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 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.

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.

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.

Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene

1992 ◽  
Vol 12 (8) ◽  
pp. 3614-3627
Author(s):  
P G Traber ◽  
G D Wu ◽  
W Wang
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Nuclear Proteins ◽  
Intestinal Cell ◽  
Specific Gene ◽  
In Vitro Mutagenesis ◽  
Primary Mouse

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

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.

scholarly journals Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.

1992 ◽  
Vol 12 (8) ◽  
pp. 3614-3627 ◽  
Author(s):  
P G Traber ◽  
G D Wu ◽  
W Wang
Keyword(s):  
Dna Binding ◽  
Binding Proteins ◽  
Nuclear Protein ◽  
Regulatory Element ◽  
Dna Binding Proteins ◽  
Nuclear Proteins ◽  
Intestinal Cell ◽  
Specific Gene ◽  
In Vitro Mutagenesis ◽  
Primary Mouse

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

scholarly journals Transcriptional regulation of the rat fatty acid synthase gene: identification and functional analysis of positive and negative effectors of basal transcription

1996 ◽  
Vol 317 (1) ◽  
pp. 257-265 ◽  
Author(s):  
Babak OSKOUIAN ◽  
Vangipuram S. RANGAN ◽  
Stuart SMITH
Keyword(s):  
Fatty Acid ◽  
Electrophoretic Mobility ◽  
Fatty Acid Synthase ◽  
Regulatory Element ◽  
Dnase I ◽  
Mobility Shift ◽  
Negative Regulatory Element ◽  
Dnase I Footprinting ◽  
Ccaat Box ◽  
Electrophoretic Mobility Shift Assays

The gene for fatty acid synthase (FAS), which contains both GC-rich sequences and a TATA box in its promoter region, is expressed in a tissue-specific manner in response to developmental, nutritional and hormonal signals. Here we report the identification of sequence elements in the 5´-flanking region responsible for modulation of basal promoter activity. Transient transfection of H4IIE hepatoma cells and 3T3-30A5 preadipocytes with plasmids containing the chloroamphenicol acetyltransferase gene driven by FAS promoter sequences of different lengths revealed that two regions between nucleotides -249 and -30 contain elements capable of enhancing transcription. One of these positive regulatory elements was localized to nucleotides -241/-236 using DNase I footprinting, electrophoretic mobility-shift assays and mutagenesis. The sequence element is a typical GC box and the nuclear protein binding to this region appears immunochemically indistinguishable from Sp1. The second positive regulatory element, an inverted CCAAT box, was localized to nucleotides -98/-92 by electrophoretic mobility-shift assays and mutagenesis. A putative negative regulatory element, initially identified by reporter gene transfection experiments, was localized between nucleotides -319 and -301 by DNase I footprinting, electrophoretic mobility-shift assays and deletion mutagenesis; this region consists of 78% G residues. In conclusion, initiation of FAS transcription from a single start site is enhanced by the presence of an adjacent TATA motif, an inverted CCAAT box and an upstream binding site for the transcription factor Sp1; further modulation of transcription is achieved through complex interactions between these promoter elements and an upstream negative regulatory element.

scholarly journals Induction of NF-kappa B-like activity by platelet-derived growth factor in mouse fibroblasts.

1992 ◽  
Vol 3 (10) ◽  
pp. 1131-1139 ◽  
Author(s):  
N E Olashaw ◽  
T F Kowalik ◽  
E S Huang ◽  
W J Pledger
Keyword(s):  
Growth Factor ◽  
Complex I ◽  
Cell Types ◽  
Complex Iii ◽  
Platelet Derived Growth Factor ◽  
Related Factor ◽  
Nf Kappa B ◽  
Mobility Shift ◽  
Mouse Fibroblasts ◽  
In Cells

Nuclear factor kappa B (NF-kappa B) modulates the expression of numerous genes via interaction with a specific DNA sequence termed the kappa B site. Its activity is modulated by a cytosolic inhibitor protein termed I kappa B, and its activation occurs in response to a variety of agents in a variety of cell types, most notably B and T lymphocytes. Data presented here show that an activity (designated complex I) that binds specifically to the kappa B site is induced in density-arrested Balb/c-3T3 mouse fibroblasts by platelet-derived growth factor (PDGF), a potent mitogen for these cells. Increased levels of complex I, as evaluated by electrophoretic mobility shift assays of nuclear extracts, were observed in cells treated for 1-4 h (but not 15 min) with the BB isoform of PDGF. 12-O-tetradecanoylphorbol 13-acetate (TPA) and the AA isoform of PDGF also stimulated this response and both isoforms, but not TPA, were effective in cells depleted of protein kinase C. Complex I most likely is authentic NF-kappa B, a p50-p65 heterodimer, or a closely related factor because it exhibited properties characteristic of those previously described for NF-kappa B including inducibility by deoxycholate and cycloheximide and sensitivity to I kappa B. A second kappa B binding activity (complex II), which apparently contained p50 homodimers, displayed limited induction by PDGF, whereas a third complex (complex III) migrated faster than but behaved similarly to complex I. These studies suggest that NF-kappa B or an NF-kappa B-like factor may participate in the expression of PDGF-inducible genes.

scholarly journals Regulation of osmC Gene Expression by the Two-Component System rcsB-rcsC inEscherichia coli

2001 ◽  
Vol 183 (20) ◽  
pp. 5870-5876 ◽  
Author(s):  
Marcela Davalos-Garcia ◽  
Annie Conter ◽  
Isabelle Toesca ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Response Regulator ◽  
Regulatory Element ◽  
In Vitro Transcription ◽  
Proximal Promoter ◽  
Two Component System ◽  
Mobility Shift ◽  
Component System ◽  
Two Component ◽  
Gel Mobility Shift

ABSTRACT The Escherichia coli osmC gene encodes an envelope protein of unknown function whose expression depends on osmotic pressure and growth phase. The gene is transcribed from two overlapping promoters, osmCp 1 andosmCp 2. Several factors regulating these promoters have been reported. The leucine-responsive protein Lrp represses osmCp 1 and activatesosmCp 2, the nucleoid-associated protein H-NS represses both promoters, and the stationary-phase sigma factor ςs specifically recognizesosmCp 2. This work reports the identification of an additional regulatory element, the two-component systemrcsB-rcsC, affecting positively the distal promoter osmCp 1. The response regulator of the system, RcsB, does not affect expression of the proximal promoter osmCp 2. Deletion analysis located the site necessary for RcsB activation just upstream ofosmCp 1. In vitro transcription experiments and gel mobility shift assays demonstrated that RcsB stimulates RNA polymerase binding at osmCp 1.

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