Activity of the human cytochrome c1 promoter is modulated by E2F

2000 ◽  
Vol 351 (1) ◽  
pp. 251-256 ◽  
Author(s):  
Katarina LUCIAKOVA ◽  
Peter BARATH ◽  
Ronggui LI ◽  
Ahmed ZAID ◽  
B. Dean NELSON
Keyword(s):  
Mammalian Cells ◽  
Promoter Activity ◽  
Mitochondrial Genes ◽  
Transcription Start ◽  
Mobility Shift ◽  
Transactivation Domains ◽  
Human Cytochrome ◽  
Cytochrome C1 ◽  
Gel Mobility Shift ◽  
E2f Proteins

The human cytochrome c1 promoter is strongly activated in transfected Drosophila SL2 cells expressing exogenous human E2F1. Transfection-deletion experiments, DNase I protection by E2F1 and gel mobility-shift experiments locate E2F1 activation sites to two regions on either side of the transcription start site. Deletion of either region prevents E2F1 activation in transfected SL2 cells, suggesting a co-operative interaction between them. E2F6, a member of the E2F family that lacks transactivation domains but contains specific suppressor domains, inhibits cytochrome c1 promoter activity when co-transfected into HeLa cells, indicating that the E2F proteins modulate the cytochrome c1 promoter in mammalian cells. However, E2F is not a general regulator of oxidative phosphorylation genes since three additional nuclear-encoded mitochondrial genes were unaffected by E2F1 or E2F6.

USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1065-C1072 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Inos Gene ◽  
E Box ◽  
Inos Promoter ◽  
Gel Mobility Shift

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.

scholarly journals 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.

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376 ◽  
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

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.

Regulation of NHE-1 promoter in mammalian myocardium

1996 ◽  
Vol 270 (1) ◽  
pp. H259-H266 ◽  
Author(s):  
W. Yang ◽  
J. R. Dyck ◽  
H. Wang ◽  
L. Fliegel
Keyword(s):  
Mammalian Cells ◽  
Ph Regulation ◽  
Integral Membrane Protein ◽  
Neonatal Rat ◽  
Mobility Shift ◽  
Rat Cardiomyocytes ◽  
Nuclear Extracts ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift ◽  
Reduced Activity

The Na+/H exchanger (NHE-1) is an integral membrane protein responsible for intracellular pH regulation in the myocardium and other tissues. The NHE-1 isoform is universally distributed in mammalian cells. We examined regulation of a 1.1-kb fragment of the NHE-1 promoter in neonatal rat cardiomyocytes. Deletion of most of the promoter up to an AP-2 site reduced activity 75%. Further deletion of the promoter or mutation of the AP-2 site reduced or eliminated activity almost completely. Gel mobility shift assay showed that purified AP-2 protein or AP-2-like protein from nuclear extracts of isolated myocytes can bind to DNA of the NHE-1 protein. External acidosis did not cause increased transcription from the promoter. Removal of serum from the medium reduced activity of the NHE-1 promoter. The elements responsible for activation of the promoter by serum were contained within both the 1.1-kb and AP-2-containing region. The results show that the cis-acting putative AP-2 site and the presence of serum are important in NHE-1 expression, whereas external acidosis had no direct effect on the promoter.

scholarly journals Ca2+ administration stimulates the binding of AP-1 factor to the 5′-flanking region of the rat gene for the Ca2+-binding protein regucalcin

1998 ◽  
Vol 329 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Tomiyasu MURATA ◽  
Masayoshi YAMAGUCHI
Keyword(s):  
Promoter Activity ◽  
Binding Protein ◽  
Luciferase Reporter ◽  
Rat Tissues ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Flanking Region ◽  
Gel Mobility Shift ◽  
Dna Complex ◽  
Regucalcin Gene

mRNA of the Ca2+-binding protein, regucalcin, is mainly expressed in the liver and only to a small extent in the kidney, and the expression of hepatic regucalcin mRNA is markedly stimulated by Ca2+ administration [Shimokawa and Yamaguchi (1992) FEBS Lett. 305, 151-154]. The existence of nuclear factors that bind to the 5ʹ-flanking region of the rat regucalcin gene was investigated. When nuclear proteins obtained from various rat tissues were used in gel mobility-shift assays, tissue-specific formation of a protein-DNA complex was found in the liver and kidney. An additional novel protein-DNA complex was formed when liver nuclear extracts obtained from Ca2+-administered rats (10 mg of Ca2+/100 g body weight) were used. Competition gel mobility-shift experiments using consensus and mutant oligonucleotides for AP-1 factor showed that the additional novel complex was formed from binding of the AP-1 factor to the regucalcin gene. Ca2+-induced binding of the AP-1 factor to the regucalcin gene was completely inhibited by simultaneous administration of trifluoperazine, an antagonist of calmodulin, suggesting that the activation of nuclear AP-1 protein is partly mediated through a Ca2+/calmodulin-dependent pathway. Moreover, the 5ʹ-flanking region of the rat regucalcin gene ligated to a luciferase reporter gene possessed the promoter activity in H4-II-E hepatoma cells. This promoter activity was enhanced by treatment with Bay K 8644, a Ca2+-channel agonist. The present study demonstrates that the Ca2+-response sequences are located within the 5ʹ-flanking region of the rat regucalcin gene.

scholarly journals The lung enriched transcription factor TTF-1 and the ubiquitously expressed proteins Sp1 and Sp3 interact with elements located in the minimal promoter of the rat Clara cell secretory protein gene

1996 ◽  
Vol 316 (2) ◽  
pp. 467-473 ◽  
Author(s):  
Ruud F. G. TOONEN ◽  
Sharon GOWAN ◽  
Colin D. BINGLE
Keyword(s):  
Promoter Region ◽  
Secretory Protein ◽  
Minimal Promoter ◽  
Clara Cell ◽  
Start Site ◽  
Transcription Start ◽  
Mobility Shift ◽  
Clara Cell Secretory Protein ◽  
Gel Mobility Shift ◽  
Minimal Promoter Region

The mechanisms that direct expression of the Clara cell secretory protein (CCSP) gene to the bronchiolar epithelial cells of the lung remain to be elucidated. Previous studies have identified a number of proteins which bind to a functionally important region (Region 1) located -132 to -76 bp from the transcription start site in the rat CCSP gene. Subsequently we have shown that while Region 1 is an important positive regulator of CCSP gene expression, sequences 3′ of this region (-75 to +38) are sufficient to confer tissue-specific expression of a reporter gene. In the present study we have used transient transfections with a deletion series of CCSP–CAT reporter plasmids (where CAT is chloramphenicol acetyltransferase) and gel mobility shift assays with a series of overlapping oligonucleotides covering the whole minimal promoter region to study protein–DNA interactions within this region. These studies have identified a conserved functional binding site for the lung and thyroid enriched homeodomain transcription factor TTF-1, located between positions -51 and -42 from the transcription start site. CCSP–CAT chimaeric reporters containing this region are specifically activated by TTF-1 in co-transfection assays, and nuclear extracts from cells which express TTF-1 bind to this region, as does in vitro translated rat TTF-1. Three additional conserved regions were identified, and in further gel mobility shift studies with an oligonucleotide spanning the conserved region immediately 5′ to the TTF-1 site we identified a binding site for the ubiquitously expressed zinc-finger-containing proteins Sp1 and Sp3. These studies suggest that cell-type-restricted and ubiquitous nuclear proteins may play a combined role in the regulation of the CCSP gene within the bronchiolar epithelium by interacting with the minimal promoter region.

Involvement of Sp1 and Sp3 in differential regulation of human NHE3 promoter activity by sodium butyrate and IFN-γ/TNF-α

2007 ◽  
Vol 293 (1) ◽  
pp. G374-G382 ◽  
Author(s):  
Md. Ruhul Amin ◽  
Pradeep K. Dudeja ◽  
Krishnamurthy Ramaswamy ◽  
Jaleh Malakooti
Keyword(s):  
Sodium Butyrate ◽  
Promoter Activity ◽  
Core Promoter ◽  
Differential Regulation ◽  
Mobility Shift ◽  
Alternative Pathways ◽  
Tnf Α ◽  
Repressive Effect ◽  
Ifn Γ ◽  
Gel Mobility Shift

Previously, we reported that IFN-γ and TNF-α downregulate the expression of the human Na+/H+ exchanger (NHE)3 gene by modulating Sp1/Sp3 transcription factors in C2BBe1 cells. It is reported that butyrate inhibits IFN-γ and TNF-α signaling pathways. In this study, we have investigated the effect of sodium butyrate (NaB) and IFN-γ/TNF-α on human NHE3 promoter activity. In transient transfection studies, NaB (5 mM) led to 10-fold stimulation of NHE3 promoter activity after incubation for 24 h. With 5′-deletion analysis, the NaB-responsive region was mapped to the NHE3 core promoter, bp −95 to + 5, which we had shown previously to confer responsiveness to IFN-γ/TNF-α. The stimulatory effect of NaB on the NHE3 promoter was reduced by 60% in the presence of IFN-γ/TNF-α. Mutually, the repressive effect of these cytokines was attenuated by NaB. Knockdown of Sp1 and Sp3 expression with small interfering RNA (siRNA) resulted in a significant resistance to NaB effects. NaB treatment showed no effect on Sp1 and Sp3 protein expression as assessed by Western blot analyses. Gel mobility shift assays with nuclear proteins from NaB-treated cells showed enhanced binding of Sp1 and Sp3 to the NHE3 promoter. The phosphatase inhibitor okadaic acid (200 nM) blocked the stimulatory effect of NaB on the NHE3 promoter. NaB effects on the NHE3 promoter were significantly attenuated by protein phosphatase (PP)1α- and PP2Aα-specific siRNA transfection. Our data suggest that the differential regulation of NHE3 gene expression by NaB and IFN-γ/TNF-α is mediated through alternative pathways that converge on Sp1/Sp3.

scholarly journals Phosphorylation of the proline-rich domain of Xp95 modulates Xp95 interaction with partner proteins

2006 ◽  
Vol 401 (2) ◽  
pp. 521-531 ◽  
Author(s):  
Robert E. DeJournett ◽  
Ryuji Kobayashi ◽  
Shujuan Pan ◽  
Chuanfen Wu ◽  
Laurence D. Etkin ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Adaptor Protein ◽  
Sh3 Domain ◽  
Phase Cell ◽  
Mobility Shift ◽  
Rich Domain ◽  
M Phase ◽  
Gel Mobility Shift ◽  
Partner Proteins

The mammalian adaptor protein Alix [ALG-2 (apoptosis-linked-gene-2 product)-interacting protein X] belongs to a conserved family of proteins that have in common an N-terminal Bro1 domain and a C-terminal PRD (proline-rich domain), both of which mediate partner protein interactions. Following our previous finding that Xp95, the Xenopus orthologue of Alix, undergoes a phosphorylation-dependent gel mobility shift during progesteroneinduced oocyte meiotic maturation, we explored potential regulation of Xp95/Alix by protein phosphorylation in hormone-induced cell cycle re-entry or M-phase induction. By MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS analyses and gel mobility-shift assays, Xp95 is phosphorylated at multiple sites within the N-terminal half of the PRD during Xenopus oocyte maturation, and a similar region in Alix is phosphorylated in mitotically arrested but not serum-stimulated mammalian cells. By tandem MS, Thr745 within this region, which localizes in a conserved binding site to the adaptor protein SETA [SH3 (Src homology 3) domain-containing, expressed in tumorigenic astrocytes] CIN85 (α-cyano-4-hydroxycinnamate)/SH3KBP1 (SH3-domain kinase-binding protein 1), is one of the phosphorylation sites in Xp95. Results from GST (glutathione S-transferase)-pull down and peptide binding/competition assays further demonstrate that the Thr745 phosphorylation inhibits Xp95 interaction with the second SH3 domain of SETA. However, immunoprecipitates of Xp95 from extracts of M-phase-arrested mature oocytes contained additional partner proteins as compared with immunoprecipitates from extracts of G2-arrested immature oocytes. The deubiquitinase AMSH (associated molecule with the SH3 domain of signal transducing adaptor molecule) specifically interacts with phosphorylated Xp95 in M-phase cell lysates. These findings establish that Xp95/Alix is phosphorylated within the PRD during M-phase induction, and indicate that the phosphorylation may both positively and negatively modulate their interaction with partner proteins.

Molecular cloning and functional analysis of the human Na+/H+ exchanger NHE3 promoter

2002 ◽  
Vol 282 (3) ◽  
pp. G491-G500 ◽  
Author(s):  
Jaleh Malakooti ◽  
V. C. Memark ◽  
Pradeep K. Dudeja ◽  
Krishnamurthy Ramaswamy
Keyword(s):  
Promoter Activity ◽  
Tissue Expression ◽  
Binding Activity ◽  
Start Codon ◽  
Nucleotide Sequencing ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Flanking Region ◽  
Gel Mobility Shift ◽  
Molecular Signals

Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3, colocalized to the brush border membrane of the epithelial cells, exhibit differences in their pattern of tissue expression and regulation by various molecular signals. To investigate the mechanisms involved in regulation of NHE3 gene expression, the human NHE3 promoter region was cloned and characterized. Primer extension experiments located the transcription start site to a position 116 nucleotides upstream from the translation start codon. The 5′-flanking region lacked a CCAAT box but contained a TATA-like sequence. Nucleotide sequencing of the 5′-flanking region revealed the presence of a number of cis elements including Sp1, AP-2, MZF-1, CdxA, Cdx-2, steroid and nonsteroid hormone receptor half sites, and a phorbol 12-myristate 13-acetate-response element. Transient transfection experiments using C2/bbe cell line defined a maximal promoter activity in −95/+5 region. The regulatory response elements clustered within this region include a potential transcription factor IID (TF IID), a CACCC, two Sp1, and two AP-2 motifs. Deletion of a fragment containing the AP-2 and Sp1 motifs resulted in a drastic decrease in promoter activity. In gel mobility shift assays, an oligonucleotide spanning from −78 to −56 bp bound a recombinant AP-2, and the corresponding binding activity in nuclear extracts was supershifted with anti-AP2α antibody. Our studies suggest that the NHE3 expression is regulated by a combination of ciselements and their cognate transcription factors that include the AP-2 and Sp1 family members.

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

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

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.

scholarly journals A novel mutation in the steroidogenic acute regulatory protein gene promoter leading to reduced promoter activity

2006 ◽  
Vol 37 (1) ◽  
pp. 71-80 ◽  
Author(s):  
Andres J Casal ◽  
Victoria J P Sinclair ◽  
Alessandro M Capponi ◽  
Jérôme Nicod ◽  
Uyen Huynh-Do ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Novel Mutation ◽  
Regulatory Protein ◽  
Gene Promoter ◽  
Wild Type ◽  
Mobility Shift ◽  
Adrenal Steroid ◽  
H295r Cells ◽  
Steroidogenic Acute Regulatory ◽  
Gel Mobility Shift

We have identified a novel cytosine/thymidine polymorphism of the human steroidogenic acute regulatory (StAR) gene promoter located 3 bp downstream of the steroidogenic factor-1 (SF-1)-binding site and 9 bp upstream of the TATA box (ATTTAAG). Carriers of this mutation have a high prevalence of primary aldosteronism. In transfection experiments, basal StAR promoter activity was unaltered by the mutation in murine Y-1 cells and human H295R cells. In Y-1 cells, forskolin (25 μM, 6 h) significantly increased wild-type promoter activity to 230±33% (P<0.05, n=4). In contrast, forskolin increased mutated promoter activity only to 150±27%, with a significant 35% reduction compared to wild type (P<0.05, n=3). In H295R cells, angiotensin II (AngII; 10 nM) increased wild-type StAR promoter activity to 265±22% (P<0.01, n=3), while mutated StAR promoter activity in response to AngII only reached 180±29% of controls (P<0.01, n=3). Gel mobility shift assays show the formation of two additional complexes with the mutated promoter: one with the transcription repressor DAX-1 and another with a yet unidentified factor, which strongly binds the SF-1 response element. Thus, this novel mutation in the human StAR promoter is critically involved in the regulation of StAR gene expression and is associated with reduced promoter activity, a finding relevant for adrenal steroid response to physiological stimulators.

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