Wilms Tumor Suppressor Gene Research Articles

Frasier Syndrome Diagnosed in a 16-Year-Old Girl Presenting for Evaluation of Amenorrhea

Primary care physicians commonly encounter patients requesting evaluation for primary and secondary amenorrhea. In most cases, the etiology can be easily determined by a complete history and physical with some laboratory tests. We discuss the interesting presentation and evaluation of amenorrhea in a 16-year-old woman with a history of end-stage renal disease resulting from focal segmental glomerulosclerosis. Sequencing of the Wilms tumor-suppressor gene (WT1) gene, located on chromosome 11p13, revealed a mutation confirming the rare diagnosis of Frasier syndrome. This article explores the current knowledge of the genetics and clinical presentation of Frasier syndrome and a related allelic disorder, Denys-Drash syndrome. As more patients with mutations of the WT1 gene are identified, our understanding of the complex functions of this protein and implications for clinical care will improve.

75 Tissue-Specific Activation of the Erythropoietin Gene by the Wilms' Tumor Suppressor Wt1(-KTS)

The mechanisms of the developmental-stage and tissue-specific regulation of erythropoietin (Epo) expression under normoxia are only partially understood yet. The Wilms tumor suppressor gene, Wt1, encodes a zinc finger transcription factor, which is required for the normal development of various organs, including the kidney, heart, and eye. Recent findings suggest a role of Wt1 in the development of the hematopoietic system. In our study, we tested the hypothesis that Wt1 is involved in Epo gene regulation. Using electrophoretic mobility shift assay and in vitro DNA-footprinting, we identified the binding of the Wt1(-KTS) variant, which acts as a transcription factor, to the minimal Epo promoter. Under normoxia, Epo mRNA and protein expression were significantly upregulated in human embryonic kidney HEK 293 cells, which had been stably transfected with Wt1. A reporter plasmid construct harboring the 117bp minimal human Epo promoter was activated more than 20-fold by the Wt1(-KTS) isoform in transient transfection assays. Mutations in the Wt1 binding site in the Epo promoter did not interfer with the hypoxic induction of reporter constructs harboring the downstream Epo enhancer. Hepatic Epo mRNA expression was significantly decreased in Wt1 −/− mouse embryos (e12.0). Immunohistochemical fluorescence-double-staining indicated also the co-expression of Wt1 and Epo in neuronal cells of the developing dorsal root ganglion. Co-expression has been also found in the adult testis were both genes may have antiapoptotic function. Preliminary data indicate furthermore that Wt1(-KTS) modulates the binding of other transcription factors, such as Sp1, to the Epo promoter. In conclusion, Wt1 is a novel transcriptional activator in the developmental-stage and tissue-specific regulation of Epo.

WT1 Activates a Glomerular-Specific Enhancer Identified from the Human Nephrin Gene

The glomerular filtration barrier separates the blood from the urinary space. Nephrin is a transmembrane protein that belongs to the immunoglobulin superfamily and is localized to the slit diaphragms that are a critical component of this filtration barrier. Mutations in the nephrin gene (NPHS1) lead to congenital Finnish nephropathy, whereas alterations in the level of nephrin expression have been identified in a wide range of acquired glomerular diseases. A 186-bp fragment from the human NPHS1 promoter is capable of directing podocyte-specific expression of a beta-galactosidase transgene when placed in front of a heterologous minimal promoter in transgenic mice. The Wilms tumor suppressor gene (WT1) is a zinc-finger-containing transcription factor that is coexpressed with NPHS1 in differentiated podocytes; gel shift binding assays demonstrate that a recombinant WT1 protein can bind and activate the 186-bp NPHS1 fragment in a sequence-specific manner. Taken together, these results suggest that WT1 may be required for regulation of the NPHS1 gene in vivo.

Wilms Tumor Gene (WT1) and p53 expression in endometrial carcinomas: a study of 130 cases using a tissue microarray

Objective. With the exception of ovarian serous carcinoma, Wilms tumor suppressor gene ( WT1) expression in common gynecologic carcinomas has not been described in detail. We studied a large number of endometrial carcinomas to determine the range of tumors that express WT1; this could have prognostic and therapeutic significance. Methods. We studied the immunohistochemical expression of WT1 and p53 in 130 primary human endometrial carcinomas of various histological subtypes, grades, and stages using a tissue microarray. The clinical data were retrieved from the medical records. Results. WT1 was expressed in a wide variety of endometrial cancers and was most marked in malignant mixed Mullerian tumors (MMMTs) (70% positive). WT1 expression was significantly correlated with high histological grade, and there was a trend toward a worse clinical outcome for patients whose tumors expressed WT1. An association between expression of WT1 and p53 and between these and outcome was noted in a univariate analysis, but only stage and p53 status remained prognostically significant independent variables. Conclusion. WT1 is expressed in appreciable numbers of endometrial cancers, particularly MMMTs. These findings support further investigation of WT1 as a possible therapeutic target in gynecologic malignancies.

Progesterone up-regulates WT1 mRNA and protein, and alters the relative expression of WT1 transcripts in cultured endometrial stromal cells.

To determine the change in expression of the Wilms tumor suppressor gene product, WT1, by progesterone alone in endometrial stromal cell culture and to study its relationship with prolactin, a marker of decidualization. In addition, to examine the change in ratio of WT1 isoforms with and without exon 5 message. Endometrial biopsies were taken from eight patients who had hysterectomy. Stromal cells were isolated and cultured in the presence of progesterone alone (12 days) or progesterone and 8-bromo-cyclic adenosine monophosphate (cAMP) (6 days). RNA was extracted from cells, and reverse transcription, real-time polymerase chain reaction (PCR), and conventional PCR were done to analyze WT1 mRNA expression. Immunocytochemistry was performed on equivalent cells to study WT1 protein expression. Decidualization was identified by increased prolactin concentrations in the media and immunocytochemical markers IGFBP-1 and collagen IV. Reverse transcription and real-time PCR revealed a significant increase in WT1 mRNA with increasing progesterone concentrations when decidualization was occurring (n = 6, P =.002). Increasing progesterone concentrations also increased the proportion of the WT1 transcript containing a 17-amino-acid insert (+ exon 5 expression); changes in WT1 exon 5 expression have been shown to be involved in control of proliferation and differentiation. Significant correlations between WT1 message and prolactin existed at physiologic progesterone concentrations (6.25, 12.5, 25, and 50 nM; P <.05) until prolactin concentrations reached a plateau at 100 nM. At concentrations of progesterone alone (> 25 nM) and progesterone with 8-bromo-cAMP, WT1 protein was localized to the nuclei of many of the decidualized stromal cells. The changing expression of WT1 isoforms in endometrial stromal cells caused by progesterone may be important for differentiation into the decidualized phenotype.

Usefulness of quantitative assessment of Wilms tumor suppressor gene expression in chronic myeloid leukemia patients undergoing imatinib therapy

The Wilms tumor suppressor gene (WT1) is overexpressed in a number of human hematological malignancies, including chronic myeloid leukemia (CML). Although at present, the biological significance of WT1 expression in CML in still unclear, this marker could represent a useful tool for molecular monitoring of CML patients prior to and post imatinib therapy. In fact, the use of real-time polymerase chaine reaction (PCR) to quantitatively measure the WT1 transcript amount may be a predictor of patient response to imatinib therapy. Semin Hematol 40(suppl 2):37-41. © 2003 Elsevier Inc. All rights reserved.

Usefulness of quantitative assessment of Wilms tumor suppressor gene expression in chronic myeloid leukemia patients undergoing imatinib therapy

The Wilms tumor suppressor gene (WT1) is overexpressed in a number of human hematological malignancies, including chronic myeloid leukemia (CML). Although at present, the biological significance of WT1 expression in CML in still unclear, this marker could represent a useful tool for molecular monitoring of CML patients prior to and post imatinib therapy. In fact, the use of real-time polymerase chaine reaction (PCR) to quantitatively measure the WT1 transcript amount may be a predictor of patient response to imatinib therapy. Semin Hematol 40(suppl 2):37-41. © 2003 Elsevier Inc. All rights reserved.

Regulation of taurine transporter gene ( TauT) by WT1

In the present study we have demonstrated that WT1 (Wilms tumor suppressor gene) enhances the expression of TauT (taurine transporter gene) in human embryonic kidney 293 cells in a dose-dependent manner. TauT promoter activity was increased five-fold by cotransfection of a full-length TauT promoter–reporter construct with WT1. Electrophoretic mobility shift assays (EMSAs) using nuclear extracts from WT1-overexpressing 293 cells showed a putative WT1-binding site in the basal promoter region of TauT, which bound to WT1 in EMSAs. Mutation of this WT1 consensus sequence abolished binding of WT1. These results demonstrate that TauT may represent a downstream target gene of WT1 during renal development.

Inhibition of Wilms Tumor 1 Transactivation by Bone Marrow Zinc Finger 2, a Novel Transcriptional Repressor

The Wilms tumor suppressor gene, wt1, encodes a zinc finger transcription factor that has been implicated in the regulation of a number of genes. Protein-protein interactions are known to modulate the transcription regulatory functions of Wilms tumor (WT1) and have also implicated WT1 in splicing. In this report, we identify a novel WT1-interacting protein, bone marrow zinc finger 2 (BMZF2), by affinity chromatography utilizing immobilized WT1 protein. BMZF2 is a potential transcription factor with 18 zinc fingers. The BMZF2 mRNA is mainly expressed in fetal tissues, and the protein is predominantly nuclear. Co-immunoprecipitation experiments are consistent with an in vivo association between WT1 and BMZF2. Glutathione S-transferase pulldown assays and far Western blots revealed that zinc fingers VI-X (amino acids 231-370) are required for interaction with the zinc finger region of WT1. Functionally, BMZF2 inhibits transcriptional activation by WT1. Moreover, a chimeric protein generated by fusion of BMZF2 to the GAL4 DNA-binding domain significantly decreases promoter activity of a reporter containing GAL4 DNA-binding sites, suggesting the presence of an active repressor domain within BMZF2. Our results suggest that BMZF2 interferes with the transactivation potential of WT1.

WT1, a multiform protein. Contribution of genetic models to the understanding of its various functions.

WT1 was initially identified as a Wilms tumour suppressor gene through the study of children presenting the contiguous gene syndrome WAGR ( W ilms tumor, A niridia, G enitourinary malformations, mental R etardation) due to large constitutional deletions of DNA in band 11p13 ([1][1],[2][2]). This 10-

The Wilms tumor suppressor WT1 regulates early gonad development by activation of Sf1.

In mammals, several genes including the Wilms tumor suppressor gene Wt1, the Lim homeobox gene Lhx9, and the gene encoding steroidogenic factor 1 (Sf1) have been implicated in the development of the indifferent gonad prior to sexual differentiation. Interactions among these genes have not yet been elucidated. Using biochemical and genetic experiments, we demonstrate here that WT1 and LHX9 function as direct activators of the Sf1 gene. Interestingly, only the -KTS form of WT1 is able to bind to and transactivate the Sf1 promoter. This observation is consistent with differential roles for the -KTS and +KTS variants of WT1 which have been postulated on the basis of human disorders such as the Frasier syndrome. Our data suggest a pathway in which the products of the Wt1 and Lhx9 genes activate expression of Sf1 and thus mediate early gonadogenesis.

Transcriptional regulation of podocyte specification and differentiation.

Glomerular visceral epithelial cells (podocytes) are highly specialized cells found in the vertebrate and invertebrate kidney and make up a major portion of the filtration barrier between blood and urinary spaces. During development, specification and differentiation of the podocyte lineage must be tightly orchestrated to produce highly specialized characteristics such as foot processes and slit diaphragms. Furthermore, podocytes are poised to direct incoming endothelial and mesangial cells during glomerular development. They express a number of growth factors that likely play a major role in these processes. Recent findings from transgenic and knockout mouse models and the identification of genes responsible for human podocyte disease have provided insight into transcriptional regulation of some of these processes. These transcription factors include Pax2, WT1 (the Wilms tumor suppressor gene), Pod1 (capsulin, epicardin), Kreisler (maf-1), lmx1b, and mf2. Furthermore, regulatory regions from a podocyte-restricted gene, NPHS1 (nephrin) that are required to direct podocyte-specific expression have been identified from both human and murine genes and provide a tool to further dissect the transcriptional regulation of podocyte-specific gene expression. This article reviews the present state of knowledge regarding transcriptional regulation of podocyte specification and differentiation.

Cloning and Characterization of Two Promoters for the Human HSAL2 Gene and Their Transcriptional Repression by the Wilms Tumor Suppressor Gene Product

HSAL2 is a member of a gene family that encodes a group of putative developmental transcription factors. The HSAL gene complex was originally identified on the basis of DNA sequence homology to a region-specific homeotic gene (SAL) in Drosophila. This study reveals a novel, functional 5' exon for HSAL2 and demonstrates that two distinct HSAL2 gene transcripts arise from two overlapping transcription units, resulting in proteins that differ by 25 amino acids. By utilizing functional luciferase reporter assays, two distinct promoters for HSAL2, P1 for the proximal promoter (upstream of exon 1) and P2 for the distal promoter (upstream of exon 1A), were identified. Evaluation of mRNA prevalence and tissue specificity, with particular focus on adult tissues, revealed that production of mRNA from P1 was selective and relatively rare. Production of mRNA from P2 was demonstrably higher and was expressed by a greater number of tissues. In contradistinction, HSAL2 expression directed by P2 was undetectable in some malignant populations as opposed to their normal human counterparts, suggesting a potential role as a tumor suppressor gene. Consensus-binding sites were identified for several transcriptional factors, with multiple sites for WT-1, and Hox-1.3 present within both the P1 and P2 regions. In transient transfection assays, transcription from both HSAL2 P1 and P2 was strikingly repressed by the WT-1 tumor suppressor protein. These findings suggest that an intracellular WT-1/HSAL2 pathway may play a role in development and hematopoiesis.

The murine Wilms tumor suppressor gene ( wt1) locus

The Wilms tumor suppressor gene WT1 plays a crucial role in the etiology of various human diseases as well as in the development of specific organs including the kidneys, gonads and the spleen. At present the human as well as the Fugu wt1 locus have been characterized. We have used a PAC clone to analyze the murine wt1 locus and report here the structure of the wt1 gene as well as a characterization of the nine wt1 introns regarding their size and sequence at the exon/intron and intron/exon boundaries. In addition we provide a restriction map of the murine wt1 locus which should prove useful for the cloning of various constructs designed for the generation of mouse models. Prompted by the existence of a WT1 antisense transcript in humans we also examined strand-specific transcription at the murine wt1 locus. Our analysis suggests that there is no detectable antisense transcription of sequences within or immediately downstream of wt1 exon 1. We find, however, evidence for a divergent transcript which encompasses sequences at and around minor transcriptional initiation sites of wt1 and which is transcribed in the opposite direction. Despite the very high degree of similarity between the human and the murine wt1 sequence and expression as well as the presence of divergent transcripts in both cases, the existence of antisense transcription does not seem to be conserved between the two species.

Absence of PPP2R1A mutations in Wilms tumor.

Evidence from genetic linkage analysis indicates that a gene located at 19q13.4, FWT2, is responsible for predisposition to Wilms tumor in many Wilms tumor families. This region has also been implicated in the etiology of sporadic Wilms tumor through loss of heterozygosity analyses. The PPP2R1A gene, encoding the alpha isoform of the heterotrimeric serine/threonine protein phosphatase 2A (PP2A), is located within the FWT2 candidate region and is altered in breast and lung carcinomas. PPP2R1B, encoding the beta isoform, is mutated in lung, colon, and breast cancers. These findings suggested that both PPP2R1A and PPP2R1B may be tumor suppressor genes. Additionally, PP2A is important in fetal kidney growth and differentiation and has an expression pattern similar to that of the Wilms tumor suppressor gene WT1. Since PPP2R1A was therefore a compelling candidate for the FWT2 gene, we analysed the coding region of PPP2R1A in DNA and RNA samples from affected members of four Wilms tumor families and 30 sporadic tumors and identified no mutations in PPP2R1A in any of these 34 samples. We conclude that PPP2R1A is not the 19q familial Wilms tumor gene and that mutation of PPP2R1A is not a common event in the etiology of sporadic Wilms tumor.

The speckling domain of the Wilms tumor suppressor WT1 overlaps with the transcriptional repression domain

The Wilms tumor suppressor gene WT1 encodes a zinc finger protein, expressed as different splicing variants, that has all the hallmarks of a transcription factor. The −KTS form of WT1 displays a homogeneous localization within the nucleus and has been shown to activate or repress the activity of various target genes. In contrast, the WT1(+KTS) variant demonstrates a speckled pattern of expression within the nucleus. This and its association with factors of the splicing machinery has led to the hypothesis that WT1(+KTS) might play a role in post-transcriptional processes. By the generation of a series of deletion constructs and subsequent immunofluorescence analysis, we have identified and characterized the domain which is responsible for the localization of WT1 variants in nuclear speckles. The speckling domain comprises amino acids 76–120 within the N-terminus of WT1 and is sufficient to target other proteins into distinct nuclear domains. Interestingly the WT1 speckling domain does not overlap with the domain required for interaction with the splicing factor U2AF65 but overlaps with the transcriptional repression domain. Thus our data challenge the view that association of WT1 with spliceosomes is responsible for the speckling phenotype.

Germline Wilms tumor suppressor gene (WT1) mutation leading to isolated genital malformation without Wilms tumor or nephropathy

Mutations of the Wilms tumor suppressor gene (WT1 ) have been described only in patients with syndromes associated with urogenital malformation and Wilms tumor or nephropathy. We present a male patient with an isolated genital malformation caused by a WT1 mutation. (J Pediatr 2001;138:421-4)

The role of NF-kappaB in the regulation of the expression of wilms tumor suppressor gene WT1.

The Wilms tumor suppressor gene, WT1, plays an important role in genitourinary development and the etiology of Wilms tumor. WT1 has a spatially and temporally defined expression in the developing genitourinary system and in specific cells of the hematopoietic system, but the regulatory pathways that control WT1 expression are not well understood. Recently, members of the NF-kappaB family of transcription factors have been proposed as potent activators of the murine WT1 promoter through binding to a NF-kappaB site. Because the human WT1 promoter contains a conserved NF-kappaB site, we investigated whether NF-kappaB also regulates the expression of the human WT1 gene. We activated NF-kappaB through cytokine stimulation or inhibited NF-kappaB through expression of a NF-kappaB "super repressor" in WT1 expressing Wilms tumor, renal carcinoma, and erythroleukemia cultures and examined the level of endogenous WT1 gene expression. Although a transfected NF-kappaB reporter construct was responsive to these manipulations, we found that altering NF-kappaB activity had no effect on endogenous WT1 expression in the cell types used in our study. We conclude that despite the presence of conserved NF-kappaB elements in the murine and human WT1 promoters, NF-kappaB is not required to regulate the expression of the WT1 gene in its natural context.

Isolated diffuse mesangial sclerosis and Wilms tumor suppressor gene

Diffuse mesangial sclerosis is a rare renal disease, occurring either in isolation or as part of Denys-Drash syndrome. Denys-Drash syndrome originates from mutations of the Wilms tumor suppressor gene (WT1 ). We describe the presence of WT1 mutations in 7 Japanese children with isolated diffuse mesangial sclerosis. (J Pediatr 2001;138:425-7)

Presence of WT1, the Wilm's Tumor Suppressor Gene Product, in Nuclear Poly(A)+ Ribonucleoprotein

The tumor suppressor gene WT1 encodes a zinc finger protein, which consists of four C-terminal C(2)-H(2) zinc fingers of the Krüppel type, and at the N terminus a Q/P-rich trans-regulatory domain, both characteristic of transcription factors. However, recent findings suggest that WT1 may also be involved in a post-transcriptional process. Specifically, WT1 isoforms containing the alternatively spliced exon 9 (+lysine-threonine-serine (KTS)) preferentially associate with nuclear speckles and co-immunoprecipitate splicing antigens (Larsson, S. H., Charlieu, J.-P., Miyagawa, K., Engelkamp, D., Rassoulzadegan, M., Ross, A., Cuzin, F., van Heyningen, V., and Hastie, N. D. (1995) Cell 81, 391-401); furthermore, WT1 has been shown to interact with the ubiquitous splicing factor U2AF65 (Davies, R. C., Calvo, C., Larsson, S. H., Lamond, A. I., and Hastie, N. D. (1998) Genes Dev. 12, 3217-3225) and binds to RNA in vitro (Caricasole, A., Duarte, A., Larsson, S. H., Hastie, N. D., Little, M., Holmes, G., Todorov, I., and Ward, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7562-7566; Bardeesy, N., and Pelletier, J. (1998) Nucleic Acids Res. 26, 1784-1792). To extend these findings, we have fractionated nuclear extracts to see if particles containing WT1 have the properties of ribonucleoprotein (RNP). In summary, WT1 is enriched by oligo(dT) chromatography, as are U2AF65, the U5 small nuclear RNP-associated protein p116 and hnRNP A1. Gel filtration and sedimentation profiles suggest that WT1 is present in RNase-sensitive particles, >2 MDa in size, peaking at approximately 60 S, and approximately 1.27 g/cm(3) on Nycodenz. Similar results were obtained from two cell lines expressing WT1, fetal kidneys (day E17), and transiently transfected cells, suggesting that the presence of WT1 protein in nuclear poly(A)(+) RNP is a general aspect of WT1 function.