Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1

Abstract

The Wilms' tumor suppressor protein WT1 is a transcriptional regulator involved in differentiation and the regulation of cell growth. WT1 is subject to alternative splicing, one isoform including a 17-amino acid region that is specific to mammals. The function of this 17-amino acid insertion is not clear, however. Here, we describe a transcriptional activation domain in WT1 that is specific to the WT1 splice isoform that contains the 17-amino acid insertion. We show that the function of this domain in transcriptional activation is dependent on a specific interaction with the prostate apoptosis response factor par4. A mutation in WT1 found in Wilms' tumor disturbs the interaction with par4 and disrupts the function of the activation domain. Analysis of WT1 derivatives in cells treated to induce par4 expression showed a strong correlation between the transcription function of the WT1 17-amino acid insertion and the ability of WT1 to regulate cell survival and proliferation. Our results provide a molecular mechanism by which alternative splicing of WT1 can regulate cell growth in development and disease.

Figure 1

A splice isoform–specific transcriptional activation domain in WT1. (A) Diagram of WT1 showing the DNA-binding domain, proline-rich region, and potential leucine zipper (LZ). The two alternate splice sites are shown in black: the 17AA alternative splice is shown with the amino acid sequence and is located between the putative leucine zipper and DNA-binding domain (designated D domain); the second alternative splice (amino acid sequence KTS) is located between zinc fingers three and four. The region of WT1 fused to GAL4 (1–93) are shown below (amino acids 245–297). These fusions included the +17AA form (GAL4 D+) and the –17AA form (GAL4 D−). (B) Analysis of purified GAL4 (1–93), GAL4 D+, and GAL4 D− by SDS-PAGE and Coomassie staining. Molecular weight markers (kD) are shown at left. (C) In vitro transcription assay using 200 and 400 ng of each GAL4 fusion protein with Hela cell nuclear extract and the G₅E4T promoter (shown above). Transcripts were analyzed by primer extension and denaturing electrophoresis. (−)no addition of a GAL4 derivative. The signals were quantified by phosphorimager analysis and are presented as fold activation compared to the basal level of transcription (−).

Figure 2

The 17AA transcriptional activation region functions in the context of the natural WT1 DNA-binding domain. (A) Diagram indicating the regions of WT1 expressed as recombinant His-tagged proteins. The 17AA alternative splice is indicated in black fill. Recombinant proteins were analyzed by SDS-PAGE and Coomassie staining (M, molecular weight markers in kD, are shown at left). (B) In vitro transcription assay using the W₅E4T (containing 5 × WT1 DNA-binding sites) and G₅E4T promoters. Transcripts were detected by primer extension. Transcription levels (quantified by phosphorimager analysis) are presented relative to G₅E4T. (C) In vitro transcription assays were performed and analyzed as for part B except that 200 and 400 ng of each recombinant His-tagged protein was added. A HeLa nuclear extract (NE) or HeLa nuclear extract that had been fractionated over a column containing WT1 DNA-binding sites (Depleted NE) was used as indicated. Transcript levels were quantified by phosphorimager analysis and are presented as fold activation relative to the transcriptional activity of the depleted extract in the absence of a WT1 derivative.

Figure 3

The 17AA motif of WT1 is sufficient for transcriptional activation. (A) Deletion mutants of WT1 (245–297) were constructed as indicated. The purified proteins (200 and 400 ng) then were used in in vitro transcription assays with the G₅E4T promoter and HeLa nuclear extract. (−) No recombinant protein was added, and fold activation is presented relative to this. (B) Recombinant His-tagged GAL4 fusions were purified and analyzed by SDS-PAGE and Coomassie staining. Molecular weight markers (kD) are shown at left. In vitro transcription assays were performed as for A.

Figure 4

The function of the 17AA activation domain is cell type specific. (A) Transcription assays were performed using 200 and 400 ng of recombinant His-tagged GAL4 fusions with the G₅E4T promoter. Four hundred nanograms of the synthetic activator GAL4 AH was added where indicated. Assays contained either HeLa, embryonic kidney 293, or HL60 nuclear extract. (−) No recombinant protein was added to the assay. Transcripts were detected as previously, quantified, and presented as fold activation relative to the basal level (−) for each nuclear extract. (B) Transcription assays were performed as in A with HeLa:293 nuclear extract (ratio of 4:1) at left and 293:HeLa nuclear extract (ratio of 4:1) at right. Fold activation was determined as in A. (NE) Nuclear extract.

Figure 5

Interaction partners for the WT1 17AA region. (A) Diagram of GST-WT1 fusion proteins (residues 245–297) either lacking (GST D−) or containing (GST D+) the 17AA motif. (B) Protein affinity chromatography of HeLa nuclear extract fractionated over a GST, GST D−, or GST D+ column. After extensive washing, the bound fraction was eluted and analyzed by immunoblottting with either anti-par4 (top) or anti-TBP (bottom) antibodies. (C) HeLa nuclear extract was fractionated over either a GST D− or a GSD D+ column, and after a brief low-salt wash (250 mM KCl) and a subsequent wash (1 M KCl), the presence of par4 was assessed in each fraction by immunoblotting with anti-par4 antibodies. (D) A GST pull-down assay was performed with immobilized GST-par4 and a bacterial lysate containing the recombinant GAL4 derivatives indicated. The bound fraction was analyzed by immunoblotting with anti-GAL4 antibodies. I is 10% of the input into each pull-down assay.

Figure 6

Par4 is required for transcriptional activation by WT1 17AA. (A) Anti-par4 and anti-TBP immunoblots of SDS-PAGE resolved HeLa, 293, and HL60 nuclear extracts (10 μg each). (B) In vitro transcription assay showing the effects of increasing amounts of anti-par4 antibodies (100, 200, and 400 ng) on transcriptional activation by GAL4 D+ (200 ng). (−) No recombinant protein was added to assay. GAL4 AH was included as a control, with 400 ng of anti-par4 antibody added where indicated. Assays were performed using HeLa nuclear extract and transcripts analyzed as before. (C) In vitro transcription assay showing that anti-par4 antibodies but not a nonrelevant rabbit antibody (anti-p65 subunit of NF-κB) inhibits transcriptional activation by GAL4 D+.

Figure 7

Par4 expression is induced by UV irradiation and is directly linked to transcriptional activation by WT1 17AA. (A) Control and UV light–treated HeLa cells were used to prepare nuclear extract that was subsequently immunoblotted (10 μg per lane) with either anti-par4 (top) or anti-TBP (bottom) antibodies. (B) In vitro transcription assay showing the effect of UV light on the transcriptional activity of the GAL4 D+ fusion (200 ng) or GAL4 AH (200 ng) in assays using the G₅E4T promoter. Anti-par4 antibodies (400 ng) were added as indicated. Transcripts were detected as before, quantified, and expressed relative to the basal level (−) in each extract.

Figure 8

WT1 17AA is a UV light responsive transcriptional activation domain in vivo. (A) Plasmids expressing GAL4, GAL4 D−, GAL4 D+, and GAL4 D+ G253A (4 μg each) were transfected into embryonic kidney 293 cells along with the reporter G₅E4CAT (1 μg). Six hours after UV light treatment of the cells, CAT activity was measured and is presented relative to the level of CAT activity with empty pCDNA3 vector alone (0 lane). The results are the mean average of four independent experiments. (B) Immunoblot with anti-par4 antibodies showing that the level of par4 is elevated in 293 cells that have been treated with UV light. (C) Coimmunoprecipitation of par4 with the WT1 17AA motif. 293 cells were transfected with 4μg of each GAL4 derivative and after UV light treatment nuclear extracts were prepared. Immunoprecipitation was performed with either anti-GAL4 or anti-TFIIH antibodies and complexes harvested with protein G Sepharose. Par4 content was assessed by immunoblotting. Input (I) is 1% of the amount of nuclear extract used in each immunoprecipitation.

Figure 9

The WT1 17AA motif confers a survival function in cells exposed to UV light. (A) Embryonic kidney 293 cells were transfected with either empty CMV expression vector or full-length WT1 (−/−), WT1 ( +/−), or WT1 (+/−) G253A under the control of a CMV promoter (4 μg in each case). Sixteen hours after UV light treatment (where indicated), the cells were photographed. (B) Cells were transfected as in A, but along with 1 μg of an expression vector containing green fluorescent protein. Cells were photographed under fluorescence to visualize only the transfected cells. (C) Immunoblot with anti-WT1 antibodies showing that the WT1 +/−, −/−, and +/− G253A derivatives are expressed at equivalent levels in 293 cells. (D) Survival curves to quantify the effect of WT1 +/− and the mutant G253 on cell number after UV light treatment. Each WT1 derivative (0.5, 1, 2, and 4 μg; balanced with empty CMV vector) was transfected into 293 cells, and 16 h after UV light treatment the cells were harvested from the plate and counted. Percentage survival is relative to a control plate of 293 cells transfected with CMV, but not subject to UV light treatment. Each point is the mean average of three independent transfection experiments with standard deviation.