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F J Rauscher

Publications and source records attributed to F J Rauscher.

At least 73 records · Page 4Linked to original sources
PubMed1994

DNA recognition by splicing variants of the Wilms' tumor suppressor, WT1.

The Wilms' tumor suppressor, WT1, is a zinc finger transcriptional regulator which exists as multiple forms owing to alternative mRNA splicing. The most abundant splicing variants contain a nine-nucleotide insertion encoding lysine, threonine, and serine (KTS) in the H-C link region between the third and fourth WT1 zinc fingers which disrupts binding to a previously defined WT1-EGR1 binding site. We have identified WT1[+KTS] binding sites in the insulin-like growth factor II gene and show that WT1[+KTS] represses transcription from the insulin-like growth factor II P3 promoter. The highest affinity WT1[+KTS] DNA binding sites included nucleotide contacts involving all four WT1 zinc fingers. We also found that different subsets of three WT1 zinc fingers could bind to distinct DNA recognition elements. A tumor-associated, WT1 finger 3 deletion mutant was shown to bind to juxtaposed nucleotide triplets for the remaining zinc fingers 1, 2, and 4. The characterization of novel WT1 DNA recognition elements adds a new level of complexity to the potential gene regulatory activity of WT1. The results also present the possibility that altered DNA recognition by the dominant WT1 zinc finger 3 deletion mutant may contribute to tumorigenesis.

Alternative Splicing↗
PubMed1994

Repression of the transforming growth factor-beta 1 gene by the Wilms' tumor suppressor WT1 gene product.

The Wilms' tumor suppressor gene (WT1) encodes a zinc finger DNA binding protein which functions as a transcriptional repressor. In this study we investigated whether the human transforming growth factor-beta 1 (TGF-beta 1) gene might be a target for transcriptional repression mediated by WT1. Using constructs of the TGF-beta 1 promoter linked to the chloramphenicol acetyl transferase gene, we have demonstrated that the WT1 protein represses expression of the TGF-beta 1 gene through a CGCCCCCGC response element spanning nucleotides -111 to -119 of the TGF-beta 1 promoter. We have also shown in a cotransfection assay that Egr-1, an immediate early growth response gene, activates transcription of the TGF-beta 1 gene through the same response element and that WT1 represses both the basal and Egr-1-induced TGF-beta 1 promoter activity in monkey kidney CV-1 cells. Moreover, WT1 and Egr-1 proteins interact directly with the WT1/Egr-1 response element of the TGF-beta 1 promoter in gel mobility shift assays. These findings provide further definition of transcriptional control of the TGF-beta 1 gene by showing that the WT1 gene product suppresses TGF-beta 1 transcription and that the WT1/Egr-1 consensus element of the human TGF-beta 1 promoter plays a critical role in this repression.

Animals↗
PubMed1994

Novel replication inhibitory function of the developmental regulator/transcription repressor protein WT1 encoded by the Wilms' tumor gene.

The tumor suppressor/developmental regulator protein WT1 encoded by the Wilms' tumor gene is a zinc finger-containing transcription factor which binds to the G+C-rich motif 5'-GCGGGGGCG-3' and represses transcription. Alternatively spliced variants of WT1 (termed+KTS) having an insertion in the zinc finger region are defective for binding to and hence for repression of transcription from promoters containing this motif. Due to the known interactions of two other tumor suppressor proteins with the simian virus 40 (SV40) oncoprotein large tumor antigen (TAg) [which in one case (p53) results in inhibition of the replication initiation activity of TAg], and because of the presence of G+C-rich sequences in the SV40 origin region, we tested the effect of WT1 on TAg- and SV40 origin-dependent DNA replication. WT1 and its alternatively spliced variants were found to be potent inhibitors of replication. Inhibition of replication by WT1 required portions of the N-terminal transcription repression domain and the C-terminal DNA binding domain, while other WT1 sequences needed for transcriptional regulation were dispensable. WT1 neither inhibited the synthesis of TAg nor formed a stable complex with it. Studies of the requirement of cis-active origin sequences in vivo and protein-DNA interactions in vitro indicated that WT1 and its alternatively spliced variants might inhibit replication by their novel binding to the GC box promoter motifs of the SV40 21 bp repeat replication-auxiliary sequence.

Alternative Splicing↗
PubMed1993

Inhibition of colony-stimulating factor-1 promoter activity by the product of the Wilms' tumor locus.

Colony-stimulating factor-1 (CSF-1) is a member of the immediate early gene family, which is expressed in mitogen-stimulated quiescent fibroblasts. The biological effects of CSF-1 are multifaceted and include stimulation of the proliferation and differentiation of myeloid progenitors and activity of circulating monocytes and tissue-specific macrophages. Ablation of circulating levels of biologically active CSF-1 in mice leads to osteopetrosis and sterility, thus implicating a role for CSF-1 in bone remodeling and implantation. Identification of regulatory elements and cognate transcription factors that bind the csf-1 promoter and mediate such diverse expression patterns is of great interest. We identified a sequence element at -273 to -265 (relative to the transcription initiation site) in the murine csf-1 promoter, which contains overlapping consensus sequences for the Wilms' tumor protein (WT1), EGR-1, SP1, and SP3 proteins. WT1 and EGR-1 proteins produced in vitro bound to this sequence, and co-transfection of wt1 with a csf-1-cat reporter plasmid resulted in repression of promoter activity. Interestingly, nuclear extracts prepared from serum-stimulated C3H10T1/2 cells contained predominantly SP1 and SP3 binding activities, which recognized the -273 to -265 site. Thus repression of the csf-1 promoter by WT1 at this site may involve competition between SP1 family transcriptional activators and the WT1 repressor. Colony-stimulating factor-1 may be a physiologically relevant target gene for regulation by the WT1 transcription factor.

Animals↗
PubMed1993

Increased expression of the insulin-like growth factor I receptor gene, IGF1R, in Wilms tumor is correlated with modulation of IGF1R promoter activity by the WT1 Wilms tumor gene product.

Wilms tumor is a pediatric neoplasm that arises from the metanephric blastema. The expression of the gene encoding insulin-like growth factor II (IGF-II) is often elevated in these tumors. Since many of the actions of IGF-II are mediated through activation of the IGF-I receptor (IGF-IR), we have measured the levels of IGF-IR mRNA in normal kidney and in Wilms tumor samples using solution hybridization/RNase protection assays. IGF-IR mRNA levels in the tumors were 5.8-fold higher than in adjacent normal kidney tissue. Among the tumors themselves, the levels of IGF-IR mRNA in those containing heterologous stromal elements were 2-fold higher (P < 0.01) than in tumors without these elements. IGF-IR gene (designated IGF1R) expression in the tumors was inversely correlated with the expression of the Wilms tumor suppressor gene WT1, whose inactivation appears to be a key step in the etiology of Wilms tumor. Cotransfection of Chinese hamster ovary cells with rat and human IGF-IR gene promoter constructs driving luciferase reporter genes and with WT1 expression vectors showed that the active WT1 gene product represses IGF-IR promoter activity in a dose-dependent manner. These results suggest that underexpression, deletion, or mutation of WT1 may result in increased expression of the IGF-IR, whose activation by IGF-II may be an important aspect of the biology of Wilms tumor.

Animals↗
PubMed1993

Physical and functional interaction between WT1 and p53 proteins.

WT1 is a tumor-suppressor gene expressed in the developing kidney, whose inactivation leads to the development of Wilms tumor, a pediatric kidney cancer. WT1 encodes a transcription factor which binds to the EGR1 consensus sequence, mediating transcriptional repression. We now demonstrate that p53, the product of a tumor-suppressor gene with ubiquitous expression, physically associates with WT1 in transfected cells. The interaction between WT1 and p53 modulates their ability to transactivate their respective targets. In the absence of p53, WT1 acts as a potent transcriptional activator of the early growth response gene 1 (EGR1) site, rather than a transcriptional repressor. In contrast, WT1 exerts a cooperative effect on p53, enhancing its ability to transactivate the muscle creatine kinase promoter.

Animals↗
PubMed1993

Tumor suppressor genes which encode transcriptional repressors: studies on the EGR and Wilms' tumor (WT1) gene products.

The study of the WT1 and EGR-1 transcription factors has provided a molecular paradigm for regulation of a coordinate set of target genes during cell growth and differentiation. The next critical questions that must be addressed are: what are the target genes which are normally regulated by WT1-EGR-1, and how does misregulation of these genes result in tumor formation?

Base Sequence↗
PubMed1993

Fusion of a fork head domain gene to PAX3 in the solid tumour alveolar rhabdomyosarcoma.

We have examined the structure and expression of the products associated with the t(2;13)(q35;q14) translocation associated with alveolar rhabdomyosarcoma. The chromosome 13 gene (FKHR) is identified as a member of the fork head domain family of transcription factors characterized by a conserved DNA binding motif. Polymerase chain reaction analysis demonstrates that a 5'PAX3-3' FKHR chimaeric transcript is expressed in all eight alveolar rhabdomyosarcomas investigated. Immunoprecipitation experiments detect the predicted fusion protein. These findings indicate that the t(2;13) generates a potentially tumorigenic fusion transcription factor consisting of intact PAX3 DNA binding domains, a truncated fork head DNA binding domain and C-terminal FKHR regions.

Amino Acid Sequence↗
PubMed1993

Wilms' tumor (WT1) gene expression in rat decidual differentiation.

The Wilm's tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract, where it is thought to play a role in the differentiation of these tissues. We have used immunocytochemistry and in situ hybridization to study WT1 expression in the rat uterus during normal development and pregnancy from 0 to 20 days post coitum (p.c.). WT1 mRNA was abundant in uterine stroma from juvenile rats, but was much less abundant in uterine tissue from sexually mature rats; WT1 expression is not affected by ovariectomy or by treatment with estradiol or estradiol plus progesterone. WT1 gene was highly expressed, however, in the endometrial cells of early pregnancy. On day 6 p.c. WT1 mRNA was detected in anti-mesometrial decidual cells, and WT1 immunoreactivity was concentrated in the nuclei of these cells. All cells of fully-developed deciduoma at 7-8 days p.c. demonstrated WT1 expression. WT1 was not detected in trophoblast/placental tissues but remained abundant in the decidua basalis until parturition. The expression of WT1 was compared with insulin-like growth factor-II (IGF-II) and its receptor in the decidual since it has been shown that IGF-II gene transcription is repressed by WT1 in vitro. However, no spatiotemporal correlation in the expression of these three genes was found in differentiation of the rat decidua. In summary, these data suggest a role for WT1 in decidualization, since its expression is activated during the differentiation of uterine stromal cells into decidual cells.

Animals↗
PubMed1993

Differential effects of rabies and borna disease viruses on immediate-early- and late-response gene expression in brain tissues.

In situ hybridization and Northern blot analysis were used to examine expression of the immediate-early-response genes (IEGs) egr-1, junB, and c-fos, and the late response gene encoding enkephalin in the brains of rats infected intranasally with Borna disease virus (BDV) or rabies virus. In both Borna disease and rabies virus infections, a dramatic and specific induction of IEGs was detected in particular regions of the hippocampus and the cortex. Increased IEG mRNA expression overlapped with the characteristic expression patterns of BDV RNA and rabies virus RNA, although relative expression levels of viral RNA and IEG mRNA differed, particularly in the hippocampal formation. Furthermore, the temporal relationship between viral RNA synthesis and activation of IEG mRNA expression in BDV infection differed markedly from that in rabies virus infection, suggesting that IEG expression is upregulated by different mechanisms. Expression of proenkephalin (pENK) mRNA was also significantly increased in BDV infection, whereas in rabies virus infection, pENK mRNA levels and also the levels of glyceraldehyde-3-phosphate dehydrogenase mRNA were reduced at terminal stages of the disease, probably reflecting a generalized suppression of cellular protein synthesis due to massive production of rabies virus mRNA. The correlation between activated IEG mRNA expression and the strong increase in viral RNA raises the possibility that IEG products induce some phenotypic changes in neurons that render them more susceptible to viral replication.

Animals↗
PubMed1993

The WT1 Wilms tumor gene product: a developmentally regulated transcription factor in the kidney that functions as a tumor suppressor.

Alteration of transcription factor function is becoming a common theme in molecular mechanisms of oncogenesis. A recent example of this trend is the isolation and characterization of the chromosome 11p13 Wilms tumor suppressor gene, WT1. The WT1 protein contains a DNA binding domain consisting of four zinc fingers of the Cys2-His2 class and a proline-glutamine rich region capable of regulating transcription. Deletions of the WT1 gene or point mutations which destroy the DNA binding activity of the protein are associated with the development of the pediatric nephroblastoma Wilms tumor and Denys-Drash syndrome. This article reviews the role of WT1 in normal kidney development processes, the known biochemical functions of the protein and the status of identifying target genes regulated by this potentially oncogenic transcription factor.

Amino Acid Sequence↗
PubMed1993

A structure-function analysis of transcriptional repression mediated by the WT1, Wilms' tumor suppressor protein.

The chromosome 11p13 Wilms' tumor locus (wt1) encodes a zinc finger-containing transcription factor (WT1). WT1 binds to the consensus sequence (5'-GCGGGGGCG-3') and represses transcription when bound to this site in vivo. The mechanism of repression is not yet defined. To investigate the mechanisms of transcriptional repression and map the domains of WT1 responsible, we constructed hybrid proteins between the yeast GAL4 1-147 DNA binding domain and WT1. Fusion of a 298 amino acid glutamine-proline-rich N-terminal segment of WT1 to the GAL4 DNA binding domain created a potent transcriptional repressor. The use of N- and C-terminal truncations of this segment demonstrated that as few as 96 amino acids were required for active repression by GAL4-WT1 hybrid proteins in NIH3T3 fibroblasts. However, the truncated GAL4-WT1 fusion proteins functioned poorly as repressors in embryonic kidney-derived 293 cells, suggesting cell type-specific requirements for transcriptional repression. Site-directed mutagenesis of the WT1 repression domain revealed that deletion of homopolymeric proline and glycine regions, as well as single amino acid changes, partially inactivated the repression function. Single repressor binding sites placed upstream of the transcription start site conferred WT1-mediated repression to a heterologous promoter, whereas multiple sites resulted in additive (non-synergistic) increases in transcriptional repression. Significant repression of transcription was observed when binding sites were placed 760 base pairs upstream or 1000 base pairs downstream relative to the site of transcription initiation. We conclude that the transcriptional repression function of WT1 is contained in the N-terminal, non-DNA binding domain of the protein and that repression can be functionally transferred to a heterologous DNA binding domain.

3T3 Cells↗
PubMed1992

Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.

Wilms tumor, an embryonic kidney malignancy, accounts for approximately 6% of all pediatric neoplasms. A gene implicated in the genesis of this tumor, the Wilms tumor suppressor gene (WT1), encodes a zinc-finger DNA-binding protein (WT1) that functions as a transcriptional repressor. In certain Wilms tumors, the platelet-derived growth factor A chain (PDGF-A) is overexpressed; it has therefore been suggested that it may play an autocrine role in development of these neoplasms. Since the PDGF-A promoter contains putative binding sites for WT1, we explored the role of WT1 in regulating A-chain expression. The major PDGF-A promoter activity was localized in transient transfection assays to a region spanning from -643 to + 8 relative to the transcription start site. WT1 bound to several sites in this region of the promoter, as demonstrated by gel-shift analysis and DNase I footprinting, and functioned as a powerful repressor of PDGF-A transcription in vivo. Maximal repression (> 50-fold) of the PDGF-A promoter was dependent on the presence of multiple WT1 binding sites in transient transfection assays. Our observations suggest a mechanism for normal downregulation of a growth factor gene and of an autocrine growth process of import in kidney development and other biological systems.

3T3 Cells↗
PubMed1992

The Wilms' tumor gene product, WT1, represses transcription of the platelet-derived growth factor A-chain gene.

The Wilms' tumor locus on chromosome 11p13 contains a tumor suppressor gene, wt1, which encodes a DNA binding protein (WT1) with four zinc fingers and a glutamine-proline-rich N terminus and which functions as a repressor of transcription. The platelet-derived growth factor (PDGF) A-chain gene encodes a potent growth factor, which is expressed in high levels in a number of tumor cell lines. We initiated a search for WT1 target genes and now report that WT1 strikingly represses transcription of the PDGF A-chain gene in transient transfection assays and that the WT1 protein interacts directly with a highly G+C-rich region of the PDGF A-chain promoter in gel mobility shift assays. The results suggest that WT1 may function to repress expression of the PDGF A-chain gene and that loss of this or related repressor activities may contribute to the abnormal growth of Wilms' tumors.

Base Sequence↗
PubMed1992

Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1.

The Wilms tumor suppressor gene wt1 encodes a zinc finger DNA binding protein, WT1, that functions as a transcriptional repressor. The fetal mitogen insulin-like growth factor II (IGF-II) is overexpressed in Wilms tumors and may have autocrine effects in tumor progression. The major fetal IGF-II promoter was defined in transient transfection assays as a region spanning from nucleotides -295 to +135, relative to the transcription start site. WT1 bound to multiple sites in this region and functioned as a potent repressor of IGF-II transcription in vivo. Maximal repression was dependent on the presence of WT1 binding sites on each side of the transcriptional initiation site. These findings provide a molecular basis for overexpression of IGF-II in Wilms tumors and suggest that WT1 negatively regulates blastemal cell proliferation by limiting the production of a fetal growth factor in the developing vertebrate kidney.

Animals↗
PubMed1992

Mutational analysis of Max: role of basic, helix-loop-helix/leucine zipper domains in DNA binding, dimerization and regulation of Myc-mediated transcriptional activation.

The Max protein forms a heterodimeric complex with the Myc family of proteins and binds to DNA in a sequence-specific manner. We investigated the role of the helix-loop-helix (HLH), leucine zipper (LZ) and basic domains of Max in protein complex formation, DNA-binding activity and transcriptional regulation. We mutagenized the basic, HLH and LZ domains of Max and studied the ability of the normal and mutant proteins to bind to DNA as both homo- and heterodimers and their ability to heterodimerize with Myc. Helix-1 and helix-2 regions of Max were found to be critical for homodimer formation and subsequent DNA binding, while the LZ was essential for heterodimer formation. In transient transfection assays the Myc protein functioned as a transcriptional activator while Max protein repressed the trans-activation observed with Myc.

Base Sequence↗
PubMed1991

Identification of a zinc finger protein that inhibits IL-2 gene expression.

Transient activation of the interleukin-2 (IL-2) gene after antigen recognition by T lymphocytes is crucial for subsequent T cell proliferation and differentiation. Several IL-2 gene regulatory elements and binding factors necessary for activation of the IL-2 gene have been defined. However, little is known about negative regulation of IL-2 expression, which is likely to be important in the rapid shut-off of IL-2 transcription. A nucleotide sequence element (NRE-A) that negatively regulates IL-2 expression has been identified within the IL-2 gene. T cell nuclear extracts contained an NRE-A binding activity. A complementary DNA was isolated that encodes a zinc finger-containing protein that suppressed IL-2 gene expression. The observation of negative regulation of the immunoglobulin heavy chain gene enhancer by an element similar to NRE-A suggests that related proteins may regulate multiple immune response genes.

Amino Acid Sequence↗