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.

Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

scholarly journals Interactions of the Antizyme AtoC with Regulatory Elements of the Escherichia coli atoDAEB Operon

2007 ◽  
Vol 189 (17) ◽  
pp. 6324-6332 ◽  
Author(s):  
Meropi K. Matta ◽  
Efthimia E. Lioliou ◽  
Cynthia H. Panagiotidis ◽  
Dimitrios A. Kyriakidis ◽  
Christos A. Panagiotidis
Keyword(s):  
Escherichia Coli ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Integration Host Factor ◽  
Chemical Mutagenesis ◽  
Dual Function

ABSTRACT AtoC has a dual function as both an antizyme, the posttranslational inhibitor of polyamine biosynthetic enzymes, and the transcriptional regulator of genes involved in short-chain fatty acid catabolism (the atoDAEB operon). We have previously shown that AtoC is the response regulator of the AtoS-AtoC two-component signal transduction system that activates atoDAEB when Escherichia coli is exposed to acetoacetate. Here, we show that the same cis elements control both promoter inducibility and AtoC binding. Chromatin immunoprecipitation experiments confirmed the acetoacetate-inducible binding of AtoC to the predicted DNA region in vivo. DNase I protection footprinting analysis revealed that AtoC binds two 20-bp stretches, constituting an inverted palindrome, that are located at −146 to −107 relative to the transcription initiation site. Analyses of promoter mutants obtained by in vitro chemical mutagenesis of the atoDAEB promoter verified both the importance of AtoC binding for the inducibility of the promoter by acetoacetate and the σ54 dependence of atoDAEB expression. The integration host factor was also identified as a critical component of the AtoC-mediated induction of atoDAEB.

scholarly journals Evidence that Oxidative Stress InducesspxA2Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

2016 ◽  
Vol 198 (21) ◽  
pp. 2902-2913 ◽  
Author(s):  
Skye Barendt ◽  
Cierra Birch ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Oxidative Stress ◽  
Bacillus Anthracis ◽  
Transcription Initiation ◽  
Bacterial Species ◽  
Negative Control ◽  
Mobility Shift ◽  
Content Type ◽  
Oxidative Protein Damage

ABSTRACTBacillus anthracispossesses two paralogs of the transcriptional regulator, Spx. SpxA1 and SpxA2 interact with RNA polymerase (RNAP) to activate the transcription of genes implicated in the prevention and alleviation of oxidative protein damage. ThespxA2gene is highly upregulated in infected macrophages, but how this is achieved is unknown. Previous studies have shown that thespxA2gene was under negative control by the Rrf2 family repressor protein, SaiR, whose activity is sensitive to oxidative stress. These studies also suggested thatspxA2was under positive autoregulation. In the present study, we show byin vivoandin vitroanalyses thatspxA2is under direct autoregulation but is also dependent on the SpxA1 paralogous protein. The deletion of eitherspxA1orspxA2reduced the diamide-inducible expression of anspxA2-lacZconstruct.In vitrotranscription reactions using purifiedB. anthracisRNAP showed that SpxA1 and SpxA2 protein stimulates transcription from a DNA fragment containing thespxA2promoter. Ectopically positionedspxA2-lacZfusion requires both SpxA1 and SpxA2 for expression, but the requirement for SpxA1 is partially overcome whensaiRis deleted. Electrophoretic mobility shift assays showed that SpxA1 and SpxA2 enhance the affinity of RNAP forspxA2promoter DNA and that this activity is sensitive to reductant. We hypothesize that the previously observed upregulation ofspxA2in the oxidative environment of the macrophage is at least partly due to SpxA1-mediated SaiR repressor inactivation and the positive autoregulation ofspxA2transcription.IMPORTANCERegulators of transcription initiation are known to govern the expression of genes required for virulence in pathogenic bacterial species. Members of the Spx family of transcription factors function in control of genes required for virulence and viability in low-GC Gram-positive bacteria. InBacillus anthracis, thespxA2gene is highly induced in infected macrophages, which suggests an important role in the control of virulence gene expression during the anthrax disease state. We provide evidence that elevated concentrations of oxidized, active SpxA2 result from an autoregulatory positive-feedback loop drivingspxA2transcription.

Identification of a new promoter upstream of the murine dihydrofolate reductase gene

1989 ◽  
Vol 9 (10) ◽  
pp. 4568-4570
Author(s):  
L J Schilling ◽  
P J Farnham
Keyword(s):  
Dihydrofolate Reductase ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Transcription Initiation Site ◽  
Deletion Mapping ◽  
Reductase Gene ◽  
Bona Fide ◽  
Translational Start

In vitro reactions identified a transcription initiation site located 740 nucleotides upstream of the dihydrofolate reductase translational start. Transcription from this site proceeded in the direction opposite to that of dihydrofolate reductase mRNA. Deletion mapping indicated that this new promoter can be separated from the dihydrofolate reductase promoter and that separation increased transcription at -740. Transcripts that initiate at -740 were also detected in cellular RNA, indicating that this is a bona fide transcription initiation site in vivo.

scholarly journals Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

1990 ◽  
Vol 10 (6) ◽  
pp. 2832-2839 ◽  
Author(s):  
A S Ponticelli ◽  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Initiation Site ◽  
Activator Proteins ◽  
Nuclear Extracts ◽  
Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

scholarly journals Transcription from a murine T-cell receptor V beta promoter depends on a conserved decamer motif similar to the cyclic AMP response element.

1989 ◽  
Vol 9 (11) ◽  
pp. 4835-4845 ◽  
Author(s):  
S J Anderson ◽  
S Miyake ◽  
D Y Loh
Keyword(s):  
Cyclic Amp ◽  
Regulatory Region ◽  
Cell Receptor ◽  
Transcription Activator ◽  
Mobility Shift ◽  
Response Element ◽  
Cyclic Amp Response Element ◽  
Gel Mobility Shift

We identified a regulatory region of the murine V beta promoter by both in vivo and in vitro analyses. The results of transient transfection assays indicated that the dominant transcription-activating element within the V beta 8.3 promoter is the palindromic motif identified previously as the conserved V beta decamer. Elimination of this element, by linear deletion or specific mutation, reduced transcriptional activity from this promoter by 10-fold. DNase I footprinting, gel mobility shift, and methylation interference assays confirmed that the palindrome acts as the binding site of a specific nuclear factor. In particular, the V beta promoter motif functioned in vitro as a high-affinity site for a previously characterized transcription activator, ATF. A consensus cyclic AMP response element (CRE) but not a consensus AP-1 site, can substitute for the decamer in vivo. These data suggest that cyclic AMP response element-binding protein (ATF/CREB) or related proteins activate V beta transcription.

Myogenic regulatory factors regulate M-cadherin expression by targeting its proximal promoter elements

2010 ◽  
Vol 428 (2) ◽  
pp. 223-233 ◽  
Author(s):  
Sheng Pin Hsiao ◽  
Shen Liang Chen
Keyword(s):  
Cell Adhesion ◽  
Transcription Initiation ◽  
Myogenic Differentiation ◽  
Initiation Site ◽  
Proximal Promoter ◽  
Myogenic Regulatory Factors ◽  
Regulatory Factors ◽  
Cell Cell

M- and N-cadherin are members of the Ca2+-dependent cell–cell adhesion molecule family. M-cadherin is expressed predominantly in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell–cell adhesion also plays an important role in skeletal myogenesis. In the present study, we found that both genes were differentially expressed in C2C12 and Sol8 myoblasts during myogenic differentiation and that the expression of M-cadherin was preferentially enhanced in slow-twitch muscle. Interestingly, most MRFs (myogenic regulatory factors) significantly activated the promoter of M-cadherin, but not that of N-cadherin. In line with this, overexpression of MyoD in C3H10T1/2 fibroblasts strongly induced endogenous M-cadherin expression. Promoter analysis in silico and in vitro identified an E-box (from −2 to +4) abutting the transcription initiation site within the M-cadherin promoter that is bound and differentially activated by different MRFs. The activation of the M-cadherin promoter by MRFs was also modulated by Bhlhe40 (basic helix–loop–helix family member e40). Finally, chromatin immunoprecipitation proved that MyoD as well as myogenin binds to the M-cadherin promoter in vivo. Taken together, these observations identify a molecular mechanism by which MRFs regulate M-cadherin expression directly to ensure the terminal differentiation of myoblasts.

scholarly journals Thymine at —5 Is Crucial for cpc Promoter Activity of Synechocystis sp. Strain PCC 6714

2003 ◽  
Vol 185 (21) ◽  
pp. 6477-6480 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Shoko Fujiwara ◽  
Ryohei Tanigawa ◽  
Kan Tanaka ◽  
Takatsugu Hirokawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Promoter Activity ◽  
Initiation Site ◽  
Pcc 7942 ◽  
Rna Polymerase Promoter ◽  
Synechocystis Sp

ABSTRACT The levels of transcripts of the cpc operon were highly reduced in a PD-1 mutant of cyanobacterium Synechocystis sp. strain PCC 6714. This was due to a substitution of C for T that occurred at 5 bp upstream of the transcription initiation site of the cpc operon. Any substitution for T at the −5 position drastically reduced both in vivo and in vitro promoter activity in cyanobacterium Synechococcus sp. strain PCC 7942 but not the in vivo activity in Escherichia coli. This suggests that the requirement of −5T appears to be specific for a cyanobacterial RNA polymerase-promoter combination.

scholarly journals Mechanism of action of a repressor of dioxin-dependent induction of Cyp1a1 gene transcription.

1992 ◽  
Vol 12 (5) ◽  
pp. 2115-2123 ◽  
Author(s):  
A J Watson ◽  
K I Weir-Brown ◽  
R M Bannister ◽  
F F Chu ◽  
S Reisz-Porszasz ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
High Salt ◽  
In Vitro Assays ◽  
Ah Receptor ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Gel Mobility Shift

A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.

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