scholarly journals Different Sp1 family members differentially affect transcription from the human elongation factor 1 A-1 gene promoter

1998 ◽  
Vol 333 (3) ◽  
pp. 511-517 ◽  
Author(s):  
S⊘ren J. NIELSEN ◽  
Morten PRÆSTEGAARD ◽  
Helle F. J. ØRGENSEN ◽  
Brian F. C. CLARK
Keyword(s):  
Hela Cells ◽  
Promoter Activity ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Elongation Factor ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Gc Box ◽  
Elongation Factor 1 ◽  
Human Elongation Factor

The GC box is an important transcriptional regulatory element present in the promoters of many mammalian genes. In the present study we examine the effect of known GC-box-binding proteins on the promoter of the human elongation factor 1 A-1 (hEF1A-1) gene in human HeLa cells and Drosophila SL2 cells. In HeLa cells co-transfection with the GC-box-binding protein BTEB resulted in a 4–10-fold increase in hEF1A-1 promoter activity. This stimulation was dependent on a single GC box located between positions -69 and -50 of the promoter. Little or no effect was observed of other GC-box-binding proteins including Sp1, Sp3, Sp4 and BTEB2. In SL2 cells stimulation by Sp1 and Sp3 through the single GC box of the proximal promoter led to 13-fold and 21-fold increases respectively in promoter activity. Inclusion of further upstream sequences resulted in high levels of expression when Sp1 or Sp3 was co-transfected with the reporter plasmid. In this setting Sp1 stimulated transcription by 750-fold, whereas Sp3 was even more potent, yielding a 1150-fold stimulation. Mobility-shift assays performed with the promoter-proximal GC box demonstrated the binding of Sp1, Sp3 and Sp4 to this sequence. To our knowledge, the present study represents the first comparison of all known GC-box-binding proteins on a natural promoter.

The Human Elongation Factor 1 A-2 Gene (EEF1A2): Complete Sequence and Characterization of Gene Structure and Promoter Activity

Genomics ◽  
2000 ◽  
Vol 68 (1) ◽  
pp. 63-70 ◽  
Author(s):  
Claus Bischoff ◽  
Søren Kahns ◽  
Ann Lund ◽  
Helle F. Jørgensen ◽  
Morten Præstegaard ◽  
...  
Keyword(s):  
Gene Structure ◽  
Promoter Activity ◽  
Elongation Factor ◽  
Complete Sequence ◽  
Elongation Factor 1 ◽  
Human Elongation Factor

scholarly journals Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.

1995 ◽  
Vol 15 (7) ◽  
pp. 3830-3839 ◽  
Author(s):  
T Yamagata ◽  
J Nishida ◽  
R Sakai ◽  
T Tanaka ◽  
H Honda ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Regulatory Element ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Proximal Promoter ◽  
Binding Motif ◽  
Mobility Shift ◽  
Cis Acting ◽  
Interleukin 5 ◽  
Mobility Shift Assay

Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.

scholarly journals Regulation of the human α2(1) procollagen gene by sequences adjacent to the CCAAT box

1997 ◽  
Vol 322 (1) ◽  
pp. 199-206 ◽  
Author(s):  
Malcolm COLLINS ◽  
Virna D. LEANER ◽  
Mziwandile MADIKIZELA ◽  
M. Iqbal PARKER
Keyword(s):  
Complex Formation ◽  
Complex I ◽  
Binding Proteins ◽  
Regulatory Element ◽  
Complex Iii ◽  
Proximal Promoter ◽  
In Vitro Mutagenesis ◽  
Mobility Shift ◽  
Pyrimidine Nucleotide ◽  
Ccaat Box

The human, rat, mouse and chicken α2(I) procollagen promoters analysed to date all contain an inverted CCAAT box at -80. In this study we have examined the binding of nuclear proteins to the proximal promoter of the human α2(I) procollagen gene, where an inverted CCAAT box is flanked by a downstream GGAGG sequence and its inverted counterpart (CCTCC) on the upstream end. Each of the GGAGG sequences is separated from the inverted CCAAT box by a single pyrimidine nucleotide (). Electrophoretic mobility-shift assays (EMSAs) revealed that two distinct DNAŐprotein complexes formed on this DNA sequence. Methylation interference analysis and in vitro mutagenesis studies revealed that the integrity of the sequence (the GGAGG/CCAAT-binding element or G/CBE) was important for the binding of the CCAAT-binding factor (CBF) (complex I). Competition studies showed that complex formation on the human G/CBE could be competed by mouse CBE and nuclear factor-Y (NF-Y) oligonucleotides, suggesting that mouse CBE and human G/CBE-binding proteins belong to the same family of CCAAT box binding proteins. Furthermore, antibodies to mouse CBF specifically supershifted the G/CBE complex (complex I) in EMSAs. The downstream GGAGG and 3ƀ-flanking sequences () or collagen modulating element (CME), however, were important for the formation of a novel DNAŐprotein complex (complex III). The formation of this complex was not competed out by CBE or NF-Y oligonucleotides, nor was DNAŐprotein complex formation affected by the anti-CBF antibody. Functional analysis of G/CBE and CME elements subjected to mutagenesis, using promoterŐchloroamphenicol acetyl transferase constructs in transient transfection assays, showed that both these elements were essential for activity of the human promoter. These experiments identified a novel regulatory element in the human α2(1) procollagen gene which is not present in the rodent gene.

scholarly journals Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity

2004 ◽  
Vol 380 (2) ◽  
pp. 455-463 ◽  
Author(s):  
Hsueh-Hsiao WANG ◽  
An-Na CHIANG
Keyword(s):  
Gene Expression ◽  
Hela Cells ◽  
Promoter Activity ◽  
Hepatoma Cell ◽  
Site Directed Mutagenesis ◽  
Tata Box ◽  
Proximal Promoter ◽  
Nuclear Factor ◽  
Mobility Shift ◽  
Glycoprotein I

β2-Glycoprotein I (β2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of β2-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of β2-GPI gene expression has not been clarified. To gain more insight into the control of β2-GPI gene expression, we cloned the 4.1-kb 5´-flanking region and characterized the proximal promoter of the β2-GPI gene in this study. Cis-acting elements required for β2-GPI promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the β2-GPI 5´-flanking sequence revealed that the region from −197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative cis-elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the β2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1α with the HNF-1 site residing downstream of the TATA box. Co-transfection of β2-GPI promoter-luciferase vector with HNF-1α expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1α on β2-GPI promoter activity. In addition, overexpression of HNF-1α enhanced the endogenous β2-GPI expression. These results suggest that the atypical TATA box and HNF-1 cis-element are critical for β2-GPI transcription and HNF-1α may play an important role in cell-specific regulation of β2-GPI gene expression.

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 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 Promoter analysis of the DHCR24 (3β-hydroxysterol Δ24-reductase) gene: characterization of SREBP (sterol-regulatoryelement-binding protein)-mediated activation

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Lidia A. Daimiel ◽  
María E. Fernández-Suárez ◽  
Sara Rodríguez-Acebes ◽  
Lorena Crespo ◽  
Miguel A. Lasunción ◽  
...  
Keyword(s):  
Cellular Response ◽  
Binding Protein ◽  
Regulatory Element ◽  
Cholesterol Biosynthesis ◽  
Regulatory Elements ◽  
Proximal Promoter ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Gene Characterization ◽  
Cis Acting

DHCR24 (3β-hydroxysterol Δ24-reductase) catalyses the reduction of the C-24 double bond of sterol intermediates during cholesterol biosynthesis. DHCR24 has also been involved in cell growth, senescence and cellular response to oncogenic and oxidative stress. Despite its important roles, little is known about the transcriptional mechanisms controlling DHCR24 gene expression. We analysed the proximal promoter region and the cholesterol-mediated regulation of DHCR24. A putative SRE (sterol-regulatory element) at −98/−90 bp of the transcription start site was identified. Other putative regulatory elements commonly found in SREBP (SRE-binding protein)-targeted genes were also identified. Sterol responsiveness was analysed by luciferase reporter assays of approximately 1 kb 5′-flanking region of the human DHCR24 gene in HepG2 and SK-N-MC cells. EMSAs (electrophoretic mobility-shift assays) and ChIP (chromatin immunoprecipitation) assays demonstrated cholesterol-dependent recruitment and binding of SREBPs to the putative SRE. Given the presence of several CACCC-boxes in the DHCR24 proximal promoter, we assessed the role of KLF5 (Krüppel-like factor 5) in androgen-regulated DHCR24 expression. DHT (dihydrotestosterone) increased DHCR24 expression synergistically with lovastatin. However, DHT was unable to activate the DHCR24 proximal promoter, whereas KLF5 did, indicating that this mechanism is not involved in the androgen-induced stimulation of DHCR24 expression. The results of the present study allow the elucidation of the mechanism of regulation of the DHCR24 gene by cholesterol availability and identification of other putative cis-acting elements which may be relevant for the regulation of DHCR24 expression.

In vivo regulation of the β-myosin heavy chain gene in hypertensive rodent heart

2001 ◽  
Vol 280 (5) ◽  
pp. C1262-C1276 ◽  
Author(s):  
Carola E. Wright ◽  
P. W. Bodell ◽  
F. Haddad ◽  
A. X. Qin ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Promoter Activity ◽  
Proximal Promoter ◽  
Future Research ◽  
Chain Gene ◽  
Direct Gene Transfer ◽  
Basal Region ◽  
Mobility Shift

The main goal of this study was to examine the transcriptional activity of different-length β-myosin heavy chain (β-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by ∼45% relative to control. Results show that β-MHC promoter activity of all tested wild-type constructs, i.e., −3500, −408, −299, −215, −171, and −71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the −71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting βe1, βe2, GATA, and βe3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the β-MHC gene. Collectively, these results indicate that three separate regions on the β-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between −408 and −3500 bp, 2) a proximal region between −299 and −215 bp, and 3) a basal region within −71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate β-MHC transcriptional regulation in response to pressure overload.

scholarly journals Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1469-1474 ◽  
Author(s):  
Yaxu WU ◽  
Johannes RUEF ◽  
N. Gadiparthi RAO ◽  
Cam PATTERSON ◽  
S. Marschall RUNGE
Keyword(s):  
Endothelial Cells ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Thrombin Receptor ◽  
Wild Type ◽  
Mobility Shift ◽  
Human Thrombin ◽  
Gc Box

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3ʹ GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3ʹ cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.

scholarly journals Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression

1993 ◽  
Vol 13 (6) ◽  
pp. 3202-3212
Author(s):  
L Guerrini ◽  
S S Gong ◽  
K Mangasarian ◽  
C Basilico
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Hela Cells ◽  
Promoter Activity ◽  
Regulatory Element ◽  
Amino Acid Starvation ◽  
Asparagine Synthetase ◽  
Start Site ◽  
Transcription Start ◽  
Nuclear Extracts

We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically up-regulated by asparagine deprivation in ts11 cells, mutants of BHK hamster cells which encode a temperature-sensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Sp1, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNA-binding properties.

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