WT1 Isoforms Research Articles

Downregulation of Wilms’ tumor gene (WT1) is not a prerequisite for erythroid or megakaryocytic differentiation of the leukemic cell line K562

The Wilms’ tumor gene (WT1) encodes a transcription factor of the zinc finger type. A high expression of WT1 has been detected in a range of acute leukemias, and WT1 is downregulated during induced differentiation of some leukemic cell lines. Overexpression of WT1 in some myeloid cell lines confers resistance to differentiation induction. These observations suggest that a high WT1 expression in hematopoietic cells is incompatible with differentiation. In this study, each of the four different isoforms of WT1 was constitutively overexpressed in the leukemic cell line K562. K562 cells express endogenous WT1, which is downregulated as a response to induced differentiation along the erythroid and megakaryocytic pathways. We now demonstrate that a forced exogenous expression of the four different isoforms of WT1 in K562 does not affect the differentiation response, as judged by accumulation of hemoglobin in response to hemin or the expression of megakaryocytic cell surface markers in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that downregulation of WT1 during induced differentiation of K562 cells is not a prerequisite for erythroid or megakaryocytic differentiation of these cells.

Ornithine Decarboxylase Is a Transcriptional Target of Tumor Suppressor WT1

The product of the Wilm's tumor suppressor gene, WT1, is a zinc-finger DNA-binding protein, which is thought to be a transcription factor. Two genes, those encoding epidermal growth factor receptor and syndecan-1, are known to be endogenous targets of WT1. Previous studies had identified binding sites for WT1 in the promoter of the ornithine decarboxylase (ODC) gene. In this paper, we tested whether the endogenous ODC gene might be a target of WT1 by establishing lines of baby hamster kidney (BHK) cells that expressed WT1 isoform A under control of a tetracycline-regulated expression system. When expression of WT1 was activated in BHK cells, the cellular level of ODC mRNA declined, with kinetics that correlated with the increase in WT1 level, demonstrating that the endogenous ODC gene was indeed responsive to cellular level of WT1. WT1 isoforms A and B inhibited the activity of the ODC promoter by approximately fivefold in transiently transfected BHK cells, while isoforms C and D, which have altered DNA binding domains, had no significant effect. The sequence CTCCCCCGC, located at nucleotides −106 to −98 relative to the site of transcriptional initiation in the ODC gene, interacted with the zinc-finger domain of isoforms A and B of WT1 with high affinity and specificity. A mutation in the binding site that disrupted this interaction partially removed the inhibition of ODC promoter activity by WT1, as did mutation of the two E-box sequences in intron I of the ODC gene. Simultaneous mutation of the WT1-binding motif and the two E-boxes completely abolished inhibition by WT1 of ODC promoter activity. These results, taken together, implicate the ODC gene as a downstream target of the tumor suppressor WT1.

Imbalanced Expression of Functionally Different WT1 Isoforms May Contribute to Sporadic Unilateral Wilms' Tumor

Functional loss of the product of the Wilm's tumor suppressor gene (wt1) has been identified in subsets of familial Wilms' tumors. Previously, four alternative splice products of WT1 were recognized and each was found to regulate transcription of effector genes differently, suggesting that disruption of the normal ratio of these spliced products will disrupt the normal expression patterns of WT1 effector genes and perhaps lead to Wilms' tumor. In support of these suggestions, we found that four of seven cases of sporadic unilateral Wilms' tumor had striking differences in the ratios of the spliced products of WT1 compared with each other and normal kidney. These data indicate that in addition to structural mutations, alterations in the relative amounts of the mature WT1 isoforms may also be important in the etiology of sporadic Wilms' tumor.

WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes.

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms' tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both -KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.

Analysis of WT1 target gene expression in stably transfected cell lines.

The Wilms' tumour suppressor gene WT1 encodes a zinc finger protein that is mutated in a subset of Wilms' tumours. Mutation screening and animal studies revealed essential roles during development and later function of the kidneys and the entire genitourinary system. Sequence similarity suggested a possible role for WT1 as a transcription factor. Indeed, sequence specific DNA binding and transcriptional activation or repression potential could be demonstrated in transient transfection assays with various reporter constructs. To identify endogenous WT1 target genes we established HEK293 cell lines expressing the different WT1 isoforms in a tetracycline dependent manner. Differential display PCR (ddPCR) was performed on RNA from stable WT1 transfected HEK293 cell lines and two other WT1 transfected lines (G401 and Saos-2). In an extended survey of several thousand ddPCR bands only few differences in intensity were seen and none of these could unambiguously be verified as being WT1 regulated by subsequent Northern blot analysis. In addition, almost none of the WT1 target genes identified to date in transient co-transfection assays could be confirmed by either ddPCR or Northern hybridization in the three stable transfected cell lines. Among the nine genes expressed, the only exceptions were CSF1 and to a lesser extent IGF1R being induced in Saos-2/G401 and HEK293 cells, respectively. At least two of the cell lines tested had previously shown clear biological effects though -- either WT1 dependent apoptosis (Saos-2) or greatly reduced tumorigenicity (G401). This suggests that WT1 may regulate only a very small set of genes that escape the detection methods used or it may not act as a transcription factor that influences steady state levels of mRNA.

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.

Overlapping RNA and DNA binding domains of the wt1 tumor suppressor gene product.

The Wilms' tumour suppressor gene (wt1) is mutated in a subset of patients with Wilms' tumour and has a critical role in urogenital development. wt1 encodes a zinc finger transcription factor which regulates expression of several genes involved in cellular proliferation and differentiation. Although a number of studies have characterized the DNA binding properties of the WT1 protein, recent evidence has suggested that WT1 may also have a role in RNA metabolism. We have used an RNA selection method to identify WT1 binding ligands from a random RNA pool. Three groups of RNA ligands specifically recognized by WT1 were identified. Mutational analysis pinpointed ribonucleotide sequences critical for binding. Analysis of truncated WT1 proteins demonstrated that three of four zinc fingers were necessary for RNA-protein interaction. The naturally occurring WT1 isoforms with insertion of lysine, threonine and serine between zinc fingers three and four were unable to bind the selected RNAs. The selected RNA ligands competed with the cognate WT1 DNA binding site for complex formation with WT1. Our findings suggest potential cellular RNA target sequences for WT1 and provide tools for studying the structural and functional properties of this tumour suppressor protein.

Constitutive expression of the Wilms' tumor gene (WT1) in the leukemic cell line U937 blocks parts of the differentiation program.

The Wilms tumor gene, WT1, encodes a zinc-finger DNA binding protein which is thought to function as a tissue specific transcription factor, regulating cell growth and differentiation. High expression of WT1 has been detected in a range of acute leukemias. To elucidate a role for WT1 in leukemogenesis, we transfected the monoblastic cell line U937, which lacks detectable levels of endogenous WT1, with two isoforms of WT1. We showed that, in contrast to U937 control cells, cells constitutively expressing either of the isoforms, WT1(-KTS) or WT1(+KTS), did not respond to differentiation induction by retinoic acid or vitamin D3, as judged by the capacity to reduce nitro blue tetrazolium and morphology. Although U937 cells expressing WT1 were hampered in their ability to differentiate on incubation with retinoic acid and vitamin D3, the induced G1/G0-accumulation was similar to differentiating control cells treated with inducers. Furthermore, distinct effects on the maturation process were indicated by downregulation of the myeloid cell surface makers CD13 and CD15, while the upregulation of CD14 and CD11c on WT1 transfected cells was similar to control cells upon incubation with retinoic acid and vitamin D3. Taken together our results demonstrate that a constitutive expression of WT1 in the leukemic cell line U937 leads to impairment of differentiation responses, indicating that a high expression of WT1 can contribute to the differentiation block of acute leukemia.

Expression of the Wilms' Tumor Suppressor Gene, WT1, Is Upregulated by Leukemia Inhibitory Factor and Induces Monocytic Differentiation in M1 Leukemic Cells

AbstractThe Wilms' tumor gene, WT1, encodes a transcription factor of the Cys2-His2 zinc finger type. The functional significance of WT1 expression in leukemias, in addition to tissues and cell lines of hematopoietic origin, has not been determined. Using the murine myeloblastic leukemia cell line M1 as a model for macrophage differentiation, expression of WT1 is shown to be activated in M1 cells 24 hours after differentiation induction by leukemia inhibitory factor (LIF). Upregulation ofWT1 in these cells is associated with cellular differentiation, coinciding with expression of the monocyte/macrophage marker c-fms, and the appearance of mature cells. WT1 isoforms lacking the KTS insert are unable to be ectopically expressed in M1 cells. Stable expression of the WT1 isoforms containing the KTS insert leads to spontaneous differentiation of the M1 myeloblasts through the monocytic differentiation pathway. These cells express c-fms,in addition to the myeloid-specific cell surface marker Mac-1. Exposure of these cells to LIF results in the rapid onset of terminal macrophage differentiation, accompanied by apoptotic cell death. These results show that the WT1 gene is an important regulator of M1 cell monocytic differentiation in vitro, and suggests a potential role for this gene in the molecular control of hematopoiesis.

Expression of the Wilms' Tumor Suppressor Gene, WT1, Is Upregulated by Leukemia Inhibitory Factor and Induces Monocytic Differentiation in M1 Leukemic Cells

The Wilms' tumor gene, WT1, encodes a transcription factor of the Cys2-His2 zinc finger type. The functional significance of WT1 expression in leukemias, in addition to tissues and cell lines of hematopoietic origin, has not been determined. Using the murine myeloblastic leukemia cell line M1 as a model for macrophage differentiation, expression of WT1 is shown to be activated in M1 cells 24 hours after differentiation induction by leukemia inhibitory factor (LIF). Upregulation of WT1 in these cells is associated with cellular differentiation, coinciding with expression of the monocyte/macrophage marker c-fms, and the appearance of mature cells. WT1 isoforms lacking the KTS insert are unable to be ectopically expressed in M1 cells. Stable expression of the WT1 isoforms containing the KTS insert leads to spontaneous differentiation of the M1 myeloblasts through the monocytic differentiation pathway. These cells express c-fms, in addition to the myeloid-specific cell surface marker Mac-1. Exposure of these cells to LIF results in the rapid onset of terminal macrophage differentiation, accompanied by apoptotic cell death. These results show that the WT1 gene is an important regulator of M1 cell monocytic differentiation in vitro, and suggests a potential role for this gene in the molecular control of hematopoiesis.

Do intronic mutations affecting splicing of WT1 exon 9 cause Frasier syndrome?

The WT1 gene, one of the genes responsible for Wilms tumour, is thought to play a crucial role in the development of the kidneys and gonads. This gene encodes four...

Regulation of renal EGF receptor expression is normal in Denys-Drash syndrome

In patients with Denys-Drash syndrome, mutations of the Wilms' tumor suppressor gene are associated with nephroblastomas and developmental abnormalities of the genital tract and renal glomerulus. Normally, the Wilms' tumor gene product (WT1) is expressed at high levels in visceral glomerular epithelial cells (VGEC) of the emerging fetal glomerulus. We demonstrate that WT1 could normally serve to suppress EGF receptor expression in VGEC, since immunoreactive EGF receptor is strikingly absent compared to epithelial cells of the emerging proximal and distal tubule, which lack WT1. When HEK293 cells were co-transfected with plasmids containing EGFR enhancer/promoter elements linked to a CAT reporter and plasmids containing WT1 cDNA, EGFR enhancer/promoter activity was suppressed by all wild-type WT1 isoforms, but not by deletion mutants of WT1 lacking normal zinc-finger or N-terminal domains. Surprisingly, plasmids expressing a Denys-Drash WT1 mutant (R394W) retained the ability to suppress EGFR promoter activity in this system. Furthermore, we found that immunoreactive EGFR was appropriately undetectable in glomeruli from a three-year-old girl with Denys-Drash syndrome and in sections of her Wilm's tumor. These data suggest that faulty suppression of EGFR cannot account for the abnormalities of glomerulogenesis seen in Denys-Drash patients.

Allelic imbalance at chromosome 1q21 in Wilms tumor

The Wilms tumor suppressor gene 1, WT1, located on chromosome 11p13 is mutated in only a subset of Wilms tumors. Cytogenetic studies of Wilms tumors show that the most frequent structural anomalies after those affecting chromosome 11p are rearrangements of 1q, suggesting that there is a gene involved in Wilms tumor etiology in this region. The WT1 target sequence +P5 (D1S3309E), isolated using whole-genome polymerase chain reaction (PCR), binds all WT1 isoforms in vitro and has been mapped to 1q21-22. As +P5 may mark a 1q Wilms tumor gene, constitutional and tumor DNA from 33 Wilms tumor patients (36 tumors) was screened for allele imbalance using microsatellite markers from 1p21 to 1q44. Although no gross rearrangements of the +P5 region were found, this study demonstrates allele imbalance for 1q in 12% of patients ( 5 36 tumors), defining a smallest region of overlap at 1q21. This finding supports a role for 1q21 in Wilms tumorigenesis.

Functional properties of WT1

WT1 encodes a zinc finger transcription factor that is inactivated in a subset of Wilms' tumors. We have recently shown that introduction of wild-type WT1 into a Wilms' tumor-derived cell line, RM1, results in growth suppression, consistent with its function as a tumor suppressor gene. WT1-mediated growth suppression was also observed in other cells derived from embryonal tumors, including two osteosarcoma cell lines, U2OS and Saos-2, notable for the respective presence or absence of wild-type p53. To further characterize the functional properties of WT1, multiple cell lines were established, expressing either wild-type WT1 splicing variants or naturally occurring mutants under control of a tightly regulated tetracycline repressable promoter. Induction of WT1 in these cells resulted in programmed cell death. This effect was preferentially mediated by WT1 isoform B (encoding alternative splice I, lacking alternative splice II “KTS”), and it was independent of p53, occurring in both U2OS and Saos-2 cells. WT1-mediated apoptosis was associated with transcriptional repression of the epidermal growth factor receptor (EGFR) and reduced synthesis of endogenous EGFR protein synthesis. Constitutive expression of EGFR abrogated WT1-mediated cell death. We conclude that wild-type WT1 can induce apoptosis in embryonal cancer cells, presumably through the withdrawal of required growth factor survival signals, and that EGFR is a physiological target gene for WT1. © 1996 Wiley-Liss, Inc.

Functional properties of WT1.

WT1 encodes a zinc finger transcription factor that is inactivated in a subset of Wilms' tumors. We have recently shown that introduction of wild-type WT1 into a Wilms' tumor-derived cell line, RM1, results in growth suppression, consistent with its function as a tumor suppressor gene. WT1-mediated growth suppression was also observed in other cells derived from embryonal tumors, including two osteosarcoma cell lines, U2OS and Saos-2, notable for the respective presence or absence of wild-type p53. To further characterize the functional properties of WT1, multiple U2OS and Saos-2 cell lines were established, expressing either wild-type WT1 splicing variants or naturally occurring mutants under control of a tightly regulated tetracycline repressable promoter. Induction of WT1 in these cells resulted in programmed cell death. This effect was preferentially mediated by WT1 isoform B (encoding alternative splice I, lacking alternative splice II "KTS"), and it was independent of p53, occurring in both U2OS and Saos-2 cells. WT1-mediated apoptosis was associated with transcriptional repression of the epidermal growth factor receptor (EGFR) and reduced synthesis of endogenous EGFR protein synthesis. Constitutive expression of EGFR abrogated WT1-mediated cell death. We conclude that wild-type WT1 can induce apoptosis in embryonal cancer cells, presumably through the withdrawal of required growth factor survival signals, and that EGFR is a physiological target gene for WT1.

An RNA recognition motif in Wilms' tumour protein (WT1) revealed by structural modelling

The Wilms' tumour suppressor gene 1 (WT1) (1,2) encodes four C2H2 zinc finger-containing proteins (3) critical for normal mammalian urogenital development (4). Mutations in this gene are observed in the childhood kidney cancer, Wilms' tumour (WT) (5). WT1 can bind specific DNA targets within the promoters of many genes (6-9) and both transcriptional repression and activation domains have been identified (10). On this basis, it has been assumed that regulation of transcription is the basis of WT1 tumour suppressor activity. However, subnuclear localization studies have revealed an association between WT1 proteins and 'speckled bodies' within the nucleus. Degradation of nuclear RNA in cells expressing WT1 abolishes this speckled localization and WT1 co-immunoprecipitates with a number of spliceosomal proteins, suggesting that it may also bind to RNA (11). Using structural rather than sequence comparison, we have now identified an evolutionarily conserved N-terminal RNA recognition motif (RRM) in all known WT1 isoforms similar to that in the constitutive splicing factor U1A. Given the association between WT1 mutations and Wilms' tumours, this study, together with other recent findings, may suggest a novel tumour suppression mechanism.

RNA processing. Wilms' tumour--the splicing connection?

Major isoforms of WT1--products of the tumour suppressor gene WT1, implicated in predisposition to Wilms' tumour--may preferentially interact with splicing factors, suggesting a role for WT1 in RNA processing.

WT1-mediated Transcriptional Activation Is Inhibited by Dominant Negative Mutant Proteins

The WT1 tumor suppressor gene encodes four isoforms of a zinc finger transcription factor with both activation and repression functions which are dependent upon promoter architecture. Using a simple HSV-tk promoter containing 5'-Egr-1/WT1-binding sites, we found that WT1 isoforms (A) and (B) strongly activated transcription. WT1(A) and (B) bound equally well to the Egr-1/WT1-binding site, but WT1(B), which contains a 17 amino acid insertion compared to WT1(A), was a consistently stronger activator of transcription than WT1(A). Transcriptional activation by wild-type WT1 was inhibited by coexpression of WT(PM) or WT(AR), genetically defined dominant negative alleles of WT1. In vitro, as well as in the yeast two-hybrid system, WT1 protein associated with itself and with dominant negative mutant proteins. The major domain required for self-association and inhibition of transcriptional activation mapped to the first 182 amino acids of WT1. Dominant negative WT1 alleles may play a role in tumorigenesis by associating with wild-type WT1 proteins and decreasing their transcriptional activity.

Expression of the wilms' tumor gene WT1 in human malignant mesothelioma cell lines and relationship to platelet‐derived growth factor A and insulin‐like growth factor 2 expression

Mutations in the WT1 tumor suppressor gene are known to contribute to the development of Wilms' tumor (WT) and associated gonadal abnormalities. WT1 is expressed principally in the fetal kidney, developing gonads, and spleen and also in the mesothelium, which lines the coelomic cavities. These tissues develop from mesenchymal components that have subsequently become epithelialized, and it has therefore been proposed that WT1 may play a role in this transition of cell types. To test the possible involvement of this gene in malignant mesothelioma, we have first studied its expression in a panel of human normal and malignant mesothelial cell lines. WT1 mRNA expression levels varied greatly between the cell lines and no specific chromosomal aberration on 11p, which could be related to the variation in WT1 expression in these cell lines, was observed. Furthermore, no gross deletions rearrangements, or functionally inactivating point mutations in the WT1 coding region were identified. All four WT1 splice variants were observed at similar levels in these cell lines. The WT1 gene encodes a zinc-finger transcription factor and the four protein isoforms are each believed to act as transcriptional repressors of certain growth factor genes. Lack of WTI expression in thus predicted to result in growth stimulation of tumor cells. Binding of one particular WT1 isoform construct to the insulin-like growth factor 2 (IGF2) and platelet-derived growth factor A (PDGFA) gene promoters has been demonstrated to result in repression of these genes in transient transfection studies. Analysis of IGF2 and PDGFA mRNA expression levels compared with WTI mRNA expression levels failed to demonstrate an inverse correlation in the mesothelial cell lines, which endogenously express these genes. Finally, the putative role of WT1 in the transition of cell types was investigated. No obvious correlation between WT1 expression levels and cell morphology of the malignant mesothelial cell lines was evident from this study. Moreover, no change in WT1 expression was observed in normal mesothelial cells which were, by alteration of culture conditions, manipulated to switch from the mesenchymal to epithelial morphology.

Does the Wilms' tumour suppressor gene, WT1, play roles in both splicing and transcription?

The Wilms' tumour suppressor gene (WT1) encodes a protein(s) with 4 zinc fingers that is essential for the development of the genitourinary system. A considerable body of evidence exists to support the idea that WT1 binds DNA and functions as a transcription factor. However, we have shown recently by confocal microscopy and immunoprecipitation studies that a significant proportion of WT1 is associated with splice factors in kidney cell lines, fetal tissues and transfected Cos cells. Different isoforms of WT1 are produced by an alternative splice that leads to the presence or absence of a 3 amino acid insertion (KTS) between zinc fingers 3 and 4. We have shown that these different forms localise differently in the nucleus. The +KTS form mainly localises with splice factors, the -KTS form mainly with transcription factors. Here we propose a model to account for these different localisations. Also, we discuss the possible significance of these findings.