Transcription factor Wilms' tumor 1 regulates developmental RNAs through 3' UTR interaction.

Wilms' tumor 1 (WT1) is essential for the development and homeostasis of multiple mesodermal tissues. Despite evidence for post-transcriptional roles, no endogenous WT1 target RNAs exist. Using RNA immunoprecipitation and UV cross-linking, we show that WT1 binds preferentially to 3' untranslated regions (UTRs) of developmental targets. These target mRNAs are down-regulated upon WT1 depletion in cell culture and developing kidney mesenchyme. Wt1 deletion leads to rapid turnover of specific mRNAs. WT1 regulates reporter gene expression through interaction with 3' UTR-binding sites. Combining experimental and computational analyses, we propose that WT1 influences key developmental and disease processes in part through regulating mRNA turnover.

WT1 regulates the expression of inhibitory chemokines during heart development.

The embryonic epicardium is an important source of cardiovascular precursor cells and paracrine factors that are required for adequate heart formation. Signaling pathways regulated by WT1 that promote heart development have started to be described; however, there is little information on signaling pathways regulated by WT1 that could act in a negative manner. Transcriptome analysis of Wt1KO epicardial cells reveals an unexpected role for WT1 in repressing the expression of interferon-regulated genes that could be involved in a negative regulation of heart morphogenesis. Here, we showed that WT1 is required to repress the expression of the chemokines Ccl5 and Cxcl10 in epicardial cells. We observed an inverse correlation of Wt1 and the expression of Cxcl10 and Ccl5 during epicardium development. Chemokine receptor analyses of hearts from Wt1(gfp/+) mice demonstrate the differential expression of their chemokine receptors in GFP(+) epicardial enriched cells and GFP(-) cells. Functional assays demonstrate that CXCL10 and CCL5 inhibit epicardial cells migration and the proliferation of cardiomyocytes respectively. WT1 regulates the expression levels of Cxcl10 and Ccl5 in epicardial cells directly and indirectly through increasing the levels of IRF7. As epicardial cell reactivation after a myocardial damage is linked with WT1 expression, the present work has potential implications in adult heart repair.

Mice lacking the 68-amino-acid, mammal-specific N-terminal extension of WT1 develop normally and are fertile.

Mutations in the Wilms' tumor 1 gene, WT1, cause pediatric nephroblastoma and the severe genitourinary disorders of Frasier and Denys-Drash syndromes. High levels of WT1 expression are found in the developing kidney, uterus, and testis--consistent with this finding, the WT1 knockout mouse demonstrates that WT1 is essential for normal genitourinary development. The WT1 gene encodes multiple isoforms of a zinc finger-containing protein by a combination of alternative splicing and alternative translation initiation. The use of an upstream, alternative CUG translation initiation codon specific to mammals results in the production of WT1 protein isoforms with a 68-amino-acid N-terminal extension. To determine the function in vivo of mammal-specific WT1 isoforms containing this extension, gene targeting was employed to introduce a subtle mutation into the WT1 gene. Homozygous mutant mice show a specific absence of the CUG-initiated WT1 isoforms yet develop normally to adulthood and are fertile. Detailed histological analysis revealed normal development of the genitourinary system.

WT1 expression in breast cancer disrupts the epithelial/mesenchymal balance of tumour cells and correlates with the metabolic response to docetaxel.

WT1 is a transcription factor which regulates the epithelial-mesenchymal balance during embryonic development and, if mutated, can lead to the formation of Wilms' tumour, the most common paediatric kidney cancer. Its expression has also been reported in several adult tumour types, including breast cancer, and usually correlates with poor outcome. However, published data is inconsistent and the role of WT1 in this malignancy remains unclear. Here we provide a complete study of WT1 expression across different breast cancer subtypes as well as isoform specific expression analysis. Using in vitro cell lines, clinical samples and publicly available gene expression datasets, we demonstrate that WT1 plays a role in regulating the epithelial-mesenchymal balance of breast cancer cells and that WT1-expressing tumours are mainly associated with a mesenchymal phenotype. WT1 gene expression also correlates with CYP3A4 levels and is associated with poorer response to taxane treatment. Our work is the first to demonstrate that the known association between WT1 expression in breast cancer and poor prognosis is potentially due to cancer-related epithelial-to-mesenchymal transition (EMT) and poor chemotherapy response.

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source.

Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes.

A universal vector for high-efficiency multi-fragment recombineering of BACs and knock-in constructs.

There is an increasing need for more efficient generation of transgenic constructs. Here we present a universal multi-site Gateway vector for use in recombineering reactions. Using transgenic mouse models, we show its use for the generation of BAC transgenics and targeting vectors. The modular nature of the vector allows for rapid modification of constructs to generate different versions of the same construct. As such it will help streamline the generation of series of related transgenic models.

A wt1-controlled chromatin switching mechanism underpins tissue-specific wnt4 activation and repression.

Wt1 regulates the epithelial-mesenchymal transition (EMT) in the epicardium and the reverse process (MET) in kidney mesenchyme. The mechanisms underlying these reciprocal functions are unknown. Here, we show in both embryos and cultured cells that Wt1 regulates Wnt4 expression dichotomously. In kidney cells, Wt1 recruits Cbp and p300 as coactivators; in epicardial cells it enlists Basp1 as a corepressor. Surprisingly, in both tissues, Wt1 loss reciprocally switches the chromatin architecture of the entire Ctcf-bounded Wnt4 locus, but not the flanking regions; we term this mode of action "chromatin flip-flop." Ctcf and cohesin are dispensable for Wt1-mediated chromatin flip-flop but essential for maintaining the insulating boundaries. This work demonstrates that a developmental regulator coordinates chromatin boundaries with the transcriptional competence of the flanked region. These findings also have implications for hierarchical transcriptional regulation in development and disease.

Wt1 is required for cardiovascular progenitor cell formation through transcriptional control of Snail and E-cadherin.

The epicardial epithelial-mesenchymal transition (EMT) is hypothesized to generate cardiovascular progenitor cells that differentiate into various cell types, including coronary smooth muscle and endothelial cells, perivascular and cardiac interstitial fibroblasts and cardiomyocytes. Here we show that an epicardial-specific knockout of the gene encoding Wilms' tumor-1 (Wt1) leads to a reduction in mesenchymal progenitor cells and their derivatives. We show that Wt1 is essential for repression of the epithelial phenotype in epicardial cells and during embryonic stem cell differentiation through direct transcriptional regulation of the genes encoding Snail (Snai1) and E-cadherin (Cdh1), two of the major mediators of EMT. Some mesodermal lineages do not form in Wt1-null embryoid bodies, but this effect is rescued by the expression of Snai1, underscoring the importance of EMT in generating these differentiated cells. These new insights into the molecular mechanisms regulating cardiovascular progenitor cells and EMT will shed light on the pathogenesis of heart diseases and may help the development of cell-based therapies.

High-efficiency Rosa26 knock-in vector construction for Cre-regulated overexpression and RNAi.

INTRODUCTION: Rosa26 is a genomic mouse locus commonly used to knock-in cDNA constructs for ubiquitous or conditional gene expression in transgenic mice. However, the vectors generally used to generate Rosa26 knock-in constructs show instability problems, which have a severe impact on the efficiency of the system. RESULTS: We have optimized the cloning procedure to generate targeting vectors for Cre-regulated expression of constructs within several days with minimal hands-on time, thereby enabling high-throughput approaches. We demonstrate that transient expression of Cre still results in expression of the construct, as shown by the expression level and via functional assays. In addition to its well-established possibilities in expressing cDNA constructs, we show that the Rosa26 locus can be used to drive expression of functional miRNA constructs from its endogenous promoter. CONCLUSION: We provide a new high-efficiency cloning system for Rosa26 knock-in constructs to express either cDNA or miRNA fragments. Our system will enable high-throughput approaches for controlled expression of cDNA or miRNA constructs, with the latter providing a potential high-speed alternative for conditional knock-out models.

The Wilms' tumour protein (WT1) shuttles between nucleus and cytoplasm and is present in functional polysomes.

Mutations of the Wilms' tumour-1 (WT1) gene in humans can lead to childhood kidney cancer, life-threatening glomerular nephropathy and gonadal dysgenesis. The WT1 protein is normally expressed in the developing genitourinary tract, heart, spleen and adrenal glands and is crucial for their development, however it's function at the molecular level is yet to be fully understood. The protein is predominantly nuclear and there is evidence that the two different isoforms of WT1 (-KTS and +KTS) are involved in two different steps of gene expression control: transcription and RNA processing. In this study we report a novel property of WT1, namely that it shuttles between the nucleus and cytoplasm. Moreover, western blot analysis showed that between 10 and 50% of total cellular WT1 can be detected in the cytoplasm depending on the cell type. A significant proportion of cytoplasmic WT1 is in association with ribonucleoprotein particles (RNPs), which strengthens the idea of its involvement in RNA metabolism. Furthermore, we report that WT1 is associated with actively translating polysomes, extending even further the potential roles of WT1 and opening the possibility that it is involved in the regulation of translation. Interestingly, despite the functional differences between two of the WT1 isoforms (+/-KTS) within the nucleus, both isoforms share the shuttling property and are found in translating polysomes.

Expression in Xenopus oocytes shows that WT1 binds transcripts in vivo, with a central role for zinc finger one.

The Wilms' tumour suppressor gene WT1 encodes a protein involved in urogenital development and disease. The salient feature of WT1 is the presence of four 'Krüppel'-type C(2)-H(2) zinc fingers in the C-terminus. Uniquely to WT1, an evolutionarily conserved alternative splicing event inserts three amino acids (KTS) between the third and fourth zinc fingers, which disrupts DNA binding. The ratio of +KTS:-KTS isoforms is crucial for normal development. Previous work has shown that WT1 (+KTS) interacts with splice factors and that WT1 zinc fingers, particularly zinc finger one, bind to RNA in vitro. In this study we investigate the role of zinc finger one and the +KTS splice in vivo by expressing tagged proteins in mammalian cells and Xenopus oocytes. We find that both full-length +/-KTS isoforms and deletion constructs that include zinc finger one co-sediment with ribonucleoprotein particles (RNP) on density gradients. In Xenopus oocytes both isoforms located to the lateral loops of lampbrush chromosomes. Strikingly, only the +KTS isoform was detected in B-snurposomes, but not when co-expressed with -KTS. However, co-expression of the C-terminus (amino acids 233-449, +KTS) resulted in snurposome staining, which is consistent with an in vivo interaction between isoforms via the N-terminus. Expressed WT1 was also detected in the RNA-rich granular component of nucleoli and co-immunoprecipitated with oocyte transcripts. Full-length WT1 was most stably bound to transcripts, followed by the C-terminus; the least stably bound was CTDeltaF1 (C-terminus minus zinc finger one). Expression of the transcription factor early growth response 1 (EGR1), whose three zinc fingers correspond to WT1 zinc fingers 2-4, caused general chromosomal loop retraction and transcriptional shut-down. However, a construct in which WT1 zinc finger one was added to EGR1 mimicked the properties of WT1 (-KTS). We suggest that in evolution, WT1 has acquired the ability to interact with transcripts and splice factors because of the modification of zinc finger one and the +KTS alternative splice.