KTS Isoform Research Articles

Abstract 3529: EWSR1-WT1 isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor

Abstract Desmoplastic small round cell tumor (DSRCT) is a rare pediatric cancer caused by the EWSR1-WT1 fusion gene. No targeted therapies have been established and multimodal therapy is ineffective with a 5-year survival rate of 15-25%. Many questions about the biology of DSRCT remain unanswered including the critical genes regulated by the EWSR1-WT1 fusion gene, the role of the - and +KTS fusion gene isoforms produced by alternative splicing, and the relationship between EWSR1-WT1 binding and gene expression alterations. To address these questions, we established four doxycycline-inducible DSRCT cell lines that selectively knockdown the expression of EWSR1-WT1. RNA-seq analysis on these cell lines with or without EWSR1-WT1 knockdown identified commonly altered genes and pathways. To illuminate the role of the EWSR1-WT1 -KTS and +KTS isoforms, the -KTS, +KTS, or both isoforms were overexpressed in a mesothelial cell line, a hypothesized cell of origin for DSRCT. Remarkably, different gene expression alterations were observed between the -KTS and +KTS isoforms. While overexpression of the -KTS isoform upregulated 765 genes and downregulated 366 genes, overexpression of the +KTS isoform upregulated 203 genes and downregulated 25 genes. Gene set enrichment analysis showed EWSR1-WT1 regulated gene signatures are recapitulated by overexpression of the -KTS isoform or both +/-KTS isoforms, but not the +KTS isoform alone, suggesting the -KTS isoform predominantly regulates gene expression in DSRCT. To interrogate the influence of direct and indirect gene regulation by EWSR1-WT1, ChIP-seq and CUT&RUN data were integrated into our RNA-seq analysis. Across each of our four DSRCT cell lines, approximately 25% of EWSR1-WT1 bound peaks led to upregulation, 10% led to downregulation, and 65% led to no gene expression alteration. EWSR1-WT1 binding accounted for 33% of EWSR1-WT1 commonly upregulated genes but only 4.2% of commonly downregulated genes. Upregulated EWSR1-WT1 bound genes were more likely to be bound in the promoter or first intron and coincide with Znf263 or Hoxb4 motifs. To evaluate the functional role of EWSR1-WT1 targets in DSRCT biology, a screen of the 24 druggable EWSR1-WT1 targets was conducted and a number of novel and known target genes were identified as important for DSRCT survival. Current work is verifying these targets with gene knockdown and evaluating their function in vivo. Citation Format: Justin W. Magrath, Ilon A. Goldberg, Alifiani B. Hartono, Sean B. Lee. EWSR1-WT1 isoform selectivity, DNA binding, and druggable targets: unpacking the biology of desmoplastic small round cell tumor. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3529.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

RNA Binding by the KTS Splice Variants of Wilms' Tumor Suppressor Protein WT1.

Wilms' tumor suppressor protein WT1 regulates the expression of multiple genes through binding of the Cys2-His2 zinc finger domain to promoter sites. WT1 has also been proposed to be involved in post-transcriptional regulation, by binding to RNA using the same set of zinc fingers. WT1 has two major splice variants, where the Lys-Thr-Ser (KTS) tripeptide is inserted into the linker between the third and fourth zinc fingers. To obtain insights into the mechanism by which the different WT1 splice variants recognize both DNA and RNA, we have determined the solution structure of the WT1 (-KTS) zinc finger domain in complex with a 29mer stem-loop RNA. Zinc fingers 1-3 bind in a widened major groove favored by the presence of a bulge nucleotide in the double-stranded helical stem. Fingers 2 and 3 make specific contacts with the nucleobases in a conserved AUGG sequence in the helical stem. Nuclear magnetic resonance chemical shift mapping and relaxation analysis show that fingers 1-3 of the two splice variants (-KTS and +KTS) of WT1 form similar complexes with RNA. Finger 4 of the -KTS isoform interacts weakly with the RNA loop, an interaction that is abrogated in the +KTS isoform, and both isoforms bind with similar affinity to the RNA. In contrast, finger 4 is required for high-affinity binding to DNA and insertion of KTS into the linker of fingers 3 and 4 abrogates DNA binding. While finger 1 is required for RNA binding, it is dispensable for binding to consensus DNA sites.

Role for first zinc finger of WT1 in DNA sequence specificity: Denys-Drash syndrome-associated WT1 mutant in ZF1 enhances affinity for a subset of WT1 binding sites.

Wilms tumor protein (WT1) is a Cys2-His2 zinc-finger transcription factor vital for embryonic development of the genitourinary system. The protein contains a C-terminal DNA binding domain with four tandem zinc-fingers (ZF1–4). An alternative splicing of Wt1 can add three additional amino acids—lysine (K), threonine (T) and serine (S)—between ZF3 and ZF4. In the −KTS isoform, ZF2–4 determine the sequence-specificity of DNA binding, whereas the function of ZF1 remains elusive. Three X-ray structures are described here for wild-type −KTS isoform ZF1–4 in complex with its cognate DNA sequence. We observed four unique ZF1 conformations. First, like ZF2–4, ZF1 can be positioned continuously in the DNA major groove forming a ‘near-cognate’ complex. Second, while ZF2–4 make base-specific interactions with one DNA molecule, ZF1 can interact with a second DNA molecule (or, presumably, two regions of the same DNA molecule). Third, ZF1 can intercalate at the joint of two tail-to-head DNA molecules. If such intercalation occurs on a continuous DNA molecule, it would kink the DNA at the ZF1 binding site. Fourth, two ZF1 units can dimerize. Furthermore, we examined a Denys–Drash syndrome-associated ZF1 mutation (methionine at position 342 is replaced by arginine). This mutation enhances WT1 affinity for a guanine base. X-ray crystallography of the mutant in complex with its preferred sequence revealed the interactions responsible for this affinity change. These results provide insight into the mechanisms of action of WT1, and clarify the fact that ZF1 plays a role in determining sequence specificity of this critical transcription factor.

Distinct global binding patterns of the Wilms tumor gene 1 (WT1) -KTS and +KTS isoforms in leukemic cells.

The zinc finger transcription factor Wilms tumor gene 1 (WT1) acts as an oncogene in acute myeloid leukemia. A naturally occurring alternative splice event between zinc fingers three and four, removing or retaining three amino acids (±KTS), is believed to change the DNA binding affinity of WT1, although there are conflicting data regarding the binding affinity and motifs of the different isoforms. Increased expression of the WT1 -KTS isoform at the expense of the WT1 +KTS isoform is associated with poor prognosis in acute myeloid leukemia. We determined the genome-wide binding pattern of WT1 -KTS and WT1 +KTS in leukemic K562 cells by chromatin immunoprecipitation and deep sequencing. We discovered that the WT1 -KTS isoform predominantly binds close to transcription start sites and to enhancers, in a similar fashion to other transcription factors, whereas WT1 +KTS binding is enriched within gene bodies. We observed a significant overlap between WT1 -KTS and WT1 +KTS target genes, despite the binding sites being distinct. Motif discovery revealed distinct binding motifs for the isoforms, some of which have been previously reported as WT1 binding sites. Additional analyses showed that both WT1 -KTS and WT1 +KTS target genes are more likely to be transcribed than non-targets, and are involved in cell proliferation, cell death, and development. Our study provides evidence that WT1 -KTS and WT1 +KTS share target genes yet still bind distinct locations, indicating isoform-specific regulation in transcription of genes related to cell proliferation and differentiation, consistent with the involvement of WT1 in acute myeloid leukemia.

Abstract 2005: Global binding pattern of the Wilms’ tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells

Abstract The aim of this study was to investigate the global DNA-binding pattern of Wilms’ tumor gene 1 (WT1) in leukemic cells. Clinical and preclinical data indicate the zinc finger transcription factor WT1 as an oncogene, but the full target gene repertoire of WT1 in leukemic cells has not been previously characterized. The -KTS isoforms (excluding the three amino acid (KTS) insert between zinc finger three and four) are considered as the most efficient DNA-binders. Among these, the 17AA isoform (including 17 amino acids encoded by exon 5) is the most abundant one. To specifically analyze the DNA-binding of WT1(+17AA/-KTS) in leukemic cells, we generated a K562 clone that stably expressed BIO-tagged WT1(+17AA/-KTS), as well as the biotinylating enzyme Bir A. From the cells chromatin immunoprecipitation (ChIP) by streptavidin capture was performed followed by sequencing with a minimum of 50 million reads per sample. After alignment to the genome and peak calling, peaks were characterized and compared to available K562 tracks in the ENCODE database. We found that 45% of identified WT1(+17AA/-KTS) peaks are in the proximity of transcription start sites (promoter area, first exon or first intron) of target genes, whereas only 11% of randomized peaks were found here. Within the peaks we show strong enrichment for three different previously published WT1-binding motifs. Comparison to ENCODE tracks showed that WT1(+17AA/-KTS) peaks are in close proximity to binding sites of other transcription factors, to histone marks for actively transcribed genes, and to binding sites of chromatin modifiers. Considering peaks within promoters and gene bodies only (for safe assignment to a target gene), Gene Ontology (GO) analysis revealed enrichment of GO groups important for proliferation, cell death, embryonic development, and cell motility. In conclusion, WT1(+17AA/-KTS) binds close to transcription start sites in areas of active transcription. The target genes implicated in proliferation, cell death, cell signaling and motility adds to the growing evidence of WT1 as an effector gene in leukemia. Citation Format: Tove Ullmark, Linnea Järvstråt, Giorgia Montano, Helena Jernmark-Nilsson, Carl Sandén, Björn Nilsson, Karina Vidovic, Urban Gullberg. Global binding pattern of the Wilms’ tumor gene 1 (WT1) +17AA -KTS isoform in leukemic cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2005.

HuR antagonizes the effect of an intronic pyrimidine-rich sequence in regulating WT1 +/−KTS isoforms

WT1 + KTS and −KTS isoforms only differ in 3 amino acids in protein sequence but show significant functional difference. The +/−KTS isoforms were generated by alternative usage of 2 adjacent 5’ splice sites at RNA level, however, how these 2 isoforms are regulated is still elusive. Here we report the identification of an intronic pyrimidine-rich sequence that is critical for the ratio of +/−KTS isoforms, deletion or partial replacement of the sequence led to full/significant shift to -KTS isoform. To identify trans-factors that can regulate +/−KTS isoforms via the binding to the element, we performed RNP assembly using in vitro transcribed RNA with or without the pyrimidine-rich sequence. Mass spectrometry analysis of purified RNPs showed that the element associated with many splicing factors. Co-transfection of these factors with WT1 reporter revealed that HuR promoted the production of −KTS isoform at the reporter level. RNA immuno-precipitation experiment indicated that HuR interacted with the pyrimidine-rich element in WT1 intron 9. We further presented evidence that transient or stable over-expression of HuR led to enhanced expression of endogenous −KTS isoform. Moreover, knockdown of HuR resulted in decreased expression of endogenous −KTS isoform in 293T, SW620, SNU-387 and AGS cell lines. Together, these data indicate that HuR binds to the pyrimidine-rich sequence and antagonize its effect in regulating WT1 +/−KTS isoforms.

Soluble expression and purification of tumor suppressor WT1 and its zinc finger domain

Full length murine WT1 and its zinc finger domain were separately inserted into Escherichia coli expression vectors with various fusion tags on either terminus by Gateway technology (Invitrogen) and expression of soluble protein was assessed. Fusion proteins including the four zinc finger domains of WT1 were used to optimize expression and purification conditions and to characterize WT1:DNA interactions in the absence of WT1:WT1 interactions. Zinc finger protein for in vitro characterization was prepared by IMAC purification of WT1 residues 321–443 with a thioredoxin–hexahistidine N-terminal fusion, followed by 3C protease cleavage to liberate the zinc fingers and cation exchange chromatography to isolate the zinc fingers and reduce the level of the truncated forms. Titration of zinc finger domain with a binding site from the PDGFA promoter gave a Kd of 100±30nM for the −KTS isoform and 130±40nM for the +KTS isoform. The zinc finger domain was also co-crystallized with a double-stranded DNA oligonucleotide, yielding crystals that diffract to 5.5Å. Using protocols established for the zinc finger domain, we expressed soluble full-length WT1 with an N-terminal thioredoxin domain and purified the fusion protein by IMAC. In electro-mobility shift assays, purified full-length WT1 bound double-stranded oligonucleotides containing known WT1 binding sites, but not control oligonucleotides. Two molecules of WT1 bind an oligonucleotide presenting the full PDGFA promoter, demonstrating that active full-length WT1 can be produced in E. coli and used to investigate WT1 dimerization in complex with DNA in vitro.

Recurrence of a dysgerminoma in Frasier syndrome

FS is an inherited disease characterized by male pseudohermaphroditism and glomerular involvement leading to end-stage renal disease during adolescence or early adulthood (J Pediatr 1964:64:740). The FS phenotype in 46,XY patients consists of female external genitalia, gonadal dysgenesis, high risk of gonadoblastoma, and development of renal failure in the second decade of life. FS is caused by heterozygous mutation in intron 9 of the WT1 leading to a change in splicing that results in loss of three amino acids (+KTS isoform), thus disrupting the normal ratio of the +KTS/-KTS isoforms that is critical for proper gonadal and renal development (Nat Genet 1997:17:467; Hum Mol Genet 1998:7:709). We report on a patient followed for FS revealed by acute peritoneal syndrome because of ovarian dysgerminoma. Therapeutic options had led to an unusual course with recurrent neoplastic disease after renal transplantation.

A GATA4/WT1 cooperation regulates transcription of genes required for mammalian sex determination and differentiation

BackgroundIn mammals, sex determination is genetically controlled. The SRY gene, located on Y chromosome, functions as the dominant genetic switch for testis development. The SRY gene is specifically expressed in a subpopulation of somatic cells (pre-Sertoli cells) of the developing urogenital ridge for a brief period during gonadal differentiation. Despite this tight spatiotemporal expression pattern, the molecular mechanisms that regulate SRY transcription remain poorly understood. Sry expression has been shown to be markedly reduced in transgenic mice harboring a mutant GATA4 protein (a member of the GATA family of transcription factors) disrupted in its ability to interact with its transcriptional partner FOG2, suggesting that GATA4 is involved in SRY gene transcription.ResultsAlthough our results show that GATA4 directly targets the pig SRY promoter, we did not observe similar action on the mouse and human SRY promoters. In the mouse, Wilms' tumor 1 (WT1) is an important regulator of both Sry and Müllerian inhibiting substance (Amh/Mis) expression and in humans, WT1 mutations are associated with abnormalities of sex differentiation. GATA4 transcriptionally cooperated with WT1 on the mouse, pig, and human SRY promoters. Maximal GATA4/WT1 synergism was dependent on WT1 but not GATA4 binding to their consensus regulatory elements in the SRY promoter and required both the zinc finger and C-terminal regions of the GATA4 protein. Although both isoforms of WT1 synergized with GATA4, synergism was stronger with the +KTS rather than the -KTS isoform. WT1/GATA4 synergism was also observed on the AMH promoter. In contrast to SRY, WT1/GATA4 action on the mouse Amh promoter was specific for the -KTS isoform and required both WT1 and GATA4 binding.ConclusionOur data therefore provide new insights into the molecular mechanisms that contribute to the tissue-specific expression of the SRY and AMH genes in both normal development and certain syndromes of abnormal sex differentiation.

The Role of Wilms' Tumor Protein 1 (WT1) in Chronic Myelogenous Leukemia Progression.

Wilms' tumor protein 1 (WT1) is a key regulatory gene whose function is required for proper development and differentiation of the heart, retina, genito-urinary, olfactory and hematopoietic systems. WT1 over expression is a characteristic observed in blasts of Chronic Myeloid Leukemia (CML). Despite this apparent co-relation, a direct link between WT1 over expression and progression of CML to blast crisis has never been established. The two major isoforms of WT1 are generated by alternative splicing; an event that involves the use of two alternative splice sites at the end of exon 9. The outcome is the insertion or not of only 3 amino acids (lysine, threonine and serine; KTS) at the c-terminus of the protein. The ratio between these isoforms is conserved among tissues and disruption of this balance can end up causing developmental abnormalities. Our group is testing if the over expression of WT1 in hematopoietic precursors can lead to progression in a murine model of CML. Over expression of WT1 has been shown to block differentiation in myeloid cell lines. However, over expression of WT1 in primary hematopoietic cells has variable effects on differentiation and proliferation. These differences may reflect differences in vector design and/or assays to evaluate. We have utilized the Murine Stem Cell Vector (MSCV), a retroviral construct that has high efficiency of transducing murine hematopoietic stem cells, to generated retroviral constructs. The vector contains an internal ribosomal entry site allowing expression of WT1+KTS or WT1-KTS isoforms with and without Bcr-Abl/GFP, the causative protein in CML. Retroviral transduction of hematopoietic progenitors was performed for 48 hours and FACS sorted GFP+Sca+lin− cells were utilized for colony forming assays in methylcellulose and in liquid culture. Our results have shown that primary murine hematopoietic progenitors transduced with Bcr-Abl/GFP and WT1+KTS demonstrate dramatically greater cytokine independent growth than progenitors transduced with Bcr-Abl/GFP alone. In contrast, progenitors transduced with Bcr-Abl/GFP and WT1-KTS demonstrate impaired growth. [Display omitted] In addition transduced cells will be used to rescue lethally irradiated mice what will provide an excellent model to determine if WT1 cooperates with BCR-ABL to induce blast crisis. The exact function of each WT1 isoform is still unknown. However, there is clear data that they have different properties and are involved in different functions. Several lines of evidence suggest a role for the +KTS isoform in splicing. During hematopoietic cell differentiation, levels of WT1 protein were determined to decrease drastically. This fluctuation in WT1 expression level may play a critical role in the process of differentiation. In conclusion, our results strongly suggest that WT1 expression influences Bcr-Abl induced growth with the isoforms WT1+KTS and WT1-KTS displaying differential effects. Our data indicate that +KTS enhances Bcr-Abl dependent cellular proliferation while -KTS isoform impairs proliferation.

The Wilms tumour suppressor protein WT1 (+KTS isoform) binds alpha-actinin 1 mRNA via its zinc-finger domain

Mutations in WT1 are associated with developmental syndromes that affect the urogenital system and neoplasms, including Wilms tumour, acute myeloid leukemia, and breast and prostate cancers. The WT1 protein belongs to the early growth response family of zinc-finger transcription factors. Uniquely to WT1, an evolutionarily conserved alternative splice event inserts the tripeptide KTS, between zinc fingers 3 and 4. Whereas -KTS isoforms bind DNA and activate or repress transcription, +KTS isoforms bind DNA less efficiently and interact with splice factors and RNA in vitro and in vivo. Although candidate DNA targets have been found, physiological mRNA targets are yet to be defined. We examined the distribution of WT1 in ribonucleoprotein (RNP) complexes in nuclear extract prepared from M15 cells, a mouse mesonephric fetal kidney cell line. WT1 cofractionated with the splice factor PSF in large RNP particles >or=2 MDa. We also found that PSF co-immunoprecipitated with WT1, suggesting a functional interaction between these 2 multifunctional proteins. Using yeast three-hybrid library constructed from the co-immunoprecipitated RNA we found that WT1 (+KTS) binds close to or at the start codon of alpha-actinin 1 (ACTN1) mRNA. A band shift assay confirmed the ability of the WT1 zinc-finger domain (+KTS) to bind this sequence in vitro. ACTN1 is the first likely physiological mRNA target of WT1.

The Wilms’ tumor 1 (WT1) gene (+KTS isoform) functions with a CTE to enhance translation from an unspliced RNA with a retained intron

The Wilms' tumor 1 (WT1) gene plays an important role in mammalian urogenital development, and dysregulation of this gene is observed in many human cancers. Alternative splicing of WT1 RNA leads to the expression of two major protein isoforms, WT1(+KTS) and WT1(-KTS). Whereas WT1(-KTS) acts as a transcriptional regulator, no clear function has been ascribed to WT1(+KTS), despite the fact that this protein is crucial for normal development. Here we show that WT1(+KTS) functions to enhance expression from RNA possessing a retained intron and containing either a cellular or viral constitutive transport element (CTE). WT1(+KTS) expression increases the levels of unspliced RNA containing a CTE and specifically promotes the association of this RNA with polyribosomes. These studies provide further support for links between different steps in RNA metabolism and for the existence of post-transcriptional operons.

Wilms’ tumor gene WT1 17AA(–)/KTS(–) isoform induces morphological changes and promotes cell migration and invasion in vitro

The wild-type Wilms' tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(-)/KTS(-) isoform induces morphological changes characterized by a small-sized cell shape in TYK-nu.CP-r (TYK) ovarian cancer cells. In the WT1 17AA(-)/KTS(-) isoform-transduced TYK cells, cell-substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector-transduced TYK cells. Constitutive expression of the WT1 17AA(-)/KTS(-) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(-)/KTS(-) isoform induced the phenotypic changes. A decrease in alpha-actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(-)/KTS(-) isoform-transduced TYK cells. In contrast, co-expression of alpha-actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(-)/KTS(-) isoform-transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(-)/KTS(-) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions.

WT1 splice-site mutations are rarely associated with primary steroid-resistant focal and segmental glomerulosclerosis

Donor splice-site de novo heterozygous mutations in intron 9 of the Wilms' tumor gene (WT1) have been reported in Frasier syndrome, which is defined by the association of focal and segmental glomerulosclerosis (FSGS), male pseudohermaphroditism, and gonadoblastoma. These splice-site mutations alter the WT1 alternative splicing leading to two WT1 isoforms, with (+) or without (-) three amino acids, lysine-threonine-serine (KTS), between zinc fingers 3 and 4. The aim of this work was to investigate the possibility that some cases of primary steroid-resistant nephrotic syndrome associated with FSGS may be caused by WT1 splice-site mutations. We analyzed WT1 exons 8 and 9 and the surrounding exon/intron boundary DNA sequences in 37 children with nonfamilial primary steroid-resistant nephrotic syndrome. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the relative ratio of +KTS/-KTS transcripts from immortalized lymphocyte RNA. One boy with FSGS and associated pathologies (diaphragmatic hernia, proximal hypospadias, and unilateral testicular ectopia) was found to carry the heterozygous 1228 +4 C-->T splice-site mutation. RT-PCR quantitation of the +KTS/-KTS transcripts from immortalized lymphocyte RNA of this patient showed a diminution of the +KTS/-KTS isoform ratio (0.43), which is identical to that reported in patients with Frasier syndrome. Using the same approach, healthy control subjects have +KTS/-KTS ratios ranging from 1.50 to 2.00. This study expands the range of the phenotypic presentation of the intron 9 splice-site WT1 mutations and adds to the already reported heterogeneity of primary steroid-resistant nephrotic syndromes. We suggest that these mutations are not likely to be a common cause of isolated steroid-resistant nephrotic syndrome, and recommend a WT1 exon 9/intron 9 splice-site study in children with primary steroid-resistant nephrotic syndrome if genital or diaphragmatic anomalies are associated. The identification of such WT1 mutations has practical implications for the management of these patients.

Alternative splicing of Wilms' tumor suppressor protein modulates DNA binding activity through isoform-specific DNA-induced conformational changes.

The Wilms' tumor suppressor protein (WT1) is a zinc finger transcription factor that appears to function differently according to the presence of a posttranscriptional modification that adds three amino acids into one of the linker regions between the zinc fingers. We have investigated the structural consequences of the insertion of the Lys-Thr-Ser (KTS) sequence by preparing recombinant protein constructs of the four zinc finger DNA-binding domain of WT1 corresponding to the two isoforms with (+KTS) and without (-KTS) the insertion, which is located in the linker region between the third and fourth zinc fingers. NMR resonance assignments were used to estimate the structural differences between the two isoforms both free in solution and in complex with a 14 base pair DNA duplex corresponding to the WT1 recognition element. The NMR spectra indicate that the two isoforms are nearly identical in structure in the absence of the DNA. Only the immediate region of the insertion showed any change in chemical shifts. Upon DNA binding, the NMR spectrum of each isoform changed to indicate greater structure formation in the linker regions. Significant differences were observed between the spectra of the DNA complexes of the +KTS and -KTS isoforms, with the -KTS construct forming a more stable complex, consistent with prior biochemical assays. The majority of the differences between the spectra of the two complexes occur in the immediate region of the insertion, which appears to be closer in structure to the free form of the protein in the case of the +KTS complex. The insertion of the KTS sequence disrupts important interactions of the linker region with the adjacent zinc fingers, thus lowering the stability of the complex. The "normal" (-KTS) sequence of the linker appears to be involved in a C-terminal helix-capping interaction with the helix of the preceding zinc finger, a stabilizing interaction which is abrogated in the +KTS isoform.

The Wilms' tumor gene product represses the transcription of thrombospondin 1 in response to overexpression of c-Jun.

Thrombospondin 1 (TSP1) is known for its significant anti-angiogenic properties. In a previous study, we have shown that transient or stable overexpression of the transcription factor c-Jun, in rat fibroblasts, leads to repression of TSP1. We now demonstrate that the c-Jun-induced repression of TSP1 does not occur directly and does not require binding of c-Jun to the TSP1 promoter. Instead, repression involves a factor secreted by c-Jun-overexpressing cells. This secreted factor triggers a signal transduction pathway from the membrane to the nucleus, and these signals lead to the binding of the product of the Wilms' tumor suppressor gene, WT1, to the -210 region of the TSP1 promoter. This region binds WT1 and SP1, but not EGR1, although its sequence fits the consensus binding site for this transcription factor. WT1 overexpression in transfected cells inhibits endogenous TSP1 gene expression and TSP1 transcription in experiments using TSP1 promoter-reporter constructs. The WT1 - KTS isoform is more active in repressing TSP1 transcription than WT1 + KTS, while EGR1 is inactive. Enhancement of WT1 binding to DNA in response to c-Jun does not require de novo protein synthesis. The above mechanism for TSP1 repression could apply to other genes, thus coordinating their regulation in the vicinity of a c-Jun-overexpressing cell. We conclude that WT1, which was discovered as a result of its tumor suppressor properties, may also possess oncogenic characteristics in the c-Jun transformation process, and thus repress the anti-angiogenic protein, TSP1.

Transcriptional Regulation of PDGF-A and TGF-β by +KTS WT1 Deletion Mutants and a Mutant Mimicking Denys-Drash Syndrome

Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are rare diseases caused by the mutations of Wilms tumor gene, WT1 The common denominator in these syndromes is a nephropathy, which is manifested by early-onset proteinuria, nephrotic syndrome and end stage renal failure. Although these syndromes are genetic models of nephropathy and the mutations of WT1 gene are characterized in these patients, the mechanism how mutations of WT1 gene affect the embryonic, kidney adversely has not been elucidated. Recently, there was a report that FS is caused by mutations in the donor splice site of WT1. These mutations predicted loss of +KTS isoform, which is one of the jour splicing variants of WT1. In this study, two +KTS deletion mutants of WT1 were made as well as a WT1 mutant mimicking a mutation found in a patient who had diffuse mesangial sclerosis, end stage renal failure and Wilms tumor. Mutant embryonic kidncy cell lines were established by transfection of 293 embryonic kidney cells with WT1 mutants. We investigated the transcription regulation of mutant WT1 among these cell lines using the reporter vectors containing PDGF-A and TGF-β promoter sequence. Our results showed that the promoter activity of PDGF-A and TGF-β, which are related to the progression of glomerular diseases, was modestly increased in the mutant cell mimicking the patent, while those activities were markedly increased in other two deletion mutant cell lines. This study demonstrated that +KTS WT1 mutation found in DDS affected the cytokine expression adversely in vitro. From these results, we suggest that the alteration of +KTS WT1 expression be responsible for the rapid progression of renal diseases in DDS and FS.

Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms.

The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.