scholarly journals Identification of a silencer module which selectively represses cyclic AMP-responsive element-dependent gene expression.

1995 ◽  
Vol 15 (11) ◽  
pp. 6139-6149 ◽  
Author(s):  
K C Chung ◽  
D Huang ◽  
Y Chen ◽  
S Short ◽  
M L Short ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Specific Binding ◽  
Regulatory Element ◽  
Inducible Promoter ◽  
Binding Activity ◽  
Mobility Shift ◽  
Responsive Element ◽  
Gel Mobility Shift ◽  
Related Proteins ◽  
Dyad Symmetry

The cyclic AMP (cAMP)-inducible promoter from the rat lactate dehydrogenase A subunit gene (LDH A) is associated with a distal negative regulatory element (LDH-NRE) that represses inherent basal and cAMP-inducible promoter activity. The element is of dyad symmetry, consisting of a palindromic sequence with two half-sites, 5'-TCTTG-3'. It represses the expression of an LDH A/chloramphenicol acetyltransferase (CAT) reporter gene in a dose-dependent, orientation- and position-independent fashion, suggesting that it is a true silencer element. Uniquely, it selectively represses cAMP-responsive element (CRE)-dependent transcription but has no effect on promoters lacking a CRE sequence. The repressing action of LDH-NRE could be overcome by cotransfection with LDH A/CAT vector oligonucleotides containing either the LDH-NRE or CRE sequence. This suggests that the reversal of repression was caused by the removal of functional active, limiting transacting factors which associate with LDH-NRE as well as with CRE. Gel mobility shift, footprinting, and Southwestern blotting assays demonstrated the presence of a 69-kDa protein with specific binding activity for LDH-NRE. Additionally, gel supershift assays with anti-CREB and anti-Fos antibodies indicate the presence of CREB and Fos or antigenically closely related proteins with the LDH-NRE/protein complex. We suggest that the LDH-NRE and CRE modules functionally interact to achieve negative modulation of cAMP-responsive LDH A transcriptional activity.

scholarly journals In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter.

1991 ◽  
Vol 11 (4) ◽  
pp. 2162-2168 ◽  
Author(s):  
E Dubois ◽  
F Messenguy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Binding ◽  
Regulatory Element ◽  
Regulatory Proteins ◽  
Arginine Metabolism ◽  
Mobility Shift ◽  
Catabolic Genes ◽  
Gel Mobility Shift ◽  
Dna Complex

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter

1991 ◽  
Vol 11 (4) ◽  
pp. 2162-2168
Author(s):  
E Dubois ◽  
F Messenguy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Binding ◽  
Regulatory Element ◽  
Regulatory Proteins ◽  
Arginine Metabolism ◽  
Mobility Shift ◽  
Catabolic Genes ◽  
Gel Mobility Shift ◽  
Dna Complex

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

Blood ◽  
2001 ◽  
Vol 98 (8) ◽  
pp. 2555-2562 ◽  
Author(s):  
Mark Loyevsky ◽  
Timothy LaVaute ◽  
Charles R. Allerson ◽  
Robert Stearman ◽  
Olakunle O. Kassim ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Regulatory Protein ◽  
Protein S ◽  
Fold Increase ◽  
Binding Activity ◽  
Mobility Shift ◽  
Iron Responsive Element ◽  
Element Binding Protein ◽  
Responsive Element ◽  
Electrophoretic Mobility Shift Assays

Abstract This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PfIRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)–like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex fromP falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated.

Lipid deprivation increases surfactant phosphatidylcholine synthesis via a sterol-sensitive regulatory element within the CTP:phosphocholine cytidylyltransferase promoter

2002 ◽  
Vol 362 (1) ◽  
pp. 81-88 ◽  
Author(s):  
Rama K. MALLAMPALLI ◽  
Alan J. RYAN ◽  
James L. CARROLL ◽  
Timothy F. OSBORNE ◽  
Christie P. THOMAS
Keyword(s):  
Specific Binding ◽  
Core Promoter ◽  
Regulatory Element ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Luciferase Reporter ◽  
Standard Diet ◽  
Flanking Sequence ◽  
Mobility Shift ◽  
Ctp:Phosphocholine Cytidylyltransferase

Lipid-deprived mice increase alveolar surfactant disaturated phosphatidylcholine (DSPtdCho) synthesis compared with mice fed a standard diet by increasing expression of CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme for DSPtdCho synthesis. We previously observed that lipid deprivation increases mRNA synthesis for CCT [Ryan, McCoy, Mathur, Field and Mallampalli (2000) J. Lipid Res. 41, 1268–1277]. To evaluate regulatory mechanisms for this gene, we cloned the proximal ∼ 1900bp of the 5′ flanking sequence of the murine CCT gene, coupled this to a luciferase reporter, and examined transcriptional regulation in a murine alveolar epithelial type II cell line (MLE-12). The core promoter was localized to a region between −169 and +71bp, which exhibited strong basal activity comparable with the simian virus 40 promoter. The full-length construct, from −1867 to +71, was induced 2–3-fold when cells were cultured in lipoprotein-deficient serum (LPDS), similar to the level of induction of the endogenous CCT gene. By deletional analysis the sterol regulatory element (SRE) was localized within a 240bp region. LPDS activation of the CCT promoter was abolished by mutation of this SRE, and gel mobility-shift assays demonstrated specific binding of recombinant SRE-binding protein to this element within the CCT promoter. These observations indicate that sterol-regulated expression of CCT is mediated by an SRE within its 5′ flanking region.

Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs

1993 ◽  
Vol 13 (7) ◽  
pp. 4167-4173
Author(s):  
J Min ◽  
H P Zassenhaus
Keyword(s):  
Binding Activity ◽  
Nucleoside Triphosphate ◽  
Yeast Mitochondria ◽  
Mobility Shift ◽  
Saccharomyces Cerevisiae Mitochondria ◽  
Molecular Masses ◽  
Labeled Rna ◽  
Gel Mobility Shift ◽  
A Site

An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.

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.

Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding

1992 ◽  
Vol 12 (5) ◽  
pp. 1940-1949
Author(s):  
A D Keller ◽  
T Maniatis
Keyword(s):  
Dna Binding ◽  
Zinc Finger ◽  
Specific Binding ◽  
Regulatory Element ◽  
Zinc Fingers ◽  
Transcriptional Repressor ◽  
Gene Promoter ◽  
Binding Activity ◽  
Necessary And Sufficient ◽  
Sequence Requirements

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.

scholarly journals Identification of single-stranded-DNA-binding proteins that interact with muscle gene elements.

1991 ◽  
Vol 11 (4) ◽  
pp. 1944-1953 ◽  
Author(s):  
I M Santoro ◽  
T M Yi ◽  
K Walsh
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Binding Activity ◽  
Sequence Motif ◽  
Mobility Shift ◽  
Specific Sequence ◽  
Single Stranded Dna ◽  
Muscle Gene

A sequence-specific DNA-binding protein from skeletal-muscle extracts that binds to probes of three muscle gene DNA elements is identified. This protein, referred to as muscle factor 3, forms the predominant nucleoprotein complex with the MCAT gene sequence motif in an electrophoretic mobility shift assay. This protein also binds to the skeletal actin muscle regulatory element, which contains the conserved CArG motif, and to a creatine kinase enhancer probe, which contains the E-box motif, a MyoD-binding site. Muscle factor 3 has a potent sequence-specific, single-stranded-DNA-binding activity. The specificity of this interaction was demonstrated by sequence-specific competition and by mutations that diminished or eliminated detectable complex formation. MyoD, a myogenic determination factor that is distinct from muscle factor 3, also bound to single-stranded-DNA probes in a sequence-specific manner, but other transcription factors did not. Multiple copies of the MCAT motif activated the expression of a heterologous promoter, and a mutation that eliminated expression was correlated with diminished factor binding. Muscle factor 3 and MyoD may be members of a class of DNA-binding proteins that modulate gene expression by their abilities to recognize DNA with unusual secondary structure in addition to specific sequence.

scholarly journals Inhibition of cannabinoid CB1 receptor upregulates Slc2a4 expression via nuclear factor-κB and sterol regulatory element-binding protein-1 in adipocytes

2012 ◽  
Vol 49 (2) ◽  
pp. 97-106 ◽  
Author(s):  
D T Furuya ◽  
A C Poletto ◽  
H S Freitas ◽  
U F Machado
Keyword(s):  
Glucose Transporter ◽  
Binding Protein ◽  
Regulatory Element ◽  
Endocannabinoid System ◽  
Cb1 Receptor ◽  
Binding Activity ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Element Binding Protein ◽  
Sterol Regulatory Element

Evidences have suggested that the endocannabinoid system is overactive in obesity, resulting in enhanced endocannabinoid levels in both circulation and visceral adipose tissue. The blockade of cannabinoid receptor type 1 (CB1) has been proposed for the treatment of obesity. Besides loss of body weight, CB1 antagonism improves insulin sensitivity, in which the glucose transporter type 4 (GLUT4) plays a key role. The aim of this study was to investigate the modulation of GLUT4-encoded gene (Slc2a4 gene) expression by CB1 receptor. For this, 3T3-L1 adipocytes were incubated in the presence of a highly selective CB1 receptor agonist (1 μM arachidonyl-2′-chloroethylamide) and/or a CB1 receptor antagonist/inverse agonist (0.1, 0.5, or 1 μM AM251, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide). After acute (2 and 4 h) and chronic (24 h) treatments, cells were harvested to evaluate: i) Slc2a4, Cnr1 (CB1 receptor-encoded gene), and Srebf1 type a (SREBP-1a type-encoded gene) mRNAs (real-time PCR); ii) GLUT4 protein (western blotting); and iii) binding activity of nuclear factor (NF)-κB and sterol regulatory element-binding protein (SREBP)-1 specifically in the promoter of Slc2a4 gene (electrophoretic mobility shift assay). Results revealed that both acute and chronic CB1 receptor antagonism greatly increased (∼2.5-fold) Slc2a4 mRNA and protein content. Additionally, CB1-induced upregulation of Slc2a4 was accompanied by decreased binding activity of NF-κB at 2 and 24 h, and by increased binding activity of the SREBP-1 at 24 h. In conclusion, these findings reveal that the blockade of CB1 receptor markedly increases Slc2a4/GLUT4 expression in adipocytes, a feature that involves NF-κB and SREBP-1 transcriptional regulation.

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