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.

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.

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Multiple silencer elements are involved in regulating the chicken vimentin gene

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.934-943.1994 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2230-2240.1992 ◽

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.2.934 ◽

1994 ◽

Vol 14 (2) ◽

pp. 934-943 ◽

Cited By ~ 14

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2230 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2230-2240 ◽

Cited By ~ 24

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.

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Olfactory expression of trace amine-associated receptors requires cooperative cis-acting enhancers

Nature Communications ◽

10.1038/s41467-021-23824-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ami Shah ◽

Madison Ratkowski ◽

Alessandro Rosa ◽

Paul Feinstein ◽

Thomas Bozza

Keyword(s):

Gene Expression ◽

Large Family ◽

Sequence Motifs ◽

Specific Expression ◽

Cis Acting ◽

Conserved Sequence ◽

Trace Amine ◽

Sequence Elements ◽

Cell Type Specific Expression ◽

Cell Type Specific

AbstractOlfactory sensory neurons express a large family of odorant receptors (ORs) and a small family of trace amine-associated receptors (TAARs). While both families are subject to so-called singular expression (expression of one allele of one gene), the mechanisms underlying TAAR gene choice remain obscure. Here, we report the identification of two conserved sequence elements in the mouse TAAR cluster (T-elements) that are required for TAAR gene expression. We observed that cell-type-specific expression of a TAAR-derived transgene required either T-element. Moreover, deleting either element reduced or abolished expression of a subset of TAAR genes, while deleting both elements abolished olfactory expression of all TAARs in cis with the mutation. The T-elements exhibit several features of known OR enhancers but also contain highly conserved, unique sequence motifs. Our data demonstrate that TAAR gene expression requires two cooperative cis-acting enhancers and suggest that ORs and TAARs share similar mechanisms of singular expression.

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Positive and negative transcriptional elements of the human type IV collagenase gene

Molecular and Cellular Biology ◽

10.1128/mcb.10.12.6524-6532.1990 ◽

1990 ◽

Vol 10 (12) ◽

pp. 6524-6532

Author(s):

S M Frisch ◽

J H Morisaki

Keyword(s):

Core Promoter ◽

Type Iv Collagenase ◽

Mobility Shift ◽

Specific Expression ◽

Enhancer Element ◽

Type Iv ◽

Dnase I Footprinting ◽

Gel Mobility Shift ◽

Transcriptional Elements ◽

Mcf 7

Proteolysis by type IV collagenase (T4) has been implicated in the process of tumor metastasis. The T4 gene is expressed in fibroblasts, but not in normal epithelial cells, and its expression is specifically repressed by the E1A oncogene of adenovirus. We present an investigation of the transcriptional elements responsible for basal, E1A-repressible, and tissue-specific expression. 5'-Deletion analysis, DNase I footprinting, and gel mobility shift assays revealed a strong, E1A-repressible enhancer element, r2, located about 1,650 bp upstream of the start site. This enhancer bound a protein with binding specificity very similar to that of the transcription factor AP-2. A potent silencer sequence was found 2 to 5 bp downstream of this enhancer. The silencer repressed transcription from either r2 or AP-1 enhancer elements and in the context of either type IV collagenase or thymidine kinase (tk) gene core promoters; enhancerless transcription from the latter core promoter was also repressed. Comprising the silencer were two contiguous, autonomously functioning silencer elements. Negative regulation of T4 transcription by at least two factors was demonstrated. mcf-7 proteins specifically binding both elements were detected by gel mobility shift assays; a protein of approximately 185 kDa that bound to one of these elements was detected by DNA-protein cross-linking. The silencer repressed transcription, in an r2 enhancer-tk promoter context, much more efficiently in T4-nonproducing cells (mcf-7 or HeLa) than in T4-producing cells (HT1080), suggesting that cell type-specific silencing may contribute to the regulation of this gene.

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

Molecular and Cellular Biology ◽

10.1128/mcb.14.4.2503-2515.1994 ◽

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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Trans-activation of the human SOX3 promoter by MAZ in NT2/D1 cells

Archives of Biological Sciences ◽

10.2298/abs0803379k ◽

2008 ◽

Vol 60 (3) ◽

pp. 379-387 ◽

Cited By ~ 2

Author(s):

Natasa Kovacevic-Grujicic ◽

Kazunari Yokoyama ◽

Milena Stevanovic

Keyword(s):

Gene Expression ◽

Neuronal Differentiation ◽

Gene Transcription ◽

Binding Sites ◽

Promoter Activity ◽

Zinc Finger Protein ◽

Mobility Shift ◽

Finger Protein ◽

Sox3 Gene

In this study, we examine the role of three highly conserved putative binding sites for Myc-associated zinc finger protein (MAZ) in regulation of the human SOX3 gene expression. Electrophoretic mobility shift and supershift assays indicate that complexes formed at two out of three MAZ sites of the human SOX3 promoter involve ubiquitously expressed MAZ protein. Furthermore, in cotransfection experiments we demonstrate that MAZ acts as a positive regulator of SOX3 gene transcription in both undifferentiated and RA-differentiated NT2/D1 cells. Although MAZ increased both basal and RA-induced promoter activity, our results suggest that MAZ does not contribute to RA inducibility of the SOX3 promoter during neuronal differentiation of NT2/D1 cells.

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Identification of a killer cell-specific regulatory element of the mouse perforin gene: an Ets-binding site-homologous motif that interacts with Ets-related proteins

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6690-6701.1993 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6690-6701

Author(s):

H Koizumi ◽

M F Horta ◽

B S Youn ◽

K C Fu ◽

B S Kwon ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Binding Site ◽

Protein Interactions ◽

Regulatory Element ◽

Killer Cell ◽

Mobility Shift ◽

Specific Expression ◽

Native Molecular Mass ◽

Mobility Shift Assay

The gene encoding the cytolytic protein perforin is selectively expressed by activated killer lymphocytes. To understand the mechanisms underlying the cell-type-specific expression of this gene, we have characterized the regulatory functions and the DNA-protein interactions of the 5'-flanking region of the mouse perforin gene (Pfp). A region extending from residues +62 through -141, which possesses the essential promoter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region between residues -411 and -566 was chosen for further characterization, since it contains an enhancer-like activity. We have identified a 32-mer sequence (residues -491 to -522) which appeared to be capable of enhancing gene expression in a killer cell-specific manner. Within this segment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designated NF-P motif), which is highly homologous to the Ets proto-oncoprotein-binding site, was found to interact with two proteins, NF-P1 and NF-P2. NF-P2 appears to be induced by reagents known to up-regulate the perforin message level and is present exclusively in killer cells. Electrophoretic mobility shift assay and UV cross-linking experiments revealed that NF-P1 and NF-P2 may possess common DNA-binding subunits. However, the larger native molecular mass of NF-P1 suggests that NF-P1 contains an additional non-DNA-binding subunit(s). In view of the homology between the NF-P motif and other Ets proto-oncoprotein-binding sites, it is postulated that NF-P1 and NF-P2 belong to the Ets protein family. Results obtained from the binding competition assay, nevertheless, suggest that NF-P1 and NF-P2 are related to but distinct from Ets proteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.

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