Human Molecular Genetics, 2002, Vol. 11, No. 15 1707-1718
© 2002 Oxford University Press
Allele-specific expression analysis by RNA-FISH demonstrates preferential maternal expression of UBE3A and imprint maintenance within 15q11– q13 duplications
1Department of Human Genetics, 2Child and Adolescent Psychiatry, Department of Psychiatry, 3Laboratory of Developmental Neuroscience and 4Department of Pediatrics, University of Chicago, Chicago, IL, USA
Received February 27, 2002; Accepted May 21, 2002
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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15q11– q13 contains many imprinted genes, and undergoes duplicon-mediated rearrangements, including deletions, duplications and triplications, and generation of marker chromosomes. Abnormal phenotypes, including language delays and autism spectrum disorders, are primarily observed with maternal 15q11– q13 duplication. To determine possible epigenetic effects on expression within duplicated 15q11– q13 regions, we utilized RNA-FISH to directly observe gene expression. RNA-FISH, unlike RT–PCR, is polymorphism-independent, and it also detects relative levels of expression at each allele. Unamplified, gene-specific RNA signals were detected using cDNA probes. Subsequent DNA-FISH confirmed RNA signals and assigned parental origin by colocalization of genomic probes. SNRPN and NDN expression was detected primarily from paternal alleles. Control Dystrobrevin transcripts were detected equally from both alleles; however, maternal-UBE3A signals were consistently larger than paternal signals in normal fibroblasts and in neural-precursor cells. Larger UBE3A signals were also observed on one or both maternal alleles in a cell line carrying a maternal interstitial duplication, on both alleles of a maternally derived marker(15) chromosome, and occasionally on a paternal allele in a cell line carrying a paternal interstitial duplication. Expression of NDNL2, just distal to the duplicated region, was not markedly altered but paralleled changes in UBE3A expression. Excess total maternal-UBE3A RNA was confirmed by Northern blot analysis of cell lines carrying 15q11– q13 duplications or triplications. These results demonstrate that: (1) UBE3A is imprinted in fibroblasts, lymphoblasts and neural-precursor cells; (2) allelic imprint status is maintained in the majority of cells upon duplication both in cis and in trans; and (3) alleles on specific types of duplications may exhibit an increase in expression levels/loss of expression constraints.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The proximal region of chromosome 15 is an extremely complex and unstable region of the genome, undergoing frequent rearrangements mediated by interactions between 0.4 Mb duplicons (1,2), and containing a large domain of imprinted genes (3), many of which are involved in brain development and function. Rearrangements of 15q11– q13 yield phenotypes dependent upon the parental origin of the affected chromosome. The most well known of these are Prader–Willi syndrome (PWS) and Angelman Syndrome (AS), caused primarily by functional loss of the paternal (PWS) or maternal (AS) 15q11– q13 region, usually by deletion (4). Other byproducts of duplicon-recombination events include duplications and triplications of this region, and the creation of marker chromosomes, the majority of which are of maternal origin (5). Triplication of the paternal 15q11– q13 region may be required for an identifiable (PWS-like) phenotype (6), but any maternal duplication results in a spectrum of phenotypes, ranging from developmental and language delays to autism (7–9). The phenotype usually appears more severe in proportion to the number of extra maternal domains, with patients carrying a large marker 15 chromosome (tetrasomy for 15q11–13; 3 maternal: 1 paternal) having AS-like features (5).
A large cluster of genes in proximal 15q11– q13 exhibit expression almost exclusively from the paternal chromosome (10–12), loss of which result in PWS. These include SNRPN, which contains the imprinting center (IC) for this region (13), and NDN, which may regulate postmitotic growth arrest in differentiated neurons (14,15). The slightly more distal AS gene, UBE3A (16,17), however, only exhibits exclusively maternal expression in human brain (18,19), and in specific neurons in mouse (20). The recent identification of ATP10C, adjacent to UBE3A and also exhibiting maternal expression in brain and, to a lesser degree, in lymphocytes, suggests the existence of a second cluster of genes also regulated by the IC (21,22). The expression status of other neural genes that may contribute to 15q11– q13 rearrangement phenotypes, including GABRß3, 
5, and 
3, is still unclear (23–25). Many non-coding RNAs in 15q11– q13, such as the snoRNA clusters (26–28), are also imprinted. Any one of these genes, genes yet to be localized in this region or combinations thereof could contribute to the autism phenotype observed with duplication of maternal 15q11– q13.
To begin to look at gene expression in a normal, biparental cell environment, we have begun RNA-FISH analysis of expression from 15q11– q13. RNA-FISH has been a successful technique modified for studying RNA processing (29) and quantitation (30), and has also been used to illustrate allele-specific expression of genes from active/inactive X-chromosomes (31) and of the imprinted IGF2/H19 genes (32). RNA-FISH is especially suited to the study of imprinted expression, because it offers the opportunity to detect expression at the source, which renders this assay polymorphism-independent. This is especially important, as many genes are not polymorphic, or may not be heterozygous in cell lines available for study. In addition, RNA-FISH allows for the detection of expression, and relative levels of expression, at each allele. This is critical in the case of duplication events, to determine whether expression occurs from each allele within a duplicated region and at what levels, and whether expression from the allele in the normal chromosome is altered. Furthermore, with the use of translocation or satellited chromosomes of known parental origin, the parental origin of expression can be assigned (33). Or, if the gene is on an imprinted chromosome, co-detection of the gene of interest with an imprinted gene can be used to assign the signal. This allows analysis of patient cell lines without the need for polymorphism screens or parental cell lines. Finally, RNA-FISH removes the influence of potential RNA-processing, transport and stability differences in allele-specific transcripts. These differences could affect allele proportions detected by RT–PCR, which utilizes total cellular or processed RNA.
Here we describe expression analysis of the genes UBE3A, SNRPN, NDN and NDNL2 in normal fibroblast, lymphoblast and neural-precursor cells by RNA-FISH or northern blot, and compare normal expression patterns with those observed in cells which carry rearrangements in 15q11– q13.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Unequal biallelic expression of UBE3A in fibroblasts
Due to the association of an abnormal phenotype with maternal but not paternal duplication of 15q11– q13, we characterized the expression of UBE3A, the only gene in this region known to be expressed exclusively from the maternal allele when this study was designed. Using a 3.3 kb UBE3A cDNA probe, we performed RNA-FISH on normal fibroblast lines, followed by denaturation and hybridization with genomic DNA clones to confirm the location of the expression signal. As the UBE3A imprint has been reported to be brain-specific, we expected to observe signals of equal intensity from each allele in fibroblasts. However, we found that expression from the two alleles was frequently very unequal, and only one signal was observed in an average of 47% of cells across several normal, cycling cell lines. To rule out the possibility that the disparate UBE3A signals were due to the imaging process, we analyzed the RNA-FISH signals of Dystrobrevin, a gene commonly used for standardizing expression levels, using a 2.5 kb cDNA/intronic probe. In contrast to UBE3A, 80% of Dystrobrevin signals were of approximately equal intensity (Fig. 1B; Fig. 2A), with fewer than 10% of cells showing a single signal.
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Sequential UBE3A RNA/DNA-FISH using a chromosome 15 paint probe demonstrated frequent chromosome 15 association, with a majority overlapping at the single-UBE3A expression site (Fig. 1A). These cells comprise the majority of single UBE3A expression signals in cycling fibroblasts. Overlapping chromosome 15 pairs containing two UBE3A signals of unequal intensity were also observed (Fig. 1A), suggesting that these pairings represent random chromosomal association. Overall, there were
7% fewer overlapping (non-imprinted) chromosome 18 pairs than chromosome 15 pairs, and these showed dual signals of equal intensity (Fig. 1C and F). Single, overlapping Dystrobrevin signals were not observed. The specificity of single, overlapping hybridization signals to the imprinted UBE3A gene in this cycling cell population supports the possibility that this association is non-random, perhaps reflecting the preferential association of the imprinted chromosome 15q11– q13 domain in late S phase (34,35). Subsequent experiments were performed on 70–90% confluent cells (G0) to limit the number of overlapping signals; those identified were not included in the final tallies. Examination of UBE3A expression patterns in the NT2 neural precursor cells demonstrated considerable similarity to the patterns seen in fibroblasts (Fig. 2A). In contrast, preliminary results on long-term cultured, partially differentiated cortical HCN-2 neurons revealed UBE3A expression from a single allele in 63% (5/8) of cells with distinctly separate alleles (data not shown). Together, these results suggest that exclusive (maternal) imprinted UBE3A expression may be related to neuronal maturation.
UBE3A expression is imprinted and derived from the maternal allele
To determine whether the unequal UBE3A signals were allele-specific, we utilized fibroblasts containing a marked chromosome 15 translocation of known parental origin (GM00118). In these cells, the larger UBE3A signals were located on the normal, maternal chromosome 15 in 93.5% of cells with unequal signals (76% of total cells scored) (Fig. 1D and G; Fig. 2B). This demonstrates that although UBE3A expression in fibroblasts is biallelic, expression levels from parental alleles are not equivalent, with the majority of UBE3A expression derived from the maternal allele.
To compare these results with expression of other imprinted genes, we analyzed expression of the predominantly paternally expressed genes SNRPN or NDN (23,36). For RNA-FISH, a 1.7 kb cDNA probe alone or in conjunction with 4.5 kb encompassing several introns was used for SNRPN, and a 1.7 kb cDNA probe was used for NDN. UBE3A probes consistently generated a better signal than either SNRPN or NDN, and NDN signal was more easily detected than SNRPN, perhaps because of lack of introns in the NDN transcript. Co-hybridization of UBE3A with either SNRPN or NDN demonstrated that, in cells with unequal UBE3A expression signals, the less intense signal was almost always (92%) associated with the same chromosome 15 as the paternally expressed gene (Fig. 1E and H; Fig. 2B). Overall percentages of cells with inconsistent expression patterns (paternal UBE3A expression > maternal expression; biallelic SNRPN expression) were similar to those published for SNRPN (36) and for the imprinted genes H19/IGF2 (32). Taken together, these results suggest that, in fibroblasts and non-terminally differentiated neuronal cells, UBE3A is imprinted and is preferentially expressed from the maternal allele.
UBE3A is expressed from each allele on duplicated maternal chromosome 15q11– q13 regions, at levels greater than from the normal, paternal 15q11– q13
Interstitial duplication of maternal 15q11– q13 is associated with autism spectrum disorders, clumsiness, and/or developmental language impairment (7–9), yet nothing was known of the effects of the 3.5–6 Mb duplication on expression of the duplicated genes within. We therefore performed RNA-FISH on fibroblasts obtained from a female with autism, BW (7), to assay whether any expression was detectable from the duplicated alleles, and whether the imprinted expression patterns of UBE3A, NDN and SNRPN were maintained in these cells. Expression of the paternally expressed genes SNRPN and NDN in maternal dup(15) cells was indistinguishable from that in normal cells, with signal detected solely on the non-duplicated paternal 15 in almost all cases. A single signal was observed on a maternal allele in 4% of cells, as is also occasionally observed in a normal cell population.
UBE3A expression was observed from both maternal alleles in 93% of cells, and both maternal UBE3A signals were equal to or greater than paternal UBE3A expression signals in all but 13% of these cells (Fig. 3C, i and iv; Fig. 4). This is reminiscent of UBE3A expression patterns in normal, non-dup(15) cells, and suggests that interstitial duplication of a maternal chromosome 15 does not substantially influence expression of a preferentially maternally expressed gene. In the remaining 7% of cells in the population, overall expression levels were low and expression was detected from only a single maternal allele. Twenty-five per cent of these cells, however, had an increase in paternal expression relative to the expression level of the single detected maternal allele. Overall, 9% of the total population exhibited substantial UBE3A expression from the paternal allele, which is similar to that observed within normal populations.
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To determine whether imprinted gene expression patterns were maintained on extra-chromosomal 15q11– q13 duplications, we analyzed GM02729, a fibroblast line containing a supernumerary maternal inv dup(15) marker chromosome, carrying two additional copies of the region. Relative expression signals from both UBE3A and NDN on the non-rearranged maternal and paternal chromosome 15s were similar to those observed in normal cell populations (Fig. 3C, iii and vi). NDN signal was not observed from either allele on the marker chromosome, but was only observed on one normal (presumably paternal) chromosome 15. UBE3A expression, on the other hand, was detected from both of the endogenous chromosome 15s with expected asymmetry, as well as from each allele on the maternal marker chromosome. However, UBE3A expression from the maternal marker inv dup(15) alleles was usually much greater than expression from the normal maternal chromosome 15 allele. Together, these results suggest that, whereas the ubiquitously paternal expression of SNRPN and NDN is not affected by duplication, paternal deletion or maternal uniparental disomy of the 15q11– q13 region (33–35,37), control of UBE3A expression levels, with an imprint subject to cell-type modification and perhaps other controls, may require paternal complement for proper regulation or maintenance.
Paternal UBE3A expression is increased upon paternal 15q11– q13 duplication
In contrast to the phenotype associated with maternal dup(15), interstitial duplication of 15q11– q13 on the paternal chromosome 15 has no consistent phenotype (9,38) [although developmental delay may be observed with triplication of this region (6)]. RNA-FISH of fibroblasts from SW, the pheno-typically normal mother of BW, who carries a paternal duplication, demonstrated that a majority (73%) of cells exhibited ‘normal’ expression patterns for UBE3A (larger maternal signal) and NDN (paternal signal only) (Fig. 4). However, 16% of the total population showed increased expression of a single paternal UBE3A allele relative to the detected signal from the (non-duplicated) maternal allele (Fig. 3C, v), and 3% had substantially elevated levels of both paternal alleles. An additional 8% of the cells with paternal UBE3A expression signal(s) exhibited low levels of overall expression, and had no detectable maternal UBE3A signal. Interestingly, of the latter group, half exhibited maternal NDN expression, as if the imprint status of the chromosome 15s were reversed in those cells; this rate of biparental expression of NDN is slightly higher than observed from non-duplicated 15s. It is not possible to determine at this time, however, whether the normal phenotype of SW is achieved because her paternal UBE3As are still completely silenced in critical neural tissues despite occasional escape from expression controls in other tissues, or whether the proportion of cells affected is too small or not localized in such a way as to result in detectable developmental delay.
NDNL2, a member of the NDN/MAGEL2 family, is located just distal to BP3, which marks the distal boundary of duplication in these dup(15q11– q13) cell lines (Fig. 3A) (this paper and 39). NDNL2 is biallelically expressed, and shows slightly preferential maternal expression in normal fibroblasts and NT2 cells by RNA-FISH (data not shown). Expression of NDNL2 in maternal dup(15) fibroblasts was indistinguishable from that in normal cells (supplementary Table 1—see HMG Online). This demonstrates that duplication of 15q11– q13 does not result in gross changes in expression of gene(s) flanking the duplication. In paternal dup(15) cells, however, preferential paternal expression of NDNL2 was most likely to co-occur within cells exhibiting increased expression from paternal UBE3A alleles (Supplementary Table 1).
Increased total cellular maternal versus paternal UBE3A expression by northern blot
To exclude the possibility of allele-specific differences in RNA-FISH probe accessibility, we performed northern blot analysis on total RNA isolated from cell lines of normal individuals and those with 15q11– q13 rearrangements, including deletion, duplication and triplication lines, to determine whether the gross RNA produced from a given number of paternal alleles is identical to that produced by the same number of maternal alleles. Signals were normalized for loading variability by correcting to beta-actin levels.
SNRPN expression levels increased directly in proportion to the number of paternal copies of the gene present in a given cell line, regardless of maternal copy number (6) (Fig. 5B). This is consistent with SNRPN signal being observed predominantly from paternal alleles by RNA-FISH. Although the levels from paternal duplications and triplications are not absolutely 2- and 3-fold, respectively, this may represent regulation of excessive expression directly or at a total-cellular RNA level. The low-level signal from PWS deletion and UPD lines, carrying only maternal alleles, is consistent with the ability to detect low levels of this gene from PWS cells by PCR amplification (not shown).
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In contrast, although expression of the
4 kb UBE3A transcript increased along with 15q11– q13 copy number, total expression levels were higher from maternal dup(15) and three independent maternal trip(15) lymphoblast cell lines than from paternal dup(15) or trip(15) lines, respectively. These results confirm the relatively higher levels of maternal UBE3A expression observed at the site of expression by RNA-FISH analysis. Similar results are obtained from the larger 5 kb UBE3A signal smear (which may reflect multiple-splice variants of UBE3A), with the exception of the paternal dup(15) cell line DL-7 (40), which shows elevated levels of the larger UBE3A transcripts only. It is possible that DL-7 has a higher proportion of cells exhibiting deregulated paternal UBE3A expression than the other cell lines carrying paternal duplications analyzed in this study. Finally, expression of UBE3A from a marker chromosome 15 was exceedingly high, confirming gross deregulation of UBE3A transcription from the inv dup(15) as observed by RNA-FISH. Indeed, inv dup(15) expression is much greater even than that from the most highly expressing interstitial maternal trip(15) line, although the two cell classes contain the same number of maternally derived 15q11– q13 regions. Therefore, northern blot and RNA-FISH observations together suggest that interstitial duplication results in increased total levels of expression because of (usually) normal levels of expression from a greater number of alleles. The elevated expression from inv dup(15) alleles, however, may reflect the lack of a complementary paternal marker chromosome in these cells, lending support to the hypothesis that pairing promotes regulation or maintenance of imprinted expression. Such regulation may be especially important in cells where expression is imprinted but not exclusively maternal. Cell- and/or developmental-specific monoallelic expression, on the other hand, may arise via the incorporation of additional, ‘all-or-nothing’ control mechanisms.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In addition to harboring the PWS and AS gene(s), linkage and transmission disequilibrium analyses have suggested that at least one autism-susceptibility gene may be located in 15q11– q13 (41,42). Furthermore, 0.5–3% of patients with autism carry a duplication of 15q11– q13 on their maternal chromosome 15 (43), and 50% of probands with a maternal dup(15) ascertained for developmental/language delay have a defined autistic spectrum disorder (9). It is also of interest that the few PWS patients with autism carry two maternal chromosome 15s (4). In contrast, paternal duplication of 15q11– q13 is not commonly associated with a phenotype (7,9,38), although paternal triplication may be (6).
The most likely way that increases in gene/region copy number could influence phenotype involves changes in gene expression levels. Excess gene expression derived directly from genomic duplication could result in brain development or functional abnormalities, especially if dosage is critical at a given point in development or in a specific cell type. In particular, the observation that imprinted expression of the AS gene, UBE3A, only occurs within brain suggests that tight regulation of its expression levels is critical for normal brain development/function. Excess, duplication-derived maternal expression might be comparable to what would occur normally without paternal silencing, which may be neurotoxic.
A second possibility is that gene duplications could silence the expression of genes in duplicated or adjacent regions by epigenetic effects, such as induced heterochromatinization (44,45), mimicking a loss-of-function phenotype. Regional duplications could also result in either trans-silencing or enhancement, as seen in U2af1-rs1 (46) and Igf2 (47). Finally, changing the ratio of maternal/paternal domains in cis or in trans could alter the interactions between oppositely imprinted domains (34); therefore, duplications could directly interfere with the proper regulation of imprinted expression.
UBE3A expression is imprinted in tissues other than brain, but the degree of preferential maternal expression varies within the cell population
The correlation between abnormalities of a maternal 15q11– q13 domain and phenotype led us to hypothesize that the candidate gene for these disorders would be imprinted and maternally expressed. Therefore, we chose to analyze UBE3A, the first gene in the region known to exhibit exclusive maternal expression. The normal function of UBE3A and its role in AS is not fully understood. UBE3A codes for an E3 ubiquitin–protein ligase, although only a few targets have been identified, including p53 (48). UBE3A also acts as a coactivator of steroid receptors, although this function is not always disrupted in AS patients with UBE3A mutations (49). UBE3A mutations have not yet been detected in autistic patients (50). However, it remains plausible that changes in the E3 or coactivator functions could alter normal brain development/function and result in an autistic spectrum disorder (51).
In this paper, we present results from RNA-FISH (Figs 1–4) and northern blot (Fig. 5) analysis of fibroblasts, lymphoblasts and undifferentiated neuronal cells showing that UBE3A expression is preferentially maternal and is therefore imprinted in tissues other than brain. In support of this finding, Kagotani et al. have recently shown that both mono- and bi-allelically expressing cells are observed for UBE3A within lymphocyte cultures (52). We also demonstrate that fibroblast populations contain cells with varying degrees of preferential maternal UBE3A expression. We have obtained similar results (data not shown) for the recently described ATP10C gene (21,22), which lies adjacent to UBE3A in 15q11– q13 and is also subject to control by the AS-IC. Recent hypotheses have suggested that the ‘exclusive’ UBE3A imprint is a result of brain-specific paternal antisense transcription (53,54). Our results suggest, however, that UBE3A expression is already selective, and that perhaps the paternal UBE3A promoter is packaged/modified in such a way as to predispose it to the ‘silencing’ effects of tissue-specific factors. Indeed, polymorphism-based expression analysis of select UBE3A transcripts in fetal brains demonstrated preferential, rather than exclusive, maternal expression (19). Our preliminary results on undifferentiated neuronal cell lines suggest that preferential maternal expression is observed in undifferentiated cells, becoming exclusively maternal as the neurons differentiate (L. Herzing, unpublished data).
Parental-specific expression levels are maintained between unduplicated and duplicated alleles in the majority of cells following 15q11– q13 duplication
SNRPN/NDN.
RNA-FISH and northern blot analysis of SNRPN or NDN expression from cell lines carrying duplications of 15q11– q13 show that duplication does not result in a loss of expression, but, rather, that expression occurs from the great majority of paternal alleles regardless of duplication status. In particular, RNA-FISH analysis demonstrated that these genes are expressed almost exclusively from paternal alleles. Northern blot confirmed that SNRPN expression levels were approximately proportional to the number of paternal 15q11– q13 regions within each cell line, as we had reported previously (6), whether carried on interstitial duplications or on a marker chromosome (Fig. 5B). A small minority of cells exhibited abnormal SNRPN/NDN expression patterns by RNA-FISH, which are expected within normal population variance. Thus, neither duplication in cis nor in trans alters the fidelity of the exclusively paternal, imprinted expression of SNRPN or NDN.
UBE3A.
UBE3A expression was also detected from most duplicated alleles (Fig. 3). Unlike SNRPN and NDN, however, variations in UBE3A expression were detected and were dependent upon the nature of the 15q11– q13 duplication. Maternal dup(15) had little influence upon the imprinted expression pattern of UBE3A as determined by RNA-FISH, with expression from both maternal alleles greater than or equal to expression from the paternal allele in roughly 85% of cells. Northern blot analysis (Fig. 5) confirmed that the sum of expression from maternal (interstitial) duplicated or triplicated alleles is greater than from similarly amplified paternal alleles for most transcripts. Some variability in allelic expression levels between cell lines with similar karyotypes was found, although preferential maternal expression was suggested in all cases. Finally, in addition to the expected small population of cells with upregulated paternal expression, RNA-FISH identified a novel class of cells in the maternal dup(15) cell line, with expression from only one duplicated maternal allele combined with normal levels of paternal expression (5%).
Increased total UBE3A expression is also seen, but to a much greater degree, in almost all cells of a fibroblast line containing a maternal large marker chromosome 15. Both RNA-FISH and northern blot analysis detected excessive amounts of UBE3A expression, arising from both marker alleles. Neither SNRPN nor NDN expression was detected from the marker, however, providing evidence that the increased UBE3A expression is not a result of gross chromatin disorganization causing a global loss of expression constraints. Relative levels of UBE3A expression from the unrearranged chromosome 15s were unchanged.
Although 73% of cells carrying a paternal (dup)15 exhibited normal UBE3A expression patterns by RNA-FISH, represented by expression from the maternal allele greater than or equal to expression from the paternal allele(s), the remaining 27% had increased paternal expression. Most of these exhibited expression from both paternal alleles, contrasting sharply with the almost unmodified UBE3A expression patterns observed in maternal (dup)15 cells. Furthermore, in the paternal (dup)15 cells with elevated paternal UBE3A expression, there is a similar shift in the expression of NDNL2, which maps adjacent to the duplication, from marginally preferentially maternal to preferentially paternal (Supplementary Table 1). Northern blot analysis demonstrated a selective increase in the larger, alternatively spliced UBE3A transcripts in a cell line with a paternal duplication, but not in the major transcript; however, this was not found in a line with a paternal triplication. Together, these results suggest that proper maintenance of low UBE3A expression levels may be susceptible to the effects of regional duplication.
The occasional deregulation of ‘suppressed’ paternal UBE3A transcription is reminiscent of the unstable imprinted expression of some paternally expressed 15q11– q13 genes such as ZNF127. For these genes, exclusive imprinted expression is susceptible to changes in chromosomal complement (23). In addition to occasional increases in paternal UBE3A expression associated with paternal duplication, unrestrained UBE3A expression from alleles of the inv dup(15) marker suggest that complete lack of a complementary chromosome might also override expression controls of a susceptible gene, even on the preferentially expressed allele.
Phenotypic consequences of 15q11– q13 duplication are a result of maternal overexpression
The results presented herein confirm that, for 15q11– q13 duplications, the resultant phenotypes are most likely due to a relative overexpression of the duplicated genes, rather than from haplo-insufficiency resulting from duplication-mediated gene silencing or because of gross changes in expression of flanking genes. Because the relatively greater maternal expression levels are maintained upon 15q11– q13 duplication, maternal duplication in particular would result in total expression levels considerably higher even than those occurring in normal cells prior to silencing of the paternal allele.
The marginal increase in total expression generated by a paternal duplication may not be sufficient to exceed a critical threshold for proper neural function, and/or this additional paternal expression may still be subject to silencing in critical tissues. Low endogenous paternal expression of conditionally imprinted genes, coupled with minimal, potentially silenced expression increases following amplification, may serve to explain the somewhat counter-intuitive finding that duplication of over 5 Mb of chromosomal material has no detrimental phenotype. The PWS-like phenotype observed with paternal triplication, however, may be a result of expression levels beyond a threshold limit, or may be a result of a 3-fold increase of paternally expressed genes (6).
We also demonstrate that amplification in cis differs from that in trans. The overabundance of expression from each UBE3A allele in a large marker chromosome may explain why the phenotype for large marker patients is considerably more severe than that of interstitial triplication patients, despite both classes carrying identical numbers of 15q11– q13 regions.
Many factors, including parental origin of affected chromosomes, total chromosomal complement within cells, and the nature of gene expression characteristics such as imprinted versus tissue-specific expression, determine the phenotypic consequences of chromosomal abnormalities. It is therefore critical to determine the potentially varied expression characteristics of regional genes under normal circumstances, to better understand the effects of chromosomal rearrangements on total gene expression levels and to relate these to phenotype.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell culture
Normal fibroblast cell lines and lines carrying chromosome 15 rearrangements were obtained from the Coriell Cell Repositories (Camden, NJ, USA) and grown under recommended conditions. Cell lines included: GM05565 and GM00242 (normal); GM00118 (46,XX,-15,+der(15)t(Y;15)(15qter>15p1::Yq11>Yqter)pat); GM02729 (47,XY,+t(15;15)(15pter>15q13::15p11>15pter). Fibroblasts were obtained from a patient carrying a maternal dup(15) (BW), her phenotypically normal mother carrying a paternal dup(15) (SW) and her normal father (7) via punch biopsy followed by fibroblast outgrowth in culture.
NT2 neuronal precursor cells were obtained from Stratagene (La Jolla, CA, USA) and grown in 1 : 1 Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 15% fetal bovine serum. Neuronal precursor HCN-2 cells (ATCC CRL 10742) were obtained from the Coriell Cell Repositories and grown in DMEM supplemented with 4.5 g/l glutamine, 1.5 g/l sodium bicarbonate, 1 mM sodium pyruvate, 1x non-essential amino acids, and 10% fetal bovine serum. HCN-2 cells were grown in flasks coated with laminin; 50% of the medium was replaced every 4–5 days, and cells were split 1 : 2 approximately every 3–4 weeks during expansion, or refed for up to 12 months.
Prior to FISH analysis, cells were cultured on 0.1% gelatin-(fibroblast, NT2) or gelatin+laminin-(HCN-2) coated Lab-TekII chamber slides (Nalge-Nunc Intl.; Fisher Scientific, Pittsburgh, PA, USA).
RNA/DNA FISH
RNA FISH was performed with modification of published techniques (55,56). All reagents were RNase-free. Cells grown on chamber slides were fixed by air-drying, permeabilized with 0.5% Triton X-100 in CSK (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES, pH 6.8) buffer and fixed with cold 4% paraformaldehyde. Slides were stored at 4°C in 70% ethanol. For RNA-FISH, slides were dehydrated through an ethanol series to 100% ethanol, air dried, and hybridized overnight at 37°C. Slides were washed and the biotin/digoxygenin signal detected using FITC-conjugated avidin or rhodamine-conjugated anti-digoxygenin (DIG) antibodies, and mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA) containing 250 ng/ml DAPI. Some slides were treated with RNaseH before signal detection as a control (not shown).
RNA signal was detected using a Zeiss Axiophot microscope with filters for separate detection of DAPI, fluorescein isothiocyanate (FITC), and rhodamine as well as a triple bandpass filter (No. 83000, Chroma Technology, Brattleboro, VT, USA) to detect signals simultaneously. Images were collected and merged using a cooled CCD camera (KAF 1400, Photometrics, Tucson, AZ, USA) and IP Lab Spectrum (Signal Analytics, Vienna, VA, USA) or SmartCapture (Digital Scientific, Cambridge, UK). To minimize the effects of intercellular differences, each field was imaged at three different focal planes and the maximum intensity signal from each allele used for scoring.
For overlaying DNA FISH, slides were washed in 4x SSC/0.1% sodium dodecylsulfate (SDS) to remove mountant, dehydrated, denatured, dehydrated again and hybridized at 37°C overnight to DNA probes labeled opposite (e.g. FITC versus rhodamine) of the RNA label. Slides were washed stringently and probe was detected as above.
cDNA probes for detection of RNA and genomic BAC/PAC probes for genomic localization were labeled using nick-translation and DIG dUTP or biotin dUTP: UBE3A, 3.3 kb insert from Image # 361377; SNRPN, 1.5 kb insert from Image # 970586, and/or PCR-generated genomic sequence spanning exons 2–8 (4.5 kb); NDN, 1.6 kb insert from Image # 39127; NDNL2; PCR-generated 1.1 kb fragment (primers WI-17399F; 5'-TGGAACTTGAACCCAAGAGC); Dystrobrevin, PCR-generated fragments from cDNA (2.5 kb) spanning bp11–2518 (57). UBE3A PAC 158H23 was kindly provided by Dr James S. Sutcliffe, and SNRPN p1-108 was obtained originally from Stuart E. Leff. NDN BAC 84N13 was isolated by P. Ungaro from a Genome Systems library and obtained, along with NDNL2 BAC RP11-18H24, and Image clones, from Genome Systems, Inc. (St Louis, MO, USA). Spectrum Green-labeled chromosome 15 paint probes were obtained from Vysis Inc. (Downer's Grove, IL, USA); Spectrum Orange-labeled chromosome 18 (Dystrobrevin) and chromosome 5 paint probes were obtained from Gibco/BRL (Frederick, MD, USA), and DIG-conjugated Y chromosome alpha-satellite probes were obtained from Oncor (Intergen, Purchase, NY, USA). DNA from genomic and Image clones was isolated using an AutoGen740 (Integrated Separation System, Natick, MA, USA).
Northern blot analysis
Total RNA was purified using TRIzol Reagent from Life Technologies Inc. (Rockville, MD, USA). Twenty micrograms of total RNA were loaded in each lane for northern blot analysis. A cDNA probe for the SNRPN region exon -1 to exon 1 (RN175, RN140) was generated as previously described (6). A 2.0 kb beta-actin cDNA probe was provided by N. Matsumoto. UBE3A was detected using a 1.9 kb NotI/EcoRI fragment from Image # 361377 spanning the 5' half of the gene. Overnight hybridization was performed using ExpressHyb Solution (Clontech, Palo Alto, CA, USA) at 65°C. The filters were washed in a solution of 0.2x& SSC, 0.1% SDS at 65°C. Relative intensities of the hybridized bands were scanned on a STORM phosphoimager and quantified using ImageQuaNT software (Molecular Dynamics, Sunnyville, CA, USA). Blots were probed sequentially as follows: UBE3A, beta-ACTIN, SNRPN.
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ACKNOWLEDGEMENTS |
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We thank S.L. Christian for critical reading of the manuscript and for sharing her depth of chromosome 15 knowledge. We thank J. Fantes for support and advice in all aspects of this project, S. Mewborn for excellent technical assistance, the BW family for generously participating in this project, W. Dobyns for obtaining skin biopsies and J. Hedrick for coordinating patient scheduling and sample acquisition. Tissue samples were obtained in part from the Miami Brain and Tissue Bank for Developmental Disorders contract # NOI-HD-8-3284, and as a generous gift from D. Driscoll. Support for L. H. was provided by a Cure Autism Now (CAN) Foundation Young Investigator Award. This work was supported in part by National Institutes of Health grants R01 HD36111 (DHL), R01 MH52223 (EHC) and K02 MH01389 (EHC).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Human Genetics, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Tel: +1 7738340525; Fax: +1 7738340505; Email: dhl{at}genetics.uchicago.edu
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REFERENCES |
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