Biomedical subjects

Z E Zehner

Publications and source records attributed to Z E Zehner.

At least 19 recordsLinked to original sources

PubMed2001

Perinuclear mRNA localisation by vimentin 3'-untranslated region requires a 100 nucleotide sequence and intermediate filaments.

The role of the vimentin 3'-untranslated region (3'-UTR) in mRNA localisation was studied in cells transfected with a reporter sequence linked to subregions of the 3'-UTR. In situ hybridisation showed that nucleotides 37-137, including a previously identified protein-binding domain, were sufficient to localise transcripts to perinuclear cytoplasm. Transfection of two SW13 cell lines that do and do not express vimentin showed that perinuclear localisation due to either the vimentin or c-myc 3'-UTR requires intermediate filaments. The data suggest that both a specific protein-binding region of the vimentin 3'-UTR and intermediate filaments themselves are required to determine the site of vimentin synthesis.

3' Untranslated Regions↗

PubMed2000

The zinc finger repressor, ZBP-89, binds to the silencer element of the human vimentin gene and complexes with the transcriptional activator, Sp1.

Vimentin is a component of the eukaryotic cytoskeleton belonging to the family of intermediate filament proteins. It exhibits a complex pattern of tissue- and development-specific expression. It is also a marker of the metastatic potential of many tumor cells. Previously, the human vimentin promoter was shown to contain several regions for the binding of positive and negative acting regulatory factors. Until now, the silencer element, which shuts down vimentin synthesis in selected tissues during development, was not precisely localized; nor was its binding protein known. In vivo DMS footprinting by ligation-mediated PCR delineated the position of guanine residues important to vimentin expression. Transient transfection assays in HeLa cells of various vimentin 5'-end promoter sequences and mutants thereof precisely defined two regulatory elements, a negative element and an adjoining positive acting element. Band shift assays, UV cross-linking, and Southwestern blot analysis confirm that the silencer element specifically binds a protein. Several lines of evidence show that ZBP-89, a zinc finger, Kruppel-like repressor protein is vimentin's silencer element binding factor. Co-immunoprecipitation and DNA affinity chromatography prove that Sp1 heterodimerizes with ZBP-89 when bound to the silencer element to yield a DNA-protein complex whose mobility is indistinguishable from that displayed by HeLa nuclear extract in band shift assays.

Base Sequence↗

PubMed2000

A Stat1alpha factor regulates the expression of the human vimentin gene by IFN-gamma.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. Here, we report an increase in vimentin gene transcription induced by the cytokine interferon-y (IFN-gamma). Northern blot analysis and reporter gene assays reveal that IFN-gamma induces vimentin gene transcription in HeLa cells. However, no increase in vimentin mRNA synthesis was observed de novo in MCF-7 cells, which do not already express vimentin. Band shift analysis shows that the Stat1alpha protein mediates vimentin induction by IFN-gamma. A human mutant fibroblast cell line (U3A), which lacks Stat1alpha but expresses vimentin mRNA, yields no increase in vimentin mRNA levels on the addition of IFN-gamma. These results suggest that the induction of vimentin gene expression might be an important part of a complex cellular response to IFN-gamma.

Adenocarcinoma↗

PubMed2000

Chromosomal localization of the genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma, and VIM in chicken by fluorescence in situ hybridization.

Six structural genes encoding ALDH, BMP-2, R-FABP, IFN-gamma, RXR-gamma and VIM were mapped in the chicken by fluorescence in situ hybridization (FISH) using genomic and cDNA clones as probes. The genes were found to be located on four different macrochromosomes: chromosome 1 (IFNG and FABP), chromosome 2 (VIM and ALDH), chromosome 3 (BMP2) and a smaller macrochromosome, most probably chromosome 7 (RXRG). With the exception of IFNG none of the newly mapped sites corresponds to known orthologous regions between chicken and human chromosomes.

Aldehyde Dehydrogenase↗

PubMed1999

A GC-box is required for expression of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene (-1416 to +73) was shown to contain two positive-acting regions, separated by a negative region, and at least eight GC-boxes as determined by sequence homology (Rittling, S.R., Baserga, R., 1987. Mol. Cell. Biol. 7, 3908-3915). We have analyzed the region -900 to +41 for protein binding by in vivo footprinting experiments using ligation-mediated PCR. For the various GC-boxes, we detect protein binding only to that GC-box (at position -64 and -55) closest to the transcriptional start site. Transient transfection assays of various vimentin 5'-end fragments and mutations thereof fused to the reporter gene cat indicate that this sequence is indispensable for promoter function regardless of the inclusion of upstream DNA sequences. In vitro binding studies confirm that this region binds protein specifically. We suggest that this GC-box and its binding factor are required for regulated expression of the human vimentin gene.

Binding, Competitive↗

PubMed1999

An antisilencer element is involved in the transcriptional regulation of the human vimentin gene.

Vimentin is an intermediate filament protein normally expressed in cells of mesenchymal origin. The promoter of the human vimentin gene was previously reported to contain two positive-acting regions, separated by a negative region (Rittling, S.R., Baserga, R., 1987. Functional analysis and growth factor regulation of the human vimentin promoter. Mol. Cell. Biol. 7, 3908-3915). Here, detailed studies reveal two additional regulatory elements, a new positive transcriptional element located between -717 and -757, and a new repressor element at -780 to -821. In transient transfections, the positive-acting element is able to completely override the effect of different silencer elements when fused to a heterologous promoter. However, this element does not enhance gene activity when the silencer element is absent and thus cannot be viewed as a true enhancer. Since it appears to overcome the effect of a silencer element, we refer to it as an antisilencer element. Gel mobility shift assays, UV-cross-linking experiments, and Southwestern blots reveal that a 105-kDa protein specifically binds to this region.

Cell Line↗

PubMed1997

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA.

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.

Animals↗

PubMed1996

Expression of rabbit ileal N3 Na+/nucleoside cotransport activity in Xenopus laevis oocytes.

To determine if the Na+/nucleoside cotransport activity in the distal rabbit intestine has either purine-selective (N1) or broad (N3) substrate specificity, Na(+)-dependent inosine uptake was expressed in Xenopus laevis oocytes. The rate of expressed Na(+)-stimulated inosine uptake saturated with increasing inosine concentration (apparent K(m) = 58.2 +/- 13.0 microM), was insensitive to inhibition by 6-(4-nitrobenzyl)thio-9-beta-D-ribofuranosylpurine, and was partially inhibited by phloridzin. Na(+)-dependent uptake was inhibited by guanosine (IC50 = 7.1 microM) and thymidine (IC50 = 5.5 microM). The Na+/nucleoside cotransport activity expressed by rabbit ileal mRNA in the Xenopus oocyte expression system is most characteristic of the N3 subclass of this family of transport proteins.

Animals↗

PubMed1995

Two homologous enhancer elements in the chicken vimentin gene may bind a nuclear factor in common with a nearby silencer element.

Vimentin, a cytoskeletal protein belonging to the intermediate filament protein family, exhibits a complex pattern of expression. In the case of the chicken vimentin gene, several regulatory elements within the 5' region of the gene have been characterized, including an enhancer activity between -160 and -320, which may contribute to the down-regulation of vimentin expression during myogenesis. In this study, sequences within this region were examined via transient transfections of various deletion constructs, and two distinct enhancer elements were found, one on either side of a previously described silencer element. These two enhancer elements also enhanced transcription when fused separately to the basal promoter region of the chicken vimentin gene. Gel mobility shift assays, UV cross-linking experiments, and DNase I protection studies indicate that these two enhancer elements and the silencer element all contain a common binding site for the previously described 95-kDa silencer element binding protein, suggesting that this regulatory protein can act as both an activator and a repressor.

Animals↗

PubMed1995

Regulation of chicken vimentin gene expression by serum, phorbol ester, and growth factors: identification of a novel fibroblast growth factor-inducible element.

Vimentin is a cytoskeletal protein that belongs to the intermediate filament protein family. It is normally expressed in cells of mesenchymal origin and is developmentally as well as cell cycle regulated. Multiple silencer elements as well as unique antisilencer element are responsible for regulating the chicken vimentin gene. The silencer elements bind a protein of M(r) 90,000 (the silencer protein), whereas the antisilencer element binds a protein of M(r) 110,000-120,000 (the antisilencer protein). In this study, we examined the effect of serum, phorbol ester, transforming growth factor beta, and fibroblast growth factor of gene expression and identify the regions in the 5'-end of the chicken vimentin gene responsible for induction. The binding activity of both the silencer and the antisilencer proteins are affected by 12-O-tetradecanoylphorbol-13-acetate treatment, whereas the antisilencer element is inducible by fibroblast growth factor.

Animals↗

PubMed1994

A negative regulatory factor is missing in a human metastatic breast cancer cell line.

The intermediate filament protein, vimentin, is differentially expressed in various tissues and stages of development and in metastatic versus nonmetastatic breast cancer cell lines. Previously, we have shown vimentin expression to be regulated at least in part by a silencer element which binds a M(r) 95,000 protein and an overriding, antisilencer element which binds a M(r) 140,000 protein. Southwestern blot (DNA-protein) analyses indicate that silencer protein binding activity is missing in the metastatic breast cancer cell line (MDA-MB-231), where vimentin is highly expressed, but is present in the nonmetastatic breast cancer cell line, MCF-7, where vimentin is not expressed. This suggests that the absence of a functional silencer protein may lead to expression of vimentin as well as other genes which contribute to the metastatic state.

Animals↗

PubMed1994

Multiple silencer elements are involved in regulating the chicken vimentin gene.

Vimentin, a member of the intermediate filament protein family, exhibits tissue- as well as development-specific expression. Transcription factors that are involved in expression of the chicken vimentin gene have been described and include a cis-acting silencer element (SE3) that is involved in the down-regulation of this gene (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we report the identification of two additional silencer elements (SE1 and SE2). We show by transfection analysis that all three silencer elements are functionally active and that optimal silencing occurs when multiple (at least two) silencer elements are present. In addition, the previously identified SE3 can be divided into three subregions, each of which is moderately active alone. By gel mobility shift assays, all three silencer elements plus SE3 subregions bind a protein which by Southwestern (DNA-protein) blot analysis is identical in molecular mass (approximately 95 kDa). DNase I footprinting experiments indicate that this protein binds to purine-rich sites. Therefore, multiple elements appear to be involved in the negative regulation of the chicken vimentin gene, which may be important in the regulation of other genes as well.

Animals↗

PubMed1993

Functional analysis of chicken vimentin distal promoter regions in cultured lens cells.

Synthesis of the cytoskeletal intermediate filament protein vimentin (Vim) in the lens is unexpected due to the mesenchymal preference of Vim-encoding gene (Vim) expression and the epithelial origin of the lens. Previous studies indicated that chicken Vim gene expression in cultured lens cells is regulated by both positive- and negative-acting sequence elements within the first -767 nucleotides (nt) of its promoter. Here, we demonstrate the existence of additional upstream chicken Vim promoter elements which function in transfected lens cells. Sequences within the nt -1360/-1156 region repressed promoter activity in transfected lens cells to levels lower than that observed for the previously defined more proximal repressor elements. The -1612/-1360 region activated promoter activity to levels similar to those observed for the strongest previously defined proximal promoter. The nt sequence analysis of the upstream promoter region revealed the presence of multiple consensus repressor and activator transcription-factor-binding sites. Several of these sites have been implicated for lens expression of enzyme-crystallin-encoding genes (cry), suggesting that Vim expression may share features with the cry genes for recruitment and high-level expression in the lens.

Animals↗

PubMed1992

Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression.

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

Animals↗

PubMed1991

Regulation of intermediate filament gene expression.

Members of the intermediate filament protein family exhibit complex patterns of development-specific and tissue-specific expression. Studies exploring the mechanisms of gene regulation are underway and key regulatory factors are currently being described and isolated for certain genes encoding intermediate filament proteins. Selected systems from this diverse group of about 50 genes will be discussed.

Animals↗

PubMed1990

Regulation of vimentin gene expression in the ocular lens.

Vimentin expression in the lens is striking due to the reported mesenchymal preference of vimentin and the epithelial origin of the lens. The amount of chicken vimentin mRNA levels determined by Northern blot analysis increased 3-fold from 7 to 14 days of embryonic lens development and then decreased 10-fold at 16 days of development, suggesting that post-transcriptional processes may contribute to the level of cytoplasmic vimentin mRNA during lens development. To analyze the mechanisms governing vimentin gene expression in the lens at the level of transcription, a series of chicken vimentin 5'-flanking region deletions were fused to the bacterial CAT gene and transfected into fibroblasts and lens cultures derived from three species. The -160 to +1 sequence conferred equal promoter activity in cultured chicken lens epithelial cells and fibroblasts. The -321 to -160 sequences increased promoter activity in all cultures, but more strongly in fibroblasts than in lens cells. Sequence elements in the region -608 to -321 repressed promoter activity in lens cells and fibroblasts. Promoter activity was partially restored in fibroblasts but not in lens cells by -767 to -608 sequences. Vimentin gene expression in the lens thus appears to be controlled by multiple positive- and negative-acting elements in its 5'-flanking sequence.

Animals↗

PubMed1990

A negative element involved in vimentin gene expression.

Vimentin is one member of the intermediate filament multigene family which exhibits both tissue- and developmental stage-specific expression. In vivo, vimentin is expressed in cells of mesenchymal origin. Previously, we identified both enhancer and promoter elements in the chicken vimentin gene which regulate gene expression in a positive manner. In this report, we have identified a 40-base-pair region at -568 base pairs between the proximal and distal enhancer elements which represses transcriptional activity. This silencer region can also repress the heterologous herpes simplex virus thymidine kinase promoter, which is comparable to the vimentin promoter. In addition, the element is able to function in a position- and orientation-independent manner, and the amount of repression is increased by multiple copies. Here we show by gel retardation assays and DNase I footprinting that this region binds a protein in nuclear extracts from HeLa cells. Southwestern (DNA-protein) blot analysis indicates this protein is approximately 95 kilodaltons in size. Moreover, protein distribution and activity mimic the expression pattern of vimentin during myogenesis, i.e., protein binding increases as vimentin gene expression decreases. The silencer region shares strong sequence similarity with 5'-flanking sequences found in both the human and hamster vimentin genes and with other characterized silencer elements, including the human immunodeficiency virus long terminal repeat, rat growth hormone, chicken lysozyme, and rat insulin genes. Thus, a negative element appears to bind a 95-kilodalton protein involved in regulating the tissue-specific expression of the chicken vimentin gene.

Animals↗

PubMed1989

Down-regulation of vimentin gene expression during myogenesis is controlled by a 5'-flanking sequence.

During myogenesis, the intermediate filament proteins vimentin and desmin are differentially expressed. While desmin levels increase dramatically, vimentin mRNA levels decrease substantially. Here, we show that transfected whole- and mini-vimentin-coding genes (Vim) are expressed in fibroblasts (mouse L cells) and down-regulated during muscle cell differentiation in culture. Functional assays with 5'-end Vim::cat constructs demonstrate that this repression is controlled by a 5'-element (nt -321 to -160). This region is distinct from Vim promoter elements (nt -160 to +71) which do not contribute to vimentin's down-regulation during myogenesis.

Animals↗