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Kathryn J. Hill,1, Marika Charalambous,2 Kate J. Wagner,1 Diya Lahiri,2 Dominic I. James,1 Jens S. Andersen,3 Valérie Schumacher,4 Brigitte Royer-Pokora,4 Matthias Mann,3 Andrew Ward,2 and Stefan G. E. Roberts1*
School of Biological Sciences, University of Manchester, Manchester M13 9PT,1 Developmental Biology Program and Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom,2 Protein Interaction Laboratory, University of Southern Denmark-Odense, DK-5230 Odense, Denmark,3 Institute of Human Genetics and Anthropology, University of Duesseldorf, D40001 Düsseldorf, Germany4
Received 25 July 2003/ Returned for modification 18 September 2003/ Accepted 20 October 2003
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ABSTRACT |
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INTRODUCTION |
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WT1 knockout mice (homozygous null) do not survive gestation, displaying absence or incorrect development of the kidney, gonads, spleen, heart, diaphragm, and retinal ganglia (12, 14, 35). These findings confirm a major role for WT1 in the formation of the genitourinary system and also a wider role in the development of other tissues.
Alternative splicing, RNA editing, and an alternative translation start codon combine to produce a plethora of WT1 isoforms (reviewed in reference 33). One alternative splice inserts three amino acids (KTS) between zinc fingers three and four, resulting in a form of WT1 that associates with RNA processing factors and localizes to regions of RNA processing in the nucleus (17). Thus, the +KTS and -KTS isoforms of WT1 have been proposed to function in RNA processing and transcription, respectively. These isoforms have both overlapping and distinct roles during development (9, 10). Interestingly, the +KTS isoform of WT1 plays the dominant role in the development of the gonad, while the -KTS isoform has a more extensive function in kidney formation.
The other alternative splice inserts 17 amino acids N terminal to the WT1 zinc fingers and has been shown to have effects on both cell division and cell survival (15, 31, 32). Specific elimination of this isoform of WT1 in mice does not result in any obvious defects in genitourinary development, suggesting that it may be required specifically for a tumor suppressor role or that it performs a subtle function (28).
Several studies have shown that WT1 contains a transcriptional activation domain that is suppressed by an N-terminal region of the protein (33). Suppression of WT1 transcriptional activation also occurs in the context of a GAL4 fusion protein, and the WT1 suppression domain can inhibit the function of other transcriptional activators (19, 22, 24, 36). A 30-amino-acid region of WT1 (residues 71 to 101) is sufficient to confer inhibition of the WT1 transcriptional activation domain. Moreover, this suppression domain is able to inhibit the transcriptional function of the activation domain of SP1 when fused in cis (24).
In this study, we use an in vitro transcription assay to provide direct evidence that the WT1 suppression domain interacts with a transcriptional cosuppressor. We identify brain acid soluble protein 1 (BASP1) as a component of the WT1 cosuppressor. BASP1 associates with WT1 in vivo, and its expression in the developing kidney is coincident with that of WT1. Our data suggest that BASP1 associates with WT1 to regulate its transcription function during development.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peptides and antibodies. Peptide synthesis was performed by Graham Bloomberg (University of Bristol, Bristol, England). The peptide was used to raise antibodies (SAPU, Edinburgh, Scotland), which were purified by peptide affinity chromatography. Antisera were raised against GST-BASP1 by Abcam (Cambridge, England). Antibodies were first purified by absorption to GST-linked beads to remove antibodies that bind GST. Anti-BASP1 antibodies were then purified by affinity chromatography with a column containing GST-BASP1 covalently linked to Sepharose. Anti-WT1 antibodies were the F6 monoclonal or N-180 polyclonal antisera from Santa Cruz. Antitubulin antibodies were from Sigma and anti-TFIIB antibodies have been described before (11).
Cell culture, transfection, and in vitro transcription assays.
Human embryonic kidney (HEK) 293, M15, G-401, HeLa, and Cos-1 cells were cultured as monolayers in Dulbecco's modified Eagle medium containing 10% fetal calf serum, 5 mM L-glutamine, 100 mg of streptomycin per ml, and 100 U of penicillin per ml. Human erythroleukemia cell line K562 was cultured in RPMI 1640 medium with L-glutamine, 10% fetal calf serum, 100 mg of streptomycin per ml, and 100 U of penicillin per ml. 293 cells, M15 cells, and Cos-1 cells were transfected as described previously (11). HeLa cells were transfected by using Lipofectamine 2000 according to the instructions of the manufacturer (Invitrogen). K562 cells were transfected with Lipofectamine 2000 by using a modified protocol for nonadherent cells (2). Chloramphenicol acetyltransferase (CAT) assays were performed and quantified by phosphorimager analysis. Luciferase activity was measured with a Promega kit as described in the manufacturer's instructions. Transfection of the RNAi vectors into HeLa cells was carried out with Lipofectamine 2000 reagent. Cells were plated out into six-well plates on the day prior to the transfection at a density such that they would reach 90 to 95% confluence on the following day. Two micrograms of either pSUPER (3) or pSUPER-BASP1 and 2 µg of CMV-WT1-/-, where appropriate, were transfected into the cells. After 24 h, the cells were split 1:2 and replated. After a further 24 h, 2 µg of either pSUPER or pSUPER-BASP1, 1 µg of pGL2-AR-A (AR) or pGL2-AR-C (AR
WRE), and 1 µg of CMV-WT1-/-, where appropriate, were transfected. Cells were harvested 48 h later.
HeLa nuclear extracts were purchased from 4C (Mons, Belgium). In vitro transcription assays were performed as described previously (24). Where indicated, antibodies or peptides were added to nuclear extract on ice and left for 30 min prior to the transcription reaction.
Immunofluorescence. Cells were grown to approximately 50% confluency in eight-well slides, fixed with 1% formaldehyde in phosphate-buffered saline (PBS), and then rinsed in PBS. Cells were incubated with primary antibodies (anti-WT1 F6 and anti-BASP1, both diluted 1:50 in a 500-µg/ml digitonin solution) for 2 h and then washed with PBS containing 1% Tween 20. The cells were then incubated for 30 min with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (1:100; Dako) or Cy3-congugated goat anti-rabbit (1:500; Jackson Immunological Research) antibodies in PBS and washed with PBS containing 1% Tween 20. Nuclei were counterstained with Hoechst stain (1 µg/ml) in PBS. Samples were mounted with Vectashield (Vector Laboratories, Inc).
Immunohistochemistry. Mouse embryos of mixed (C57BL/6:CBA) genetic background were recovered 15 days after detection of a copulation plug. Slides from samples fixed in 4% paraformaldehyde were dewaxed and rehydrated, and then antigen retrieval was carried out by boiling in 10 mM sodium citrate buffer (pH 6.0) three times for 5 min each. The slides were washed for 10 min in PBS plus 0.1% Tween 20, and then endogenous peroxidase activity was quenched by incubation in 3% H2O2 in tap water for 5 min, followed by 0.5% H2O2 in methanol for 30 min. Slides were washed three times for 10 min each in PBS plus 0.1% Tween 20 and then blocked and incubated with primary and secondary antibodies. The primary antibody (anti-BASP1 or anti-WT1 [Santa Cruz]) was used at a concentration of 1:500. After the secondary antibody (biotin-conjugated anti-rabbit; Sigma), streptavidin-horseradish peroxidase complex (ABC kit; Vector) and diaminobenzidine (DAB kit; Vector) were used for detection. The slides were then counterstained with Mayer's hematoxylin (Sigma), dehydrated, and mounted.
Paraffin-embedded sections from mouse or human (Novagen) adult kidney were placed in xylene overnight and then washed with ethanol. Endogenous peroxidases were inactivated by treating the samples with 0.3% H2O2 in methanol, and the samples were rehydrated. Target retrieval was achieved by boiling the samples for 10 min in 10 mM Tris-400 µM EGTA (pH 9). Cooled sections were placed in 50 mM NH4Cl in PBS for 30 min and then blocked with 1% bovine serum albumin-0.2% gelatin-0.05% saponin solution in PBS. Sections were incubated with antibodies as described above.
Affinity chromatography and binding assays. HeLa nuclear extract was dialyzed into buffer D (20 mM HEPES [pH 8], 20% [vol/vol] glycerol, 100 mM KCl, 0.2 mM EDTA, 1 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride). Ten milligrams of HeLa nuclear extract was precleared over a glutathione agarose column and then fractionated over columns containing 1 mg of glutathione-linked GST fusion protein. The columns were washed with 10 volumes of buffer D, and the bound fraction was eluted with buffer D (with 1 M KCl). GST pull-down assays were performed as described before (24). Immunoprecipitation assays were performed with nuclear extracts prepared from M15 cells in buffer D containing 100 mM KCl. Protein A-Sepharose was used to collect the immune complexes, and washes were performed with the same buffer.
Two-dimensional electrophoresis and protein identification. For protein precipitation, 0.25 volume of trichloroacetic acid solution (100% [wt/vol] trichloroacetic acid, 0.4% [wt/vol] sodium deoxycholate) was added and the proteins were collected by centrifugation for 30 min at 13,000 x g followed by washing with acetone. Two-dimensional electrophoresis was performed with the Pharmacia system, and the second dimension was an 8 to 18% acrylamide gradient gel.
Proteins were identified by mass spectrometry as previously described (34). The unseparated trypsin peptide mixture was analyzed by matrix-assisted laser desorption ionization, using a Bruker Reflex III matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (Bruker Daltronics). Samples for tandem mass spectrometry analysis were prepared essentially as described previously. After in-gel digestion, the supernatant was loaded onto a Poros R2 microcartridge (39) and eluted into nanoelectrospray needles (MDS Proteomics, Odense, Denmark). Nanoelectrospray tandem mass spectrometry analysis was performed on a QSTAR quadrupole time-of-flight mass spectrometer (Applied Biosystems), and fragmentation spectra were obtained for as many peptides as possible. PepSea software (MDS Proteomics) was used to search publicly available sequence databases maintained by the National Center for Biotechnology Information with a list of peptide masses or with peptide sequence tags from fragmentation spectra.
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RESULTS |
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Previous studies have shown that overexpression of an N-terminal fragment of WT1 (containing the suppression domain) causes a normally inactive intact WT1 protein to activate transcription of a promoter containing WT1 DNA-binding sites (37). It was proposed that this manifestation of activator function resulted from titration of a WT1 cosuppressor by the free suppression domain. We sought to determine if this finding could be recapitulated in an in vitro transcription assay. HeLa cell nuclear extract was incubated with a synthetic 18-amino-acid peptide containing the minimal suppression domain (as identified above) or a control peptide sequence derived from the RNA polymerase III-specific transcription factor hRRN3 (25). These peptide-treated extracts were then used in an in vitro transcription assay as in Fig. 1 (Fig. 2A). When we tested the HeLa nuclear extract that had been treated with the WT1 suppression domain peptide, GAL4-SD-SP1 was now able to activate transcription. This was not observed with a nuclear extract treated with the control peptide. Thus, consistent with previous observations, the isolated WT1 suppression domain is able to titrate a cosuppressor, thereby rendering the DNA-bound suppression domain inactive.
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The WT1 suppression domain acts by recruiting a transcriptional cosuppressor. We next sought direct evidence that the WT1 suppression domain interacts with a cosuppressor. We purified either GST or a GST fusion of the WT1 suppression domain (GST-SD) linked to glutathione agarose and checked their purity by SDS-PAGE (Fig. 2C). These beads were then assembled into columns, over which HeLa cell nuclear extract was fractionated. The flowthrough fractions from the columns were then tested in transcription assays with GAL4, GAL4-SD-SP1, or GAL4-SP1 (Fig. 2D). The flowthrough from the GST column showed the same transcription effects with the GAL4 fusion proteins that we observed with the untreated HeLa cell nuclear extract. When we examined the flowthrough from the GST-SD column, the activities of GAL4 and GAL4-SP1 were the same as that observed with the flowthrough from the GST column. In contrast, however, GAL4-SD-SP1 was able to activate transcription in the flowthrough from the GST-SD column, indicating that the GST-SD column had depleted a cosuppressor from the HeLa nuclear extract.
After extensive washing, we eluted the proteins that were bound to the GST and GST-SD columns with buffer containing 1 M salt and dialyzed the eluates into buffer containing 100 mM salt. The eluates from the GST and GST-SD columns were then added to a HeLa cell nuclear extract that had been fractionated over a GST-SD column (Fig. 2E). Addition of the 1 M eluate from the GST column had no effect on the activities of the GAL4 fusion proteins in the GST-SD flowthrough. However, addition of the 1 M eluate from the GST-SD column rendered GAL4-SD-SP1 inactive but did not reduce the level of transcriptional activation by GAL4-SP1. Thus, cosuppressor activity is present in the 1 M salt eluate from the GST-SD column but not in the GST column eluate.
Identification of BASP1 as a component of the WT1 transcriptional cosuppressor. The 1 M eluates from the GST and GST-SD column were analyzed by two-dimensional gel electrophoresis followed by silver staining (Fig. 3A). Analysis of the profiles revealed three spots that were present in the GST-SD eluate but not the GST eluate. Spot A was positively identified by nanoelectrospray tandem mass spectrometry as containing only BASP1. The group of spots designated B could not be identified, but it is possible that these are related to spot A (see below). Spot C contained GST, and it is therefore likely that this is GST-SD that has shed from the column.
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We obtained an EST that contained the entire BASP1-coding sequence. A GST-BASP1 fusion protein was produced and analyzed by SDS-PAGE and Coomassie blue staining (Fig. 3C). GST and GST-BASP1 linked to glutathione agarose beads were then used in binding assays with the recombinant GAL4 fusion proteins that were used in our in vitro transcription assays (Fig. 3D). Neither GAL4 or GAL4-SP1 interacted with GST-BASP1. However, we observed a robust interaction of GAL4-SD-SP1 with GST-BASP1, but not with GST, confirming that the WT1 suppression domain is indeed able to interact directly with BASP1.
We next transfected a plasmid containing HA-tagged BASP1-coding sequence under the control of a CMV promoter into mouse kidney M15 cells (Fig. 3E). M15 cells are derived from the mouse mesonephros, a progenitor of the kidney, and naturally express WT1 (17). Immunofluorescence was performed on the transfected cells to detect both the endogenous WT1 and HA-tagged BASP1. The cells were counterstained with Hoechst stain to show the nucleus. WT1 was present within the nuclei of all of the cells. In addition, HA-BASP1 was present entirely in the nuclei of all of the transfected cells (Fig. 3E and data not shown).
BASP1 expression and association with WT1. We next raised polyclonal antisera that recognize BASP1 and purified them by affinity chromatography with a column containing immobilized BASP1. The purified anti-BASP1 antibodies were then used to immunoblot a panel of cells lines (Fig. 4A). HeLa cell nuclear extract shows two major forms of BASP1, migrating at 52 and 40 kDa. Whole-cell extracts derived from HEK 293 cells and monkey kidney Cos-1 cells also contain the 52- and 40-kDa forms of BASP1. G-401 cells, derived from a human pediatric kidney tumor, contain predominantly the 40-kDa form of BASP1. Two different M15 whole-cell extracts are shown to represent a heterogeneity that we observed with extracts from these cells. M15 cells can contain both the 52- and 40-kDa forms, although in variable ratios. In addition, M15 cells contain a much more slowly migrating immunoreactive protein. The 52- and 40-kDa BASP1 products were produced by in vitro translation of either HA-BASP1 or BASP1 in a rabbit reticulocyte lysate, suggesting that they arise from natural processing of BASP1 (Fig. 4B). We also transfected the plasmid driving expression of HA-BASP1 into either Cos-1 cells or M15 cells and immunoblotted with the anti-BASP1 antibody (Fig. 4C). In both cell lines, transfection of the HA-BASP1 construct caused a significant increase in both the 52- and 40-kDa immunoreactive bands.
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We next performed immunofluorescence to localize the endogenous BASP1 in M15 cells (Fig. 4E). As we had observed with HA-BASP1, endogenous BASP1 in M15 cells is located largely within the nucleus along with WT1. Nuclear extracts were prepared from M15 cells and subjected to immunoprecipitation with either anti-WT1 antibodies (Fig. 4F, left panel) or anti-BASP1 antibodies (Fig. 4F, right panel). Precipitates were resolved by SDS-PAGE and immunoblotted to detect both WT1 and BASP1. The results show that an anti-WT1 monoclonal antibody, but not an anti-Flag monoclonal antibody, coprecipitates both WT1 and the 40-kDa form of BASP1. Neither the myristoylation-dependent form of BASP1 or a further-truncated BASP1 product were immunoprecipitated with anti-WT1 antibodies (left panel). In addition, purified anti-BASP1 polyclonal antibodies, but not GAL4 polyclonal antibodies, coimmunoprecipitated BASP1 and WT1. Taken together, the data suggest that endogenous WT1 and BASP1 can be associated within the same complexes in M15 cells.
We also performed immunofluorescence experiments to detect endogenous BASP1 in human pediatric kidney tumor G-401 cells (Fig. 4G), finding that BASP1 was again located entirely within the nucleus. When we examined Cos-1 cells, BASP1 was also present in the nucleus (Fig. 4H). However, the cytoplasm of Cos-1 cells showed a greater level of staining, which was particularly prominent around the nuclear periphery. Thus, the cellular localization of BASP1 is cell type specific; in the cell lines that we have analyzed, BASP1 is present in the nuclei of M15, G-401, and Cos-1 cells but showed a significant presence only in the cytoplasm in Cos-1 cells.
BASP1 is present in the developing mouse kidney. WT1 is a transcription factor that plays a central role in development. In light of the limited information regarding the biology of BASP1, we sought to determine the expression pattern of BASP1 in the mouse embryo. Sections from the mouse embryo recovered at 15.5 days postcoitum were probed with either anti-BASP1 antiserum or preimmune serum (control) (Fig. 5A). BASP1 was present in many regions of the mouse embryo. It was highly expressed in the central nervous system and also in the lungs, heart, thymus, liver, and tongue. WT1 is also expressed in several regions of the developing embryo, and its expression pattern in the developing kidney is well characterized (1). Figure 5B shows the kidney region of the mouse embryos from Fig. 5A. Significantly, BASP1 was present within the forming nephron structures of the developing kidney, which are also regions of high WT1 expression. Consistent with this, immunoblotting of an HEK tissue sample confirmed the presence of the 52-kDa form of BASP1 (Fig. 5C).
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BASP1 exhibits WT1 transcriptional cosuppressor activity.
WT1 can repress transcription at highly active promoters. We therefore considered that if BASP1 is indeed part of a WT1 transcriptional cosuppressor, it might augment the transcriptional repression of an active promoter in the presence of WT1. A reporter containing five WT1 DNA-binding sites was transfected into 293 cells either alone or with a plasmid driving expression of intact WT1 (-/- isoform) or a WT1 deletion mutant that lacks the minimal suppression domain (92-101) (Fig. 7A). The results show that increased coexpression of HA-BASP1 significantly augmented transcriptional repression by intact full-length WT1. In comparison, BASP1 showed a small effect in the absence of WT1 and also in the presence of the WT1 derivative lacking a suppression domain (92-101). We also analyzed a GAL4 fusion protein containing WT1 residues 71 to 250, which has previously been shown to repress the "high basal" thymidine kinase (tk) promoter linked to GAL4 DNA-binding sites (G5tkCAT) (21, 24, 37). G5tkCAT was transfected into HEK 293 cells along with a plasmid that expresses either the GAL4 DNA-binding domain, GAL4-WT1 (residues 71 to 250), or a GAL4-WT1 (residues 99 to 250) which lacks the suppression domain. In the absence of HA-BASP1, the GAL4-WT1(71-250) construct caused a modest level of transcriptional repression, consistent with that previously reported by us (24) (Fig. 7B), but the GAL4-WT1 derivative lacking an intact suppression domain (residues 99 to 250) did not. Cotransfection of HA-BASP1 augmented transcriptional repression by GAL4-WT1 (residues 71 to 250) but had no effect when cotransfected with the plasmid expressing either GAL4 or GAL4-WT1 (residues 99 to 250). Taken together, the results in Fig. 7A and B suggest that BASP1 can cooperate with the suppression domain of WT1 to augment transcriptional repression at an active promoter. Consistent with such a cosuppressor function, a GAL4-BASP1 fusion protein was able to repress transcription to a level similar to that observed with the well-characterized repression domain from Even Skipped (GAL4-EVE) (Fig. 7C) (24).
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During our analysis of different cell lines, we found that K562 cells lack detectable levels of BASP1 compared to others (Fig. 7E, left panel). K562 cells normally express WT1 (30), and we therefore speculated that, due to the absence of significant levels of BASP1, the endogenous WT1 would exhibit transcriptional activator function. The results in Fig. 7E show that the high activity of the amphiregulin-luciferase reporter in K562 cells is dependent upon the region of the promoter containing the WT1 response element (WRE). Significantly, transfection of a plasmid driving expression of BASP1 causes a downregulation of the amphiregulin reporter that is also dependent upon the presence of the WRE. Thus, BASP1 can regulate the activity of endogenous WT1 at the amphiregulin promoter.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We identified BASP1 as a factor present in a functional WT1 transcriptional cosuppressor fraction. WT1 and BASP1 associate in cells that naturally express both proteins and show temporal and spatial coexpression during kidney development. Downregulation of endogenous BASP1 by RNA interference elicited transcriptional activation of the amphiregulin promoter by WT1. Moreover, ectopic expression of BASP1 in a cell line with naturally low levels of BASP1 suppresses transcriptional activation of the amphiregulin promoter by endogenous WT1. Taken together, our results strongly suggest that BASP1 is a natural regulator of WT1 transcription function. BASP1 shows a more diverse expression pattern in the embryo than that observed for WT1, signifying an additional (but as-yet-unknown) function(s) for this protein. Consistent with this, our immunoprecipitation data suggest that only a portion of BASP1 is associated with WT1.
BASP1 was previously isolated from neuronal cells and was found to be a myristoylated protein (27). This does not make a role in transcription unlikely. Indeed, the Oct1/2-specific transcriptional coactivator, Bob1, exists as both myristoylated and nonmyristoylated forms (40). Only the nonmyristoylated isoform acts as a coactivator for Oct1. Our results suggest that BASP1 also exists in both myristoylated and nonmyristoylated forms and that myristoylation produces a form of BASP1 that does not interact with WT1. We found that BASP1 was present in the nuclei of mouse M15 and human G-401 cells. In Cos-1 cells we observed significant staining of BASP1 in the cytoplasm, particularly around the nuclear membrane. Transfection of WT1 into Cos-1 cells does not cause BASP1 to localize to the nucleus (data not shown). In addition, the presence of myristoylated BASP1 in M15 cells suggests that the cell line-dependent cellular localization of BASP1 cannot be explained simply by myristoylation. Thus, further studies will be required to understand how BASP1 distributes between the nucleus and the cytoplasm.
WT1 plays a critical role in kidney development and is required for the condensation of the metanephric mesenchyme onto the ureteric bud, its proliferation, and then differentiation into the epithelia of the nephron. BASP1 expression overlaps with WT1 within these same intermediate structures in the developing kidney. WT1 expression levels decrease in the latter stages of kidney development and are very low in the adult kidney, where WT1 expression has been confined to a very specialized cell type, the podocytes. Expression of WT1 is required for the maintenance of podocyte function (8). BASP1 expression is also confined to the podocytes of the adult kidney. These data strongly suggest that WT1 and BASP1 are involved in the same pathway of kidney development and perhaps in the maintenance of podocyte function. The -KTS isoform of WT1 plays a greater role in podocyte development and maintenance than the +KTS isoform (9, 10). This suggests that the podocytes are a critical site for the function of WT1 as a transcriptional regulator. Perhaps the presence of BASP1 in the podocytes provides temporal specificity to the transcriptional activity of WT1 in these cells. The WT1 target amphiregulin gene shows the same expression profile within the developing and adult kidney (18). It is therefore possible that BASP1 modulates transactivation of the amphiregulin promoter by WT1 during kidney development. This mechanism perhaps affords a tighter control of WT1 target gene expression and may be critical in regulating proliferation and differentiation. Further insights into such a mechanism will require a determination of WT1, BASP1, and amphiregulin expression at different stages of kidney development.
As mentioned above, an attractive role for the WT1 suppression domain is that it provides specificity to the function of the WT1 transcriptional activation domain. In a specific cell context, the WT1-BASP1 interaction may be disrupted or BASP1 may be downregulated, hence releasing the function of the WT1 transcriptional activation domain. It is tempting to speculate that the PEST sequences within BASP1 may be part of this regulatory process. Our data also suggest that BASP1 cellular localization could be a potential point of regulation. For example, exclusion of BASP1 from the nucleus could relieve suppression of the WT1 transcriptional activation domain. Finally, a role for BASP1 as a modulator of transcriptional activity may help to explain the variability of WT1 in the regulation of proposed target genes in different cell lines (33). Further studies of BASP1 will shed light on the function of the WT1 cosuppressor and the specific transcriptional activator function of WT1.
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ACKNOWLEDGMENTS |
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This work was funded by the AICR, BBSRC, Cancer Research UK (SP2410/0101), MRC, and Wellcome Trust. S.G.E.R. is a Wellcome Trust Senior Research Fellow.
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FOOTNOTES |
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B.C. and K.J.H contributed equally to this paper.
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