Human Molecular Genetics, 2000, Vol. 9, No. 15 2231-2239
© 2000 Oxford University Press
Identification of WTAP, a novel Wilms’ tumour 1-associating protein
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, UK
Received 13 April 2000; Revised and Accepted 17 July 2000.
DDBJ/EMBL/GenBank accession no. AJ276706.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The Wilms’ tumour suppressor gene WT1 is essential for the normal development of the genitourinary system. It appears to play a role in both transcriptional and post-transcriptional regulation of certain cellular genes. However, the mechanisms behind WT1 function are not clearly understood despite the identification of numerous potential target genes and the isolation of several WT1-binding proteins. This study therefore sets out to identify other WT1-associating proteins to help to unravel how WT1 interacts with the cellular machinery. We report the identification of a novel human WT1-associating protein, WTAP, which was isolated using the yeast two-hybrid system. Both in vitro and in vivo assays have shown that the interaction between WTAP and WT1 is specific and occurs endogenously in cells. The mouse homologue of WTAP was isolated and found to be >90% conserved at the nucleotide and protein levels. The human and mouse genes were mapped using fluorescence in situ hybridization to regions in chromosomes 6 (which is thought to harbour a tumour suppressor gene) and 17, respectively. The expression pattern of WTAP was investigated and shown to be ubiquitous, perhaps reflecting a housekeeping role. WTAP is a nuclear protein, which like WT1 localizes throughout the nucleoplasm as well as in speckles and partially co-localizes with splicing factors. Although the significance of this interaction is not yet known, WTAP promises to be an interesting WT1-binding partner.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The paediatric nephroblastoma Wilms’ tumour, affecting 1 in 10 000 children (1), is a disease in which the disruption of the normal events of kidney development leads to tumour formation (1). Mutations in the Wilms’ tumour suppressor gene, WT1, have been shown to be involved in the development of Wilms’ tumour as well as in Denys–Drash syndrome and Frasier syndrome, in which symptoms include genital malformation and nephropathy (2,3). WT1 is expressed in the developing genitourinary system and plays a critical role in the development of the kidneys and gonads as shown by knockout experiments (4).
The WT1 gene can encode >16 different isoforms (5) by means of alternative start sites (6,7), RNA editing (8) and alternative splicing (9). There are two alternative splice sites: the first includes/excludes 17 amino acids encoded by exon 5; and the second adds/removes the three amino acids lysine, threonine and serine (KTS) encoded by a complex splice junction between exons 9 and 10. The four major WT1 isoforms are designated as follows: (i) WT1+/+ if it includes both the 17 amino acids and the KTS; (ii) WT1+/– if it includes the 17 amino acids but not the KTS; (iii) WT1–/+ if it does not have the 17 amino acids but does have the KTS; and (iv) WT1–/– if it contains neither the 17 amino acids nor the KTS. The WT1 protein contains four zinc fingers of the Krüppel type at the C-terminus (10), of which the last three show close homology to those found in the EGR1 family of transcription factors. WT1 was shown to bind to DNA through its zinc finger domain (11), but the affinity and specificity are greatly reduced in the +KTS isoforms due to the disruption of the structure of the zinc fingers by the insertion of the three amino acids (12). WT1 has since been shown to be a transcription factor, as it can both activate and repress transcription from reporter constructs, depending on the promoter, cell line and expression vector used (13–15). The strongest candidate for a physiological target is the amphiregulin gene which is transcriptionally activated by WT1 (16).
The initial experiments carried out all pointed towards WT1 being a classical tumour suppressor gene whose function was to regulate the transcription of genes involved in proliferation. It has since been postulated that there may be a further function for WT1 as a post-transcriptional regulator. When the cellular localization of WT1 was examined, it was found that it is a nuclear protein which not only has a diffuse staining pattern, as is common for transcription factors, but is also present in speckles, which is more characteristic of splicing factors (17). Intriguingly it was shown by transfecting individual isoforms into cells that the –KTS isoforms generally display diffuse nucleoplasm staining, whereas the +KTS isoforms are found predominantly in the speckles (17). Then not only was WT1 shown to interact with the splicing factor U2AF65, and that this interaction was stronger for the +KTS isoform compared with the –KTS isoform, but also it was shown that it could become incorporated into spliceosomes (18). These experiments point towards WT1 playing a role in splicing. Further evidence to support the idea that WT1 may not function solely as a transcription factor but may have a role in post-transcriptional regulation came from the findings that WT1 may possess an RNA recognition motif as shown by structural modelling (19) and that it can bind to RNA through its zinc fingers (20,21). Recently WT1 was shown to be a constituent of complexes with the properties of ribonucleoprotein (22).
The genuine roles of WT1 are not yet clear, but it is known that for it to carry out its various functions, it must interact with other cellular proteins. Several have already been identified and p53, par-4, Ciao 1 and SF-1 have all been shown to regulate the transcriptional activity of WT1 (23–26). UBC9 and Hsp70 have also been shown to interact with WT1 and affect its role in cell cycle regulation (27,28). It is clear that WT1 must interact with yet more proteins of the cellular machinery to carry out its postulated roles, and thus this study set out to identify more WT1-interacting proteins in the hope that it will shed some light on the way in which WT1 acts at the molecular level.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Isolation of human WT1-associating protein (WTAP)
In order to search for WT1-interacting proteins, a yeast two-hybrid screen was carried out. A human fetal kidney cDNA library was screened using mouse WT1+/+ as the bait. One of the clones identified was shown to be specific for WT1 as the protein did not interact with negative controls used in the screen (Fig. 1B). The yeast two-hybrid system was also used to show that the clone could interact with all four isoforms of WT1 but more specifically with the –KTS isoforms. By using WT1 deletion constructs, (Fig. 1A), the domain of interaction within WT1 was shown to be at the C-terminus, and again the +KTS form of the C-terminus construct had a weaker interaction compared with the same construct which lacked the KTS (Fig. 1B). The interaction between the clone and the C-terminus is stronger compared with the full-length WT1 equivalents, possibly due to these deletion constructs not having the repression domain, which may repress Gal4 activation domain activity.
|
The isolated clone was sequenced and run through the GenBank database. The clone was found to have no homology to any known proteins and, being novel, was designated WT1-associating protein (WTAP; EMBL accession no. AJ276706). The WTAP clone isolated does not contain the full coding region as there is no initiation codon.
Isolation of mouse Wtap
The mouse homologue of WTAP was found by screening an 11-day-old mouse embryo cDNA library using the human WTAP clone as a probe. The isolated mouse clone (EMBL accession no. AJ276707) was shown to be 94% identical to WTAP at the nucleotide level and 96% identical at the amino acid level (Fig. 2). This high level of sequence conservation suggests that the entire structure of WTAP is likely to be important for function. The mouse clone isolated was not full-length as it did not have a start codon, but it did contain a termination codon, five amino acids after the end of the human WTAP clone (Fig. 2).
|
Mapping studies
The chromosomal location of both mouse and human WTAP was determined. Fluorescence in situ hybridization (FISH) analysis mapped the human WTAP gene to chromosome 6q25–27 (Fig. 3A). Interestingly this region of human chromosome 6 has been associated with several malignancies and therefore it is thought to contain a tumour suppressor gene (29). The mouse Wtap gene was also mapped by FISH to chromosome 17, near the centromere (Fig. 3B). The gene was then mapped to a higher resolution using the European collaborative interspecific mouse back-cross [EUCIB (30)]. Wtap was shown to be linked to marker D17Mit25,
4 cM away from the centromere (data not shown). This region of the mouse chromosome 17 has conserved synteny with the region of human chromosome 6 to which WTAP mapped.
|
In vitro and in vivo interactions between WTAP and WT1
The yeast two-hybrid screen identified WTAP as potentially binding to WT1, and thus further binding assays had to be performed to ensure that the interaction was specific. Glutathione S-transferase (GST) binding assays were carried out, using bacterially purified GST–WTAP and in vitro translated 35S-labelled WT1. As shown in Figure 4A, all four WT1 isoforms bind to WTAP above background binding to GST alone. Although there may be preferential binding of the –KTS isoforms, it is not as pronounced as in the yeast two-hybrid system.
|
To enable further characterization of WTAP an anti-WTAP antibody was required. For this purpose bacterially expressed GST–WTAP was raised in rabbits, and its specificity for WTAP confirmed by western blotting (Fig. 4B). It is of interest to note that the endogenous WTAP in the M15 cell line runs just below the transfected haemagglutinin (HA)-tagged WTAP, suggesting that the WTAP clone isolated from the yeast two-hybrid screen is likely to be very close to full length.
To ensure that the interaction between WTAP and WT1 occurs under endogenous conditions, without either protein being overexpressed, co-immunoprecipitation experiments were carried out using M15 nuclear lysates, since this mesonephric cell line is known to express WT1 (18) and also, as shown by RT–PCR experiments, endogenous WTAP (data not shown). The immunoprecipitations were carried out and after subsequent western blot analysis with the anti-WTAP antibody, WTAP could be detected not only in the immunoprecipitation carried out with the anti-WTAP antibody as expected, but could also be detected in the anti-WT1 immunoprecipitation, thus indicating that WTAP can bind to WT1 in vivo (Fig. 4C). The control precipitation performed with mouse IgG, on the other hand, showed no WTAP co-immunoprecipitation demonstrating that the interaction between WTAP and WT1 is specific.
Expression pattern of WTAP
Since the interaction between WTAP and WT1 was shown to occur in vivo, more information about WTAP had to be obtained to understand the significance behind the interaction of the two proteins. Thus, to investigate whether WTAP has a tissue-specific expression pattern which may follow that of WT1, RT–PCR experiments were carried out on adult mouse tissues. Figure 5 shows that WTAP has a ubiquitous expression pattern as it is expressed in all tissues tested. This is unlike WT1 which in the adult is only expressed at low levels in the spleen, heart, gonad and kidney (31,32). The RT–PCR results also show that perhaps WTAP may have alternative splicing forms, since additional products were found for the thymus, lung and heart. Further tests would have to be carried out to confirm whether WTAP does have alternative transcripts, and to see how they differ from the full length.
|
The RT–PCR experiment showed that WTAP is expressed in the adult kidney. WT1 is also expressed in the adult kidney but at low levels, whereas it is more highly expressed during the development of the kidney and it is known to play a crucial role in kidney and gonadal development (4). It was thus important to establish whether WTAP is also expressed in the developing kidneys and testes, and if so whether its expression pattern is confined to the sites where WT1 is expressed (31,33). Thus immunohistochemistry was performed on cryosections of kidneys and testes isolated at embryonic day (E) 17.5. The sections were stained for WT1 and for WTAP, using the H2 anti-WT1 antibody and the anti-WTAP antibody, respectively. Figure 6 shows that the expression pattern seen for WT1 is as expected, with the expression being confined to the developing nephrons of the kidney and the Sertoli cells of the gonad (reviewed in ref. 34). WTAP on the other hand is seen to be expressed in all cells of the kidney and testis, again showing its ubiquitous expression. It is possible that within the testes there is elevated expression of WTAP in the Sertoli cells. However, this may be due to there being a greater cell density in these structures leading to an apparently more intense signal. A more detailed study would be required to address this point, but it is clear that WTAP is found in structures that express WT1 but is not confined to them.
|
Cellular localization of WTAP
The cellular distribution of WTAP was looked at to investigate whether it is found in the same compartment as WT1. Immunofluorescence carried out on M15 cells, using anti-WT1 and anti-WTAP antibodies, showed that WTAP is a nuclear protein and thus is found in the same cellular compartment as WT1 which allows for a physical association (Fig. 7A). Both WT1 and WTAP seem to be present both diffusely throughout the nucleoplasm, excluding the nucleolus, and in speckles (Fig. 7B). This specific nuclear distribution of WT1 has been reported before (17) and thus it is of interest to find that WTAP matches this localization.
|
WTAP partially co-localizes with splicing factors
The speckled structures in which WT1 is present, correspond to sites which co-localize with splicing factors (17). Thus, to determine whether WTAP also co-localizes with splicing factors, immunofluorescence was repeated, this time using the antibody 3C5. 3C5 detects proteins in interchromatin granule clusters (IGCs), the structures in which splicing factors are stored (35,36). Figure 7C shows the staining pattern produced with the 3C5 antibody. The pattern is predominantly speckled, with some diffuse staining. The immunofluorescence obtained for WTAP shows that there is more diffuse staining compared with 3C5 but, nevertheless, the speckle structures obtained for WTAP do co-localize with those of 3C5, as seen in the merged output (Fig. 7C).
The IGCs are thought to be sites where splicing factors are stored and/or assembled until they are required for processing pre-mRNAs. The speckles are therefore very dynamic, as splicing factors shuttle between the speckles and the site of active transcription (37,38). When transcription is inhibited, the splicing factors are not required and so remain in the IGCs, causing these structures to become larger and more uniform in shape. However, not all splicing factors return to the IGC when transcription is inhibited. For example, U170K and U2AF65 are found in small foci around the remnants of the nucleolus after the inhibition of transcription (39). The same is true for WT1, as when M15 cells are treated with the transcription inhibitor actinomycin D, WT1 localizes to small foci and not with the majority of other snRNPs (17). The nuclear localization of WTAP was therefore looked at after M15 cells had been treated with actinomycin D, to see whether the treatment affected the nuclear distribution of WTAP. When M15 cells are treated with actinomycin D, WTAP re-distributes into large foci which overlap with the large speckles of 3C5 (Fig. 7D). The staining seen for WTAP, however, is not identical to that seen for 3C5. Even after actinomycin D treatment, WTAP continues to have a substantial amount of nucleoplasm staining, whereas for 3C5 there is hardly any nucleoplasm staining at all, the splicing factors becoming localized almost exclusively to the speckles. These results therefore suggest that WTAP does partially co-localize with splicing factors and that its nuclear distribution is altered by the transcription inhibitor actinomycin D.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
These studies have identified a novel protein, WTAP, which associates with the tumour suppressor protein WT1. WTAP was isolated through a yeast two-hybrid screen and when sequenced was found to be a novel protein with no known motifs. Although the sequence of WTAP provided no immediate clues to its function or how it might affect that of WT1, it is still of interest as its interaction with WT1 was shown to be specific. WTAP not only binds to WT1 in the yeast two-hybrid system, but also in vitro as shown by the GST pull-down experiments and in vivo as shown by co-immunoprecipitation from cells endogenously expressing both proteins.
The mouse homologue of WTAP was found to be 96% identical to human WTAP at the protein level. The WTAP gene was mapped by FISH to human chromosome 6q25–27 and to the conserved syntenic region of mouse chromosome 17 near the centromere. Human chromosome 6q25–27 has been associated with several malignancies, including ovarian cancer, renal cell carcinoma and leukaemia (40–42) and it is thought that a tumour suppressor gene may lie within this region (29). It would be of interest to investigate whether WTAP is indeed associated with these diseases. The mapping of the mouse gene was further refined by using the EUCIB resource to 4 cM away from the centromere, a region which may be part of the T-locus. The T-locus in the mouse is a region of chromosome 17 identified by sets of dominant and recessive mutations, some of which have profound effects on embryonal development, sperm production and function and genetic recombination in a large region of their chromosome (43). So far no T-alleles have been found to map as close to the centromere of chromosome 17 as Wtap does and thus it is not clear whether Wtap is part of the T-locus. However, it would be very interesting if Wtap is involved in embryonal development or sperm production since it interacts with WT1 and it is known that WT1 is essential for the development of the genitourinary system (4) and is thought to play a role in sperm production (44). The immunohistochemistry carried out on testes showed that perhaps Wtap had a higher expression level in the Sertoli cells (Fig. 6), which could be indicative of a role in sperm production. Wtap knockout experiments would have to be carried out to determine whether Wtap is essential for development.
The expression of WTAP was examined and compared with the expression pattern previously documented for WT1. RT–PCR experiments showed that WTAP is ubiquitously expressed, unlike the tissue-specific expression pattern of WT1 (31,32). These experiments also showed that in some tissues there may be alternative isoforms of WTAP; however, further experiments would have to be performed to confirm these findings. The immunohistochemistry carried out on kidneys and testes showed that in these organs, where both WTAP and WT1 are expressed, the expression pattern of WTAP is not identical to that of WT1. Whereas WT1 is confined to certain structures within these organs, WTAP is expressed throughout with perhaps an elevated expression seen in the Sertoli cells, although this could be an effect of cell density. Thus, WTAP expression is not restricted to sites where WT1 is located. Due to its ubiquitous expression, WTAP may have a housekeeping role, and in cells which also express WT1, the function of WTAP may be altered due to its interaction with WT1, and/or WTAP may in turn affect the function of WT1.
Although the major fragment detected by RT–PCR in the majority of tissues corresponded to what was predicted from the cDNA sequence, several smaller, less abundant fragments were detected in a few tissues. It is likely that these represent alternatively spliced transcripts. As yet we have not characterized these alternative transcripts nor detected their potential protein products. Only a single full-length WTAP protein was detected in the M15 kidney cell line which seemed to correlate with the RT–PCR pattern. Further studies will be required to characterize any alternative products and their interaction with WT1.
The cellular localization of WTAP was also studied to investigate whether WTAP and WT1 are in the same cellular compartment. Immunofluorescence studies carried out on M15 cells showed that, like WT1, WTAP is a nuclear protein. More specifically WTAP was distributed throughout the nucleoplasm, excluding the nucleolus, as well as being present in speckled domains. This is very similar to the nuclear localization of WT1 originally described by Larsson et al. (17). It is thought that the –KTS isoforms of WT1 mainly produce the diffuse staining, while the +KTS isoforms are localized in the speckles. Thus, the in vitro data showing that WTAP can interact with all four WT1 isoforms may have some in vivo relevance, since in the nucleoplasm WTAP might interact with predominantly –KTS isoforms, whereas in the speckles it might interact with predominantly +KTS isoforms. The RT–PCR results indicated that there may be alternative transcripts of WTAP and perhaps, like the WT1 isoforms, they localize to different compartments of the nucleus, thus carrying out different roles.
The yeast two-hybrid data suggested that WTAP may bind with greater affinity to the –KTS isoforms of WT1, and this may correlate with the fact that a lot of WTAP is found to be diffuse in the nucleoplasm just like these WT1 isoforms. The –KTS isoforms are thought to play a role in transcription and thus, by interacting with WT1, WTAP may alter its transcriptional activity. However, preliminary results showed that WTAP had no effect on either the transcriptional activation or repression by WT1 as measured using reporter constructs (data not shown). Preliminary studies also found no obvious effect of WTAP on growth suppression by WT1 (data not shown).
The +KTS isoforms of WT1 are mainly located in the speckles, where a proportion of WTAP is also found. The immunofluorescence studies showed that the speckled structures in which WTAP was found correspond to the sites where splicing factors are stored. Furthermore, the distribution of WTAP within the nucleus was affected when cells were treated with the transcription inhibitor actinomycin D. The treatment caused WTAP to remain in the speckles thus enlarging them, which is also what happens to the majority of splicing factors. WT1 is thought to have a role in post-transcriptional regulation due to its having an RNA recognition motif (19); binding RNA in vitro (20–22); being present in speckles (17); directly interacting with the splicing factor U2AF65 (18); and being part of RNP complexes (22). It is therefore of great interest to find that this novel protein, WTAP, which interacts with WT1, is also associated with these speckles. However, care must be taken when interpreting these results as it must be noted that apart from the components of the splicing machinery other proteins, which have no known direct function in splicing, are found in the speckles. For example, the C-terminal domain from hyper-phosphorylated RNA polymerase II has been shown to be associated with splicing factors in the speckles but has not been shown to have a direct role in splicing (45). It is also worth noting that in the study carried out to characterize proteins components of the spliceosome, WTAP was not identified (46). However, it is unlikely that all splicing factors would have been purified and characterized in this assay and thus it does not rule out the possibility of WTAP being a potential splicing factor. Thus, if WTAP is eventually shown to participate in splicing then it may help in turn to clarify the role of WT1 in splicing.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Plasmids
The WT1 and deletion constructs used as baits in the yeast two-hybrid system were as described previously (18). PGEX-WTAP was made by digesting WTAP from pGAD10 (Clontech) as an EcoRI fragment and cloned into pGEX (Pharmacia Biotech). HA–WTAP was made by cloning WTAP, with added XbaI–SalI sites, into pCI-neo vector (Promega) which had had three N-terminal HA motifs engineered into it (a gift from Dr Ruggiu).
Yeast two-hybrid analysis
The yeast strain Y190 [MATa, gal4, gal80, his3, trp1–901, ade2–101, ura3–52, leu2–3, –112 URA3::GAL–lacZ, LYS2::GAL(UAS)–HIS3, cyh.r] was used as a host for the interaction experiments. A human fetal kidney library (Clontech) was screened with the bait pAS2-WT1+/+. DNA was introduced into yeast using lithium acetate trans-formation, and interactions tested by growth on leu–trp–his– medium supplemented with 50 mM 3-aminotriazole (Sigma). To assess ß-galactosidase activity filter lifts (from leu–trp– plates) and liquid assays (in leu–trp– medium) were performed as described (47). ß-galactosidase activity was calculated as 1000 x A420/(volume x time x protein concentration). At least three independent colonies from two transformations were tested.
Isolating the mouse Wtap
Approximately 1 x 106 clones from an 11-day-old mouse embryo 5' stretch plus cDNA library (Clontech) were screened using a random prime 32P-labelled WTAP probe (Boehringer Mannheim). Hybridization of the filters was carried out at 65°C in 4x SSC, 0.4% SDS, 0.2% NaPPi, 100 µg/ml salmon sperm DNA and 2x Denhart’s. Washes were carried out at 65°C in 2x SSC, 0.4% SDS and 0.2% NaPPi. Positive clones were subjected to secondary and tertiary screening. The vectors containing the Wtap inserts were then recovered by using a Qiagen lambda prep kit, and finally the inserts were subcloned into Bluescript vector (Stratagene) and sequenced.
FISH
FISH was performed as described by Fantes et al. (48), using a WTAP cDNA probe.
RT–PCR
The organs required for RNA extraction were dissected from an adult mouse and stored on ice. Three millilitres of 3 M LiCl/6 M urea was added to the tissues, which were immediately homogenized. The samples were sonicated for 1 min and the RNA precipitated overnight at 4°C. The RNA was pelleted by spinning at 12 000 r.p.m. at 4°C for 20 min and all traces of LiCl/urea were removed. The pellet was re-suspended in 0.3 ml of 10 mM Tris/0.5% SDS, and sequential phenol–chloroform extractions were carried out. The RNA was precipitated and re-suspended in 50–100 µl DEPC-H2O. RNA (5 µg) in a total of 5 µl of water was denatured by heating to 90°C. After cooling on ice the following were added: 10 µl of 5x RT-buffer, 5 µl of 10 mM dNTPs, 1 µl of RNase inhibitor, 1 µl of random hexamers, 27 µl of DEPC-H20 and 1 µl of reverse transcriptase. This was incubated at 37°C for 1 h after which time a PCR reaction was carried out using primers 5'-CTGAGTGGCTGGAAGTTTAC-3' and 5'-ACTTCTGAACGTGGCGGGAACCCACAGTTC-3'.
Transient transfections
The HA–WTAP was transfected into M15 cells, which were seeded at a confluency of 50–80%, using LipofectAmine (Gibco BRL) according to the manufacturer’s instructions. The cells were incubated for 24 h at 37°C after which time the transfection medium was replaced with complete medium and cells were incubated for a further 24 h before being lysed.
Antibodies
For immunoprecipitation or western blotting the following antibodies were used: 12CA5 m
HA tag (Boehringer Mannheim); H2 m WT1 (Dako, Copenhagen, Denmark); 474 r WTAP [this study: produced at the Scottish Antibody Production Unit (SAPU), raised against GST–WTAP]; normal mouse IgG (Sigma). For immunofluorescence studies splicing proteins were detected using 3C5 (36) followed by fluorescein isothiocyanate (FITC) anti-mouse IgM; WT1 was detected by using H2 m WT1 (Dako) followed by Texas Red anti-mouse IgG; and WTAP was detected by 474 r WTAP (this study) followed by either Texas Red or anti-rabbit IgG–FITC.
In vitro and in vivo binding assays
In vitro and in vivo binding assays were performed as detailed by Davies et al. (18), using medium salt association buffer (250 mM NaCl,100 mM Tris pH 8, 0.1% NP40).
Immunohistochemistry
Immunohistochemistry was carried out on E17.5 mouse kidneys and testes. The organs were dissected and fixed immediately in 4% paraformaldehyde for 1–1.5 h. They were washed twice for 20 min in phosphate-buffered saline (PBS), and placed overnight in a 30% sucrose solution made in PBS. The organs were embedded in OCT, frozen in liquid nitrogen and stored at –70°C. Sections, 10 µm thick, were cut onto TESPA slides using the JUNG CM3000 cryostat (Leica). The slides were left to dry at room temperature for 1 h before immunohistochemistry was performed.
The sections were blocked for 1 h in blocking solution (2% bovine serum albumin, 0.2% Tween 20, 6.7% glycerol, 0.02% sodium azide, in PBS). If the primary antibody to be used was monoclonal, then the sections were incubated with goat IgG Fab arms (1:25 dilution) for 1 h to remove any background. The sections were washed three times for 5 min each in Tris-buffered saline with Tween 20 (TBST) followed by the primary antibody, diluted according to a total volume of 150 µl in block. After 1 h incubation, three 5 min washes with TBST were carried out. The secondary antibody, coupled to either FITC or Texas Red, was added for 45 min and the slides kept in the dark. The slides were washed three times for 5 min each in TBST before being mounted with Vectashield (Vector Laboratories) containing 4',6 diamidino-2-phenylindole (DAPI; Vectra) to stain the nucleus. The fluorescence was then examined under an Axioplan fluorescent microscope. Images were captured using Digital Scientific Smartcapture software.
Immunofluorescence
M15 cells were grown on chamber slides (Nunc) until they had reached 80% confluency, after which time the cells were rinsed twice in PBS and fixed for 10 min at room temperature in an acetone:methanol (1:1) mix. The immunofluorescence was carried out as described for the immunohistochemistry.
|
ACKNOWLEDGEMENTS |
|---|
We thank M. Lee for carrying out the FISH studies; I. Jackson for providing the EUCIB DNA; and V. Sharnhorst and A.G. Jochemsen for providing the WT1 reporter constructs.
|
FOOTNOTES |
|---|
+ Present address: Sylvius Laboratory, Wassenaarseweg 72, 2333AL Leiden, The Netherlands
§ To whom correspondence should be addressed. Tel: +44 131 3322471; Fas: +44 131 3432620; Email: n.hastie@hgu.mrc.ac.uk
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Pritchard Jones, K. and Hastie, N.D. (1990) Wilms’ tumour as a paradigm for the relationship of cancer to development. Cancer Surv., 9, 555–578.
2 Barbaux, S., Niaudet, P., Gubler, M.-C., Grunfeld, J.-P., Jaubert, F., Kuttenn, F., Fekete, C.N., Souleyreau-Therville, N., Thibaud, E., Fellous, M. et al. (1997) Donor splice-site mutations in WT1 are responsible for Frasier syndrome. Nature Genet., 17, 467–470.[Web of Science][Medline]
3 Little, M. and Wells, C. (1997) A clinical overview of WT1 gene mutations. Hum. Mutat., 9, 209–225.
4 Kreidberg, J.A., Sariola, H., Loring, J.M., Maeda, M., Pelletier, J., Housman, D. and Jaenisch, R. (1993) WT-1 is required for early kidney development. Cell, 74, 679–691.[Web of Science][Medline]
5 Schedl, A. and Hastie, N. (1998) Multiple roles for the Wilms’ tumour suppressor gene, WT1 in genitourinary development. Mol. Cell Endocrinol., 140, 65–69.
6 Bruening, W. and Pelletier, J. (1996) A non-AUG translational initiation event generates novel WT1 isoforms. J. Biol. Chem., 271, 8646–8654.
7 Scharnhorst, V., Dekker, P., van der Eb, A.J. and Jochemsen, A.G. (1999) Internal translation initiation generates novel WT1 protein isoforms with distinct biological properties. J. Biol. Chem., 274, 23456–23462.
8 Sharma, P.M., Bowman, M., Madden, S.L., Rauscher, F.J. and Sukumar, S.L. (1994) RNA editing in the Wilms’ tumor susceptibility gene, WT1. Genes Dev., 8, 720–731.
9 Haber, D.A., Sohn, R.L., Buckler, A.J., Pelletier, J., Call, K.M. and Housman, D.E., (1991) Alternative splicing and genomic structure of the Wilms tumor gene WT1. Proc. Natl Acad. Sci. USA, 88, 9618–9622.
10 Call, K.M., Glaser, T., Ito, C.Y., Buckler, A.J., Pelletier, J., Haber, D.A., Rose, E.A., Kral, A., Yeger, H., Lewis, W.H. et al. (1990) Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms’ tumor locus. Cell, 60, 509–520.[Web of Science][Medline]
11 Rauscher, F.J., Morris, J.F., Tournay, O.E., Cook, D.M. and Curran, T. (1990) Binding of the Wilms’ tumor locus zinc finger protein to the EGR-1 consensus sequence. Science, 250, 1259–1262.
12 Bickmore, W.A., Oghene, K., Little, M.H., Seawright, A., van Heyningen, V. and Hastie, N.D. (1992) Modulation of DNA binding specificity by alternative splicing of the Wilms’ tumour WT1 gene transcript. Science, 257, 235–237.
13 Wang, Z.Y., Qiu, Q.Q. and Deuel, T.F. (1993) The Wilms’ tumor gene product WT1 activates or suppresses transcription through separate functional domains. J. Biol. Chem., 268, 9172–9175.
14 Moshier, J.A., Skunca, M., Wu, W., Boppana, S.M., Rauscher III, F.J. and Dosescu, J. (1996) Regulation of ornithine decarboxylase gene expression by the Wilms’ tumor suppressor WT1. Nucleic Acids Res., 24, 1149–1157.
15 Reddy, J.C., Hosono, S. and Licht, J.D. (1995) The transcriptional effect of Wt1 is modulated by choice of expression vector. J. Biol. Chem., 270, 29976–29982.
16 Lee, S.B., Huang, K., Palmer, R., Truong, V.B., Herzlinger, D., Kolquist, K.A., Wong, J., Paulding, C., Yoon, S.K., Gerald, W. et al. (1999) The Wilms tumor suppressor WT1 encodes a transcriptional activator of amphiregulin. Cell, 3, 663–73.
17 Larsson, S.H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V. and Hastie, N.D. (1995) Subnuclear localization of WT1 in splicing or transcription domains is regulated by alternative splicing. Cell, 81, 391–401.[Web of Science][Medline]
18 Davies, R.C., Calvio, C., Bratt, E., Larsson, S.H., Lamond, A.I. and Hastie, N.D. (1998) WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes. Genes Dev., 12, 3217–3225.
19 Kennedy, D., Ramsdale, T., Mattick, J. and Little, M. (1996) An RNA recognition motif in Wilms’ tumour protein (WT1) revealed by structural modelling. Nature Genet., 12, 329–332.[Web of Science][Medline]
20 Caricasole, A., Duarte, A., Larsson, S.H., Hastie, N.D., Little, M., Holmes, G., Todorov, I. and Ward, A. (1996) RNA binding by the Wilms Tumour suppressor zinc finger proteins. Proc. Natl Acad. Sci. USA, 93, 7562–7566.
21 Bardeesy, N. and Pelletier, J. (1998) Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product. Nucleic Acids Res., 26, 1784–1792.
22 Ladomery, M.R., Slight, J., McGhee, S. and Hastie, N.D. (1999) Presence of WT1, the Wilms’ tumour suppressor gene product, in nuclear poly(A)+ RNP. J. Biol. Chem., 274, 36520–36526.
23 Maheswaran, S., Park, S., Bernard, A., Morris, J.F., Rauscher III, F.J., Hill, D.E. and Haber, D.A., (1993) Physical and functional interaction between WT1 and p53 proteins. Proc. Natl Acad. Sci. USA, 90, 5100–5104.
24 Johnstone, R.W., See, R.H., Sells, S.F., Wang, J., Muthukkumar, S., Englert, C., Haber, D.A., Licht, J.D., Sugrue, S.P., Roberts, T. et al. (1996) A novel repressor, par-4, modulates transcription and growth suppression functions of the Wilms’ tumor suppressor WT1. Mol. Cell. Biol., 16, 6945–6956.[Abstract]
25 Johnstone, R.W., Wang, J., Tommerup, N., Vissing, H., Roberts, T. and Shi, Y. (1998) Ciao 1 is a novel WD40 protein that interacts with the tumour suppressor protein WT1. J. Biol. Chem., 273, 10880–10887.
26 Nachtigal, M.W., Hirokawa, Y., Enyeart-VanHouten, D.L., Flanagan, J.N., Hammer, G.D. and Ingraham, H.A. (1998) Wilms’ tumor 1 and Dax-1 modulate the orphan nuclear receptor SF-1 in sex-specific gene expression. Cell, 93, 445–454.[Web of Science][Medline]
27 Wang, Z.-Y., Qiu, Q.-Q., Seufert, W., Taguche, T., Testa, J.R., Whitmore, S.A., Callen, D.F., Welsh, D., Shenk, T. and Deuel, T.F. (1996) Molecular cloning of the cDNA and chromosome localization of the gene for human ubiquitin-conjugating enzyme 9. J. Biol. Chem., 271, 24811–24816.
28 Maheswaran, S., Englert, C., Zheng, G., Lee, S.B., Wong, J., Harkin, D.P., Bean, J., Ezzell, R., Garvin, A.J., McCluskey, R.T. et al. (1998) Inhibition of cellular proliferation by the Wilms tumour suppressor WT1 requires association with the inducible chaperone Hsp70. Genes Dev., 12, 1108–1120.
29 Theile, M., Seitz, S., Arnold, W., Jandrig, B., Frege, R., Schlag, P.M., Haensch, W., Guski, H., Winzer, K.-J., Barrett, J.C. et al. (1996) A defined chromosome 6q fragment (at D6S310) harbors a putative tumor suppressor gene for breast cancer. Oncogene., 13, 677–685.[Web of Science][Medline]
30 The European Backcross Collaborative Group (1994) Towards high resolution maps of the mouse and human genome—a facility for ordering markers to 0.1 cM resolution. Hum. Mol. Genet., 3, 621–627.
31 Armstrong, J.F., Pritchard Jones, K., Bickmore, W.A., Hastie, N.D. and Bard, J.B. (1993) The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo. Mech. Dev., 40, 85–97.[Web of Science][Medline]
32 Buckler, A.J., Pelletier, J., Haber, D.A., Glaser, T. and Housman, D.E. (1991) Isolation, characterization, and expression of the murine Wilms’ tumor gene (WT1) during kidney development. Mol. Cell. Biol., 11, 1707–1712.
33 Pritchard Jones, K., Fleming, S., Davidson, D., Bickmore, W., Porteous, D., Gosden, C., Bard, J., Buckler, A., Pelletier, J., Housman, D. et al. (1990) The candidate Wilms’ tumour gene is involved in genitourinary development. Nature, 346, 194–197.[Medline]
34 Hastie, N.D. (1994) The genetics of Wilms’ tumour—a case of disrupted development. Annu. Rev. Genet., 28, 523–558.[Web of Science][Medline]
35 Misteli, T. and Spector, D.L. (1998) The cellular organization of gene expression. Curr. Opin. Cell Biol., 10, 323–331.[Web of Science][Medline]
36 Turner, B.M. and Franchi, L. (1987) Identification of protein antigens associated with the nuclear matrix and with clusters of interchromatin granules in both interphase and mitotic cells. J. Cell Sci., 87, 269–282.
37 Spector, D.L. (1996) Nuclear organization and gene expression. Exp. Cell Res., 229, 189–197.[Web of Science][Medline]
38 Misteli, T., Caceres, J.F. and Spector, D.L. (1997) The dynamics of a pre-mRNA splicing factor in living cells. Nature, 387, 523–527.[Medline]
39 Carmo-Fonseca, M., Pepperkok, R., Carvalho, M.T. and Lamond, A.I. (1992) Transcription-dependent colocalization of the U1, U2, U4/U6, and U5 Snrnps in coiled bodies. J. Cell Biol., 117, 1–14.
40 Cooke, I.E., Shelling, A.N., Le Meuth, V.G., Charnock, M.L. and Ganesan, T.S. (1996) Allele loss on chromosome arm 6q and fine mapping of the region at 6q27 in epithelial ovarian cancer. Genes Chromosomes Cancer, 15, 223–233.[Web of Science][Medline]
41 Morita, R., Saito, S., Ishikawa, J., Ogawa, O., Yoshida, O., Yamakawa, K. and Nakamura, Y. (1991) Common regions of deletion on chromosomes 5q, 6q, and 10q in renal cell carcinoma. Cancer Res., 51, 5817–5820.
42 Menasce, L.P., Orphanos, V., Santibanez-Koref, M., Boyle, J.M. and Harrison, C.J. (1994) Common region of deletion on the long arm of chromosome 6 in non-Hodgkin’s lymphoma and acute lymphoblastic leukaemia. Genes Chromosomes Cancer, 10, 286–288.
43 Bennett, D. (1975) The T-locus of the mouse. Cell, 6, 441–454.
44 Rio-Tsonis, K.D., Covarrubias, L., Kent, J., Hastie, N. and Tsonis, P.A. (1996) Regulation of the Wilms’ tumor gene during spermatogenesis. Dev. Dyn., 207, 372–381.[Web of Science][Medline]
45 Kim, E., Bregman, D.B. and Warren, S.L. (1997) Splicing factors associate with hyperphosphorylated RNA polymerase II in the absence of pre-mRNA. J. Cell Biol., 136, 19–28.
46 Neubauer, G., King, A., Rappsilber, J., Calvio, C., Watson, M., Ajuh, P., Sleeman, J., Lamond, A. and Mann, M. (1998) Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex. Nature Genet., 20, 46–50.[Web of Science][Medline]
47 Harshman, K.D., Moye-Rowley, W.S. and Parker, C.S. (1988) Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor that is similar to AP-1 that is distinct from GCN4. Cell, 53, 321–330.[Web of Science][Medline]
48 Fantes, J.A., Oghene, K., Boyle, S., Danes, S., Fletcher, J.M., Bruford, E.A., Williamson, K., Seawright, A., Schedl, A., Hanson, I. et al. (1995) A high-resolution integrated physical, cytogenetic, and genetic map of human chromosome 11: distal p13 to proximal p15.1. Genomics., 25, 447–461.[Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. W. Small, Z. Bolender, C. Bueno, C. O'Neil, Z. Nong, W. Rushlow, N. Rajakumar, C. Kandel, J. Strong, J. Madrenas, et al. Wilms' Tumor 1-Associating Protein Regulates the Proliferation of Vascular Smooth Muscle Cells Circ. Res., December 8, 2006; 99(12): 1338 - 1346. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Horiuchi, M. Umetani, T. Minami, H. Okayama, S. Takada, M. Yamamoto, H. Aburatani, P. C. Reid, D. E. Housman, T. Hamakubo, et al. Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA PNAS, November 14, 2006; 103(46): 17278 - 17283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Huang, Y. Guo, Y. Shui, S. Gao, H. Yu, H. Cheng, and R. Zhou Multiple Alternative Splicing and Differential Expression of dmrt1 During Gonad Transformation of the Rice Field Eel Biol Reprod, November 1, 2005; 73(5): 1017 - 1024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. B. Srichai, M. Konieczkowski, A. Padiyar, D. J. Konieczkowski, A. Mukherjee, P. S. Hayden, S. Kamat, M. A. El-Meanawy, S. Khan, P. Mundel, et al. A WT1 Co-regulator Controls Podocyte Phenotype by Shuttling between Adhesion Structures and Nucleus J. Biol. Chem., April 2, 2004; 279(14): 14398 - 14408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Vespa, G. Vachon, F. Berger, D. Perazza, J.-D. Faure, and M. Herzog The Immunophilin-Interacting Protein AtFIP37 from Arabidopsis Is Essential for Plant Development and Is Involved in Trichome Endoreduplication Plant Physiology, April 1, 2004; 134(4): 1283 - 1292. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Discenza and J. Pelletier Insights into the physiological role of WT1 from studies of genetically modified mice Physiol Genomics, February 13, 2004; 16(3): 287 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. O. F. Penalva and L. Sanchez RNA Binding Protein Sex-Lethal (Sxl) and Control of Drosophila Sex Determination and Dosage Compensation Microbiol. Mol. Biol. Rev., September 1, 2003; 67(3): 343 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ladomery, J. Sommerville, S. Woolner, J. Slight, and N. Hastie Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one J. Cell Sci., April 15, 2003; 116(8): 1539 - 1549. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lalli, K. Ohe, E. Latorre, M. E. Bianchi, and P. Sassone-Corsi Sexy splicing: regulatory interplays governing sex determination from Drosophila to mammals J. Cell Sci., February 1, 2003; 116(3): 441 - 445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ortega, M. Niksic, A. Bachi, M. Wilm, L. Sanchez, N. Hastie, and J. Valcarcel Biochemical Function of Female-Lethal (2)D/Wilms' Tumor Suppressor-1-associated Proteins in Alternative Pre-mRNA Splicing J. Biol. Chem., January 24, 2003; 278(5): 3040 - 3047. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||