Human Molecular Genetics, 2001, Vol. 10, No. 19 2049-2059
© 2001 Oxford University Press
Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK and 1Department of Biochemistry, Medical Sciences Institute/Wellcome Trust Building Complex, University of Dundee, Dundee DD1 5EH, UK
Received June 22, 2000; Revised and Accepted July 13, 2001.
DDBJ/EMBL/GenBank accession no. AJ276371.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The transcription factor PAX6 plays a critical, evolutionarily conserved role in eye, brain and olfactory development. Homozygous loss of PAX6 function affects all expressing tissues and is neonatally lethal; heterozygous null mutations cause aniridia in humans and the Small eye (Sey) phenotype in mice. Several upstream and intragenic PAX6 control elements have been defined, generally through transgenesis. However, aniridia cases with chromosomal rearrangements far downstream of an intact PAX6 gene suggested a requirement for additional cis-acting control for correct gene expression. The likely location of such elements is pinpointed through YAC transgenic studies. A 420 kb yeast artificial chromosome (YAC) clone, extending well beyond the most distant patient breakpoint, was previously shown to rescue homozygous Small eye lethality and correct the heterozygous eye phenotype. We now show that a 310 kb YAC clone, terminating just 5' of the breakpoint, fails to influence the Sey phenotypes. Using evolutionary sequence comparison, DNaseI hypersensitivity analysis and transgenic reporter studies, we have identified a region, >150 kb distal to the major PAX6 promoter P1, containing regulatory elements. Components of this downstream regulatory region drive reporter expression in distinct partial PAX6 patterns, indicating that the functional PAX6 gene domain extends far beyond the transcription unit.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Gene regulation is hierarchical, but mechanisms acting above the level of promoters, enhancers and silencers, such as locus control regions (LCRs), remain difficult to define (1–5). It has been proposed that the default state for chromatin is the ‘closed’ DNaseI-resistant, repressive conformation, in which transcription factors cannot bind and most genes are not transcribed (6,7). To achieve spatially, temporally and quantitatively correct gene expression, the chromatin conformation needs to be reset to ‘open’ at required loci. Classically, LCRs are thought to play a key role in the long range tissue-specific chromatin opening around a gene locus. Once the permissive state is established, local control of expression is exerted by the concerted binding of tissue-specific and ubiquitous transcription factors at a number of promoter, enhancer and silencer sites (8). These regulatory DNA elements are commonly found in the region immediately upstream of the transcription unit and within the first few introns (9). Most gene regulation studies are confined to these gene-proximal regions. However, we were led by the presence of distant chromosomal breakpoints in several individuals with classical aniridia (10 and unpublished data) (Fig. 1A), to search for PAX6 regulatory regions a long way downstream of the gene.
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Heterozygous PAX6 mutations result in human aniridia and in the homologous mouse Small eye (Sey) phenotype. The aniridia and Sey phenotypes are similar and variable. Aniridia often involves not only reduction or absence of the iris but also foveal/macular hypoplasia, cataracts, ciliary body abnormalities, corneal limbal stem cell deficiency and glaucoma. A high proportion of classical aniridia cases (11; http://www.hgu.mrc.ac.uk/Softdata/PAX6/), as well as the documented mouse Sey phenotypes (in Pax6SeyEd/+ and Pax6Sey1Neu/+ mice) (12), are caused by haploinsufficiency—heterozygous loss-of-function mutations, mostly due to premature protein truncation. Correct PAX6 (Pax6) gene dosage is critical for eye development. Increased Pax6 expression, as well as haploinsufficiency, leads to reduced eye size (13). Pax6 is expressed not only in the retina, lens and cornea of the developing vertebrate eye, but also at different developmental stages in regions of the forebrain, hindbrain, cerebellum, the ventral neural tube, the olfactory system and pancreatic islet cells (14,15). The homozygous Sey phenotype reflects this expression pattern more closely. Pax6SeyEd/Pax6SeyEd animals die immediately after birth with no eyes, no nasal structures and severe brain abnormalities (12), a phenotype very like the only reported human case with functional loss of both copies of PAX6 (16). It is not surprising therefore that complex spatio-temporal and quantitative control are required for normal developmental expression of PAX6.
Loss of gene expression can be elicited in many different ways. Disruption of long range control is predicted to cause some null mutations, although such mutations are not often assessed in the course of mutation analysis. However, the mechanism has been implicated in a number of developmental anomalies for which recurrent mutations within a specific gene have been established as the usual cause of disease, but where some cases have been associated with chromosomal rearrangements that are outside the gene transcription unit. These breakpoints have been shown to lie some distance upstream or downstream of the gene (17) and, by partial analogy with some Drosophila translocation cases, are said to lead to loss of gene expression through position effects mediated by altered chromatin organization (18). Such ‘extragenic’ translocation and deletion breaks have now been defined downstream of the PAX6 gene in several aniridia cases (10,19,20).
The most distant breakpoints, in two familial cases with the classical aniridia phenotype (10), were identified (19) at 125 kb (SGL) and 150 kb (SIMO) downstream of the PAX6 P1 promoter site (21). PAX6 exonic sequences were shown to be intact in these cases (19). Several additional aniridia-associated breakpoints have since been identified in the interval between the PAX6 poly(A)-addition site and the SGL/SIMO breaks (Fig. 1A) (20 and unpublished data). These cases suggest that aniridia can arise by disruption of long range control of PAX6 expression.
To test the hypothesis that loss of a cis-regulatory region is the cause of aniridia in these patients with breakpoints downstream of the PAX6 transcription unit, we made transgenic mice with yeast artificial chromosome (YAC) Y589, a human YAC that mimics the furthest patient breakpoint. In contrast to the rescue of Pax6SeyEd/Pax6SeyEd lethality and full phenotypic correction in both heterozygous and homozygous Sey mice by the 420 kb human PAX6-containing YAC Y593 (13), Y589 fails to rescue lethality or correct the mutant phenotypes. This suggested the presence of essential regulatory elements in the 80 kb region beyond the SIMO breakpoint between the ends of Y589 and Y593. Through DNaseI hypersensitive site (HS) mapping and evolutionary sequence comparison we have identified multiple regulatory elements over an 
25 kb region. Transgenic reporter studies reveal that some component elements drive distinct tissue-specific expression in a partial PAX6 expression pattern. We have designated this distant control complex the PAX6 ‘downstream regulatory region’ (DRR).
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation and molecular analysis of Y589 transgenic mice
The exact extent of YACs Y593 and Y589 was defined by YAC end sequencing and database comparison. The SIMO breakpoint was pinpointed by sequence analysis. Transgenic mice were generated (13) by pronuclear injection of DNA prepared from the human YAC Y589, with 160 kb upstream and 150 kb downstream of the PAX6 promoter P1, terminating 2.75 kb 5' of SIMO, the furthest patient breakpoint. Four transmitting lines (589-1 to 4) were obtained and analysed for transgene content (Fig. 1A and B). Hybridization with a human PAX6 cDNA probe plus probe SfiX, which includes promoter P0, revealed the presence of all the expected human-specific EcoRI fragments in all four lines (Fig. 1B) indicating that no major rearrangements had taken place in the transcribed gene. Probes p60 (
150 kb 5' of P1) and p5 (50 kb 5' of P1) (10) were both present in lines 589-2 and 589-3, but absent in lines 589-1 and 589-4. SX3, located 55 kb downstream of P1 is present in all four lines, but probe EH3, 140 kb from P1, is absent in line 589-3. PCR analysis with YAC vector arm primers revealed the presence of the upstream Trp1 arm in 589-2 and 589-3, and the downstream Ura3 arm in 589-1, 589-2 and 589-4 (Fig. 1A and B). FISH analysis (data not shown), using cosmids p60, FAT5 and H1281 (10 and Fig. 1B), confirmed transgene position, integrity, copy-number and orientation. 589-2 carries an intact copy of Y589, 589-1 and 589-4 each carry one upstream truncated copy, and 589-3 carries two head-to-head copies truncated >55 kb downstream of P1 (Fig. 1B).
Phenotypic analysis of Y589 transgenic mice
To study the functional capacity of the Y589 transgenes (Fig. 1A), mice compound heterozygous for each transgene and the Pax6SeyEd allele (Tg/+//Sey/+) were produced and mated together to generate Pax6SeyEd homozygotes carrying the transgene (Tg/+//Sey/Sey). In contrast to transgenesis for the intact Y593, which fully corrects the heterozygous Sey phenotype and rescues the homozygous Sey lethality (13 and Fig. 2A–C), no homozygous rescue or significant amelioration of the heterozygous phenotype was found with any Y589 transgenic lines (Fig. 2D and E). For each Y589 transgenic line at embryonic day (E)18.5, molecularly confirmed Tg/+//Sey/Sey fetuses were present in the expected proportions, but indistinguishable from their non-transgenic Sey/Sey littermates; no prolonged postnatal survival was observed. The adult ocular phenotype in Tg/+//Sey/+ mice was not significantly different from the iris hypoplasia and cataracts observed in non-transgenic Sey mice (22) (Fig. 2F). No eye phenotype was conferred by the presence of any Y589 transgene on the wild-type background, unlike the variable microphthalmia observed in the multicopy Y593 line, 593-1 (i.e. PAX77) (13).
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These results strongly suggest that elements essential for normal PAX6 expression lie in the genomic region between the telomeric ends of Y589 and Y593.
RNA in situ studies for human PAX6 expression from the YAC transgenes
To assess PAX6 expression capability from different YAC transgenes, we took advantage of the ability to discriminate between human and mouse transcripts. Using whole-mount in situ hybridization, we compared human PAX6 expression at E10.5 in the lethality-rescuing 593-1 transgenic mice with the non-rescuing 589 transgenes on a wild-type mouse background. No expression above background was observed in lines 589-3 and 589-4 (data not shown). Qualitative and quantitative differences were seen between 593-1 (Fig. 2G) and 589-2 (Fig. 2H), and the truncated 589-1 (Fig. 2I). Quantitative variation, indicated by the >16-fold longer time required for comparable staining of 589- and 593-transgenic embryos, may stem partly from transgene copy number differences. However, in the Y589 transgenic mice severe reduction or absence of expression in the diencephalon is observed, compared with expression in telencephalon and rhombencephalon. Y589-driven eye expression is also reduced to a partial pattern (inserts in Fig. 2G–I). We deduce that incomplete PAX6 expression from the 589-2 and 589-1 transgenes, and the inability of Y589 to rescue Sey/Sey lethality, is due to the absence of regulatory elements in the 80 kb region between the ends of Y593 and Y589.
DNaseI HS analysis in human PAX6-expressing cells
To identify the presence of regulatory elements within this region, we undertook DNaseI HS analysis of a >40 kb region around and distal to the SIMO breakpoint, using chromatin isolated from PAX6-expressing and non-expressing human cell lines. HS mapping is commonly used to reveal the position of DNA elements available for the binding of proteins required for gene regulation (23). DNaseI HS represent nucleosome-free regions of DNA that are readily accessible to regulatory proteins. Three PAX6-expressing human cell lines were used: lens-derived CD5a (Materials and Methods), retinal pigment epithelium-derived ARPE (24) and glioblastoma line U87 (ATCC no. HTB-14), with the colon carcinoma cell line HT-29 (ATCC no. HTB-38) as non-expressing control. The availability of finished sequence for the human PAX6 region (http://www.sanger.ac.uk/cgi-bin/humchr?chr=11) allowed us to predict restriction sites and produce the probes shown (Fig. 3A) for comprehensive HS analysis. Isolated nuclei were treated with increasing concentrations of DNaseI. The efficiency of DNaseI treatment was assessed by first hybridizing blots with a probe from the PAX6 P1 promoter region, which forms a strong HS in the expressing cell lines (data not shown). Using CD5a, no HS were observed with probes EH3, EI/EN proximally, or with the more distal probes HS3 and 7K2, but seven HS specific to PAX6-expressing cells were defined in the intervening region (Fig. 3A and B). The presence and position of these sites were confirmed with probes from opposite ends of each restriction fragment, and by using two different restriction enzymes (XbaI or EcoRI) following DNase treatment. Sites HS1–7 were found specifically in CD5a, but not HT-29, and preliminary analysis of cell lines U87 and ARPE confirms the presence of HS2 and HS3 in both these cell lines, while the HS6 site is strong in U87 and much weaker in ARPE (data not shown). HS8 is present only in HT-29. HS5A and 5B, present in both CD5a and HT-29, are common to PAX6-expressing and non-expressing cells. Overall, these findings point to the presence of a complex, PAX6 expression-associated DRR, distal to the furthest patient breakpoint.
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Comparative sequence analysis: human, mouse, Fugu
To identify the location of possible regulatory elements, we assessed the evolutionary conservation of available genomic sequence in the PAX6 downstream region, from human, mouse and Fugu (25–28). Human sequence analysis predicted the presence of a 10-exon protein-encoding gene immediately downstream of PAX6 and transcribed in the opposite direction (D.A.Kleinjan, A.Seawright, G.Elgar and V.van Heyningen, manuscript in preparation). The position and exon/intron structure of this gene, designated PAX6 neighbour gene (PAXNEB; official nomenclature C11ORF19) are depicted (Figs 1A and B and 3). All of the patient breakpoints (10,19,20) (Fig. 1A), and the HS1–4 region of the DRR (Fig. 3A), fall within the large terminal intron of the PAXNEB gene. The gene contains no recognizable motifs suggestive of function, but is highly conserved across vertebrates and significant stretches of amino acid homology are present in many distant phyla.
Sequencing of the Fugu Wilms tumour, aniridia, genitourinary anomalies and mental retardation region demonstrates maintenance of synteny between Fugu, man and mouse (26 and G.Elgar, personal communication) both upstream and downstream of PAX6, well beyond the PAXNEB gene. Using PAXNEB exons as landmarks allowed us to compare human and Fugu sequence in the putative DRR region. No significant sequence conservation was found other than over the coding exons of PAXNEB (D.A.Kleinjan, A.Seawright, G.Elgar and V.van Heyningen, manuscript in preparation; G.Elgar, unpublished data).
In contrast, mammalian sequence conservation at the SIMO breakpoint had been indicated by early zoo-blot studies (19,29) suggesting the presence of a possible regulatory element despite the absence of DNaseI hypersensitivity in this region in the PAX6-expressing cell lines. A 7.5 kb EcoRI fragment isolated from a 129/Sv bacterial artificial chromosome (BAC) clone containing the breakpoint-homologous region was partly sequenced (EMBL no. AJ276371). Mouse–human sequence conservation around the SIMO breakpoint revealed 85% nucleotide identity over a 1400 bp fragment, with a 500 bp core region showing 96% identity (BESTFIT result available at http://www.hgu.mrc.ac.uk/Research/Cellgen/), with no obvious open reading frame (ORF), suggesting a role as a control element.
Recent availability of C57Bl/6 sequence from the UK Mouse Sequencing Programme (ftp://ftp.sanger.ac.uk/pub/mouse/Chr_2/unfinished_sequence/bM431C3) has permitted a more widespread human–mouse sequence comparison over the HS1–4 region of the DRR. The following conservation levels are observed: HS1, 75% over 425 bp; HS2–3, 88% over a 1250 bp region; HS4, 71% over 540 bp, with additional homology of 75% over 730 bp between HS1 and HS2 (see http://www.hgu.mrc.ac.uk/Research/Cellgen/).
Transgenic reporter studies of DRR region enhancer function
The functional significance of the control elements predicted within the DRR by PAX6-specific DNaseI hypersensitivity and human–mouse sequence conservation was explored by mouse transgenic reporter analysis.
A 4.5 kb fragment, HS234Z, containing HS2, HS3 and HS4 (Fig. 3A) inserted in both orientations into an Hsp68-LacZ reporter cassette (30) (Fig. 4A), was used to produce four independent transgenic lines and two sets of transients analysed as primary embryos. LacZ expression was consistently observed in the following pattern: (i) from E8.0 in the neural folds (Fig. 4B); (ii) at E8.5 in the optic primordium of the telencephalon (Fig. 4C); (iii) at E9.5, in the dorsal optic vesicle (Fig. 4D,E); and (iv) from E10.5 encompassing the whole retina (neural and pigmented) (Fig. 4G and H) and showing some olfactory region expression (Fig. 4F). This expression pattern, confined to the retina and sparing the lens, continues into adulthood (Fig. 4I). Additional ectopic expression was seen in one or two lines (Fig. 4F). The consistent retinal expression represents a highly restricted subset of the normal Pax6 pattern.
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A second series of transgenic lines was produced using a construct, EI–Z, containing a 2.5 kb genomic fragment (Fig. 4J), including the 1400 bp highly conserved sequence spanning the SIMO breakpoint. Ten out of eleven independent transgenic lines expressed the reporter. These 10 lines all consistently expressed in the lens, diencephalon and hindbrain. Diencephalon expression was seen constantly throughout development from E9.5 (Fig. 4K, M, O and P). The hindbrain expression pattern is more dynamic, at E9.5 rhombomeres 2 and 6–8 are seen to express (Fig. 4K and L). By E10.5 rhomobomere 2 expression has broadened into rhombomere 1, 2 and 3, with peak levels in 2 (Fig. 4O), while expression in the caudal hindbrain has become restricted to rhombomere 6 (Fig. 4O and P). Subsequently, rostral hindbrain expression is maintained while caudal hindbrain expression diminishes (Fig. 4P). Eye expression is also observed from E9.5, first in the rostral optic vesicle (Fig. 4K), then becoming confined to the lens (Fig. 4N, O and Q). From E14.5 some retinal expression appears in the proximal optic cup (Fig. 4R). These consistently observed expression patterns with different transgenic constructs indicate that components of the DRR can act as distinct tissue-specific enhancer elements.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Distant extragenic chromosomal breaks lead to PAX6 haploinsufficiency and aniridia
Intragenic loss-of-function mutations account for the vast majority of aniridia cases analysed (11; http://www.hgu.mrc.ac.uk/Softdata/PAX6/), implying that PAX6 is the only gene implicated in this haploinsufficiency anomaly. It is likely therefore that the seven classical aniridia cases with chromosomal rearrangements up to 130 kb downstream of the PAX6 poly(A) signal (10,20 and unpublished data) and (Figs. 1 and 3) are also caused by loss of PAX6 function. The 22 kb PAX6 transcription unit apparently needs to be present within a much larger intact genomic region for normal developmental expression. Loss of expression from the disrupted genomic copy is difficult to demonstrate directly in humans where expressing tissues are unavailable. However, somatic cell hybrid work has recently confirmed that PAX6 expression is lost from similar rearranged chromosomes (20). Detailed mechanisms leading to haploinsufficiency in these cases are not yet understood, but here we show the identification of a distant DRR containing several regulatory elements beyond the furthest of the patient breakpoints.
Recapitulating the human genomic rearrangement in the mouse
The importance of the distant downstream region for correct PAX6 expression is initially shown through YAC transgenesis. Unlike Y593 (extending 80 kb beyond the SIMO breakpoint) (13), the shorter Y589 (terminating just PAX6-proximal to the SIMO breakpoint) is unable to correct the Sey heterozygote phenotype or rescue the homozygous lethality. Homozygous Pax6SeyEd/Pax6SeyEd mice carrying the Y589 transgene still die immediately after birth and the heterozygote phenotype is not significantly ameliorated. Y589 can therefore be said to mimic the SIMO rearrangement. Comparison of PAX6 expression from the Y593 and Y589 transgenes (Fig. 2G–I) shows that Y589, although it can direct some expression from an intact PAX6 transcription unit, can support only a partial expression pattern and at significantly reduced levels, clearly insufficient to rescue the mutant phenotypes. This suggests that control elements essential for normal PAX6 expression are present in the region between the telomeric end-points of Y593 and Y589.
The DRR is located within introns of a novel neighbouring gene
The minimum extent of the DRR was initially defined by mapping the YAC end sequences onto the known human genomic sequence for the PAX6 locus (http://www.sanger.ac.uk/cgi-bin/humchr?chr=11). Analysis of the PAX6 downstream region also led to the identification of a new gene, PAXNEB or C11ORF19 (D.A.Kleinjan, A.Seawright, G.Elgar and V.van Heyningen, manuscript in preparation). The centromeric end (i.e. the Y589 end) of the DRR is positioned within the large final intron 9, and its telomeric end (i.e. the Y593 end) in intron 3 of the PAXNEB gene, which is transcribed in the opposite direction to PAX6 (D.A.Kleinjan, A.Seawright, G.Elgar and V.van Heyningen, manuscript in preparation). The ubiquitously expressed PAXNEB gene is disrupted in all seven of the chromosomal rearrangement cases. However, heterozygous disruption of PAXNEB is unlikely to be the cause of aniridia because: (i) a very high proportion of aniridia cases tested (11) are accounted for by documented PAX6 mutations, suggesting it is the sole gene involved; (ii) PAXNEB is ubiquitously expressed; (iii) no aniridia-associated breakpoints are found elsewhere in the 250 kb genomic PAXNEB gene; and (iv) Y593, carrying an intact PAX6 gene but lacking the first three exons of the PAXNEB gene, corrects the heterozygote phenotype in the Sey1H deletion mouse (31) with only one intact copy of the PAXNEB homologue present (D.A. Kleinjan, A. Seawright, G. Elgar and V. van Heyningen, manuscript in preparation). The presence of distant regulatory elements for one gene within the transcription unit of an unrelated neighbouring gene is not unique, but has been observed for several genes such as the alpha globin (32) and human growth hormone clusters (6).
Sequence comparison identifies likely regulatory regions
Evolutionary sequence comparison in non-coding genomic regions has been revealed as a powerful objective method for identifying potential functionally significant elements (25–28,32). This has been borne out very clearly for the upstream and intragenic promoters and enhancers of PAX6, where functional analysis has confirmed, or in some cases heralded, the presence of sequence-conserved regulatory regions upstream of the gene and within introns in a variety of species (33–38). Most of the downstream region sequence analysis described here was carried out after the studies of DNaseI hypersensitivity and enhancer function were initiated, providing an opportunity to validate the method of finding regulatory elements through sequence conservation.
While a new Fugu sequence downstream of PAX6 (G.Elgar, S.Warner, D.Goode and P.Snell, manuscript in preparation) shows continuing synteny conservation to PAXNEB and beyond, PIP plot analysis reveals very little extragenic sequence conservation; none, for example, at the SIMO breakpoint or over the HS1–8 region. The DRR would thus have been missed based on Fugu to human sequence comparison. The absence of sequence conservation may indicate that in this region control element sequences have diverged too much to be recognisable, or that the elements present in the mammalian DRR are absent in fish, which would suggest differences in gene regulation between the vertebrate classes. However, like human PAXNEB, the Fugu homologue contains an unusually large intron at the 3' end of the gene (18 kb in Fugu versus 134 kb in man) (G.Elgar, personal communication), which suggests a functional role for this intron that may not be mediated via conserved sequence. Such functional conservation without sequence conservation has been documented between Drosophila and mouse (36).
In contrast to the absence of sequence conservation with Fugu, mouse–human comparison reveals clear confirmation of the experimental data. The region around the SIMO breakpoint shows a very high degree of conservation over 1400 bp, a much larger region than observed normally for regulatory elements at other loci (27,28). This may reflect the fact that the region functions as a compound enhancer, driving expression in multiple tissues (see below). Large regions of sequence conservation are also seen, however, over the HS2–4 region, which is observed to drive reporter expression only in the retina. The presence of defined HS suggests there is a core element where regulatory proteins bind, while flanking sequences have also been under evolutionary pressure.
DNaseI hypersensitivity analysis confirms some regulatory regions
Seven tissue-specific DNaseI HS (HS1–7), spread over an 20 kb region, were identified in the PAX6-expressing lens cell line CD5a. Most of these have now also been found in two other PAX6-expressing human cells, the retinal pigment epithelium line ARPE and the glioblastoma U87. None of these sites was observed in HT-29. Two minor sites HS5a and 5b were common to all cell lines tested. In contrast a strong site (HS8) telomeric to HS1–7 was found only in HT-29. The presence of an HS at the distal end of the HS region, in a non-expressing cell line, may signify function as a negative control element for PAX6 in tissues where no expression is required, either directly or by limiting the influence of the DRR to specific tissues. HS in the TCR alpha LCR have been shown to restrict the dominant chromatin opening activity of the LCR to specific tissues (38). It is also possible that a boundary or insulator element (39) might be required between a ubiquitously expressed gene like PAXNEB and the spatially and temporally controlled PAX6 gene.
The conserved sequence at the SIMO breakpoint revealed no DNaseI hypersensitivity in either the available PAX6-expressing cells or in HT-29. As the formation of HS is often tissue-specific this may signify that the element is not functional in CD5a, ARPE, U87 or HT-29, although the fragment does drive expression in the lens and brain of transgenic embryos (Fig. 4). Transformed cell lines like CD5a (lens-derived) and U87 (brain-derived) may not be an accurate model for expression control; HS analysis of endogenous tissue may be required to resolve this inconsistency.
The topological arrangement of several HS spaced over a 20 kb region is reminiscent of the organization of many LCRs, suggesting that the DRR may constitute part of a PAX6 LCR system. Individual HS, validated as part of an LCR, have been shown by transgenic analysis to function as enhancers in some cases, e.g. HS2 and HS3 of the globin LCR (40,41). The possible role of the PAX6 DRR as an LCR will need to be tested in large fragment transgenic systems and by specific deletion studies.
The role of downstream regulatory elements in controlling PAX6 function
The ability to drive consistent patterns of transgenic reporter gene expression is a clearly validated functional test for control elements. Testing for enhancer function in transgenic reporter studies, elements HS234 and EI recapitulate distinct parts of the endogenous PAX6 expression pattern. Expression in rhombomeres 2 and 6 elicited by EI is a subset of the wider endogenous hindbrain expression pattern and forms an intriguing area for further investigation since the role of PAX6 in the hindbrain is still poorly understood. PAX6 expression is expected to be controlled by a particular spectrum of transcription factors in different tissues. In line with this, several distinct enhancers with some overlap in tissue specificity have been identified in the upstream regions and within the introns of the PAX6 gene (33–37). Overlap is now also seen with some of the downstream elements. In particular, enhancer elements capable of driving lens and neuroretina expression in transgenic mice have already been identified upstream (34–36) and in intron 4 (33,35) of PAX6. This apparent redundancy of enhancer elements may signify the requirement for interaction of upstream and downstream control elements through combinatorial binding to the same regulator proteins or complexes. In line with this, the same set of transcription factor binding targets are found in the HS of the LCR and the promoters of the globin locus (40). This would be consistent with current suggestions for a looping model to explain long range control of gene expression (1,39). It is impossible to deduce the normal interactive role of such control elements by assessing their enhancer functions in isolation.
In conclusion, we show that a complex regulatory region, the DRR, is present 130 kb downstream of the PAX6 poly(A) addition site. Separation of the DRR from the PAX6 transcription unit results in the haploinsufficiency-associated aniridia phenotype, underlining the need for long range cis control of PAX6 expression, although the mechanisms of such transcriptional regulation remain to be elucidated. The genesis of this study starting from the association of aniridia with distant chromosomal rearrangements highlights how human mutation analysis can reveal novel aspects of gene function. It also illustrates that current definitions of a gene often underestimate the genomic extent of the functional gene domain.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation and analysis of YAC transgenic mice
YAC Y589, isolated like Y593 from the human ICI library (42), was shown to cover 310 kb by pulsed field gel electrophoresis. YAC end sequences were identified by inverse PCR, followed by sequencing. Sequences identified were superimposed on the known human genomic sequence [EMBL nos: Z95332, Z83307, Z83001, Z83306, Z83309, Z83308, Z86001 (43)]. The telomeric end of Y593 was shown to be at nucleotide 9279 in cosmid SRL9A13 (EMBL no. Z86001); and the telomeric end of Y589 was found to be at nucleotide 12 125 in cosmid SRL11M20 (EMBL no. Z83308). The SIMO breakpoint was defined by PCR analysis to be at nucleotide 14 880 in cosmid SRL11M20 (EMBL no. Z83308).
Y589 was retrofitted with a conditional centromere to allow improved DNA amplification and gel-purification. DNA was prepared for microinjection as previously described (13), and injected into pronuclei from oocytes of F1 mice from a (C57/Bl6 x CBA) cross. Injected oocytes were replaced in pseudopregnant CD1 fosters. Transgenic animals were identified by tailtip analysis. They were crossed with CBA females for one generation. All subsequent crosses were onto a CD background. Rescue of the Sey phenotype was assessed by crossing the transgenic lines with CD1 SeyEd/+ mice, and by intercrossing mice heterozygous for both the PAX6SeyEd allele and for the transgene.
Southern blot analysis of YAC transgenic lines was performed on DNA isolated from tailtips according to standard procedures. Primers used to assay for the presence of YAC arms in the transgenic lines are: YR1, 5'-ATATAGGCGCCAGCAACCGCACCTGTGGCG-3'; YR2, 5'-GTAATCTTGAGATCGGGCGTTCGA-3'; YL1, 5'-CACCCGTTCTCGGAGCACTGTCCGACCGC-3; and YL2, 5'-CCTTAAACCAACTTGGCTACCGAGA-3'.
For FISH analysis, spleens were dissected from transgenic mice, washed in PBS, punctured repeatedly with a sterile needle and splenocytes flushed out by forcing through 2 ml of RPMI. Bacterial lipopolysaccharide was added and the culture incubated for 46 h. FISH was performed as described (42).
RNA in situ hybridization
A human PAX6-specific RNA in situ protocol was developed by Penny Rashbass (Sheffield, UK). Part of the human PAX6 3'-UTR is PCR amplified using primers 5'-GCTCTAGACTCATTTCCCCTGGT-3' and 5'-TAATACGACTCACTATAGGATTGTTCCAACTG-3', the latter containing a T7 polymerase binding site. The digoxigenin (Dig)-labeled PAX6 anti-sense human-specific riboprobe was synthesized according to the manufacturers protocol (Boehringer) using the PCR product as template. Embryos dissected from timed gestations were fixed overnight at 4°C in a 4% paraformaldehyde (PFA) solution in PBS. This was followed by stepwise transfer to 100% methanol and storage at –20°C if required. Embryos were rehydrated to PBS, bleached with 6% H2O2 in PBT (PBS/0.1% Triton X-100) for 4 h, proteinase K (10 µg/ml) digested for 15 min and refixed in 0.2% glutaraldehyde/4% PFA for 20 min at room temperature. Prehybridization was overnight in hybridization solution [50% ultrapure formamide (Gibco-BRL); 5x SSC pH 8, 50 µg/ml heparin, 0.5% CHAPS, 5 mM EDTA, 100 µg tRNA, 0.1% Triton X-100, 2% Boehringer blocking reagent]. For hybridization, the prehybridization solution was replaced with fresh hybridization solution containing 0.1 µg/ml Dig-labelled probe and incubated overnight at 65°C. Following hybridization, embryos were washed at 65°C in solution A (50% ultrapure formamide, 5x SSC pH 8, 0.5% CHAPS, 0.1% Triton X-100), followed by washes in 2x SSC, 0.1% CHAPS and 0.2x SSC, 0.1% CHAPS. Embryos were washed in TBST (0.14 M NaCl, 2.7 mM KCl, 25 mM Tris–HCl pH 7.5, 0.1% Triton X-100), preblocked with 10% sheep serum in TBST for 3 h at room temperature, and incubated overnight at 4°C with 1:2000 Anti-Dig Fab fragment (Boehringer) in 1% heat-inactivated sheep serum in TBST. After antibody binding, embryos were washed for 1 or 2 days in TBST. For staining, the embryos were washed in NTMT (100 mM NaCl, 100 mM Tris–HCl pH 9.5, 50 mM MgCl2, 0.1% Triton X-100), and alkaline phosphatase activity was visualized using substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate in NTMT. After staining the embryos were washed in PBS pH 5.5, 1% Triton X-100 for several hours and post-fixed in 4% PFA/0.1% glutaraldehyde for 20 min at room temperature before storage in PBS at 4°C.
DNaseI HS analysis
Human PAX6-positive cell lines used were derived from endogenously expressing tissue: CD5a was immortalized from lens epithelium using replication-deficient SV40-adeno hybrid virus (44), ARPE derived from retinal pigment epithelium (24) and U87 is a human glioblastoma cell line (ATCC no. HTB-14). Continuing high-level PAX6 expression was demonstrated by immunohistochemical analysis (data not shown). The colon cancer line HT-29 (ATCC no. HTB-38) was used as a PAX6-negative control cell. Near confluent CD5a, ARPE, U87 and HT-29 cell cultures, still attached to the flask surface, were washed twice with cold PBS. Cells were harvested by trypsinization, washed and resuspended using 3.6 ml cold buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) by gently pipetting up and down with a 200 µl micropipette tip. Cells were allowed to swell on ice for 15 min, after which NP-40 was added to 0.06% final concentration. The tube was vortexed vigorously for 15 s, and the disruption monitored by examining a drop of the suspension under the microscope. Nuclei were collected by centrifugation for 30 s at 4°C. Nuclei were resuspended in cold buffer B (15 mM Tris–HCl pH 7.4, 60 mM KCl, 15 mM NaCl, 0.2 mM EDTA, 0.2 mM EGTA and 5% glycerol, supplemented with 1 mM DTT, 0.15 mM spermine and 0.5 mM spermidine just before use) at a concentration of 5 x 107 nuclei per ml. DNaseI treatment was carried out in a total volume of 1 ml of buffer B containing 2.5 x 107 nuclei, 5 mM MgCl2 and DNaseI varying in amount from 0 to 400 U (DNase fade-out). The reactions were incubated on ice for 25 min, and stopped by adding 20 µl 0.5 M EDTA, 25 µl 20% SDS and 100 µl of a 10 mg/ml Proteinase K solution, after which the mixture was further incubated overnight at 37°C. After phenol/chloroform extraction the DNA was collected by isopropanol precipitation. The DNA was resuspended in TE buffer and digested with XbaI or EcoRI. Restriction fragments were resolved on a 0.8% agarose gel and blotted onto a nylon membrane (Hybond/Zetaprobe). The blots were hybridized with probes indicated in Figure 3.
Sequence analysis
Gene prediction studies and comparative sequence analysis were carried out using routine methods (BLAST, Dot-plot, BESTFIT). Human sequence was obtained by the Sanger Centre (43). The SIMO breakpoint had been identified previously (29) (but no sequence was available). Using the SIMO breakpoint clone EI (Figs 1 and 4), a BAC clone was identified from a mouse 129/Sv library (Genome Systems). A 7.5 kb EcoRI fragment was subcloned and partly sequenced (EMBL no. AJ276371). BESTFIT sequence analysis with the homologous human sequence is posted at (http://www.hgu.mrc.ac.uk/Research/Cellgen/). No significant ORF was identified within the available sequence.
Fugu sequencing over a large conserved synteny region around the Pax6 gene was carried out by G.Elgar’s group at MRC HGMP Resource Centre (26 and G.Elgar, S.Warner, D.Goode and P.Snell, manuscript in preparation).
Production of reporter constructs and generation and analysis of transgenic mice
The Hsp68–LacZ reporter construct p610Za (29) was modified by inserting a short linker containing SphI-NruI-NotI-SalI sites into the SphI and SalI sites of the multiple cloning site to obtain vector p610+. HS234Z constructs were made as follows: a 4.5 kb SpeI fragment, containing DNaseI HS 2, 3 and 4, was isolated from cosmid SRL11M20 and subcloned into the SpeI site of pBluescriptII (Stratagene) to obtain two clones with HS234Z in each orientation. Inserts from each plasmid were liberated as NotI/SalI fragments and cloned into vector p610+. Microinjection fragments were digested with NotI and Asp718I and isolated from gels using a Qiagen gel extraction kit.
Construct EI-Z was made as follows: a 4.8 kb EcoRI fragment containing the conserved breakpoint region EI, whose position is marked by probe EI (Fig. 4A) was subcloned into pBluescriptII (Stratagene) resulting in pF5.4. From this plasmid a 2.5 kb SphI/SalI fragment containing the conserved region was isolated and cloned into the SphI and SalI sites of p610+. The microinjection fragment was isolated following digestion with SphI and Asp718I as described above.
Microinjection of LacZ reporter constructs was performed according to standard procedures. Transgenic mice and embryos were identified by PCR and/or Southern blot analysis. Embryos were collected at the appropriate stages, washed in PBS and fixed for 1 h in a solution of 1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA and 0.02% NP-40 in PBS. After fixation the embryos were washed in PBS containing 0.02% NP-40, before being stained for several hours at 37°C in the dark in a solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6.3H2O, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40 and 0.1% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal).
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ACKNOWLEDGEMENTS |
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We thank Nick Hastie for continuing help and support; Muriel Lee for the FISH analysis; Greg Elgar for Fugu sequence data and discussions; Manfred Gessler for information on the PAXNEB gene; Penny Rashbass for RNA in situ techniques; and the patients and their families who participated in this study. We also thank clinicians Kalle Simola (Helsinki/Tampere), Yoshimitsu Fukushima (Saitama), Cynthia Tifft (Washington), Judith Goodship (Newcastle), Michel Vekemans (Paris), for samples from cases with translocation/inversion-associated aniridia. We are grateful to Brendan Doe and staff for expert technical help and to Sandy Bruce and Douglas Stewart for patient figure preparation. This work was supported in part by EU shared cost grant BMH4-CT96-1428 and initiated while V.v.H. was an HHMI International Research Scholar.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 131 3322471; Fax: +44 131 3322620; Email: v.vanheyningen@hgu.mrc.ac.uk Present address: Andreas Schedl, Max Delbruck Centrum fur Molekulare Medizin, Robert Rossle Strasse 10, 13122 Berlin Buch, Germany
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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