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

1992 ◽  
Vol 12 (5) ◽  
pp. 2230-2240 ◽  
Author(s):  
D M Stover ◽  
Z E Zehner
Keyword(s):  
Gene Expression ◽  
Developmental Regulation ◽  
Gene Activity ◽  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Silencer Element ◽  
Vimentin Gene

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.

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

1992 ◽  
Vol 12 (5) ◽  
pp. 2230-2240
Author(s):  
D M Stover ◽  
Z E Zehner
Keyword(s):  
Gene Expression ◽  
Developmental Regulation ◽  
Gene Activity ◽  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Silencer Element ◽  
Vimentin Gene

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.

Multiple silencer elements are involved in regulating the chicken vimentin gene

1994 ◽  
Vol 14 (2) ◽  
pp. 934-943
Author(s):  
R J Garzon ◽  
Z E Zehner
Keyword(s):  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Specific Expression ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Silencer Elements ◽  
Gel Mobility Shift ◽  
Filament Protein ◽  
Silencer Element ◽  
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.

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

1994 ◽  
Vol 14 (2) ◽  
pp. 934-943 ◽  
Author(s):  
R J Garzon ◽  
Z E Zehner
Keyword(s):  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Specific Expression ◽  
Cis Acting ◽  
Dnase I Footprinting ◽  
Silencer Elements ◽  
Gel Mobility Shift ◽  
Filament Protein ◽  
Silencer Element ◽  
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.

scholarly journals A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376 ◽  
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

scholarly journals A soluble transcription factor, Oct-1, is also found in the insoluble nuclear matrix and possesses silencing activity in its alanine-rich domain.

1996 ◽  
Vol 16 (8) ◽  
pp. 4366-4377 ◽  
Author(s):  
M K Kim ◽  
L A Lesoon-Wood ◽  
B D Weintraub ◽  
J H Chung
Keyword(s):  
Nuclear Matrix ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Homeodomain Protein ◽  
Mobility Shift ◽  
Rich Domain ◽  
Gel Mobility Shift ◽  
Silencer Element

Expression of the human thyrotropin beta (hTSHbeta) gene is restricted to thyrotrophs, at least in part, by silencing. Using transient-transfection assays, we have localized a silencer element to a region between -128 and -480 bp upstream of the transcription initiation site. The silencing activity was overcome in a thyrotroph-specific manner by an unknown enhancer located in the sequences at -approximately 10000 to -1200 bp. The ubiquitous POU homeodomain protein Oct-1 recognized the A/T-rich silencer element at multiple sites in gel mobility shift assays and in vitro footprinting analyses. The silencing activity of Oct-1 was localized in its C-terminal alanine-rich domain, suggesting that Oct-1 plays a role in silencing of the hTSHbeta promoter. Further, a significant fraction of Oct-1 was shown to be associated with the nuclear matrix, and the hTSHbeta silencer region was tethered to a nuclear matrix of human cells in vivo, suggesting a possible role of the Oct-1-hTSHbeta silencer region interaction in chromatin organization.

scholarly journals Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 861-864 ◽  
Author(s):  
N Raich ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos ◽  
T Enver
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Negative Element ◽  
Cis Acting

The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

scholarly journals Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice

Blood ◽  
1992 ◽  
Vol 79 (4) ◽  
pp. 861-864 ◽  
Author(s):  
N Raich ◽  
T Papayannopoulou ◽  
G Stamatoyannopoulos ◽  
T Enver
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Developmental Regulation ◽  
Globin Gene ◽  
Negative Element ◽  
Cis Acting

Abstract The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

Transcriptional regulation of the type I myosin heavy chain promoter in inactive rat soleus

2002 ◽  
Vol 282 (3) ◽  
pp. C528-C537 ◽  
Author(s):  
K. A. Huey ◽  
R. R. Roy ◽  
F. Haddad ◽  
V. R. Edgerton ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Gene Promoter ◽  
Type I ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Transcription Enhancer ◽  
Enhancer Factor

Chronic muscle inactivity with spinal cord isolation (SI) decreases expression of slow type I myosin heavy chain (MHC) while increasing expression of the faster MHC isoforms, primarily IIx. The purpose of this study was to determine whether type I MHC downregulation in the soleus muscle of SI rats is regulated transcriptionally and to identify cis-acting elements or regions of the rat type I MHC gene promoter involved in this response. One week of SI significantly decreased in vivo activity of the −3500-, −408-, −299-, −215-, and −171-bp type I MHC promoters. The activity of all tested deletions of the type I MHC promoter, relative to the human skeletal α-actin promoter, were significantly reduced in the SI soleus, except activity of the −171-bp promoter, which increased. Mutation of the βe3 element (−214/−190 bp) in the −215- and −408-bp promoters and deletion of this element (−171-bp promoter) attenuated type I downregulation with SI. Gel mobility shift assays demonstrated a decrease in transcription enhancer factor-1 binding to the βe3 element with SI, despite an increase in total binding to this region. These results demonstrate that type I MHC downregulation with SI is transcriptionally regulated and suggest that interactions between transcription enhancer factor-1 and the βe3 element are likely involved in this response.

The Aspergillus nidulans abaA gene encodes a transcriptional activator that acts as a genetic switch to control development

1994 ◽  
Vol 14 (4) ◽  
pp. 2503-2515
Author(s):  
A Andrianopoulos ◽  
W E Timberlake
Keyword(s):  
Aspergillus Nidulans ◽  
Transcriptional Activation ◽  
Expression System ◽  
Regulatory Gene ◽  
Binding Motif ◽  
Mobility Shift ◽  
Genetic Switch ◽  
Regulatory Regions ◽  
Cis Acting ◽  
Gel Mobility Shift

The Aspergillus nidulans abaA gene encodes a protein containing an ATTS DNA-binding motif and is required for the terminal stages of conidiophore development. Results from gel mobility shift and protection, missing-contact, and interference footprint assays showed that AbaA binds to the sequence 5'-CATTCY-3', where Y is a pyrimidine, making both major- and minor-groove contacts. Multiple AbaA binding sites are present in the cis-acting regulatory regions of several developmentally controlled structural genes as well as those of the upstream regulatory gene brlA, the downstream regulatory gene wetA, and abaA itself. These cis-acting regulatory regions confer AbaA-dependent transcriptional activation in a heterologous Saccharomyces cerevisiae gene expression system. From these observations, we propose that the AbaA transcription factor establishes a novel set of feedback regulatory loops responsible for determination of conidiophore development.

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