Human Molecular Genetics, 2001, Vol. 10, No. 4 383-394
© 2001 Oxford University Press
Large-scale evaluation of imprinting status in the Prader–Willi syndrome region: an imprinted direct repeat cluster resembling small nucleolar RNA genes
1Core Research for Evolutional Science and Technology (CREST) project, Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori 683-8503, Japan, 2Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan and 3Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
Received 11 October 2000; Revised and Accepted 12 December 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Loss of paternal gene expression at the imprinted domain on proximal human chromosome 15 causes Prader–Willi syndrome (PWS), a complex multiple-anomaly disorder involving variable mental retardation, hyperphasia leading to obesity and infantile hypotonia with failure to thrive. Although numerous paternally expressed transcripts have been identified that reside in the candidate region, the individual contributions to the development of PWS have not been firmly established. Recent studies of mouse models carrying a cytogenetic deletion suggest that paternal deficiency of the SNRPN-IPW interval is critical for perinatal lethality of potential relevance to PWS. Here we determined the allelic expression profiles of a total of 118 cDNA clones using monochromosomal hybrids retaining either a paternal or maternal human chromosome 15. Our results demonstrated a preponderance of unusual transcripts lacking protein-coding potential that were expressed exclusively from the paternal copy of the critical interval. This interval was also found to encompass a large direct repeat (DR) cluster displaying a potentially active chromatin conformation of paternal origin, as suggested by enhanced sensitivity to nuclease digestion. Database searches revealed an unexpected organization of tandemly repeated consensus elements, all of which possessed well-defined box C and D sequences characteristic of small nucleolar RNAs (snoRNAs). Southern blot analysis further demonstrated a considerable degree of phylogenetic conservation of the DR locus in the genomes of all mammalian species tested, but not in chicken, Xenopus and Drosophila. These findings imply a potential direct contribution of the DR locus, representing a cluster of multiple snoRNA genes, to certain phenotypic features of PWS.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Prader–Willi syndrome (PWS; OMIM 176270) and Angelman syndrome (AS; OMIM 105830) are two distinct neurobehavioral disorders that involve molecular defects in the imprinting cluster on human chromosome 15q11–q13. It has now been established that PWS results from the deficiency of paternal gene expression, whereas AS is caused by the deficiency of maternal gene expression, both of which can be due to cytogenetic deletions, uniparental disomy (UPD), imprinting mutations or rare balanced translocations (1,2). To date, it has also been evident that most familial cases of AS can be attributed to loss-of-function mutations in the UBE3A/E6-AP gene, which exhibits tissue- and region-specific imprinted expression with paternal silencing in brain tissues (3–8). Loss of maternal expression of UBE3A has therefore been implicated as a causative event in AS, representing a single gene disorder of ubiquitin-mediated proteolysis that is indispensable for long-term potentiation (LTP) in the limbic system (8). Although additional genes or regulatory elements in the candidate interval also appear to contribute to the development of AS, it is widely believed that the major phenotypic effects can be explained by the maternal deficiency of UBE3A in all subgroups (8–10).
In contrast to AS, no familial cases of PWS that display inheritance suggestive of a single gene mutation have been described as yet, implying that multiple, contiguous paternally expressed genes may each have additive roles in the phenotype of PWS (1,2,9,11). In agreement with the discernible genetic aspects of these syndromes, four paternally expressed protein-coding genes, MKRN3/ZNF127, NDN/NECDIN, NDNL1/MAGEL2 and SNURF-SNRPN, have so far been shown to reside in the PWS candidate region (9,12). This interval also involves a number of RNA transcripts that apparently lack protein-coding potential, including ZNF127-AS, PAR-SN, PAR-5, IPW, PAR-1 and UBE3A-AS (12–14). Although each of the paternally expressed genes or transcripts has been assumed to be a candidate for PWS, mice deficient for Zfp127, Ndn, Snurf, Snrpn and Ipw are phenotypically normal and do not represent perinatal lethality associated with feeding difficulties equivalent to infants with PWS (15). Conflicting results describing Ndn-deficient mice have been reported, with a more recent study showing that paternal deletion results in partial lethality and a failure-to-thrive phenotype. These discrepancies are presumably due to differences in targeting strategies or genetic backgrounds (16).
To further narrow down the PWS candidate region, Tsai et al. (17) have produced mice with a large paternal deletion extending from Snrpn to Ube3a, which exhibit severe growth retardation, hypotonia and postnatal lethality. This finding, coupled with the fact that a cytogenetic deletion at the pink-eyed dilution (p) locus, which includes Ipw, is not linked to perinatal lethality when paternally inherited (18), suggests that paternal deficiency of the Snrpn–Ipw interval may be responsible for the severe phenotypic aspects that are a likely model of PWS. Extensive molecular analysis of familial PWS and AS patients displaying small microdeletions within the 5' portion of the SNRPN gene has identified an imprinting center (IC), which is proposed to exert epigenotype switching through the entire cluster in the germline (19–22). Additionally, recent studies of a rare PWS family and chimeric mice with imprinting mutations showed clearly that de novo IC deletion on the paternal chromosome results in postzygotic acquisition of a maternal epigenotype in somatic cells (23). Although it has emerged that the IC exhibits bifunctional activities, i.e. somatic maintenance of parental epigenotypes as well as in switching the gametic imprint, this also raises the possibility that imprinting defects caused by IC deletion can be explained simply as the consequence of a failure to maintain a gametic imprint in the parental soma (24).
Carrel et al. (25; and see http://genetics.gene.cwru.edu/willard/data.htm) have previously demonstrated non-random distribution of a substantial number of transcripts that escape X inactivation with the use of mouse/human somatic cell hybrids carrying either the active or inactive human X chromosome. This expression-based assay has established an extensive X-inactivation profile of the entire chromosome, providing considerable insight not only into mechanistic aspects of epigenetic organization of the X chromosome but also into clinical implications regarding heterogeneous manifestations in individuals with X chromosome abnormalities (26). Likewise, we have developed a series of human monochromosomal hybrids retaining single individual chromosomes of defined parental origin as an in vitro resource for the investigation of imprinted loci in humans (27). This has recently led to the identification of LIT1/KvLQT1-AS/KCNQ1OT1, a paternally expressed imprinted antisense RNA in the human KvLQT1/KCNQ1 locus (28). In addition, targeting experiments using the DT40 cell shuttle system demonstrate that the human LIT1 locus can act as a negative regulator in cis for the coordination of imprinting at the chromosomal domain, thereby defining a novel imprinting control element on chromosome 11p15.5 (29).
Recent advances in the Human Genome Project have provided the means to facilitate rapid and precise studies of gene expression on large numbers of transcripts derived from numerous tissues (30). In an attempt to perform a large-scale evaluation of epigenetic states of individual autosomal alleles, this study focused on proximal human chromosome 15 spanning the PWS/AS candidate region. Expression profiles of a total of 118 cDNA clones were determined successfully using human monochromosomal hybrids containing a paternal or maternal chromosome 15. Yeast artificial chromosome (YAC) and cosmid contig-based physical mapping of individual loci that exhibited differential expression between the two parental alleles revealed a number of imprinted, unusual transcripts within the PWS critical interval, most of which appear to lack protein-coding potential. This critical interval was also shown to encompass a large direct repeat (DR) cluster associated with multiple paternally expressed transcripts. This AT-rich DR locus exhibited a considerable degree of phylogenetic conservation in mammals but not in non-mammals and included tandemly repeated copies of a consensus element resembling a box C/D small nucleolar RNA (snoRNA), implying a functional significance of the repetitive domains. These findings are also discussed in relation to the possible function of unusual transcripts that have been identified on chromosome 15q11–q13 in the postzygotic process of imprint maintenance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Identification of differentially expressed transcripts
To explore human transcripts that are expressed differentially between the two parental alleles of the PWS/AS critical region, we selected 131 expressed sequence tags (ESTs) that have been assigned to the interval delimited by the genetic markers D15S1035 and D15S165 from the GeneMap database, including some representing known imprinted genes. The expression of each of the 131 corresponding transcripts was evaluated using previously established mouse A9 hybrids retaining a single paternal or maternal human chromosome 15. The allelic expression profiles of 118 clones were determined by RT–PCR analysis, whereas no expression could be detected for the remaining 13 clones. Of the 118 clones that were expressed in A9 hybrids, 17 showed a significant and consistent allelic expression bias in which the paternal allele was predominant. As shown in Figure 1A, exclusive paternal expression of NDN, SNRPN and IPW was exhibited in A9 hybrids. This contrasted with the observation that RT–PCR products were readily detected from both parental alleles of the UBE3A gene, which has previously been reported to exhibit tissue-specific imprinting, with predominant expression of the maternal allele in brain, but not in various other cell types including fibroblasts and lymphoblasts (5,6,31). For MLSN1, which has been assigned to the 15q13–q14 region, no significant allelic expression bias was detected, suggesting biallelic expression of MLSN1 in certain human tissues.
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To perform allele-specific expression analysis in human populations for transcripts that were differentially expressed in A9 hybrids, we sought to identify transcribed sequence polymorphisms by direct sequencing in 86 individuals from 27 families. A single nucleotide polymorphism (SNP) was identified in the cDNA sequence corresponding to the UniGene cluster Hs.22543, whereas the remaining cDNA sequences represented no SNPs (Fig. 1B). Direct sequencing analysis of RT–PCR products using the polymorphism in the Hs.22543 cDNA sequence revealed expression of only the paternal allele in normal human lymphoblasts (Fig. 1C). Since the C
A substitution created a polymorphic MaeIII restriction site, we also verified paternal or monoallelic expression of the transcript by PCR-based restriction analysis (Fig. 1D). These results further validated the value of human monochromosomal hybrids as a resource for the efficient detection of imprinted transcripts in humans.
YAC and cosmid contig-based physical mapping
A total of 17 ESTs exhibiting differential expression in monochromosomal hybrids were assembled using a combination of conventional BLASTN alignments and EST assembly programs (Materials and Methods). This allowed us to demonstrate that two ESTs belonged to the same transcript, as they were derived from distinct but overlapping EST clones. At the same time, seven ESTs were found to be identical to previously identified imprinted transcripts and were excluded from further analysis. After sequencing nine cDNA clones that corresponded to ESTs of interest, listed in Table 1, we performed a new run of sequence assembly using the full sequence of the insert of each clone to extend the transcribed sequence of a given EST. To establish a physical map of the differentially expressed transcripts, we then performed PCR-based screening of a series of YAC and cosmid clones encompassing the PWS/AS candidate region, using primer pairs designed for each EST (Fig. 2). Of these, seven ESTs were localized successfully to YACs within the interval between SNRPN and UBE3A, although PCR analysis of Hs.268983 failed to yield amplification products, presumably due to the comparatively high content of common interspersed repeats. Four ESTs could be further sublocalized to the cosmid contigs covering the SNRPN–IPW interval. Three other ESTs, corresponding to Hs.43052, Hs.141364 and Hs.269257, were excluded from the cosmid contigs and could be expected to be assigned to the clone gaps that were placed telomeric to IPW (Fig. 2). The remaining EST, W89101, was assigned to YAC 307A12 located centromeric to SNRPN, but could not be more finely mapped because no cosmid clones were available. Consequently, eight independent clones were physically mapped between genetic markers D15S10 and D15S11. It should be noted that seven of the eight clones were localized within the critical interval flanked by the imprinted genes SNRPN and UBE3A, a paternal deficiency which has been reported to result in postnatal lethality in mice (17).
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Northern blot analysis
To gain insight into the potential significance of the differentially expressed transcripts, the expression of the nine independent cDNA clones was examined in human tissues by northern blot analysis. Using a cDNA probe corresponding to EST H59928, mRNA was detected in various tissues with high levels in fetal heart and lesser but significant amounts in fetal kidney, small intestine, brain, lung and skeletal muscle as well as adult brain and kidney (Fig. 3).The predominant transcript was
1.4 kb in length, which is compatible with the length of the cDNA clone analyzed here. In heart, kidney and pancreas, hybridization also detected a faint higher-molecular-weight signal suggestive of related transcripts, which have yet to be investigated. Despite the exceptionally high repeat content of the cDNA sequence, northern blot analysis of Hs.141364 revealed a single 3.0 kb transcript in heart, pancreas, skeletal muscle and brain (data not shown). The expression of the remaining seven clones was not detected by northern blot analysis. PCR amplification products were, however, readily detected at least in fetal brain by RT–PCR analysis (data not shown). This is consistent with the fact that most of these ESTs do not represent ‘singletons’ that are not identical to any other ESTs in the data set, implying that they represent transcriptional units. In addition, database searches of Hs.22543 identified multiple transcripts that span over 7 kb of DNA and are colinear with genomic sequence (data not shown). Therefore, it is suggested that the transcripts were beyond the limit of resolution, rather than below the limit of detection by northern blot analysis, as shown for Air transcripts as well as for LIT1 RNA (32,33). Complete sequencing of the insert of each cDNA clone revealed that no open reading frame (ORF) of significant length was encoded by any transcript and that most of these carried an excess of interspersed repeats (Table 1). These observations suggest that unusual continuous RNAs are expressed exclusively from the paternal allele of the PWS/AS critical region, in addition to the multiple smaller RNAs revealed by northern blot analysis.
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Large DR clusters associated with multiple paternally expressed transcripts
Database searches using BLASTN alignment revealed an unexpected feature of the H59928 cDNA sequence. Although no common interspersed repeat was detected using the RepeatMasker program, we found that the cDNA sequence shared a considerable degree of similarity to multiple genomic sequences from human BAC clone RP11-131I21 (GenBank accession no. AC009696), which included the entire coding region of the SNRPN gene. As illustrated in Figure 4A, alignment of the genomic sequence to itself indicated two adjacent large DR clusters, in which AT-rich repetitive elements were organized in a head-to-tail tandem array. The 23 kb long centromeric cluster (DR1) contained 8.5 copies of a 2.7 kb repeat, clearly discerning multiple copies of an
1.3 kb repeat in the telomeric cluster of 21 kb (DR2). The telomeric repeats were relatively heterogeneous with respect to both length and sequence, also showing a contrast to the centromeric repeats. Although these two clusters bear no apparent homology to each other, detailed similarity searches of the nucleotide sequence revealed the presence of a consensus element with >87% identity for 98 bp or more (Fig. 4B). Although the DR2 repeat unit was around half the size of that of DR1, the consensus element was found in each unit (Fig. 4A). The DR locus comprises a total of 24 tandemly repeated copies of this motif, all of which contained box C, D' and D sequences characteristic of a particular class of snoRNAs. Thus, we ascertained that this was the only putative box C/D snoRNA gene per unit and that the box H/ACA snoRNA gene was not included in the DR locus.
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Through the analysis of EST databases, H59928 and five additional ESTs were found to be localized within or close to these unique DR clusters (Fig. 4A). As expected, RT–PCR analysis also showed that the paternal allele was preferentially expressed in A9 hybrids (Fig. 5A). It is noted that exclusive paternal expression was demonstrated for the cDNA corresponding to EST AI968076 that was localized within the DR1 locus, implying that the DR1 unit can be actively transcribed, as is the case for the DR2 locus. This was consistent with the results of RT–PCR analysis, showing that no expression was detected in lymphoblasts from typical PWS patients with a paternal 15q11–q13 deletion, whereas the expression was readily detectable in those from typical AS patients with a 15q11–q13 deletion (data not shown). The absence of intronic sequences was also evident and the lack of any extended ORFs in the cDNA sequences was dictated by a high density of stop codons in all three reading frames, except for EST AF017338, which has been identified as a differentially expressed transcript in individuals with neuropsychiatric diseases (34). Since the corresponding cDNA included an ATG-initiated ORF encoding 138 amino acids with unknown function, it cannot be excluded that the AF017338 cDNA encodes a small peptide. Using a cDNA probe corresponding to a putative precursor snoRNA, we detected two major signals with estimated sizes of
1.4 and 7.5 kb on northern blot analysis of polyadenylated RNA (Fig. 3), consistent with previous studies (35). In addition to these transcripts, de los Santos et al. (35) detected a smaller 140 nucleotide transcript using a probe corresponding to a putative mature snoRNA. It is therefore suggested that precursor snoRNAs might be processed to the small 140 nucleotide RNAs corresponding to mature, stable snoRNA molecules. As illustrated in Figure 4A, the primers for EST AI902294 would detect a single mature snoRNA and the remaining three cDNA clones (AI968076, H59928 and AF017338) localized within the DR locus may correspond to a region of the precursor. In conclusion, these results further suggest that the 15q11–q13 critical region harbors multiple paternally expressed transcripts that may lack protein coding potential.
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Next, we examined parent-of-origin-specific methylation modification using a methylation-sensitive PCR assay. Genomic DNA from A9 hybrids and control cell lines was digested with the methylation-sensitive restriction enzyme HpaII followed by PCR amplification to distinguish parental alleles. Using primers spanning two HpaII recognition sequences located in the 5' CpG island of the SNRPN gene, amplification products were detected in A9 hybrids with a maternal chromosome but not with a paternal chromosome (Fig. 5B). This is consistent with the previous observation of maternal methylation at the SNRPN CpG island (19), so that the underlying mechanism by which imprinted expression of the gene is exerted appears to be maintained in a mouse background. Conversely, all seven HpaII sites located in the UBE3A CpG island were shown to be methylated on the paternal chromosome, demonstrating that the allele-specific methylation imprint at the 5' CpG island is not accompanied by differential expression of UBE3A in A9 hybrids. The CpG methylation of the DR locus was analyzed using the primers Cla1F/R, which spanned an HpaII site. Figure 5B shows that methylated fragments were readily amplified from both parental chromosomes, indicating biallelic methylation at this site. This was consistent with the well-known observation that the majority of CpG-poor regions of the genome of adult mammals undergo de novo methylation during early development (36). Since further methylation analysis failed to reveal differential methylation at the DR locus, possibly due to the highly repetitive sequences, this assay did not exclude the possibility that other CpG dinucleotides displayed allele-specific differential methylation.
To further explore the nucleosomal arrangement that could reflect sequence-specific protein–DNA interaction in cell nuclei, the parental chromosome-specific chromatin organization of the DR locus was assessed subsequently by nuclease sensitivity assay. Nuclei isolated from A9 hybrids retaining either a paternal or maternal human chromosome 15 were digested with various concentrations of KpnI. Following organic extraction, genomic DNA was restricted with EcoRI. The samples were analyzed by Southern blotting using a probe from the unique internal portion of the DR locus. As shown in Figure 5C, two major signals with estimated sizes of 6.3 and 9.0 kb were generated by KpnI treatment of nuclei from A9 hybrids with a paternal chromosome. As these signals were seen only at substantially higher nuclease concentrations in nuclei from A9 hybrids with a maternal chromosome, the maternal copy of the DR locus appeared to be more resistant to cleavage by the endonuclease. Although the control probe hybridization indicated that the two KpnI digestion series were fully comparable, we could not determine the precise mapping positions of the prominent paternal nuclease-hypersensitive sites because of the extraordinary extent of the repetitive sequence. Despite this, these results suggest that the paternal chromosome is potentially accessible to nuclear factors involved in protein–DNA interactions, consistent with predominant paternal expression of the multiple closely linked transcripts in A9 hybrids.
Evolutionary conservation of the DR clusters
The results of the nuclease sensitivity assay implied a functional significance of the repetitive domains and prompted us to investigate the extent of phylogenetic conservation of the DR locus. Genomic DNA from nine eukaryotic species was digested with EcoRI and hybridized to probes A and B as indicated in Figure 4A. Major signals with estimated sizes of 13.6 and 15.8 kb were detected in the human genome using probes A and B, respectively (Fig. 6A). Both probes detected homologous genomic fragments in rhesus, porcine, bovine, rat and mouse, but failed to detect hybridization fragments in chicken, Xenopus and Drosophila, suggesting that the DR clusters are well conserved among mammals but absent in non-mammals. Since the sizes of hybridizing fragments that were observed using probe A appear to differ slightly from those observed using probe B, we further performed a co-hybridization using probes A and B, and ascertained that the these two probes did not hybridize to the same EcoRI fragments (Fig. 6A). One or two EcoRI fragments hybridized strongly with both probes in human, rhesus, porcine, bovine, rat and mouse. In addition, Southern blot analysis using different restriction enzymes, XbaI and SpeI, demonstrated one major hybridizing signal in both human and rhesus (Fig. 6B and data not shown). Taken together, these findings imply that the conserved repetitive region exists as a single copy in the mammalian genome. Moreover, extended database searches using the sequence of the human consensus element identified one ruminant and two rodent homologous sequences that exhibited >85% internal sequence similarity for 45–99 bp (Fig. 4B). Additionally, the mouse repetitive sequences were found to be included in the chromosome 7 BAC clone RP23-309E15 (GenBank accession no. AC026683), which encompasses the entire transcribed region of the mouse Ipw gene. This indicates that the repetitive region of human chromosome 15q11–q13 may be syntenic to an orthologous distal region of mouse chromosome 7, further suggesting that the DR clusters appear to share unusual similarity among mammals.
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The genomic organization of AT-rich repetitive sequences in the human genome are suggestive of clusters of multiple snoRNAs in maize (37). Indeed, inspection of these substantially conserved elements revealed that all possessed well-defined sequence elements known as box C (consensus RUGAUGA) and box D (consensus CUGA), which are positioned near the 5' and 3' ends of the box C/D snoRNAs (Fig. 4B). These conserved elements included multiple translation stop signals in all three reading frames. It has been known that the pre-mRNA is spliced to release the snoRNA but the resultant mRNA has no ORF and contains many stop codons (38), which may be the most analogous situation of DR locus. C and D boxes were also found to be adjacent to terminal inverted repeats, suggesting a potent secondary structure of stem–loops in RNA molecules, which is proposed to block cleavage by endonucleases in the formation of mature snoRNA. Another feature of box C/D snoRNAs is that the majority contain long stretches of complementarity to the universal core regions of rRNA lying upstream to either box D or an internal box D sequence, termed D' (consensus CUGA) (39,40). In vertebrates, these so-called antisense snoRNAs have been shown to target the rRNA segments to be methylated through the interaction of complementary sequences (41). However, since extensive complementarity to rRNA was not observed, it was not obvious that these elements are involved in ribose methylation. It should also be noted that the 13 nucleotides upstream of box D' were absolutely conserved in the sequences from the four species (Fig. 4B). Interestingly, but with little relevance, a consecutive sequence of 9 of 13 is identical to part of the guide sequence of U14 that is the second most abundant snoRNA in humans (37). In addition, Huettenhofer et al. (42) identified an 18 nucleotide complementarity to an mRNA for a neurotransmitter receptor, implying a biological significance of this motif. In any case, our observations suggest that large DR clusters might represent functionally important domains, especially in mammals.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A series of human monochromosomal hybrids retaining single individual chromosomes of defined parental origin has been developed via microcell-mediated chromosome transfer (27). This in vitro assay system has recently led to the identification of an imprinted locus, LIT1, which can act as a negative regulator in cis for the coordination of the local control of imprinting on chromosome 11p15.5, thereby defining an IC for this domain (28,29). Using these human monochromosomal hybrids, we have now shown that another well-documented imprinting cluster on chromosome 15q11–q13 harbors numerous paternally expressed transcripts, most of which are assigned to the PWS critical interval. The current study, however, does not exclude the existence of a second maternally expressed imprinted gene in this cluster, as suggested by the lack of marked differences in expression levels of the transferred chromosomal alleles of the UBE3A gene that exhibits tissue-specific imprinting with paternal silencing in brain tissues. Despite this, analysis of monochromosomal hybrids also demonstrated differential methylation at the 5' CpG island of this gene, although the physiological significance of this still remains to be clarified. Recent comparative analysis of this imprinting cluster in human and mouse suggested that CpG islands represent one of the characteristic features of imprinted domains (43). Accordingly, identification of differentially methylated CpG residues, especially those located within CpG islands, may allow us to recapitulate more precisely the epigenetic status of individual loci that are subject to tissue- or region-specific imprinting.
There is a well-established experimental precedent for a differential methylation-based assay using mouse/human somatic cell hybrids carrying either the active or inactive human X chromosome (44,45). With this hybrid system, Carrel et al. (25) have recently demonstrated non-random distribution of a substantial number of genes that escape X inactivation using an expression-based assay of individual X-linked transcripts, providing considerable insights into the chromosomal basis of epigenetic organization of the X chromosome. The extensive X-inactivation profile of the entire chromosome also offers direct implications concerning severity of clinical, heterogeneous manifestations in individuals with X chromosome aneuploidy. A further large-scale evaluation of epigenetic states of individual chromosomal alleles should be facilitated by the recent worldwide efforts to complete sequencing of the human genome and to produce a high-resolution map of the growing number of cDNAs within the framework of the Human Genome Project.
Similar to the situation with unusual transcripts that had previously been reported by others to be localized within the 15q11–q13 region (12–14), most of the transcripts identified in this study have neither significant ORFs nor introns, suggesting that they might lack protein-coding potential and function as RNA transcripts. Although it is not yet known how important a role RNA transcripts play in either the mechanisms of imprinting or human disorders, it is worth noting that certain non-coding RNAs are included in imprinted domains and accept a part in the regulation of the imprinted expression of coding transcripts. Such cases have been well described for the imprinted gene pairs Igf2/H19 and Igf2r/Air, in which the two closely linked transcription units would compete for their shared enhancers on a single chromosome (46–48). Recently, Schmidt et al. (49) have identified the Dlk1/Gtl2 gene pair as a novel imprinting cluster which is highly reminiscent of the reciprocal imprinting of Igf2/H19. Evidence from targeted disruption experiments suggests that methylation at the 5' CpG island of LIT1 RNA can activate multiple transcription units on the maternal chromosome (29). These observations highlight general mechanisms in which allele-specific silencing of coding genes could be an indirect consequence of expression in cis of non-coding RNAs that are directly imprinted as primary targets of the imprint (46–48). In this model, differential methylation at the CpG-rich residues upstream of non-coding RNAs has emerged as a key mediator in the imprinting process. The essential role for non-coding RNAs in epigenetic mechanisms is also supported by the previous observation that stable production of Xist RNA, which can coat the X chromosome to be inactivated, may itself be regulated in cis by antisense Tsix RNA, whereas differential methylation at the 5' CpG island has not yet been described (50). This, however, cannot necessarily be applied to the imprinting cluster on chromosome 15q11–q13. In contrast to the non-coding RNAs with the presumed functions described above, no RNA transcripts identified within the 15q11–q13 region appear to be closely linked to CpG islands, suggesting another unique property of the transcripts. In addition, no sequence element on chromosome 11p15.5 has been identified where disruption results in an imprinting defect that is accompanied by regional alteration of the methylation status across the entire imprinted domain in the germline. This is also a contrast to imprint-switching exerted by ICs at the SNRPN locus (19–22).
Our results suggest that the imprinted domain on chromosome 15q11–q13 harbors multiple independent transcription units which are additional examples of atypical continuous transcripts such as Air transcript and LIT1 RNA (32,33). Recent studies of a rare PWS family and chimeric mice carrying a de novo IC deletion demonstrate that a paternal chromosome can postzygotically acquire a maternal epigenotype with the general trend toward hypermethylation (23). The striking prevalence and unique property of multiple unusual RNAs on chromosome 15q11–q13 as described above lead us to the assumption that stable production of multiple unusual RNAs from the paternal allele may be required to maintain the paternal chromosome in a largely unmethylated state in somatic cells. Alternatively, paternal repression caused by the IC deletion would be unable to counteract postzygotic methylation throughout the PWS region. The repressed state of the maternal chromosome thereby results in the acquisition of de novo methylation, which can stabilize the inactive state of chromatin as a ‘molecular lock’ (35,51).
Although the most common genetic aberration in PWS and AS is a large chromosomal deletion including proximal chromosome 15q11–q13, these syndromes represent apparently different genetic aspects (1,2). In contrast to the substantial number of AS patients without a detectable deletion or UPD, no familial cases of PWS that display inheritance suggestive of a single gene mutation have yet to be reported (9,11). This implies that the paternal deficiency of two or more imprinted loci could contribute to the phenotypes of PWS, thereby representing a contiguous gene syndrome. Consistent with this, numerous paternally expressed genes and transcripts have been identified and localized in the candidate region (9,12). This is also supported by previous studies of mouse models of PWS, with the exception of Ndn-deficient mice (15,16). Recently, Tsai et al. (17) have generated mice in which a genomic segment from Snrpn to Ube3a was deleted on the paternal chromosome, which exhibited severe growth retardation, hypotonia and partial lethality. Taken together with the results from lethal mutations at the pink-eyed dilution locus (18), it has been proposed that a paternally expressed structural gene located within the Snrpn–Ipw critical interval might be responsible for perinatal lethality that are a likely model for PWS. Therefore, the work reported here mainly focused on this critical interval on chromosome 15q11–q13 and identified a highly conserved DR locus associated with multiple paternally expressed transcripts.
The presence of a DR locus closely associated with imprinted transcripts in humans and mice implies that it could function as a structural regulatory element. In this regard, differentially methylated domains upstream of H19 have recently been shown by four groups to carry enhancer-blocking activity that is possibly mediated by the inhibition of the binding of the insulator protein CTCF to conserved GC-rich repetitive elements by methylation (52). A similar mechanism is postulated to explain the transcription competition at the centromeric domain on human chromosome 11p15.5 (29). In contrast, no differentially methylated domain appears to be involved in the AT-rich DR locus identified within the PWS critical interval. Alternatively, the large DR locus was found to contain multiple domains that share some, but not all, of the well-defined characteristics of the box C/D family of snoRNAs. In addition to the three primary sequence motifs (C, D and D' boxes), they encoded the potential for terminal base pairing but did not have regions complementary to the rRNA species, which functions in specifying the position of ribose methylation of the rRNAs. In yeast, certain box C/D snoRNAs are essential for cell growth due to their major roles in rRNA processing (39). The critical function of animal snoRNA is manifested in the form of ribonucleoproteins (RPNs), as demonstrated by the fact that mutated forms of the nucleolar protein MFL result in developmental defects in Drosophila and that the human homolog of MFL is mutated in patients with dyskeratosis congenita (53,54). The results of zooblot analysis revealed extraordinary conservation of these DR clusters among mammalian species. Moreover, database searches demonstrated the presence of these consensus elements in both bovine and mouse genomes. Given these observations it is conceivable that, although further experimental evidence is still required, the DR clusters that likely encode multiple snoRNA genes could contribute to some of the complex phenotypes of PWS.
The phenotypic role of the DR locus in PWS is supported by previous studies of two PWS patients with a rare balanced translocation of paternal origin (55,56). Methylation and expression analyses showed that the translocations had breakpoints between SNRPN and IPW and did not affect paternal SNRPN and PAR5, whereas expression of IPW and PAR1 was undetectable. It is therefore tempting to speculate that these translocations may disrupt the DR locus itself or may separate the target transcription units from regulatory elements. The majority of the coding sequences for the box C/D snoRNAs can be found in pre-mRNA introns of a host gene in various higher eukaryotes, with the exception of the clustering of multiple snoRNA genes in plants (37). Our results therefore provide the first evidence suggesting the organization of vertebrate snoRNA genes into clusters, although the physiological function of these remain unresolved due to the lack of potential to base pair with pre-rRNA. In both vertebrates and yeast, the box C/D motif has also been shown to direct nucleolar localization by specific binding to target nucleolar proteins (57). In this regard, it should be noted that the human telomerase RNA component (hTR) contains the box H/ACA motif shared with another class of snoRNA, which is essential for accumulation of the mature RNA as well as telomerase catalytic activity (58). The existence of snoRNAs that belong to the box C/D class but act on the spliceosomal small nuclear RNA (snRNA) also imply functional diversity of the vertebrate snoRNAs in the nucleolus (59). In the meantime, knockout and transgenic mouse models of potential relevance to PWS can now be used to further explore the phenotypic and physiological roles of the DR locus in this disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Differential EST screen
To identify differentially expressed transcripts in human monochromosomal hybrids, PCR-based expression analysis was performed on 131 ESTs within the first 20 cM of chromosome 15 in the GeneMap database (http://www.ncbi.nlm.nih.gov/genemap/). Mouse A9 hybrids containing either a paternal or maternal human chromosome 15 tagged with pSV2bsr were maintained in the presence of 3 µg/ml blasticidin S as described by Meguro et al. (60). Total RNA was prepared from cultured cell lines using the single-step acid guanidinium isothiocyanate procedure (61). First strand cDNA synthesis was carried out with a random hexamer primer (Roche Molecular Systems) and M-MLV reverse transcriptase (Life Technologies). A negative control was designed so that first strand synthesis was conducted without reverse transcriptase and used later in the PCR to ensure that no genomic DNA contamination gave false-positive results. An aliquot of prepared cDNA was amplified with a step-down PCR approach to obtain gene specificity, as essentially described by Mitsuya et al. (28). The reaction products were analyzed by electrophoresis in a 2% agarose gel and staining with SYBR Green I (Molecular Probes). Primers corresponding to the human ESTs were obtained from Research Genetics or Amersham Pharmacia Biotech. Primer sequences are given at the above web page.
Assessment of allelic expression
cDNAs from B lymphocytes transformed by Epstein–Barr virus were amplified by PCR using primers 22543F (5'-GGATAACAATTTCACACAGGAGTGCATACCATCACTAAAACATAGT-3') and 22543R (5'-CACGACGTTGTAAAACGACCAAGCACAGTATAACAAGGCCA-3'). Reactions contained 0.5 µM of each primer, 0.2 µM of each dNTP, 1x PCR buffer and 0.5 U of Taq DNA polymerase (Perkin-Elmer) in a final volume of 20 µl. The thermocycling conditions used were 24 cycles of 95°C for 20 s, 56°C for 20 s and 72°C for 45 s. Genomic DNA was subjected to a total of 18 cycles under the same conditions as cDNA. PCR products were purified using a QIAquick column (Qiagen) and directly sequenced as described below, using M13 sequencing primers. Confirmation of the allelic expression was obtained by PCR–RFLP analysis with the restriction enzyme MaeIII (New England Biolabs). The restriction digests were analyzed on a 5% acrylamide gel and visualized with SYBR Green I.
Mapping of cDNA clones to YAC and cosmid contigs
DNA and EST database searches were performed using the BLAST programs on the NCBI server (http://www.ncbi.nlm.nih.gov). Human ESTs were assembled with the ESTblast programs (http://www.hgmp.mrc.ac.uk/ESTBlast/) and the selected representative cDNA clones corresponding to each UniGene cluster or EST were obtained from the IMAGE Consortium through Genome Systems. The inserts were sequenced directly in both directions using a Thermo Sequenase Cycle Sequencing kit (Amersham Pharmacia Biotech) and vector-specific primers, which were run on a Li-Cor automated sequencer 4200 (MWG-Biotech). The sequences were analyzed for interspersed repeat content and repetitive sequences were masked using the RepeatMasker2 program (http://ftp.genome.washington.edu/RM/RepeatMasker.html). The contiguous masked sequence was then used for BLASTN alignments against the EST database and for primer selection. Primer sequences used for PCR-based screening for YAC and cosmid clones are available on request. YAC DNA was prepared by using a Puregene DNA Isolation kit (Gentra Systems) according to the manufacturer’s specifications. Purification of cosmid DNA was performed using a QIAprep spin miniprep kit (Qiagen). In general, PCR reactions were carried out with 25 ng of bacterial DNA in PCR buffer for 18 cycles under the following conditions: 95°C for 20 s, 60°C for 20 s and 72°C for 30 s. The sequencing of cosmid ends was also performed as described above using standard sequencing primers.
Northern blot analysis
An 1.4 kb cDNA fragment corresponding to EST H59928 (IMAGE clone 204357; Genome Systems) was labeled with [
-32P]dCTP by random priming and hybridized to a Gene Hunter mRNA blot (BioChain). Hybridizations were performed in 50% formamide, 5x SSPE, 5x Denhardt, 0.5% SDS and 200 µg/ml salmon sperm DNA at 42°C. The final wash of the mRNA blot was in 0.1x SSC and 0.1% SDS at 50°C. Exposure to BioMax film (Eastman Kodak) was for 24 h at –80°C. Control hybridizations were carried out with a 724 bp ß-actin cDNA probe.
Methylation analysis
The DNA methylation status at CpG residues was determined by methylation-sensitive PCR assay. Genomic DNA from cultured cell lines was prepared by standard procedures. Five micrograms of each DNA sample was incubated with or without the methylation-sensitive enzyme HpaII and then amplified by PCR in the presence of GC-Melt (Clontech) to destabilize the GC-rich DNA secondary structure. Primer sequences were as follows: for the SNRPN CpG island, 5'-GTACCCACCTCCACCCATGT-3' and 5'-GCCTGACGCATCTGTCTGAG-3'; for the UBE3A CpG island, 5'-CCACCGCCTCGTTCTCTTT-3' and 5'-GCTCGGGGTGACTACAGGAG-3'; for the DR locus, 5'-AGGGTTGTGTATGGCTGGTC-3' and 5'-CACTCACTTGCCCAGAGACA-3'.
Nuclease sensitivity assay
The chromatin conformation of the DR locus was analyzed using the endonuclease hypersensitivity assay. Nuclei from mouse A9 hybrid cells were isolated by a modification of the method of Wu (62). Cultured cells were washed in ice-cold phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml aprotinin and 1 µg/ml pepstatin (Roche Molecular Systems) and detached from the dish with a rubber policeman. Cell pellets were suspended in 12 ml of 0.3 M sucrose buffer [0.3 M sucrose, 15 mM Tris–HCl pH 7.6, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA pH 7.8, 0.5 mM dithiothreitol, 0.1 mM PMSF and 5 mM sodium butyrate] containing 0.2% Nonidet P-40. Samples were then layered onto 6 ml of 1.5 M sucrose buffer and centrifuged at 32 000 r.p.m. for 40 min at 4°C using an SW41 TI rotor (Beckman). Nuclear pellets were resuspended at a concentration of 1 x 107 nuclei/ml in KpnI reaction buffer. Digestion mixtures containing 100 µl of crude nuclei were incubated with 0, 100, 500 or 1000 U of KpnI for 1 h at 37°C. Subsequently, DNA was purified by two rounds of phenol–chloroform extraction, followed by ethanol precipitation. RNA was removed by treatment with RNase A for 1 h at 37°C. DNA (15 µg) was digested with EcoRI and subjected to Southern blot analysis. The probe used for nuclease sensitivity assay was generated by PCR using primers 3F (5'-GCCTCCACTTTGGCTTATTG-3') and 3R (5'-TGGCTTCACATATCTGTGTTCA-3').
Southern blot analysis for evolutionary conservation
Radiolabeled DNA probes were hybridized to Southern blot membranes containing 10 µg of EcoRI-, XbaI- and SpeI-digested DNA from human, rhesus monkey, porcine, bovine, rat, mouse, chicken, Xenopus laevis and Drosophila melanogaster. Hybridizations were performed in 5x SSPE, 0.5% SDS and 200 µg/ml salmon sperm DNA at 55°C and a final wash in 0.1x SSC and 0.1% SDS at 60°C. Autoradiograms were analyzed with a BAS-2500 phosphoimager (Fuji Film). Probes used for zooblot analysis were generated by PCR amplification of human genomic DNA. PCR primers were as follows: For probe A, 5'-AGGGGAGGAGGCATGTATCT-3' and 5'-GAACCAACCCCACTAAACCA-3'; for probe B, 5'-CGTGTCCATGCATTTCTGTG-3' and 5'-CATTCACCTGGCTCCTGCATA-3'.
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ACKNOWLEDGEMENTS |
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We are grateful to Yasuaki Shirayoshi and Motonobu Katoh for helpful discussions, Hiroshi Komoda for valuable advice on nuclei isolation and Chiga Okita for technical assistance. K.M. is a fellow of the Japanese Society for the Promotion of Science. This work was supported by CREST of Japan Science and Technology Corporation (JST), and grants from the Ministry of Education, Science, Sports and Culture of Japan and the Human Frontier Science Program Organization (HFSPO).
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FOOTNOTES |
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+ Present address: Laboratory of Developmental Genetics and Imprinting, Developmental Genetics Programme, The Babraham Institute, Cambridge CB2 4AT, UK
§ To whom correspondence should be addressed. Tel: +81 859 34 8260; Fax: +81 859 34 8134; Email: oshimura@grape.med.tottori-u.ac.jp
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REFERENCES |
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