Human Molecular Genetics, 2000, Vol. 9, No. 12 1829-1841
© 2000 Oxford University Press
Sequence conservation and variability of imprinting in the Beckwith–Wiedemann syndrome gene cluster in human and mouse
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 27 March 2000; Revised and Accepted 15 May 2000.
DDBJ/EMBL/GenBank accession nos AJ251835, AJ271092, AJ279791–AJ279797.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
In human and mouse most imprinted genes are arranged in chromosomal clusters. This linked organization suggests coordinated mechanisms controlling imprinted expression. We have sequenced 250 kb in the centre of the mouse imprinting cluster on distal chromosome 7 and compared it with the orthologous Beckwith–Wiedemann gene cluster on human chromosome 11p15.5. This first comparative imprinting cluster analysis revealed a high structural and functional conservation of the six orthologous genes identified. However, several striking differences were also discovered. First, compared with the mouse the human sequence is ~40% longer, mostly due to insertions of two large repetitive clusters. One of these clusters encompasses an additional gene coding for a homologue of the ribosomal protein L26. Second, pronounced blocks of unique direct repeats characteristic of imprinted genes were only found in the human sequence. Third, two of the orthologous gene pairs Tssc4/TSSC4 and Ltrpc5/LTRPC5 showed apparent differences in imprinting between human and mouse, whereas others like Tssc6/TSSC6 were not imprinted in either organism. Together these results suggest a significant functional and structural variability in the centre of the imprinting cluster. Some genes escape imprinting in both organisms whereas others exhibit tissue- and species-specific imprinting. Hence the control of imprinting in the cluster appears to be a highly dynamic process under fast evolutionary adaptation. Intriguingly, whereas imprinted genes within the cluster contain CpG islands the non-imprinted Ltrpc5 and Tssc6/TSSC6 do not. This and additional comparisons with other imprinted and non-imprinted regions suggest that CpG islands are key features of imprinted domains.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Imprinted genes are those that are predominantly expressed from only one of the parental alleles (1–6). The imprinting mechanism involves epigenetic modifications such as DNA methylation and altered chromatin structures (7). 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, such as the Prader–Willi/Angelman syndromes (8) and Beckwith–Wiedemann syndrome (BWS) (9), which is associated with fetal overgrowth and a predisposition to childhood tumours.
A significant number of imprinted genes are organized in clusters that are characterized by epigenetic features such as differences in allele-specific DNA replication, chromosome pairing of the two parental copies and sex-specific meiotic recombination frequencies (10–13). This suggests an epigenetic co-regulation of neighbouring imprinted genes. Some aspects of regulation of imprinting could affect the whole cluster, either by the chromatin structure itself or by cis-acting elements such as imprinting centres or cis-acting transcripts.
One prominent imprinting cluster in the human is the BWS region on chromosome 11p15.5. In the mouse the imprinting cluster on distal chromosome 7 is homologous to the human BWS region. We and others have identified a number of mouse genes in this region at the same relative positions as their human orthologues, indicating a syntenic organization in both organisms (14–19). In the mouse and in the human the imprinting clusters contain the paternally expressed Igf2/IGF2 (20–22) and Ins2/INS genes (23), the maternally expressed H19 (24,25), Mash2/ASCL2 (HASH2) (26,27), Cdkn1c/CDKN1C (28–31), Tssc3/TSSC3 (14), Tssc5/TSSC5 (15,16) and Kcnq1/KCNQ1 (17–19,32) genes and Cd81/CD81, whose maternal expression has so far only been shown in mouse (19,33,34). Antisense transcripts have been identified for mouse Igf2 (35), for human TSSC5 (15,16,36) and in both organisms for Kcnq1/KCNQ1 (37–39). The Igf2 and Kcnq1/KCNQ1 antisense transcripts Kcnq1ot1/KCNQ1OT1 (Lit1/LIT1) are expressed from the paternal allele; imprinting of the TSSC5 antisense transcript has not yet been investigated. The imprinted region is flanked by the non-imprinted genes Nap1l4/NAP1L4 upstream of Tssc3/TSSC3 (17,40) and Rrpl23l/RPL23L downstream of H19 (41). In the mouse the Nctc1 gene between H19 and Rrpl23l has also been shown to be biallelically expressed (42).
Regionally coordinated control of imprinting has been shown for the Ins2, Igf2 and H19 genes in the mouse (43) and is likely in the human as well (44–46). However, the Kcnq1 and Cdkn1c genes are apparently not affected by the Igf2–H19 controls (19). On the other hand, CDKN1C mutations (29,47–49), loss of imprinting of KCNQ1OT1 (37) and loss of imprinting of IGF2 (46,50) are all associated with BWS, suggesting that there might be control mechanisms that involve the whole cluster. Indeed, translocations in BWS patients in the KCNQ1 gene are associated with loss of imprinting of IGF2 (39,51). Nevertheless, there seems to be a tripartite structure of the cluster as judged by the fact that the human TSSC4 and TSSC6 genes, which are located between KCNQ1 and ASCL2, are not imprinted (52). Similarly, the mouse Cd81 gene, which is located in the same region, only shows imprinting in very early development (19).
This region between the Kcnq1 and Mash2 genes therefore seems to be of particular interest in carrying out a systematic analysis of sequence features which may be involved in either the maintenance of or escape from imprinting in the cluster. This question lends itself to a comparative sequence analysis between mice and humans, which has been shown to be highly valuable for the detection of genes, organizational features and potential regulatory sequences (53,54).
Here we have carried out the first comparative sequence analysis in an imprinting cluster. Based on a previously generated BAC contig in the mouse (17) we sequenced a 250 kb region between Kcnq1 and Mash2. We compared the gene organization between mouse and human and identified six orthologous genes in this region. Three of these were identified as novel genes in the mouse and were examined for their imprinting status. The results provide insights into the structural organization and the control mechanisms of imprinted expression in the BWS gene cluster. In particular, they highlight the importance of CpG islands for imprinted genes.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Sequencing of BAC 300P2 and identification of genes
In a previous work we generated a contig of BAC clones (17) covering 1 Mb of the imprinting cluster on distal chromosome 7 in the mouse between genes Nap1l4 and Mash2. A 250 kb large BAC (300P2) covering a gene-rich region between exon 9 of Kcnq1 and Mash2 was chosen for sequencing. Analysis in the human suggested that some of the genes within this region, like KCNQ1, LTRPC5 and ASCL2, are imprinted, whereas others, like TSSC4 and TSSC6, are not (27,32,52,55). Our aim was to identify the mouse orthologues of these human genes, to analyse their imprinting status and to identify sequence features that characterize imprinted versus non-imprinted genes/regions. Therefore, the complete sequence of BAC 300P2 was determined by assembling 4205 sequence reads obtained from a ‘shotgun’ pUC18 library with an average insert size of 1.0–1.5 kb. The finished high quality sequence (GenBank accession no. AJ251835) consists of a single contig with a length of 249 487 bp starting 1887 bp 3' of exon 9 of Kcnq1 and ending 18 997 bp 3' of the last exon of Mash2 (Fig. 1). Genes in the BAC 300P2 sequence were identified either by homology searches against the partially annotated human genomic sequence (GenBank accession nos AC002536, AC003693 and AJ006345), transcribed sequences in the GenBank databases for non-redundant sequences and expressed sequence tags (ESTs) or by using a series of gene prediction programs (see Materials and Methods). The genomic locations and structures of five complete genes and ~90 kb of the 5' part of the Kcnq1 gene (including exon 9) were thus identified. Cd81, Mash2 and Kcnq1 have been described previously (17–19,26). Ltrpc5, Tssc4 and Tssc6 were newly identified in the mouse.
|
Sequence comparison between human and mouse
The first intriguing observation when comparing the mouse BAC 300P2 with the corresponding human (GenBank accession nos AC002536A, AC003693 and J006345) sequence is the almost completely conserved order of the genes located in the respective genomic regions (Fig. 1). The comparison revealed that not only the relative order of genes, Kcnq1/KCNQ1–Ltrpc5/LTRPC5 (MTR1)–Tssc4/TSSC4–Cd81/CD81 (Tapa1/TAPA1)–Tssc6/TSSC6–Mash2/ASCL2 (HASH2), but also their relative transcriptional orientation and, for the majority of genes, the size and distribution of exons are highly conserved. This conservation is directly visible at the level of the genomic sequences, which can be aligned along the entire region with the exception of two major gaps (Fig. 2). These are caused by two remarkably large insertions of A/T-rich repeated sequences in the human chromosome (Figs 1 and 2), one in the 5' part of the KCNQ1 gene and the other between CD81 and TSSC6. The latter includes an additional intronless gene coding for a homologue of the ribosomal protein L26 (Fig. 1). The absence of this gene in the mouse sequence and the fact that it is flanked by a number of retrotransposable elements suggests that it is a recently integrated copy of the already identified RPL26 gene residing on chromosome 17p (GenBank accession no. AF083248) (56). Both repetitive insertions are rich in L1 elements and contribute to the overall higher proportion of repetitive elements (38.5% in the human sequence compared with only 23.5% in the mouse sequence). The two insertions are the main cause of the size difference of the homologous regions in human (344 kb) and mouse (249 kb).
|
Using prediction software (MarFinder) a few strong putative matrix attachment regions (MARs) (Fig. 1) were localized in both genomes. These MARs bracket the central part of the sequenced region including the Kcnq1/KCNQ1 CpG island and the entire Ltrpc5/LTRPC5, Tssc4/TSSC4 and Cd81/CD81 genes.
Characteristic unique repetitive elements have previously been identified in the vicinity of some known imprinted genes and it has been argued that they might play a role in the epigenetic control of expression (57). When screening the entire region in human and mouse for such short unique tandemly arranged sequence elements (motif size >7, element size >200), nine repeated motifs could be identified in the human but no such elements were detected in the mouse (Table 1 and Fig. 1). The repeated motifs in the human extend to a total length of up to 1.3 kb, with the longest repeat unit being 56 bp. Except for one of the motifs (VI) no homologies to other genomic regions were detected in the GenBank database. The absence of such unique tandem repeats in the mouse sequence and the finding that these are not necessarily linked to imprinted genes in the human do not support the hypothesis that such repeats are defining hallmarks of imprinted genes (57).
|
Comparison of the gene structures in human and mouse
Cd81, Mash2 and Kcnq1.
The precise placements of exons of the Cd81 (34) and Mash2 genes (58) and the 5' part (up to exon 9) of Kcnq1 (17) are shown in Figure 1. The exact coordinates of the exon–intron structures are deposited with the BAC 300P2 sequence (GenBank accession no. AJ251835). A comparison with the human sequence revealed a strong conservation of gene structures in mouse and human. In the human, however, the ASCL2 gene has only been partially identified and its 5'-end was missing. A comparison of human sequences with mouse and rat cDNAs and genomic sequences (data not shown) suggests the location of the putative 5' exon of the ASCL2 gene (see Fig. 1 and legend).
In contrast to the majority of genes in this region, whose relative exon–intron structure is quite conserved between human and mouse, the 5'-ends of the Kcnq1/KCNQ1 genes show remarkable differences between the two species (Fig. 1). The second intron in the human is ~40 kb larger than the corresponding intron in the mouse. This increase mainly results from the large insertion of repetitive sequences (Fig. 1) between exons 1b and 1c in the human. Exons homologous to the mouse exons 1ß–1
are not present in the human sequence (17). Conversely, homologues of the human exons 1b, 1c and 2a (32) were not found in the mouse. The differences in the 5' portions of the mouse and human Kcnq1/KCNQ1 genes may contribute to differences in expression and function in both organisms (17,32). In contrast to the differences in the 5' region of Kcnq1, the sequences 3' of exon 1 are highly homologous between mouse and human.
Tssc4.
Tssc4 is located 1.4 kb 3' of the last exon of Cd81 and transcribed in the same orientation; the distance to the 3'-end of the oppositely orientated Ltrpc5 is 0.7 kb. The Tssc4 cDNA sequence of 1394 bp (GenBank accession no. AJ279796) was assembled from 73 ESTs matching the BAC 300P2 sequence. The complete cDNA sequence was verified by sequencing one of the ESTs (GenBank accession no. AA268143) and RT–PCR products. A putative polyadenylation signal is present 20 bp upstream of the 3'-end. The human orthologue of Tssc4 has previously been identified by a subtractive cDNA screen and is regarded as a potential tumour suppressor gene (52). Both mouse Tssc4 and human TSSC4 are very compact genes. Although the mouse Tssc4 gene is composed of three exons spanning 1.7 kb of genomic sequence, the published cDNA sequence of the human orthologue consists of only two exons (52). However, an additional exon at the 5'-end is indicated by an overlapping EST sequence (GenBank accession no. AW249431). Based on this finding, the finally assembled human TSSC4 gene contains three exons spanning 3131 bp of genomic DNA and the cDNA has a length of 1503 bp. In the human only the first exon is located in a CpG island, whereas in the mouse the first and the second exons are positioned in a 1.3 kb long CpG island. Sequence similarity (61% sequence identity) is confined to exon 3 of Tssc4/TSSC4. This exon harbours an open reading frame (ORF) of 951 bp that potentially encodes a peptide of 317 amino acids. Although syntenically placed and actively transcribed, Tssc4/TSSC4 show a significantly lower level of sequence conservation between human and mouse compared with the flanking genes Cd81/CD81 and Ltrpc5/LTRPC5. Compared with the deduced human TSSC4 peptide (329 amino acids) the overall similarity value of 67.8% results from only six highly conserved stretches of 15–20 amino acids (Fig. 3). This patterned homology suggests a conservation of important functional domains. A motif search using these homologous regions against sequences in public databases did not reveal significant similarities to known proteins. RT–PCR revealed the existence of an alternative Tssc4 transcript (Tssc4-2) which is 498 bp shorter as a result of an internal splicing event in exon 3. This alternative splicing generates a translational shift and a truncated peptide of 114 amino acids (Fig. 3A).
|
Tssc6.
The mouse homologue of the human TSSC6 gene, which was isolated as a transcript with potential tumour suppressor activity (52), is located between Cd81 and Mash2 (Fig. 1) and transcribed in the same direction as Cd81 and Tssc4. The Tssc6 gene is split into eight exons covering 13.8 kb of genomic sequence. Based on two clusters of ESTs matching to the 3'- and 5'-ends of the gene, respectively, a 1657 bp long cDNA sequence (GenBank accession no. AJ279791) was generated by combining these EST sequences with sequences of overlapping RT–PCR products. The cDNA sequence encompasses an ORF of 678 bp potentially coding for a peptide of 226 amino acids. Similar to Tssc4, the primary sequence of Tssc6 does not show any significant similarities to other proteins in public databases except to the human TSSC6 peptide and some weak similarity (29%) to the peptide of the neighbouring Cd81 gene, a transmembrane protein. The human- and mouse-derived peptide sequences are only 62.8% identical (Fig. 4). In contrast to the Tssc4/TSSC4 orthologues, the homologies between Tssc6/TSSC6 are equally distributed along the entire length of the sequence. However, the mouse Tssc6 peptide is 68 amino acids shorter than the human and this dissimilarity is also reflected in the DNA sequences. Whereas the sequences of the first seven exons of both mouse and human Tssc6/TSSC6 genes are quite similar (68–81% identity), Tssc6 exon 8 does not show any similarity to the published human TSSC6 cDNA and genomic sequences.
|
Analysis of RT–PCR products between exons 1 and 8 from RNA isolated from tissues (i.e. lung and spleen tissues) of newborn mice detected four additional Tssc6 splice variants (Tssc6-2–Tssc6-5) (GenBank accession nos AJ279792–AJ279795). The shorter splice variants lack various internal exons (Fig. 4). Consequently, the alternative splicing shortens the ORFs or leads to translational frameshifts and truncated ORFs with a stop codon shortly after exon 5. The putative peptides have the following sizes: Tssc6-2, 198 amino acids; Tssc6-3, 176 amino acids; Tss6-4, 164 amino acids; Tssc6-5, 142 amino acids.
Ltrpc5.
Ltrpc5 is located 17.8 kb upstream of the first exon of Kcnq1 and encompasses at least 17.8 kb of genomic sequence. The human orthologue of this gene was identified on the basis of sequence similarity and one human EST (GenBank accession no. AA577486) described in the database. Expression of the human LTRPC5 (MTR1) gene has recently been described (55). The genes should be named Ltrpc5/LTRPC5 (large transient receptor potential-related channel 5) in accordance with the unifying nomenclature (59).
Ltrpc5 is transcribed in the opposite direction to the Kcnq1, Tssc4, Cd81 and Tssc6 genes. As no matching mouse ESTs (and only one human) were found in the databases our initial discovery of Ltrpc5 was based on exon predictions and sequence similarities between human and mouse genomic sequences. The similarity within the predicted 24 exons ranges from 86 to 92% identity on the DNA level. Transcription of the mouse Ltrpc5 gene was examined by RT–PCR between exons 1 and 24 and an almost full-length cDNA sequence (GenBank accession no. AJ271092) was assembled. The 5'-end of the first exon is predicted by sequence similarity to the human cDNA. The 3'-end, containing one potential polyadenylation signal, was identified by 3'-RACE. Alternative splicing resulting in an additional transcript was observed within the last two exons of the gene (Fig. 5A). No alternative splicing involving exon 18 was detected as described for the human LTRPC5 gene (55). The start of the ORF was predicted by its sequence similarity to the human and the proposed translation start point matches perfectly to a Kozak consensus sequence. The two Ltrpc5 transcripts comprise ORFs potentially coding for peptides of 1148 and 1138 amino acids length, respectively. Both isoforms contain the typical TRPC topology of six transmembrane domains. The peptide sequences deduced from the long mouse Ltrpc5 transcript and human LTRPC5 are 82% identical (Fig. 5). Within the transmembrane domains the similarity rises to 98% (Fig. 5). Database searches revealed that the Ltrpc5 protein shows highest similarities to human melastatin (30% identity) and LTRPC7 (37% identity) as well as to a hypothetical Caenorhabditis elegans TRPC-like protein (GenBank accession no. CAB05572) (26% identity). These similarities suggest that Ltrpc5 codes for a potential membrane-spanning protein regulating Ca2+ entry into cells. The predicted structure of the Ltrpc5 protein assigns it to a subfamily of long transient receptor potential proteins (LTRPCs) (59).
|
Imprinting analysis of Tssc4, Tssc6 and Ltrpc5
Kcnq1, Cd81 and Mash2 have previously been shown to exhibit imprinted expression (17–19,26). Imprinting of these genes appears to be restricted to specific tissues and developmental stages. Here we describe the imprinting status of the remaining three novel mouse genes within the sequenced region. To monitor possible developmental and tissue-specific differences the studies were performed on RNA preparations from whole embryos and placentae at two developmental stages (12.5 and 16.5 d.p.c.) and a variety of organs isolated from embryos (16.5 d.p.c.) and newborn mice. A Tssc4 cDNA probe (GenBank accession no. AA646792) hybridized on a northern blot to various transcripts of ~1.7–1.9 kb length (data not shown). We were able to amplify RT–PCR products for all three genes from all tissues analysed, suggesting a ubiquitous expression pattern of Tssc4, Tssc6 and Ltrpc5, with the exception of Ltrpc5 transcripts in placentae (12.5 d.p.c.).
To perform an allele-specific expression analysis we used RNA derived from progeny of C57BL/6 x SD7 crosses as well as from reciprocal crosses. SD7 is a congenic mouse strain which carries a defined segment of distal chromosome 7 of Mus spretus (SEG) on an M.musculus background (60). In all three genes we found DNA polymorphisms in restriction sites that allowed us to determine the allelic origin of the transcripts. RT–PCR products were cut with the relevant restriction endonucleases and the relative intensity ratios of the allele-specific DNA fragments were visualized by agarose gel electrophoresis and staining. At all three developmental stages, i.e. 12.5 d.p.c., 16.5 d.p.c. and newborn mice, and in all different tissues we detected RT–PCR products from both parental alleles for Tssc6 and Ltrpc5, indicating biallelic expression (Fig. 6). A biased, i.e. strain-specific, amplification was ruled out by control experiments using a 1:1 mixture of cDNAs of both parental strains (data not shown). Tssc4 was also biallelically expressed in embryos and neonatal tissues. However, we observed a significant bias towards the maternal allele in placentae at 12.5 and 16.5 d.p.c. for both Tssc4 transcripts (Fig. 6). This indicates that Tssc4 is imprinted in the placenta.
|
Imprinted genes are associated with CpG islands
It was remarkable that the non-imprinted Tssc6/TSSC6 genes and the non-imprinted mouse Ltrpc5 gene did not have CpG islands, whereas the imprinted human LTRPC5 did have an island and so did all other imprinted genes in the cluster. We therefore decided to compare CpG island density in the cluster with the reported averages in the mouse and human genomes (61), to the density in other non-imprinted gene-rich genomic regions (53,54) and to the frequency of CpG islands in all imprinted genes (http://www.mgu.har.mrc.ac.uk/imprinting/imptables.html#impgene ).
First, the CpG island per gene ratio in the cluster (86% in human and 66% in mouse) was significantly above average (56% in human and 47% in mouse; P = <0.01 and 0.001, respectively). Second, when comparing two randomly selected gene-rich regions, one on human chromosome 12p13/mouse chromosome 6 (12p/6), the other the X-linked BTK locus (BTK), the CpG island per gene ratios were found to be even lower than the genome averages, with 53 (12p/6) and 25% (BTK) for human and 41 (12p/6) and 25% (BTK) for mouse, respectively.
The CpG dinucleotide content in the imprinted and non-imprinted regions was, however, not significantly different (imprinting cluster, human 2.4%, mouse 1.8%; 12p/6 region, human 2.1%, mouse 1.7%). Third, of all imprinted genes in mouse (both those which are in clusters and those outside clusters) for which information was available, 88% had CpG islands. These results demonstrate that one of the key features of imprinted genes is their association with CpG islands.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The determination of a 250 kb sequence between the imprinted Kcnq1 and Mash2 genes in the central part of the BWS region in the mouse offered the opportunity to compare the structural organization of six genes within the BWS gene cluster in two mammalian species, as the human DNA sequence was already available in the GenBank database. The purpose of the comparative sequencing effort was to obtain information on sequence conservation and on the conservation of gene regulation and imprinting. This enhances our understanding of the genetic and epigenetic control mechanisms in the whole region and of pathological mechanisms leading to BWS in the human.
The key findings of this study are, first, that the organization of the human and mouse regions is highly conserved except for two large insertions in the human sequence comprising repetitive sequences and a homologue of the ribosomal RPL26 gene. Second, whereas some genes in this region such as Tssc6/TSSC6 apparently escape imprinting in mouse and human, others like Tssc4/TSSC4 are either imprinted in mouse placenta but not in the human or, conversely, like Ltrpc5/LTRPC5 are not imprinted in the mouse but are in the human. Hence the central region of the cluster shows considerable variability in imprinting patterns between the two species. Lastly, imprinted genes in the cluster and elsewhere in the genome have a CpG island frequency which is remarkably above genome average, indicating that CpG islands may be necessary for imprinted gene silencing.
The chromosomal organization of the region in both species exhibits nearly complete conservation as all orthologues (Kcnq1/KCNQ1, Ltrpc5/LTRPC5, Tssc4/TSSC4, Cd81/CD81, Tssc6/TSSC6 and Mash2/ASCL2) have the same relative order and identical transcriptional orientation in mouse and human. The only major difference is the presence of an additional copy of the RPL26 gene that codes for a full-length ribosomal-like protein in the human and resides between TSSC6 and CD81 in the middle of a large insertion of repetitive elements. It remains unclear whether the unique presence of this gene in the human sequence reflects a recent evolutionary event of a de novo insertion or whether RPL26 has been deleted in the mouse. Its presence within a cluster of repetitive sequences favours the first explanation.
With the exception of the two large insertions in the human sequence the distribution of repetitive elements, like LINE/L1, SINE/B1 and ALU/Alu elements and microsatellites, is similar in both organisms (Fig. 1). Major clusters of these repetitive elements are present 5' of Cd81/CD81 and Mash2/ASCL2 in human and mouse, minor clusters in the upstream region of Kcnq1/KCNQ1 and 3' of Mash2/ASCL2. This conserved distribution of major clusters of repetitive elements and the presence of strong predicted MARs in the 5' region of Kcnq1/KCNQ1 and around Tssc6/TSSC6 may be a reflection of a structural subdivision of the region (Fig. 1). Thus, there appear to be two peripheral domains with stably maintained and conserved imprinting patterns. The first domain is demarcated by Tssc3/TSSC3 and Kcnq1/KCNQ1 and the second by Mash2/ASCL2 and H19. In contrast, the central domain is characterized by variable, relaxed or lack of imprinting and is located between Ltrpc5/LTRPC5 and Tssc6/TSSC6. The functional analysis of human and mouse genes in this region also supports this notion (19,52,55). Examples of relaxation of imprinting are the mouse Cd81 gene (19) and the Tssc6 and Ltrpc5 genes identified here, which were found to be biallelically expressed during mid-gestation and late-stage embryonic development as well as after birth. It remains open whether Tssc6 and Ltrpc5 are imprinted at earlier developmental stages (i.e. before 12.5 d.p.c.), as observed for Cd81 (19). In contrast, all other genes in the cluster are either not subject to relaxation of imprinting, such as Cdkn1c, Mash2, Igf2 and H19 (22,25,26,28), or, as in the cases of Tssc5 and Kcnq1, the onset of relaxation is much later, around the time of birth (17,19).
Examples of species-specific variation in imprinting are Tssc4/TSSC4 and Ltrpc5/LTRPC5. Whereas Tssc4 was biallelically transcribed in fetal tissues, it was maternally expressed in placenta. These results indicate that Tssc4/TSSC4 is imprinted in mouse but not in human (52). We note that imprinting of mouse Tssc3 and Tssc5 is also most pronounced in extraembryonic tissues (14,15). The other genes showing species-specific differences in imprinted gene expression are Ltrpc5/LTRPC5. Ltrpc5 was not imprinted in the mouse but human LTRPC5 showed exclusive paternal expression in monochromosomal hybrid cell lines (55). Imprinting in normal somatic tissues, however, as well as during embryonic stages, has not been analysed. Lack of conservation of imprinting between human and mouse has also been described for the Igf2r/IGF2R gene (62), which is imprinted in mouse but not consistently imprinted in the human.
Because of the relaxation in the centre of the imprinting cluster, previously postulated boundaries of the imprinting cluster need to be treated with caution as these are based only on the biallelic expression of Nap1l4 (17), upstream of Tssc3, and Nctc1 and Rpl23l, downstream of H19 (41,42). It cannot be excluded that more imprinted genes are present in the vicinity of the cluster. Furthermore, although most of our own and previous results (52,55) suggest that the whole imprinting cluster can be separated into (at least) three subdomains with different characteristics, such a division does not exclude the possibility that some functional aspects, like the germline establishment or maintenance of imprinting, are controlled at the level of the cluster as a whole. This view is supported by the observation that translocations in KVLQT1 in BWS patients are associated with loss of imprinting in IGF2 (39,51) and that at least some BWS patients show loss of imprinting of both KCNQ1OT1 (LIT1) and IGF2 (37,39).
One of the structural features investigated, the distribution and quality of CpG islands, seems to integrate the division into subdomains and gene-specific effects with the general aspects of imprinting in the cluster. The comparison of imprinting patterns in the cluster with the distribution and characteristics of CpG islands thus revealed intriguing associations. Cdkn1/CDNK1C and Mash2/ASCL2 (26–30) are tightly imprinted and are linked to the most pronounced CpG islands in this region. In the region between Kcnq1 and Mash2 there seems to be a connection between the quality (the length) of CpG islands and the timing of relaxation or the extent of imprinting. Kcnq1 is biallelically expressed after birth and possesses a more pronounced CpG island than Tssc4, which is already biallelically expressed at 12.5 d.p.c. in the embryo and only imprinted in the mouse placenta. The non-imprinted Tssc6 and Ltrpc5 do not have CpG islands, whereas the imprinted human LTRPC5 gene (55) has one. The only exception to this rather consistent picture is the human TSSC4 gene which, while still maintaining a CpG island, is not imprinted. Hence, although CpG islands and their quality may be linked to the extent of imprinted expression, their presence seems to be necessary but not sufficient to establish/maintain imprinting. However, the functional association of CpG islands with imprinted expression is further strengthened by the observation that regions that have a similar gene density but are not imprinted have a significantly lower density of CpG islands. Hence we suggest that establishment and stable maintenance of imprinting patterns in mammalian genes requires the presence of CpG islands and is possibly influenced by their quality.
The conservation of major parts of the genomic sequence and the pronounced relaxation of imprinting in this region in human and mouse indicate that the mouse will serve as a very useful model to study general mechanisms of imprinting and the aetiology of BWS. Systematic comparative analysis should now be extended to the whole cluster and to the study of epigenetic modifications throughout the cluster region using new functional genomics technologies.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
DNA sequencing
DNA of BAC clone 300P2 (CITB Library, Research Genetics, http://resgen.com ; B. Birren et al., unpublished data) was prepared according to a standard protocol (63) which included two CsCl gradient ultracentrifugations. Aliquots of 10 µg DNA were sonicated for 15 s using a cell disruptor B-30 (output control setting 5; Branson Sonic Power, Danbury, CT). After treatment with Klenow and T4 DNA polymerases (MBI Fermentas, Vilnius, Lithuania) 1.0–1.5 kb long DNA fragments were purified from 1% agarose gels (1x TBE) using the QiaexII kit (Qiagen, Hilden, Germany), 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- to 6-fold sequence coverage of the entire BAC insert, the inserts of 3042 clones were unidirectionally sequenced on ABI 377 automatic sequencers using standard protocols. The sequences were assembled using 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, 4205 sequences were assembled; per consensus character 6.6 average reading characters were obtained.
Sequence analysis
The human and the mouse sequences were compared by dot plot analysis using PipMaker (Pennsylvania State University, http://globin.cse.psu.edu/cgi-bin/pipmaker ) and the software package MacMolly (match length 35, mismatches 12, gap penalty 3, mismatch penalty 1). Exon prediction was performed with GRAIL (Oak Ridge National Laboratory, http://compbio.ornl.gov ), Genscan (Massachusetts 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 Center 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 Censor software (http://charon.girinst.org/~server/censor.html ). CpG and G+C distribution were obtained using the ‘window’ function of the GCG package (window size 500, shift increment 3; Genetics Computer Group, Madison, WI) (64). 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 and tandem repeats with motifs longer than 6 bp were detected with the ‘compare’ function of the same software package. The searches for MARs were performed using MarFinder (http://www.ncgr.org/MarFinder ).
RNA preparations
Total RNAs of various mouse tissues and developmental stages were prepared according to a standard protocol (65). 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, 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 of 94°C at 30 s, 30 s annealing at 53–68°C (for temperatures see Table 2) and 1 min at 75°C and a final elongation at 72°C for 5 min.
|
Transcription of both Tssc4 transcripts (GenBank accession no. AJ279796) was verified by RT–PCR using primers 1 and 2. For Tssc6 overlapping RT–PCR products were generated using the primer pairs 3 + 4 (exons 1–4), 5 + 6 (exons 4–7) and 7 + 8 (exons 7–8).
Overlapping RT–PCR products of Ltrpc5 were generated using the following primer combinations: 9 + 10 (exons 1–2), 11 + 12 (exons 2–4), 13 + 14 (exons 4–8), 15 + 16 (exons 8–9), 17 + 18 (exons 9–13), 19 + 20 (exons 13–16), 21 + 22 (exons 16–19), 23 + 24 (exons 19–21) and 25 + 26 (exons 21–22).
The obtained RT–PCR products were cloned into vector pGEM-T (Promega) and sequenced using standard protocols. The 3'-RACE of Ltrpc5 was performed with primer 27 using the SMART RACE cDNA amplification kit from Clontech (Heidelberg, Germany). The products were cloned and sequenced.
Imprinting analysis
Strain-specific sequence polymorphisms were detected by sequencing PCR products derived from Mus mus domesticus C57BL/6 and M.spretus DNA templates, respectively. For Tssc4 PCRs were performed on M.m.domesticus C57BL/6 and M.spretus DNAs using the primer pairs 28 + 29 and 30 + 31. For Tssc6 (GenBank accession no. AJ279791) and Ltrpc5 (GenBank accession no. AJ271092) RT–PCR products were amplified and sequenced as described above using M.m.domesticus C57BL/6 and SD7 cDNAs.
For the subsequent imprinting analysis of Tssc4-1 (GenBank accession no. AJ279796) RT–PCR was performed using primers 32 and 33. The 811 bp long RT–PCR products obtained were purified from a 1.5 % agarose gel (1x TBE) using the QiaexII kit and cut with TaiI. In SD7-derived RT–PCR products a TaiI site is present at nucleotide 126 932 (GenBank accession no. AJ251835) generated by an A
G transition, therefore TaiI digestion results in 298 and 513 bp DNA fragments specific for SD7.
The imprinting status of Tssc4-2 (GenBank accession no. AJ279797) was analysed using primer pair 34 + 35. The 268 bp long RT–PCR products were gel purified from a 1.5% agarose gel (1x TBE) and cut with AluI, at an SD7-specific restriction site generated by a GT transition at nucleotide 126 351 (GenBank accession no. AJ251835). The obtained SD7-specific restriction fragments were 215 and 53 bp long.
For Tssc6 RT–PCR was performed with primer pair 36 + 37. The 756 bp long product obtained was gel purified and cut with TaiI at an SD7-specific site, generated by an AG exchange at nucleotide 178 113 (GenBank accession no. AJ251835). This resulted in 128 and 628 bp long DNA fragments specific for the SD7 allele.
Ltrpc5 RT–PCR products were amplified with primer pair 21 + 22. The imprinting status of Ltrpc5 was analysed using a strain-specific polymorphism at nucleotide 117 174 (GenBank accession no. AJ251835) which results in a BamHI restriction site in M.m.domesticus C57BL/6 and a DdeI restriction site in SD7.
|
ACKNOWLEDGEMENTS |
|---|
We would like to thank the members of the MPI-MG sequencing department, in particular Sven Klages, Katja Heitmann and Katja Borzym, for their technical support, Juliane Ramser and Steffen Hennig for help with database searches and technical tips, C. Harteneck for helpful comments on the LTRPC family, Angelika Daser for her support, and Patricia Ruiz and Lucy Bowden for technical help. We also gratefully acknowledge Prof. T.A. Trautners support for this work and many helpful discussions. Part of the work was supported by the Deutsche Forschungsgemeinschaft (WA1031-2), HFSP (RG0088/99), BBSRC, MRC and CRC.
|
FOOTNOTES |
|---|
+ These authors contributed equally to this work
§ To whom correspondence should be addressed. Tel: +49 30 84131274; Fax: +49 30 84131385; Email: walter@molgen.mpg.de
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Reik, W. and Walter, J. (1998) Imprinting mechanisms in mammals. Curr. Opin. Genet. Dev., 8, 154–164.[Web of Science][Medline]
2 Feil, R. and Khosla, S. (1999) Genomic imprinting in mammals: an interplay between chromatin and DNA methylation? Trends Genet., 15, 431–435.[Web of Science][Medline]
3 Brannan, C.I. and Bartolomei, M.S. (1999) Mechanisms of genomic imprinting. Curr. Opin. Genet. Dev., 9, 164–170.[Web of Science][Medline]
4 Tilghman, S.M. (1999) The sins of the fathers and mothers: genomic imprinting in mammalian development. Cell, 96, 185–193.[Web of Science][Medline]
5 Barlow, D.P. (1997) Competition—a common motif for the imprinting mechanism? EMBO J., 16, 6899–6905.[Web of Science][Medline]
6 Surani, M.A. (1998) Imprinting and the initiation of gene silencing in the germ line. Cell, 93, 309–312.[Web of Science][Medline]
7 Constancia, M., Pickard, B., Kelsey, G. and Reik, W. (1998) Imprinting mechanisms. Genome Res., 8, 881–900.
8 Nicholls, R.D., Saitoh, S. and Horsthemke, B. (1998) Imprinting in Prader-Willi and Angelman syndromes. Trends Genet., 14, 194–200.[Web of Science][Medline]
9 Reik, W. and Maher, E.R. (1997) Imprinting in clusters: lessons from Beckwith-Wiedemann syndrome. Trends Genet., 13, 330–334.[Web of Science][Medline]
10 Kitsberg, D., Selig, S., Brandeis, M., Simon, I., Keshet, I., Driscoll, D.J., Nicholls, R.D. and Cedar, H. (1993) Allele-specific replication timing of imprinted regions. Nature, 364, 459–463.[Medline]
11 Knoll, J.H.M., Cheng, S.D. and Lalande, M. (1994) Allele specificity of DNA replication timing in Angelman/Prader-Willi syndrome imprinted chromosomal region. Nature Genet., 6, 41–46.[Web of Science][Medline]
12 Pàldi, A., Gyapay, G. and Jami, J. (1995) Imprinted chromosomal regions of the human genome display sex-specific meiotic recombination frequencies. Curr. Biol., 5, 1030–1035.[Web of Science][Medline]
13 Robinson, W.P. and Lalande, M. (1995) Sex-specific meiotic recombination in the Prader-Willi/Angelman syndrome imprinted region. Hum. Mol. Genet., 4, 801–806.
14 Qian, N., Frank, D., O’Keefe, D., Dao, D., Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. and Tycko, B. (1997) The IPL gene on chromosome 11p15.5 is imprinted in humans and mice and is similar to TDAG51, implicated in Fas expression and apoptosis. Hum. Mol. Genet., 6, 2021–2029.
15 Dao, D., Frank, D., Qian, N., O’Keefe, D., Vosatka, R.J., Walsh, C.P. and Tycko, B. (1998) IMPT1, an imprinted gene similar to polyspecific transporter and multi-drug genes. Hum. Mol. Genet., 7, 597–608.
16 Cooper, P.R., Smilinich, N.J., Day, C.D., Nowak, N.J., Reid, L.H., Pearsall, R.S., Reece, M., Prawitt, D., Landers, J., Housman, D.E. et al. (1998) Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain. Genomics, 49, 38–51.[Web of Science][Medline]
17 Paulsen, M., Davies, K.R., Bowden, L.M., Villar, A.J., Franck, O., Fuermann, M., Dean, W., Moore, T.F., Rodrigues, N., Davies, K.E. et al. (1998) Syntenic organization of the mouse distal chromosome 7 imprinting cluster and the Beckwith-Wiedemann syndrome region in chromosome 11p15.5. Hum. Mol. Genet., 7, 1149–1159.
18 Gould, T.D. and Pfeifer, K. (1998) Imprinting of mouse Kvlqt1 is developmentally regulated. Hum Mol. Genet., 7, 483–487.
19 Caspary, T., Cleary, M.A., Baker, C.C., Guan, X.-J. and Tilghman, S.M. (1998) Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster. Mol. Cell. Biol., 18, 3466–3474.
20 Ohlsson, R., Nystrom, A., Pfeifer-Ohlsson, S., Tohonon, V., Hedbourg, F., Schofield, P., Flam, F. and Ekström, T.J. (1993) IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome. Nature Genet., 4, 94–97.[Web of Science][Medline]
21 Giannoukakis, N., Deal, C., Paquette, J., Goodyer, C.G. and Polychronakis, C. (1993) Parental genomic imprinting of the human IGF2 gene. Nature Genet., 4, 98–101.[Web of Science][Medline]
22 Ferguson-Smith, A.C., Cattanach, B.M., Barton, S.C., Beechey, C.V. and Surani, M.A. (1991) Embryological and molecular investigations of parental imprinting on mouse chromosome 7. Nature, 351, 667–670.[Medline]
23 Deltour, L., Montagutelli, X., Guenet, J.L., Jami, J. and Pàldi, A. (1995) Tissue- and developmental stage-specific imprinting of the mouse proinsulin gene, Ins2. Dev. Biol., 168, 686–688.[Web of Science][Medline]
24 Rainier, S., Johnson, L.A., Dobry, C.J., Ping, A.J., Grundy, P.E. and Feinberg, A.P. (1993) Relaxation of the imprinted genes in human cancer. Nature, 362, 747–749.[Medline]
25 Bartolomei, M.S., Zemel, S. and Tilghman, S.M. (1991) Parental imprinting of the mouse H19 gene. Nature, 351, 153–155.[Medline]
26 Guillemot, F., Caspary, T., Tilghman, S.M., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Anderson, D.J., Joyner, A.L., Rossant, J. and Nagy, A. (1995) Genomic imprinting of Mash2, a mouse gene required for trophoblast development. Nature Genet., 9, 235–242.[Web of Science][Medline]
27 Alders, M., Hodges, M., Hadjantonakis, A.-K., Postmus, J., van Wijk, I., Bliek, J., de Meulemeester, M., Westerveld, A., Guillemot, F., Oudejans, C. et al. (1997) The human Achaete-Scute homologue 2 (ASCL2, HASH2) maps to chromosome 11p15.5, close to IGF2 and is expressed in extravillus trophoblasts. Hum. Mol. Genet., 6, 859–867.
28 Hatada, I. and Mukai, T. (1995) Genomic imprinting of p57KIP2, a cyclin-dependent kinase inhibitor, in mouse. Nature Genet., 11, 204–206.[Web of Science][Medline]
29 Hatada, I., Ohashi, H., Fukushima, Y., Kaneko, Y., Inoue, M., Komoto, Y., Okada, A., Ohishi, S., Nabetani, A., Morisaki, H. et al. (1996) An imprinted gene p57KIP2 is mutated in Beckwith-Wiedemann syndrome. Nature Genet., 14, 171–173.[Web of Science][Medline]
30 Matsuoka, S., Thompson, J.S., Edwards, M.C., Barletta, J.M., Grundy, P., Kalikin, L.M., Harper, J.W., Elledge, S.J. and Feinberg, A.P. (1996) Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15. Proc. Natl Acad. Sci. USA, 93, 3026–3030.
31 Hoovers, J.M.N., Kalkin, L.M., Johnson, L.A., Alders, M., Redeker, B., Law, D.J., Bliek, J., Steenman, M., Benedict, M., Wiegant, J. et al. (1995) Multiple genetic loci within 11p15 defined by Beckwith-Wiedemann syndrome rearrangement breakpoints and subchromosomal transferable fragments. Proc. Natl Acad. Sci. USA, 92, 12456–12460.
32 Lee, M.P., Hu, R.-J., Johnson, L.A. and Feinberg, A.P. (1997) Human KVLQT1 gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements. Nature Genet., 15, 181–185.[Web of Science][Medline]
33 Reid, L.H., Davies, C., Cooper, P.R., Crider-Miller, S.J., Sait, S.N.J., Nowak, N.J., Evans, G., Stanbridge, E.J., deJong, P., Shows, T.B. et al. (1997) A 1-Mb physical map and PAC contig of the imprinted domain in 11p15.5 that contains TAPA1 and the BWSCR1/WT2 region. Genomics, 43, 366–375.[Web of Science][Medline]
34 Andria, M.L., Hsieh, C.-L., Oren, R., Francke, U. and Levy, S. (1991) Genomic organization and chromosomal localization of the TAPA-1 gene. J. Immunol., 147, 1030–1036.[Abstract]
35 Moore, T., Constancia, M., Zubair, M., Bailleul, B., Feil, R., Sasaki, H. and Reik, W. (1997) Multiple imprinted sense and antisense transcripts, differential methylation and tandem repeats in a putative imprinting control region upstream of mouse Igf2. Proc. Natl Acad. Sci. USA, 94, 12509–12514.
36 Crider-Miller, S.J., Reid, L.H., Higgins, M.J., Nowak, N.J., Shows, T.B., Futreal, P.A. and Weissman, B.E. (1997) Novel transcribed sequences within the BWS/WT2 region in 11p15.5: tissue-specific expression correlates with cancer type. Genomics, 46, 355–363.[Web of Science][Medline]
37 Lee, M.P., DeBaun, M.R., Mitsuya, K., Galonek, H.L., Brandenburg, S., Oshimura, M. and Feinberg, A.P. (1999) Loss of imprinting of a paternally expressed transcript, with antisense orientation to KvLQT1, occurs frequently in Beckwith-Wiedemann syndrome and is independent of insulin-like growth factor II imprinting. Proc. Natl Acad. Sci. USA, 96, 5203–5208.
38 Mitsuya, K., Meguro, M., Lee, M.P., Katoh, M., Schulz, T.C., Kugoh, H., Yoshida, M.A., Nikawa, N., Feinberg, A.P. and Oshimura, M. (1999) LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids. Hum. Mol. Genet., 8, 1209–1217.
39 Smilinich, N.J., Day, C.D., Fitzpatrick, G.V., Caldwell, G.M., Lossie, A.C., Cooper, P.R., Smallwood, A.C., Joyce, J.A., Schofield, P.N., Reik, W. et al. (1999) A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome. Proc. Natl Acad. Sci. USA, 96, 8064–8069.
40 Hu, R.-J., Lee, M.P., Johnson, L.A. and Feinberg, A.P. (1996) A novel human homologue of yeast nucleosome assembly protein, 65 kb centromeric to the p57KIP2 gene, is biallelically expressed in fetal and adult tissues. Hum. Mol. Genet., 5, 1743–1748.
41 Tsang, P., Gilles, F., Yuan, L., Kuo, Y.-H., Lupu, F., Samara, G., Moosikasuwan, J., Goye, A., Zelenetz, A.D., Selleri, L. and Tycko, B. (1995) A novel L23-related gene 40 kb downstream of the imprinted H19 gene is biallelically expressed in mid-fetal and adult tissues. Hum. Mol. Genet., 4, 1499–1507.
42 Ishihara, K., Kato, R., Furuumi, H., Zubair, M. and Sasaki, H. (1998) Sequence of a 42-kb mouse region containing the imprinted H19 locus: identification of a novel muscle-specific transcription unit showing biallelic expression. Mamm. Genome, 9, 775–777.[Web of Science][Medline]
43 Leighton, P.A., Ingram, R.S., Eggenschwiler, J., Efstratiadis, A. and Tilghman, S.M. (1995) Disruption of imprinting caused by deletion of the H19 gene region in mice. Nature, 375, 34–39.[Medline]
44 Steenman, M.J., Rainier, S., Dobry, C.J., Grundy, P., Horon, I.L. and Feinberg, A.P. (1994) Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms’ tumour. Nature Genet., 7, 433–439.[Web of Science][Medline]
45 Moulton, T., Crenshaw, T., Hao, Y., Moosikasuwan, J., Lin, N., Dembitzer, F., Hensle, T., Weiss, L., McMorrow, L., Loew, T. et al. (1994) Epigenetic lesions at the H19 locus in Wilms’ tumour patients. Nature Genet., 7, 440–447.[Web of Science][Medline]
46 Reik, W., Brown, K.W., Schneid, H., Le Bouc, Y., Bickmore, W. and Maher, E.R. (1995) Imprinting mutations in the Beckwith-Wiedemann syndrome suggested by altered imprinting pattern in the IGF2-H19 domain. Hum. Mol. Genet., 4, 2379–2385.
47 O’Keefe, D., Dao, D., Zhao, L., Sanderson, R., Warburton, D., Weiss, L., Anyane-Yeboa, K. and Tycko, B. (1997) Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms tumours. Am. J. Hum. Genet., 61, 295–303.[Web of Science][Medline]
48 Lee, M.P., DeBaun, M., Randhawa, G., Reichard, B.A., Elledge, S.J. and Feinberg, A.P. (1997) Low frequency of p57(KIP2) mutation in Beckwith-Wiedemann syndrome. Am. J. Hum. Genet., 61, 304–309.[Web of Science][Medline]
49 Lam, W.W., Hatada, I., Ohishi, S., Mukai, T., Joyce, J.A., Cole, T.R., Donnai, D., Reik, W., Schofield, P.N. and Maher, E.R. (1999) Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation. J. Med. Genet., 36, 518–523.
50 Weksberg, R., Shen, D.R., Fei, Y.L., Song, Q.L. and Squire, J. (1993) Disruption of insulin-like growth factor 2 imprinting in Beckwith-Wiedemann syndrome. Nature Genet., 5, 143–150.[Web of Science][Medline]
51 Brown, K.W., Villar, A.J., Bickmore, W., Clayton-Smith, J., Catchpoole, D., Maher, E.R. and Reik, W. (1996) Imprinting mutation in the Beckwith-Wiedemann syndrome leads to biallelic IGF2 expression through an H19-independent pathway. Hum. Mol. Genet., 5, 2027–2032.
52 Lee, M.P., Brandenburg, S., Landes, G.M., Adams, M., Miller, G. and Feinberg, A.P. (1999) Two novel genes in the center of the 11p15.5 imprinted domain escape genomic imprinting. Hum. Mol. Genet., 8, 683–690.
53 Oeltjen, J.C., Malley, T.M., Muzny, D.M., Miller, W., Gibbs, R.A. and Belmont, J.W. (1997) Large-scale comparative sequence analysis of the human and murine Bruton’s tyrosine kinase loci reveals conserved regulatory domains. Genome Res., 7, 315–329.
54 Ansari-Lari, M.A., Oeltjen, J.C., Schwartz, S., Zhang, Z., Muzny, D.M., Lu, J., Gorrell, J.H., Chinault, A.C., Belmont, J.W., Miller, W. and Gibbs, R.A. (1998) Comparative sequence analysis of a gene-rich cluster at human chromosome 12p13 and its syntenic region in mouse chromosome 6. Genome Res., 8, 29–40.
55 Prawitt, D., Enklaar, T., Klemm, G., Gärtner, B., Spangenberg, C., Winterpacht, A., Higgins, M., Pelletier, J. and Zabel, B. (2000) Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression. Hum. Mol. Genet., 9, 203–216.
56 Kenmochi, N., Kawaguchi, T., Rozen, S., Davis, E., Goodman, N., Hudson, T.J., Tanaka, T. and Page, D.C. (1998) A map of 75 human ribosomal protein genes. Genome Res., 8, 509–523.
57 Neumann, B., Kubicka, P. and Barlow, D.P. (1995) Characteristics of imprinted genes. Nature Genet., 9, 12–13.[Web of Science][Medline]
58 Tanaka, M., Puchyr, M., Gertsenstein, M., Harpal, K., Jaenisch, R., Rossant, J. and Nagy, A. (1999) Parental origin-specific expression of Mash2 is established at the time of implantation with its imprinting mechanism highly resistant to genome-wide demethylation. Mech. Dev., 87, 129–142.[Web of Science][Medline]
59 Harteneck, C., Plant, T.D. and Schultz, G. (2000) From worm to man: three subfamilies of TRP channels. Trends Neurosci., 23, 159–166.[Web of Science][Medline]
60 Hemberger, M., Redies, C., Krause, R., Oswald, J., Walter, J. and Fundele, R.H. (1998) H19 and Igf2 are expressed and differentially imprinted in neuroectoderm-derived cells in the mouse brain. Dev. Genes Evol., 208, 393–402.[Web of Science][Medline]
61 Antequera, F. and Bird, A. (1993) Number of CpG islands and genes in human and mouse. Proc. Natl Acad. Sci. USA, 90, 11995–11999.
62 Kalscheuer, V.M., Mariman, E.C., Schepens, M.T., Rehder, H. and Ropers, H.-H. (1993) The insulin-like growth factor type-2 receptor gene is imprinted in the mouse but not in humans. Nature Genet., 5, 74–78.[Web of Science][Medline]
63 Sambrook, J., Fritsch, E. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
64 Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res., 12, 387–395.
65 Chomczynski, P. and Sacchi, N. (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156–159.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  I. Zaitoun and H. Khatib Comparative genomic imprinting and expression analysis of six cattle genes J Anim Sci, January 1, 2008; 86(1): 25 - 32. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Crowder, M. S. Saha, R. W. Pace, H. Zhang, G. D. Prestwich, and C. A. Del Negro Phosphatidylinositol 4,5-bisphosphate regulates inspiratory burst activity in the neonatal mouse preBotzinger complex J. Physiol., August 1, 2007; 582(3): 1047 - 1058. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lewis, K. Green, C. Dawson, L. Redrup, K. D. Huynh, J. T. Lee, M. Hemberger, and W. Reik Epigenetic dynamics of the Kcnq1 imprinted domain in the early embryo Development, November 1, 2006; 133(21): 4203 - 4210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Johnstone, A. J. DuBose, C. R. Futtner, M. D. Elmore, C. I. Brannan, and J. L. Resnick A human imprinting centre demonstrates conserved acquisition but diverged maintenance of imprinting in a mouse model for Angelman syndrome imprinting defects Hum. Mol. Genet., February 1, 2006; 15(3): 393 - 404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Choi, L. A. Underkoffler, A. J. Wood, J. N. Collins, P. T. Williams, J. A. Golden, E. F. Schuster Jr., K. M. Loomes, and R. J. Oakey A Novel Variant of Inpp5f Is Imprinted in Brain, and Its Expression Is Correlated with Differential Methylation of an Internal CpG Island Mol. Cell. Biol., July 1, 2005; 25(13): 5514 - 5522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Cerrato, A. Sparago, I. D. Matteo, X. Zou, W. Dean, H. Sasaki, P. Smith, R. Genesio, M. Bruggemann, W. Reik, et al. The two-domain hypothesis in Beckwith-Wiedemann syndrome: autonomous imprinting of the telomeric domain of the distal chromosome 7 cluster Hum. Mol. Genet., February 15, 2005; 14(4): 503 - 511. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Yokomine, H. Shirohzu, W. Purbowasito, A. Toyoda, H. Iwama, K. Ikeo, T. Hori, S. Mizuno, M. Tsudzuki, Y.-i. Matsuda, et al. Structural and functional analysis of a 0.5-Mb chicken region orthologous to the imprinted mammalian Ascl2/Mash2-Igf2-H19 region Genome Res., January 1, 2005; 15(1): 154 - 165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Paulsen, T. Khare, C. Burgard, S. Tierling, and J. Walter Evolution of the Beckwith-Wiedemann syndrome region in vertebrates Genome Res., January 1, 2005; 15(1): 146 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Thakur, V. K. Tiwari, H. Thomassin, R. R. Pandey, M. Kanduri, A. Gondor, T. Grange, R. Ohlsson, and C. Kanduri An Antisense RNA Regulates the Bidirectional Silencing Property of the Kcnq1 Imprinting Control Region Mol. Cell. Biol., September 15, 2004; 24(18): 7855 - 7862. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-Y. Park, E. A. Sellars, A. Grinberg, S.-P. Huang, and K. Pfeifer The H19 Differentially Methylated Region Marks the Parental Origin of a Heterologous Locus without Gametic DNA Methylation Mol. Cell. Biol., May 1, 2004; 24(9): 3588 - 3595. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. BRAIDOTTI, T. BAUBEC, F. PAULER, C. SEIDL, O. SMRZKA, S. STRICKER, I. YOTOVA, and D.P. BARLOW The Air Noncoding RNA: An Imprinted cis-silencing Transcript Cold Spring Harb Symp Quant Biol, January 1, 2004; 69(0): 55 - 66. [Abstract] [PDF]
|
|
|
|
|
|
J. Walter and M. Paulsen The potential role of gene duplications in the evolution of imprinting mechanisms Hum. Mol. Genet., October 15, 2003; 12(90002): R215 - 220. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kiyosawa, I. Yamanaka, N. Osato, S. Kondo, and Y. Hayashizaki Antisense Transcripts With FANTOM2 Clone Set and Their Implications for Gene Regulation Genome Res., June 1, 2003; 13(6): 1324 - 1334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Zhang, X. Chu, Q. Tong, J. Y. Cheung, K. Conrad, K. Masker, and B. A. Miller A Novel TRPM2 Isoform Inhibits Calcium Influx and Susceptibility to Cell Death J. Biol. Chem., April 25, 2003; 278(18): 16222 - 16229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. J. Smith, W. Dean, G. Konfortova, and G. Kelsey Identification of Novel Imprinted Genes in a Genome-Wide Screen for Maternal Methylation Genome Res., April 1, 2003; 13(4): 558 - 569. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Kaplan, K. Luettich, A. Heguy, N. R. Hackett, B.-G. Harvey, and R. G. Crystal Monoallelic Up-Regulation of the Imprinted H19 Gene in Airway Epithelium of Phenotypically Normal Cigarette Smokers Cancer Res., April 1, 2003; 63(7): 1475 - 1482. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Reik, M. Constancia, A. Fowden, N. Anderson, W. Dean, A. Ferguson-Smith, B. Tycko, and C. Sibley Regulation of supply and demand for maternal nutrients in mammals by imprinted genes J. Physiol., February 15, 2003; 547(1): 35 - 44. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Srivastava, E. Frolova, B. Rottinghaus, S. P. Boe, A. Grinberg, E. Lee, P. E. Love, and K. Pfeifer Imprint Control Element-mediated Secondary Methylation Imprints at the Igf2/H19 Locus J. Biol. Chem., February 14, 2003; 278(8): 5977 - 5983. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. L. Mahy, P. E. Perry, and W. A. Bickmore Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH J. Cell Biol., December 9, 2002; 159(5): 753 - 763. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yatsuki, K. Joh, K. Higashimoto, H. Soejima, Y. Arai, Y. Wang, I. Hatada, Y. Obata, H. Morisaki, Z. Zhang, et al. Domain Regulation of Imprinting Cluster in Kip2/Lit1 Subdomain on Mouse Chromosome 7F4/F5: Large-Scale DNA Methylation Analysis Reveals That DMR-Lit1 Is a Putative Imprinting Control Region Genome Res., December 1, 2002; 12(12): 1860 - 1870. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Tarrant, J. Groom, D. Metcalf, R. Li, B. Borobokas, M. D. Wright, D. Tarlinton, and L. Robb The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses Mol. Cell. Biol., July 15, 2002; 22(14): 5006 - 5018. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Paulsen, S. Takada, N. A. Youngson, M. Benchaib, C. Charlier, K. Segers, M. Georges, and A. C. Ferguson-Smith Comparative Sequence Analysis of the Imprinted Dlk1-Gtl2 Locus in Three Mammalian Species Reveals Highly Conserved Genomic Elements and Refines Comparison with the Igf2-H19 Region Genome Res., December 1, 2001; 11(12): 2085 - 2094. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Zwart, F. Sleutels, A. Wutz, A. H. Schinkel, and D. P. Barlow Bidirectional action of the Igf2r imprint control element on upstream and downstream imprinted genes Genes & Dev., September 15, 2001; 15(18): 2361 - 2366. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Ferguson-Smith and M. A. Surani Imprinting and the Epigenetic Asymmetry Between Parental Genomes Science, August 10, 2001; 293(5532): 1086 - 1089. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. John, J. F. -X. Ainscough, S. C. Barton, and M. A. Surani Distant cis-elements regulate imprinted expression of the mouse p57 Kip2 (Cdkn1c) gene: implications for the human disorder, Beckwith-Wiedemann syndrome Hum. Mol. Genet., July 1, 2001; 10(15): 1601 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Casimiro, B. C. Knollmann, S. N. Ebert, J. C. Vary Jr., A. E. Greene, M. R. Franz, A. Grinberg, S. P. Huang, and K. Pfeifer Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange- Nielsen Syndrome PNAS, February 27, 2001; 98(5): 2526 - 2531. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Meguro, K. Mitsuya, N. Nomura, M. Kohda, A. Kashiwagi, R. Nishigaki, H. Yoshioka, M. Nakao, M. Oishi, and M. Oshimura Large-scale evaluation of imprinting status in the Prader-Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes Hum. Mol. Genet., February 1, 2001; 10(4): 383 - 394. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Engemann, M. Strodicke, M. Paulsen, O. Franck, R. Reinhardt, N. Lane, W. Reik, and J. Walter Sequence and functional comparison in the Beckwith-Wiedemann region: implications for a novel imprinting centre and extended imprinting Hum. Mol. Genet., November 1, 2000; 9(18): 2691 - 2706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Vu and A. R. Hoffman Comparative Genomics Sheds Light on Mechanisms of Genomic Imprinting Genome Res., November 1, 2000; 10(11): 1660 - 1663. [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||