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F J Rauscher

Publications and source records attributed to F J Rauscher.

At least 37 records · Page 2Linked to original sources
PubMed1997

Overexpression, purification, characterization, and crystallization of the BTB/POZ domain from the PLZF oncoprotein.

The BTB/POZ domain defines a conserved region of about 120 residues and has been found in over 40 proteins to date. It is located predominantly at the N terminus of Zn-finger DNA-binding proteins, where it may function as a repression domain, and less frequently in actin-binding and poxvirus-encoded proteins, where it may function as a protein-protein interaction interface. A prototypic human BTB/POZ protein, PLZF (promyelocytic leukemia zinc finger) is fused to RARalpha (retinoic acid receptor alpha) in a subset of acute promyelocytic leukemias (APLs), where it acts as a potent oncogene. The exact role of the BTB/POZ domain in protein-protein interactions and/or transcriptional regulation is unknown. We have overexpressed, purified, characterized, and crystallized the BTB/POZ domain from PLZF (PLZF-BTB/POZ). Gel filtration, dynamic light scattering, and equilibrium sedimentation experiments show that PLZF-BTB/POZ forms a homodimer with a Kd below 200 nM. Differential scanning calorimetry and equilibrium denaturation experiments are consistent with the PLZF-BTB/POZ dimer undergoing a two-state unfolding transition with a Tm of 70.4 degrees C, and a DeltaG of 12.8 +/- 0.4 kcal/mol. Circular dichroism shows that the PLZF-BTB/POZ dimer has significant secondary structure including about 45% helix and 20% beta-sheet. We have prepared crystals of the PLZF-BTB/POZ that are suitable for a high resolution structure determination using x-ray crystallography. The crystals form in the space group I222 or I212121 with a = 38.8, b = 77.7, and c = 85.3 A and contain 1 protein subunit per asymmetric unit with approximately 40% solvent. Our data support the hypothesis that the BTB/POZ domain mediates a functionally relevant dimerization function in vivo. The crystal structure of the PLZF-BTB/POZ domain will provide a paradigm for understanding the structural basis underlying BTB/POZ domain function.

Amino Acid Sequence↗
PubMed1997

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

Animals↗
PubMed1996

Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins.

The expression of a number of human paired box-containing (PAX) genes has been correlated with various types of tumors. Novel fusion genes encoding chimeric fusion proteins have been found in the pediatric malignant tumor alveolar rhabdomyosarcoma (RMS). They are generated by two chromosomal translocations t(2;13) and t(1;13) juxtaposing PAX3 or PAX7, respectively, with a forkhead domain gene FKHR. Here we describe that specific down-regulation of the t(2;13) translocation product in alveolar RMS cells by antisense oligonucleotides results in reduced cellular viability. Cells of embryonal RMS, the other major histiotype of this tumor, were found to express either wild type PAX3 or PAX7 at elevated levels when compared with primary human myoblasts. Treatment of corresponding embryonal RMS cells with antisense olignucleotides directed against the mRNA translational start site of either one of these two transcription factors similarly triggers cell death, which is most likely due to induction of apoptosis. Retroviral mediated ectopic expression of mouse Pax3 in a PAX7 expressing embryonal RMS cell line could partially rescue antisense induced apoptosis. These data suggest that the PAX3/FKHR fusion gene and wild-type PAX genes play a causative role in the formation of RMS and presumably other tumor types, possibly by suppressing the apoptotic program that would normally eliminate these cells.

Animals↗
PubMed1996

Transcriptional activation of the syndecan-1 promoter by the Wilms' tumor protein WT1.

The Wilms' tumor suppressor gene (wt1) encodes a zinc finger DNA binding protein (WT1) which functions as a transcriptional regulator and is essential for normal urogenital development. WTI has previously been shown to repress the transcription of a variety of target genes whose products stimulate growth, such as growth factors, growth factor receptors and other transcription factors. In this study, we identify syndecan-1 as a target gene for WT1-mediated activation. Syndecan-1 is a cell surface proteoglycan whose induction is coincident with epithelial differentiation during kidney development and whose loss of expression is correlated with the loss of the epithelial phenotype and malignant transformation. The murine syndecan-1 promoter contains several potential binding sites for WT1. We demonstrate that both WT1 (-KTS) and WT1 (+KTS) isoforms bind to multiple sites in this highly G + C-rich region, as detected by gel-shift analyses. These WT1 isoforms function as transcriptional activators of syndecan-1 expression in transient transfection assays. Activation of syndecan-1 by WT1 is dependent on an intact zinc-finger region as well as a 179 amino acid proline-rich region in the amino terminus of the protein. Moreover, the endogenous syndecan-1 gene is activated by WT1 in a novel inducible cell line based upon the sheep metallothionein promoter. These results highlight an emerging role for WT1 as an activator of genes like syndecan-1 which may potentiate epithelial differentiation and maintenance in the developing kidney.

3T3 Cells↗
PubMed1996

Effect of DNA-binding drugs on early growth response factor-1 and TATA box-binding protein complex formation with the herpes simplex virus latency promoter.

Adjacent binding sites for early growth response factor-1 (EGR1) and TATA box-binding protein (TBP) were identified on the herpes simplex virus latency promoter in previous work. The binding of EGR1 to the GC-rich region prevented TBP binding to the AT-rich region. With the simultaneous addition of both EGR1 and TBP, the intercalator nogalamycin prevented EGR1 complex formation, resulting in a dose-dependent increase of the TBP.DNA complex. The minor groove binder chromomycin A3 inhibited EGR1 complex formation but resulted in a smaller increase of the TBP complex. In contrast, an alkylating intercalator hedamycin strongly inhibited binding of both proteins. The ability of these GC-binding drugs to prevent EGR1.DNA complex formation was in the following order: hedamycin > nogalamycin > chromomycin A3, and the specificity was nogalamycin > chromomycin A3 > hedamycin. With transcription factor IIA (TFIIA) in the assay, TBP was able to bind the promoter whereas formation of the EGR1.DNA complex was reduced. An AT minor groove-binding drug, distamycin A, disrupted the TBP.TFIIA.DNA complex and restored the EGR1.DNA complex. We conclude that the binding motif and sequence preference of DNA-interactive drugs are manifested in their ability to inhibit the transcription factor-DNA complexes.

Alkylating Agents↗
PubMed1996

The hybrid PAX3-FKHR fusion protein of alveolar rhabdomyosarcoma transforms fibroblasts in culture.

Pediatric alveolar rhabdomyosarcoma is characterized by a chromosomal translocation that fuses parts of the PAX3 and FKHR genes. PAX3 codes for a transcriptional regulator that controls developmental programs, and FKHR codes for a forkhead-winged helix protein, also a likely transcription factor. The PAX3-FKHR fusion product retains the DNA binding domains of the PAX3 protein and the putative activator domain of the FKHR protein. The PAX3-FKHR protein has been shown to function as a transcriptional activator. Using the RCAS retroviral vector, we have introduced the PAX3-FKHR gene into chicken embryo fibroblasts. Expression of the PAX3-FKHR protein in these cells leads to transformation: the cells become enlarged, grow tightly packed and in multiple layers, and acquire the ability for anchorage-independent growth. This cellular transformation in vitro will facilitate studies on the mechanism of PAX3-FKHR-induced oncogenesis.

Animals↗
PubMed1996

KAP-1, a novel corepressor for the highly conserved KRAB repression domain.

The KRAB repression domain is one of the most widely distributed transcriptional effector domains yet identified, but its mechanism of repression is unknown. We have cloned a corepressor, KAP-1, which associates with the KRAB domain but not with KRAB mutants that have lost repression activity. KAP-1 can enhance KRAB-mediated repression and is a repressor when directly tethered to DNA. KAP-1 contains a RING finger, B boxes, and a PHD finger; the RING-B1-B2 structure is required for KRAB binding and corepression. We propose that KAP-1 may be a universal corepressor for the large family of KRAB domain-containing transcription factors.

Amino Acid Sequence↗
PubMed1996

The IGF-I receptor gene promoter is a molecular target for the Ewing's sarcoma-Wilms' tumor 1 fusion protein.

Desmoplastic small round cell tumor (DSRCT) is an abdominal malignancy in children which is characterized by a recurrent chromosomal translocation, t(11;22)(p13;q12). This rearrangement results in the fusion of the ubiquitously expressed EWS1 gene to the Wilms' tumor suppressor (WT1) gene. The chimeric protein contains the N-terminal domain of EWS1 fused to the DNA-binding domain of WT1, including zinc fingers 2-4. Because WT1 has been shown previously to bind and repress the insulin-like growth factor I (IGF-I-R) promoter, we investigated whether this promoter is, in addition, a target for the aberrant EWS/WT1 transcription factor. EWS/WT1 activated the IGF-I-R promoter approximately 340%, whereas a fusion protein containing a three-amino acid insert (KTS) between zinc fingers 3 and 4 had no effect. On the other hand, expression vectors encoding either WT1 or EWS1 reduced the activity of the promoter to 46 and 58% of control values, respectively. Results of gel shift assays indicate that the binding affinity of EWS/WT1 to a fragment of the 5'-flanking region of the receptor promoter was higher than the affinity of WT1 itself. Consistent with the results of functional assays, the binding of EWS/WT1(+KTS) was significantly reduced. Due to the central role of the IGF-I-R in tumorigenesis, activation of the receptor promoter by EWS/WT1 may constitute a potential mechanism for the etiology and/or progression of DSRCT.

Base Sequence↗
PubMed1996

Wild-type and mutant p53 differentially regulate transcription of the insulin-like growth factor I receptor gene.

The insulin-like growth factor I receptor (IGF-I-R) plays a critical role in transformation events. It is highly overexpressed in most malignant tissues where it functions as an anti-apoptotic agent by enhancing cell survival. Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. p53 is the most frequently mutated gene in human cancer. Cotransfection of Saos-2 (os-teosarcoma-derived cells) and RD (rhabdomyosarcoma-derived cells) cells with IGF-I-R promoter constructs driving luciferase reporter genes and with wild-type p53 expression vectors suppressed promoter activity in a dose-dependent manner. This effect of p53 is mediated at the level of transcription and it involves interaction with TBP, the TATA box-binding component of TFIID. On the other hand, three tumor-derived mutant forms of p53 (mut 143, mut 248, and mut 273) stimulated the activity of the IGF-I-R promoter and increased the levels of IGF-I-R/luciferase fusion mRNA. These results suggest that wild-type p53 has the potential to suppress the IGF-I-R promoter in the postmitotic, fully differentiated cell, thus resulting in low levels of receptor gene expression in adult tissues. Mutant versions of p53 protein, usually associated with malignant states, can derepress the IGF-I-R promoter, with ensuing mitogenic activation by locally produced or circulating IGFs.

Base Sequence↗
PubMed1996

Differential function of Wilms' tumor gene WT1 splice isoforms in transcriptional regulation.

The Wilms' tumor gene, WT1, encodes a zinc finger transcription factor that can repress transcription of a number of genes. WT1 mRNA undergoes alternative splicing at two locations, yielding four different mRNA species and protein products. One alternative splice alters the zinc finger region of WT1, resulting in the addition of three amino acids, Lys-Thr-Ser (KTS), between zinc fingers 3 and 4, altering the binding of WT1 to DNA. Here, we show that the WT1 protein with and without the KTS tripeptide can repress transcription from the human full-length WT1 promoter. Repression of transcription by WT1 has been shown to require two WT1 binding sites. We examined WT1 repression of the human minimal WT1 promoter, which contains two potential WT1 binding motifs. WT1 lacking the KTS tripeptide (WT1-KTS) was unable to repress transcription from a minimal WT1 promoter of 104 base pairs, whereas WT1 containing the KTS tripeptide (WT1+KTS) repressed transcription from the minimal promoter. The ability of WT1+KTS to repress transcription where WT1-KTS could not provided a functional assay to define differential WT1 binding motifs based on the presence or the absence of the KTS tripeptides. We present data defining the differential consensus DNA binding motifs for WT1-KTS and WT1+KTS. We demonstrate that WT1 zinc finger 1 plays a role in the differential DNA binding specificity of WT1-KTS and WT1+KTS.

Alternative Splicing↗
PubMed1996

Regulation of nov by WT1: a potential role for nov in nephrogenesis.

The nov gene encodes a putative Insulin-like-Growth Factor-Binding-Protein (IGFBP) of a novel type which is structurally related to a family of growth-factors likely to play a role in the control of cell proliferation. In the kidney, nov is expressed essentially at the embryonic stage and alterations of nov expression, relative to the normal kidney, have been detected in both avian nephroblastomas and human Wilms' tumors. The levels of human nov (novH) and WT1 mRNA in individual Wilms' tumors have been shown to be inversely correlated, suggesting that the expression of novH could be under the negative control of WT1. We have now established the nucleotide sequence of the 5' flanking region and identified two transcription start sites by RNase protection assays and primer extension. We report that in transient cotransfection experiments the transcription activity of novH promoter constructs was repressed by two isoforms of WT1 proteins (WT1 and WT1+KTS). Repression of the novH promoter required both intact zinc finger regions and the NH2 transcription repression domain of WT1. Inasmuch as the minimal region of novH promoter required to mediate WT1 repression in vivo failed to bine recombinant WT1 protein in in vitro footprinting assays this repression may be mediated by either (i) low affinity sites cooperative interactions or (ii) indirectly via protein-protein interactions with another factor(s). Furthermore, constitutive expression of wild type WT1 into 293 cells resulted in a decrease of endogenous NOVH protein levels, suggesting that novH may be a physiological target for WT1. The downregulation of novH expression by WT1 might represent a key element in normal and tumoral nephrogenesis.

3T3 Cells↗
PubMed1996

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1.

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.

3T3 Cells↗
PubMed1996

Wilms' tumor 1 splice variants have opposite effects on the tumorigenicity of adenovirus-transformed baby-rat kidney cells.

The Wilms' Tumor 1 gene (WT1) encodes a transcription factor of the zinc-finger family. As a result of alternative RNA splicing, the gene can be expressed as four polypeptides which differ in the presence or absence of two stretches of amino acids: one of 17 residues (17aa) just N-terminal of the four zinc-fingers and of three residues (K-T-S) between zinc finger 3 and 4. In this study, four human cDNA constructs encoding the Wilms' tumor 1 splice variants were stably transfected into adenovirus-transformed baby rat kidney (Ad-BRK) cells. The in vivo produced WT1 proteins that lacked the KTS residues were found to bind efficiently to both the Egr-1 consensus sequence and the recently described WTE DNA sequence, as determined by electrophoretic mobility shift assays. Our studies show distinct effects of the different WT1 isoforms. Expression of the WT1 (-/+) protein, lacking the 17aa insert, strongly suppressed the tumorigenic phenotype of the Ad-BRK cells. Intriguingly, expression of the WT1 (-/-) protein, lacking both inserts, increased the tumor growth rate. In contrast to the growth in vivo, the growth rate of the transfectants in tissue culture is not influenced by any of the WT1 isoforms. However, the suppression of tumorigenicity appears to be correlated with a reduced ability of the cells to grow in serum-free medium.

Alternative Splicing↗
PubMed1996

Fusion of the EWS1 and WT1 genes as a result of the t(11;22)(p13;q12) translocation in desmoplastic small round cell tumors.

The isolation and molecular analysis of genes which cause and/or predispose to Wilms' tumor have yielded fascinating insights into the role of tissue-specific gene regulation in both development and disease processes. Analysis of the WT1 transcription factor has clearly established its role in Wilms' tumorigenesis and a broader role in both urogenital organogenesis and mesenchymal cell differentiation events. Clearly, loss of function mutations in WT1 is correlated with aberrant function as a regulator of gene expression, ultimately resulting in neoplastic transformation in the developing kidney. A question we have pursued is whether alterations of WT1 structure and/or function can be associated with other types of malignancies, possibly reflecting WT1's broader role in mesenchymal differentiation. To this end, we have analyzed a rare solid tumor designated Intra-Abdominal Desmoplastic Small Round Cell Sarcoma (IADSRCT) which often displays a recurrent chromosomal translocation t(11;22)(p13;q12) involving the WT1 genomic locus. We have shown that the EWS1 gene fron chromosome 22q12 is fused to the WT1 gene in IADSRCT and that a fusion protein is produced which functions as a potent activator of transcription. Our results suggest that WT1 has sustained a gain-of function alteration as a results of this fusion and that the fusion gene functions as a dominant oncogene in this disease. Thus, the WT1 locus may be the target for both gain- and loss-of-function mutations resulting in different disease outcomes. A summary of our ongoing analysis of the EWS-WT1 fusion gene is presented.

Adult↗
PubMed1996

Hematopoietic transcriptional regulation by the myeloid zinc finger gene, MZF-1.

Transcriptional regulators control much of hematopoiesis. One such transcriptional regulator is the myeloid zinc finger gene MZF-1. MZF-1 has been localized to the telomere of chromosome 19q, where a large number of related zinc finger genes reside. It has been found to be essential in granulopoiesis. It is a bi-functional transcriptional regulator, repressing transcription in non-hematopoietic cells, and activating transcription in cells of hematopoietic origins. Its consensus DNA binding site has been isolated, and sites in several promoters of myeloid-specific genes, such as CD34, lactoferrin, and myeloperoxidase, have been defined. In co-transfection experiments MZF-1 has been found to regulate transcription from the CD34 promoter.

Base Sequence↗
PubMed1996

Regulation of insulin-like growth factor I receptor gene expression by the Wilms' tumor suppressor WT1.

THe insulin-like growth factor I receptor (IGF-I-R) has been implicated in the etiology and/or progression of Wilms' tumor, or nephroblastoma, a pediatric neoplasm of the kidney that is often associated with deletion or mutation of the WT1 tumor suppressor gene. The levels of IGF-I-R mRNA in the tumors were sixfold higher than in normal adjacent kidney tissue and were inversely correlated to the levels of WT1 mRNA, suggesting that the expression of the IGF-I-R gene is under inhibitory control by WT1. Cotransfection of an IGF-I-R promoter-luciferase reporter construct together with a WT1 expression vector resulted in a dose-dependent suppression of promoter activity. Multiple WT1 binding sites were mapped in the 5'-flanking and 5'-untranslated regions of the IGF-I-R gene using gel retardation and DNaseI footprinting assays. Thus, suppression of the IGF-I-R promoter by WT1 involves multiple interactions of its zinc finger domain with sites located both upstream and downstream of the transcription initiation site. Finally, we showed that expression of the endogenous IGF-I-R gene is decreased in G401 cells stably transfected with a WT1 expression vector. Reduction in expression of the IGF-I-R gene is associated with a decrease in a number of IGF-I-mediated biological effects. Thus, deletion or mutation of the WT1 gene in Wilms' tumor and other malignancies can result in overexpression of the receptor, with enhanced autocrine/paracrine activation by locally produced or circulating IGFs.

Animals↗