Norwalk, CT) under standardized conditions (0.5 µM each primer, 1.5 mM MgCl2, 0.2 mM dNTPs). A standard amplification protocol was as follows: denaturation at 95°C for 3 min followed by 35 cycles at 94°C for 30 s, 59°C for 30 s, 72°C for 1 min and final elongation at 72°C for 5 min (for primer sequences, see Table 1).
Transcription of the Tnfrh genes was verified by RT–PCR using the primers Tnfhr1 forward + Tnfhr1 reverse (exons 1–6) and Tnfrh2 forward + Tnfrh2 reverse (exons 1–7). For Obph1 overlapping RT–PCR products were generated using the following primer pairs: Obhp1 forward 1 + Obph1 reverse 1 (exons 1–8), Obph1 forward 2 + Obph1 reverse 2 (exons 3–16), Obph1 forward 3 + Obph1 reverse 3 (exons 14–21). For the determination of the Obph1 mRNA sequence also the following mouse EST clones, whose sequences matched perfectly to the genomic mouse sequence, were taken into account (GenBank accession nos AI510611, AA796535, AI020221, AI448476, AI462538, AA960333, AA146175, AV007840, AW121299, AW121165, AI466945, AA266394, AI508655, AA386733, AI120028 and AI661946).
Cars RT–PCR products were amplified with the primer pair Cars forward + Cars reverse (exons 2–4). To determine the complete mRNA sequence in addition a partial mRNA sequence (GenBank accession no. AB015589) and numerous EST-derived sequences were used (GenBank accession nos AA207992, AA268840, AA415212, AA472497, AA510477, AA590230, AA607246, AA611302, AA620004, AA623507, AA867594, AA879866, AI020819, AI226371, AI317241, AI327054, AI464295, AI596185, AI663126, AI876265, AU066757, AU067082, AV274086, AV287629, AW492362, AW494371, D21456, D77468, D77689, W12552, W18507, W61713 and W89602).
The alternative 5' exon of Tssc5 was identified by RT–PCR analysis using the primer pair Tssc5 forward (in exon 2) + Tssc5 reverse (in the alternative exon 1).
For all genes the RT–PCR products obtained were cloned into pGEM-T vector (Promega) and sequenced using standard protocols.
Identification of the KCNQ1OT1/Kcnq1ot1 transcripts
BLAST searches in the EST databases revealed the following EST clusters presumably belonging to the KCNQ1OT1/Kcnq1ot1 transcripts in human and mouse (listed in 5'
3' order). For the human, GenBank accession nos AA359588, U77321, AI983908, AW515751, BE143913, AW613101, AA889050, R21635, AA693940, BE169636, AA029517, AI378251, AA359331, R10486, AI867155, AI933351, AL118587, AA329719, AA622687 and AA602136. For the mouse, GenBank accession nos AI509736, AA867058, AI552525, AA671389, AW538408, AW244283, AW824704, AW825420, AI642731, AA756179, AI047781, AU041933, AI035516, AA216796 and AU017917.
Imprinting analysis
Strain-specific sequence polymorphisms were detected by sequencing RT–PCR products derived from C57BL/6 and SD7 DNA templates, respectively. SD7 is a congenic mouse strain carrying a defined segment of distal chromosome 7 of Mus spretus on a Mus musculus domesticus background (67). For Obph1 PCRs were performed on C57BL/6 and SD7 DNAs using the following primer pairs: Obph1 forward 4 + Obph1 reverse 4 (exons 5–6). The 268 bp RT–PCR products were cut with BfaI (New England Biolabs, Beverly, MA). In C57BL/6-derived RT–PCR products a BfaI site is present at nucleotide 540 (GenBank accession no. AJ278263) generated by a CT transition; therefore, BfaI digestion results in 41 and 227 bp DNA fragments specific for C57BL/6.
The imprinting status of Cars was analysed using the primer pair Cars forward + Cars reverse (exons 2–7). The 534 bp RT–PCR products were digested with TaiI, which cuts only in C57BL/6 at a specific restriction site generated by an AG transition at nucleotide position 453 (GenBank accession no. AJ276796). The obtained restriction fragments are 229 and 305 bp long.
The imprinting analysis of Nap1l4 was performed as described previously (25).
Bisulphite analysis
Oocytes and sperm samples were derived from (C57BL/6 x CBA/Ca) F1 mice, zygotes were gained from F1 x SD7 crosses, and ES cells from F1 x M.spretus crosses (68). The preparation of zygotes (20–22 h after injection of human choriogonadotropin) and the bisulphite-based sequence analysis were carried out according to Oswald et al. (69). Nested PCR reactions were carried out with primers Kcnq1ot1 outer forward + Kcnq1ot1 outer reverse and Kcnq1ot1 inner forward + Kcnq1ot1 inner reverse, using the following PCR conditions: denaturation at 94°C for 2 min followed by 30 cycles at 94°C for 60 s, 52°C for 90 s, 72°C for 60 s and a final elongation step of at 72°C for 6 min.
The parental origin of the alleles was identified by an extra segment of sequence (5'-ACGGTCGTGAAACGAGG-3') which is present only in the F1 mice, carrying three additional CpG dinucleotides (CpGs 4, 5 and 6). These are missing in the SD7 alleles which also lack CpG 20 due to a CA transversion. Except for the ES cells the bisulphite-based sequence data of the different samples presented in this work are each derived from at least two independent bisulphite treatments.
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ACKNOWLEDGEMENTS |
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We would like to thank the members of the MPI-MG sequencing department, particularly Sven Klages, Katja Heitmann and Katja Borzym, for their technical support. We also want to thank Patricia Ruiz (MPI-MG) and Robert Feil for providing cDNAs and ES cells, and Wendy Dean (Babraham Institute) for experimental help and advice with embryology. We gratefully acknowledge T.A. Trautners’ support for this work. Part of the work was supported by the Deutsche Forschungsgemeinschaft (WA1031-2), HFSP (RG0088/99), BBSRC, MRC and CRC.
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NOTE ADDED IN PROOF |
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After submitting this paper, Yatsuki et al. (DNA Res., 7, 195–206) independently reported a comparative sequence analysis in the BWS cluster. Furthermore, data published by Horike et al. (Hum. Mol. Genet., 9, 2075–2083) support the notion that the KCNQ1OT1 CpG island constitutes a second imprinting centre in human 11p15.5.
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FOOTNOTES |
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+ These authors contibuted equally to this work
§ To whom correspondence should be addressed. Tel: +49 30 84131274; Fax: +49 30 84131385; Email: walter@molgen.mpg.de
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REFERENCES |
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