Target Of WT1 Research Articles

Epicardial cell shape and maturation are regulated by Wt1 via transcriptional control of Bmp4.

The epicardium plays a crucial role in embryonic heart development and adult heart repair; however, the molecular events underlying its maturation remain unknown. Wt1, one of the main markers of the embryonic epicardium, is essential for epicardial development and function. Here, we analyse the transcriptomic profile of epicardial-enriched cells at different stages of development and from control and epicardial-specific Wt1 knockout (Wt1KO) mice. Transcriptomic and cell morphology analyses of epicardial cells from epicardial-specific Wt1KO mice revealed a defect in the maturation process of the mutant epicardium, including sustained upregulation of Bmp4 expression and the inability of mutant epicardial cells to transition into a mature squamous phenotype. We identified Bmp4 as a transcriptional target of Wt1, thus providing a molecular basis for the retention of the cuboidal cell shape observed in the Wt1KO epicardium. Accordingly, inhibition of the Bmp4 signalling pathway both ex vivo and in vivo rescued the cuboidal phenotype of the mutant epicardium. Our findings indicate the importance of the cuboidal-to-squamous transition in epicardial maturation, a process regulated by Wt1.

The Wilms tumor protein WT1 stimulates transcription of the gene encoding insulin-like growth factor binding protein 5 (IGFBP5)

Insulin-like growth factor (IGF) binding proteins (IGFBPs) constitute a family of six secreted proteins that regulate the signaling of insulin-like growth factors (IGFs). IGFBP5 is the most conserved family member in vertebrates and the major IGF binding protein in bone. IGFBP5 is required for normal development of the musculoskeletal system, and various types of cancer frequently express high levels of IGFP5. Here we identify the gene encoding IGFBP5 as a novel downstream target of the Wilms tumor protein WT1. IGFBP5 and WT1 are expressed in an overlapping pattern in the condensing metanephric mesenchyme of embryonic murine kidneys. Down-regulation of WT1 by transfection with antisense vivo-morpholino significantly decreased Igfbp5 transcripts in murine embryonic kidney explants. Likewise, silencing of Wt1 in a mouse mesonephros-derived cell line reduced Igfbp5 mRNA levels by approximately 80%. Conversely, induction of the WT1(−KTS) isoform, whose role as transcriptional regulator has been firmly established, significantly increased IGFBP5 mRNA and protein levels in osteosarcoma cells. IGFBP5 expression was not significantly changed by WT1(+KTS) protein, which exhibits lower DNA binding affinity than the WT1(−KTS) isoform and has a presumed role in post-transcriptional gene regulation. Luciferase reporter constructs harboring 0.8 and 1.6 kilobases of the murine Igfbp5 promoter, respectively, were stimulated approximately 5-fold by co-transfection of WT1(−KTS). The WT1(+KTS) variant had no significant effect on IGFBP5 promoter activity. Binding of WT1(−KTS), but not of WT1(+KTS) protein, to the IGFBP5 promoter in human osteosarcoma cells was proven by chromatin immunoprecipitation (ChIP) and confirmed by electrophoretic mobility shift assay. These findings demonstrate that WT1 activates transcription of the IGFBP5 gene with possible implications for kidney development and bone (patho)physiology.

The Telomeric Protein TRF2 Regulates Angiogenesis by Binding and Activating the PDGFRβ Promoter

Telomeric repeat binding factor 2 (TRF2), which plays a central role in telomere capping, is frequently increased in human tumors. We reveal here that TRF2 is expressed in the vasculature of most human cancer types, where it colocalizes with the Wilms' tumor suppressor WT1. We further show that TRF2 is a transcriptional target of WT1 and is required for proliferation, migration, and tube formation of endothelial cells. These angiogenic effects of TRF2 are uncoupled from its function in telomere capping. Instead, TRF2 binds and transactivates the promoter of the angiogenic tyrosine kinase platelet-derived growth factor receptor β (PDGFRβ). These findings reveal an unexpected role of TRF2 in neoangiogenesis and delineate a distinct function of TRF2 as a transcriptional regulator.

Ubiquitin specific protease 18 (Usp18) is a WT1 transcriptional target

Wilms tumor gene WT1 encodes a zinc finger-containing transcription factor which is required for renal development. Mutations in WT1 are observed in 20% of Wilms tumors (a pediatric kidney cancer), but the in vivo WT1 targets and associated molecular pathways involved in the etiology of Wilms tumor are still elusive. To identify WT1 targets we performed genome-wide comprehensive expression profiling using Affymetrix Gene Chip Mouse Genome 430 2.0 Arrays, comparing E13.5 mouse kidneys in which Wt1 had been somatically ablated with littermate controls. We identified Usp18 as the most differentially expressed gene in mutant kidney. Using tetracycline inducible cells we demonstrated a repressive effect of WT1 on USP18 expression. Conversely, knockdown of WT1 led to the upregulation of Usp18. Furthermore, direct binding of WT1 to the Usp18 promoter was demonstrated by ChIP assay. Overexpression of USP18 in murine and human cell lines resulted in cell proliferation. Additionally, Usp18 upregulation was observed in a mouse model of Wilms tumor. Taken together our data demonstrate that Usp18 is a transcriptional target of WT1 and suggest that increased expression of USP18 following WT1 loss contributes to Wilms tumorigenesis.

Wt1 controls retinoic acid signalling in embryonic epicardium through transcriptional activation of Raldh2.

Epicardial-derived signals are key regulators of cardiac embryonic development. An important part of these signals is known to relate to a retinoic acid (RA) receptor-dependent mechanism. RA is a potent morphogen synthesised by Raldh enzymes, Raldh2 being the predominant one in mesodermal tissues. Despite the importance of epicardial retinoid signalling in the heart, the molecular mechanisms controlling cardiac Raldh2 transcription remain unknown. In the current study, we show that Wt1-null epicardial cells display decreased expression of Raldh2 both in vivo and in vitro. Using a RA-responsive reporter, we have confirmed that Wt1-null epicardial cells actually show reduced synthesis of RA. We also demonstrate that Raldh2 is a direct transcriptional target of Wt1 in epicardial cells. A secondary objective of this study was to identify the status of RA-related receptors previously reported to be critical to epicardial biology (PDGFRα,β; RXRα). PDGFRα and PDGFRβ mRNA and protein levels are downregulated in the absence of Wt1, but only Pdgfra expression is rescued by the addition of RA to Wt1-null epicardial cells. RXRα mRNA levels are not affected in Wt1-null epicardial cells. Taken together, our results indicate that Wt1 critically regulates epicardial RA signalling via direct activation of the Raldh2 gene, and identify a role for Wt1 in the regulation of morphogen receptors involved in the proliferation, migration, and differentiation of epicardial and epicardially-derived cells (EPDC).

An integrated genome screen identifies the Wnt signaling pathway as a major target of WT1

WT1, a critical regulator of kidney development, is a tumor suppressor for nephroblastoma but in some contexts functions as an oncogene. A limited number of direct transcriptional targets of WT1 have been identified to explain its complex roles in tumorigenesis and organogenesis. In this study we performed genome-wide screening for direct WT1 targets, using a combination of ChIP-ChIP and expression arrays. Promoter regions bound by WT1 were highly G-rich and resembled the sites for a number of other widely expressed transcription factors such as SP1, MAZ, and ZNF219. Genes directly regulated by WT1 were implicated in MAPK signaling, axon guidance, and Wnt pathways. Among directly bound and regulated genes by WT1, nine were identified in the Wnt signaling pathway, suggesting that WT1 modulates a subset of Wnt components and responsive genes by direct binding. To prove the biological importance of the interplay between WT1 and Wnt signaling, we showed that WT1 blocked the ability of Wnt8 to induce a secondary body axis during Xenopus embryonic development. WT1 inhibited TCF-mediated transcription activated by Wnt ligand, wild type and mutant, stabilized beta-catenin by preventing TCF4 loading onto a promoter. This was neither due to direct binding of WT1 to the TCF binding site nor to interaction between WT1 and TCF4, but by competition of WT1 and TCF4 for CBP. WT1 interference with Wnt signaling represents an important mode of its action relevant to the suppression of tumor growth and guidance of development.

WT1-Mediated Gene Regulation in Early Urogenital Ridge Development

The Wilms tumor protein WT1 is involved in the development of several organs, including the gonads. WT1 mutations in humans lead to syndromes associated with impaired sexual development and Wt1 knockout mice show regression of gonad anlagen. As a transcription factor, WT1 fulfills its function by regulating a set of target genes. With respect to gonad development only few in vivo WT1 targets, e.g. steroidogenic factor 1 (SF1) have been identified so far. To get a comprehensive view of WT1 targets in the gonad, we compared gene expression in urogenital ridges of wild-type and Wt1^–/– embryos. We found almost 150 genes differentially expressed higher than factor three, using microarray analysis. To confirm these results we performed quantitative real-time RT-PCR for many genes and observed a high degree of concordance between microarray and real-time RT-PCR results. Employing in situ hybridization we found ‘WT1 activated genes’ to be expressed in gonads, mesonephroi and coelomic epithelium – those parts of the urogenital ridge with Wt1 expression. Interestingly, many of the differentially expressed genes are known to show sex-specific expression at later time-points. These results provide a basis for investigation of developmental pathways in the urogenital ridge downstream of WT1 and for identification of new candidate genes involved in early urogenital ridge development. For example we provide a first potential target of WT1 in the coelomic epithelium – Muc16, and a gene regulated by the WT1 target SF1 – Gata4.

The Wilms' Tumor Suppressor Wt1 Activates Transcription of the Erythropoietin-Receptor in Hematopoietic Progenitor Cells.

The Wilms' tumor suppressor, Wt1, has been implicated in the stage-specific quiescence and differentiation of hematopoietic progenitor cells. Since Wt1 deficiency compromises the proliferation and differentiation of erythroid blast-forming units (BFU-E) and colony-forming units (CFU-E), we analyzed the potential role of the transcriptionally active Wt1 isoform, Wt1(-KTS), in regulating the expression of the erythropoietin receptor (EpoR). CD117-positive hematopoietic progenitor cells isolated from the liver of Wt1-deficient murine embryos (Wt1−/−) at 11.5 dpc exhibited a 10-fold lower proliferative responsiveness to recombinant erythropoietin than CD117-positive cells from wild-type embryos (Wt1+/+), or embryos with heterozygous Wt1-deletion (Wt1+/−). Benzidine staining revealed a reduced fraction of hemoglobin-containing cells in Wt1-deficient cells in comparison to Wt1+/+ cells after 48 hrs of culture of CD117-positive cells in the presence of 6 U rhEpo/ml medium. Consistently, expression of the EpoR was significantly reduced in hematopoietic progenitor cells that lack Wt1 compared to Wt1+/+ cells. The decrease of EpoR mRNA was not due to a general down-regulation of gene expression in Wt1−/− CD117-positive cells, since levels of c-kit and c-mpl expression were normal. In the heart of wild-type and Wt1−/− embryos, which was analyzed as a non-hematopoietic tissue expressing both EpoR und Wt1, no difference in Epo-R expression was found. Transient transfection of Wt1(-KTS) into human hepatoma-derived HepG2 cells increased EpoR transcripts approximately 2-fold. A luciferase reporter plasmid carrying 309 bp of the proximal human EpoR promoter was stimulated 8-fold by co-transfection with Wt1(-KTS). The responsible cis-element in the EpoR promoter was identified by mutation analysis, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay. In summary, we identified the EpoR gene to be the first downstream target of Wt1 within the hematopoietic cytokine receptor superfamily. Our data indicate that activation of the EpoR gene by Wt1 is a critical mechanism in normal murine hematopoiesis. However, our data do not support a role for Wt1 in EpoR expression in non-erythoid tissues, such as the heart.

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.

Sprouty1 Is a Critical Regulator of GDNF/RET-Mediated Kidney Induction

Intercellular signaling molecules and their receptors, whose expression must be tightly regulated in time and space, coordinate organogenesis. Regulators of intracellular signaling pathways provide an additional level of control. Here we report that loss of the receptor tyrosine kinase (RTK) antagonist, Sprouty1 (Spry1), causes defects in kidney development in mice. Spry1(-/-) embryos have supernumerary ureteric buds, resulting in the development of multiple ureters and multiplex kidneys. These defects are due to increased sensitivity of the Wolffian duct to GDNF/RET signaling, and reducing Gdnf gene dosage correspondingly rescues the Spry1 null phenotype. We conclude that the function of Spry1 is to modulate GDNF/RET signaling in the Wolffian duct, ensuring that kidney induction is restricted to a single site. These results demonstrate the importance of negative feedback regulation of RTK signaling during kidney induction and suggest that failures in feedback control may underlie some human congenital kidney malformations.

Anlaysis of complementary expression profiles following WT1 induction versus repression reveals the cholesterol/fatty acid synthetic pathways as a possible major target of WT1.

The Wilms' tumour suppressor gene, WT1, encodes a zinc-finger protein that is mutated in Wilms' tumours and other malignancies. WT1 is one of the earliest genes expressed during kidney development. WT1 proteins can activate and repress putative target genes in vitro, although the in vivo relevance of such target genes often remains unverified. To better understand the role of WT1 in tumorigenesis and kidney development, we need to identify downstream target genes. In this study, we have expression profiled human embryonic kidney 293 cells stably transfected to allow inducible WT1 expression and mouse mesonephric M15 cells transfected with a WT1 antisense construct to abolish endogenous expression of all WT1 isoforms to identify WT1-responsive genes. The complementary overlap between the two cell lines revealed a pronounced repression of genes involved in cholesterol biosynthesis by WT1. This pathway is transcriptionally regulated by the sterol responsive element-binding proteins (SREBPs). Here, we provide evidence that the C-terminal end of the WT1 protein can directly interact with SREBP, suggesting that WT1 may modify the transcriptional function of SREBPs via a direct protein-protein interaction. Therefore, the tumour suppressor activities of WT1 may be achieved by repressing the mevalonate pathway, thereby controlling cellular proliferation and promoting terminal differentiation.

The Receptor Tyrosine Kinase Regulator Sprouty1 Is a Target of the Tumor Suppressor WT1 and Important for Kidney Development

WT1 encodes a transcription factor involved in kidney development and tumorigenesis. Using representational difference analysis, we identified a new set of WT1 targets, including a homologue of the Drosophila receptor tyrosine kinase regulator, sprouty. Sprouty1 was up-regulated in cell lines expressing wild-type but not mutant WT1. WT1 bound to the endogenous sprouty1 promoter in vivo and directly regulated sprouty1 through an early growth response gene-1 binding site. Expression of Sprouty1 and WT1 overlapped in the developing metanephric mesenchyme, and Sprouty1, like WT1, plays a key role in the early steps of glomerulus formation. Disruption of Sprouty1 expression in embryonic kidney explants by antisense oligonucleotides reduced condensation of the metanephric mesenchyme, leading to a decreased number of glomeruli. In addition, sprouty1 was expressed in the ureteric tree and antisense-treated ureteric trees had cystic lumens. Therefore, sprouty1 represents a physiologically relevant target gene of WT1 during kidney development.

Upregulation of c-MYC in WT1-mutant tumors: assessment of WT1 putative transcriptional targets using cDNA microarray expression profiling of genetically defined Wilms' tumors.

The Wilms' tumor suppressor gene, WT1, functions as a transcriptional regulator that represses or activates the expression of a variety of putative target genes. However, it is not clear which genes are the biological targets of WT1, nor which cellular pathway(s) is critically altered in tumors as a result of WT1 mutation. To investigate in vivo the role of WT1 as a transcription factor in Wilms' tumors, we used cDNA microarrays to compare the expression of putative WT1 target genes in a set of 15 primary Wilmstumors carrying WT1-inactivating mutations versus a set of 16 tumors with no WT1 mutations. We hypothesized that the expression of direct downstream targets of WT1 that are relevant to tumor development would differ between these two genetically distinct sets of tumors. Using the Atlas Human Cancer 1.2 cDNA arrays to quantitate gene expression in the 31 tumors, we found that the expression of one WT1 putative target gene, c-MYC, statistically significantly differed between the two sets of tumors and was upregulated in WT1-mutant tumors. This increase of relative gene expression for c-MYC was confirmed using real-time reverse transcription-polymerase chain reaction. The differential expression of another putative target gene, EGR1, approached significance and was also upregulated in WT1-mutant tumors. These data, in addition to the coexpression of c-MYC and WT1 in embryonic renal mesenchyme, strongly suggest that c-MYC is a biologically relevant target of WT1 in Wilmstumors.

Functional characterization of WT1 binding sites within the human vitamin D receptor gene promoter.

The Wilms' tumor suppressor gene, wt1, encodes a zinc finger transcription factor that can regulate gene expression. It plays an essential role in tumorigenesis, kidney differentiation, and urogenital development. To identify WT1 downstream targets, gene expression profiling was conducted using a cDNA array hybridization approach. We confirm herein that the human vitamin D receptor (VDR), a ligand-activated transcription factor, is a WT1 downstream target. Nuclear run on experiments demonstrated that the effect of WT1 on VDR expression is at the transcriptional level. Transient transfection assays, deletion mutagenesis, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays suggest that, although WT1 is presented with a possibility of three binding sites within the VDR promoter, activation of the human VDR gene appears to occur through a single site. This site differs from a previously identified WT1-responsive site in the murine VDR promoter (Maurer U, Jehan F, Englert C, Hübinger G, Weidmann E, DeLucas HF, and Bergmann L. J Biol Chem 276: 3727-3732, 2001). We also show that the products of a Denys-Drash syndrome allele of wt1 inhibit WT1-mediated transactivation of the human VDR promoter. Our results indicate that the human VDR gene is a downstream target of WT1 and may be regulated differently than its murine counterpart.

The Human Sex-determining Gene SRY Is a Direct Target of WT1

The product of the Wilms' tumor gene, WT1, is essential for male sex determination and differentiation in mammals. In addition to causing Wilms' tumor, mutations in WT1 often cause two distinct but overlapping urogenital defects in men, Denys-Drash syndrome and Frasier syndrome. In this study we investigated the regulation of the sex determination gene SRY by WT1. Our results showed that WT1 up-regulates the SRY gene through the proximal early growth response gene-1-like DNA-binding sequences in the core promoter. Mutant WT1 proteins in Denys-Drash syndrome patients were unable to activate this promoter. These mutants did not act in a dominant negative manner, as expected over the wild-type WT1 in this promoter. We also found that WT1 could transactivate the endogenous SRY gene. These observations, together with the overlapping expression patterns of WT1 and SRY in human gonads, led us to propose that WT1 regulates SRY in the initial sex determination process in humans and activates a cascade of genes ultimately leading to the complete organogenesis of the testis.

The Wilms Tumor Suppressor WT1 Encodes a Transcriptional Activator of amphiregulin

WT1 encodes a zinc finger transcription factor implicated in kidney differentiation and tumorigenesis. In reporter assays, WT1 represses transcription from GC- and TC-rich promoters, but its physiological targets remain uncertain. We used hybridization to high-density oligonucleotide arrays to search for native genes whose expression is altered following inducible expression of WT1. The major target of WT1 was amphiregulin, a member of the epidermal growth factor family. The WT1(−KTS) isoform binds directly to the amphiregulin promoter, resulting in potent transcriptional activation. The in vivo expression profile of amphiregulin during fetal kidney development mirrors the highly specific pattern of WT1 itself, and recombinant Amphiregulin stimulates epithelial branching in organ cultures of embryonic mouse kidney. These observations suggest a model for WT1 as a transcriptional regulator during kidney differentiation.

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.