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PubMed2002

Structural and functional characterization of the phytoene synthase promoter from Arabidopsis thaliana.

The expression of the gene coding for the carotenogenic enzyme phytoene synthase is highly regulated. To study this, its promoter and truncated versions thereof were translationally fused to the luciferase gene as a reporter and these constructs were used to transform Arabidopsis thaliana. The full-length promoter was shown to be active in the dark, but mediated positive responses towards different light qualities (far-red, red, blue and white light). Among the herbicides tested, norflurazon and gabaculine showed no notable effects, while CPTA abolished light induction completely. Response towards different light qualities was mediated by a TATA box-proximal promoter region up to position -300, containing G-box-like elements involved in the distinction of different monochromatic light qualities applied. This is detected in electrophoretic mobility shift assays (EMSAs), which reveal differential complex formation. A TATA box distal region of the promoter was shown to be responsible for a high basal promoter activity that was not modulated by different light qualities. Using EMSAs, a novel cis-acting element ATCTA occurring in tandem between positions -854 and -841 proved to be decisive in this respect. The motif was found in several other promoter regions involved in carotenoid and tocopherol biosynthesis, as well as in the promoter regions mediating the expression of photosynthesis-related genes. The functional equivalence of the motifs was shown by successfully using the respective regions in EMSAs. We conclude that the ATCTA motif represents an element capable of mediating a coordinated regulation of these pathways at the transcriptional level.

Alkyl and Aryl Transferases↗

PubMed1995

NF beta A, a factor required for maximal interleukin-1 beta gene expression is identical to the ets family member PU.1. Evidence for structural alteration following LPS activation.

We have previously identified and characterized the macrophage-, neutrophil- and B cell-specific nuclear factor beta A (NF beta A), which is involved in transcriptional regulation of the interleukin-1 beta (IL-1 beta) gene. NF beta A binds to a highly conserved sequence element located 6 bp upstream of the TATA motif within the IL-1 beta promoter and is required for maximal expression of the IL-1 beta gene. Here we show that NF beta A is identical to the previously identified ets gene family member PU.1. The NF beta A binding element shares 100% sequence identity with a novel PU.1 binding element recently found in the immunoglobulin J-chain promoter. Methylation interference DNA footprinting data demonstrated that NF beta A and PU.1 make identical protein/DNA contacts. In vitro synthesized PU.1 possesses a mobility and binding specificity identical to NF beta A as determined by electrophoretic mobility shift analysis (EMSA). Antisera directed against amino acids 39-55 of PU.1 recognizes NF beta A in a manner indistinguishable from PU.1 in EMSA 'supershift' studies. NF beta A and PU.1 also possess similar protein structure as determined by proteolytic clipping bandshift analysis. Furthermore, we show that PU.1 is able to transactivate an NF beta A-dependent promoter when co-transfected into HeLa cells which lack PU.1/NF beta A. EMSA studies using recombinant TATA binding protein (TBP) and PU.1 suggest that PU.1 may induce assembly of a distinct TBP-dependent complex on the IL-1 beta promoter. Finally, immunohistochemical confocal laser scanning microscopy studies suggest that LPS stimulation of RAW macrophages induces a structural change in the N-terminal transcriptional activation domain of PU.1.

Base Sequence↗

PubMed2004

Calcyclin (S100A6) expression is stimulated by agents evoking oxidative stress via the antioxidant response element.

Calcyclin (S100A6) is a cell-specific, calcium binding protein of the S100 family whose expression is augmented in many types of cancer. By means of luciferase activity assays, RT-PCR and Northern blot hybridization, we established that transcription of S100A6 gene is increased by agents known to evoke oxidative stress. Mutation of the antioxidant response element (ARE) located at position -290/-281 of the calcyclin gene promoter, and overlapping the E-box sequence recognized by the upstream stimulatory factor (USF), led to inhibition of calcyclin gene promoter activity stimulated by cadmium ions. Electrophoretic mobility shift assays (EMSA) with the -302/-260 calcyclin gene promoter fragment revealed, apart from USF binding, the presence of another protein complex (N) shown by competitive EMSA to be bound to ARE. DNA affinity chromatography followed by Western blot showed the binding of Nrf2 transcription factor to the immobilized calcyclin gene promoter fragment and concomitant appearance of complex N in EMSA of the eluted fractions. The results indicate that agents evoking oxidative stress activate calcyclin gene via the ARE sequence in its promoter.

Antioxidants↗

PubMed2005

Proximal HNF1 element is essential for the induction of human UDP-glucuronosyltransferase 1A1 by glucocorticoid receptor.

Previous study showed noinduction of the reporter gene (-3174/+14) of UGT1A1 in HepG2 by bilirubin, but induction by dexamethasone (DEX). This induction was enhanced seven-fold by the co-expression of human glucocorticoid receptor (GR) and was inhibited by a GR antagonist, RU486, indicating stimulation by DEX-GR. Meanwhile, we could not detect stimulation by beta-estradiol, phenobarbital or rifampicin (RIF) in the presence of GR. We investigated the position playing a role in this induction by GR in the promoter region of UGT1A1 using deletion mutants, and clarified the essential sequence (-75/-63) for the binding site of hepatocyte nuclear factor 1 (HNF1). However, GR did not bind directly to this sequence, because UGT-PE2 did not compete for binding to a glucocorticoid responsive element (GRE) probe in an electrophoretic mobility shift assay (EMSA) method. Labeled [(32)P]DNA probe of HNF1 binds with nuclear extracts as shown by the EMSA. This shift of the complex of probe-protein was not inhibited by unlabeled GRE but was inhibited by unlabeled HNF1 element. This shift was not influenced by the addition of anti-GR, but was super-shifted by the addition of anti-HNF1. GR did not stimulate the induction of HNF1, because we detected no-elevation of the mRNA level of HNF1 by reverse transcription-polymerase chain reaction (RT-PCR). Therefore, the induction of UGT1A1 by DEX-GR did not depend on the elevation of HNF1 but on the interaction of GR with HNF1 or the activation of HNF1 through the transcription of other proteins. Also given the lack of evidence of binding of DEX-GR to HNF1 in the EMSA, the data suggest that the mechanism of DEX-GRE effect on HNF1 is indirect by whatever mechanisms.

Base Sequence↗

PubMed2005

Human interleukin-1 alpha gene expression is regulated by Sp1 and a transcriptional repressor.

The regulation of the human IL-1alpha gene was studied using a series of 5' deletion promoter chloramphenicol acetyltransferase (CAT) reporter constructs. The IL-1alpha promoter from -967 to +64 produced no significant expression of CAT. Progressive 5' deletion indicated the presence of a repressor binding site between -477 and -305 bp as deletion in this region resulted in CAT expression. Electrophoretic mobility shift assay (EMSA) analysis confirmed that protein(s) bound to this region and DNaseI footprinting localized the binding site to between -448 and -435. Deletion of the IL-1alpha promoter to -42 resulted in reduced CAT expression suggesting the presence of a positive regulatory element in this region. EMSA experiments using IL-1alpha promoter DNA from -163 to +64 demonstrated protein binding to this region and DNaseI footprinting demonstrated protection between -59 and -40. Transcriptional activity of the IL-1alpha promoter was also tested using an in vitro transcription assay. Reactions using -163, -100 and -52 promoter templates all produced a correctly sized transcript but deletion to -42 resulted in no transcript production. Analysis of the promoter indicated that a potential Sp1 binding site existed in the region from -52 to -45. An EMSA using an anti-Sp1 antibody indicated that Sp1 specifically bound to the -52 to +64 region.

5' Flanking Region↗

PubMed2004

Crustacean retinoid-X receptor isoforms: distinctive DNA binding and receptor-receptor interaction with a cognate ecdysteroid receptor.

We have identified cDNA clones that encode homologs of the ecdysteroid receptor (EcR) and retinoid-X receptor (RXR)/USP classes of nuclear receptors from the fiddler crab Uca pugilator (UpEcR and UpRXR). Several UpRXR cDNA splicing variants were found in coding regions that could potentially influence function. A five-amino acid (aa) insertion/deletion is located in the "T" box in the hinge region. Another 33-aa insertion/deletion is found inside the ligand-binding domain (LBD), between helix 1 and helix 3. Ribonuclease protection assays (RPA) showed that four UpRXR transcripts [UpRXR(+5+33), UpRXR(-5+33), UpRXR(+5-33) and UpRXR(-5-33)] were present in regenerating limb buds. UpRXR(-5+33) was the most abundant transcript present in regenerating limb buds in both early blastema and late premolt growth stages. Expression vectors for these UpRXR variants and UpEcR were constructed, and the proteins expressed in E. coli and in vitro expression systems. The expressed crab nuclear receptors were then characterized by electrophoretic mobility shift assay (EMSA) and glutathione S-transferase (GST) pull down experiments. EMSA results showed that UpEcR/UpRXR(-5+33) heterocomplexes bound with a series of hormone response elements (HREs) including eip28/29, IRper-1, DR-4, and IRhsp-1 with appreciable affinity. Competition EMSA also showed that the affinity decreased as sequence composition deviated from a perfect consensus element. Binding to IRper-1 HREs occurred only if the heterodimer partner UpRXR contained the 33-aa LBD insertion. UpRXR lacking both the 5-aa and 33-aa insertion bound to a DR-1G HRE in the absence of UpEcR. The results of GST-pull down experiments showed that UpEcR interacted only with UpRXR variants containing the 33-aa insertion, and not with those lacking the 33-aa insertion. These in vitro receptor protein-DNA and receptor protein-protein interactions occurred in the absence of hormone (20-hydroxyecdysone and 9-cis retinoid acid, 9-cis RA). Transactivation studies using a hybrid UpEcR ligand-binding domain construct and UpRXR (+/-33) ligand-binding domain constructs also showed that the 33-aa insertion was indispensable in mediating ecdysteroid stimulated transactivation.

Alternative Splicing↗

PubMed2005

Activation of transcription factor AP-1 and mitogen-activated protein kinases in aniline-induced splenic toxicity.

Signaling mechanisms in aniline-induced fibrogenic and/or tumorigenic response in the spleen are not known. Previous studies have shown that aniline exposure leads to iron accumulation and oxidative stress in the spleen, which may cause activation of redox-sensitive transcription factors and regulate the transcription of genes involved in fibrosis and/or tumorigenesis. To test this, male SD rats were treated with 0.5 mmol/kg/day aniline via drinking water for 30 days, and activation of transcription factor AP-1 was determined in the splenocyte nuclear extracts (NEs). AP-1 DNA-binding activity in the NEs of freshly isolated splenocytes from aniline-treated rats increased in comparison to the controls, as determined by electrophoretic mobility shift assay (EMSA). AP-1 binding was also determined in the NEs of cultured splenocytes (2 h and 24 h), which showed even a greater increase in binding activity at 2 h. The specificity of AP-1 binding for relevant DNA motifs was confirmed by competition EMSA and by supershift EMSA using antibodies specific to c-Jun and c-Fos. To further explore the signaling mechanisms in the AP-1 activation, phosphorylation patterns of mitogen-activated protein kinases (MAPKs) were pursued. Aniline exposure induced increases in the phosphorylation of the three classes of MAPKs: extracellular-signal-regulated kinase (ERK 1/2), c-Jun N-terminal kinase (JNK 1/2), and p38 MAPKs. Furthermore, TGF-beta1 mRNA expression showed a 3-fold increase in the spleens of aniline-treated rats. These observations suggest a strong association among MAPK phosphorylation, AP-1 activation, and enhanced TGF-beta1 gene expression. The observed sequence of events subsequent to aniline exposure could regulate genes that lead to fibrogenic and/or tumorigenic response in the spleen.

Aniline Compounds↗

PubMed2005

Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts.

To determine the molecular mechanisms by which N-cadherin transcription is regulated, we cloned and sequenced a 3681-bp of the 5'-flanking region of the human N-cadherin gene. Deletion analysis of the proximal region identified a minimal 318-bp region with strong promoter activity in human osteoblasts. The cryptic promoter is characterized by high GC content and a GA-rich binding core that may bind zing finger transcription factors. Electrophoretic mobility shift assays (EMSA), competition and supershift EMSA revealed that an Sp1/Sp3 binding site acts as a basal regulatory element of the promoter in osteoblasts. Incubation of osteoblast nuclear extracts with -163/-131 wild-type probe containing the GA-rich binding core revealed another specific complex, which was not formed with a -163/-131 probe mutated in the GA repeat. EMSA identified the nuclear factor involved as myeloid zinc finger-1 (MZF1). Mutation analysis showed that Sp1/Sp3 and MZF1 binding sites contribute to basal promoter activity. Cotransfection analyses showed that Sp1 and MZF1 overexpression increases whereas Sp3 antagonizes Sp1-induced N-cadherin promoter activity in osteoblasts. RT-PCR analysis showed that human osteoblastic cells express MZF1 and that Sp1/MZF1 overexpression increased N-cadherin expression. These results indicate that Sp1/Sp3 and MZF1 are important transcription factors regulating N-cadherin promoter activity and expression in osteoblasts.

5' Flanking Region↗

PubMed2003

Differentiation-dependent up-regulation of p47(phox) gene transcription is associated with changes in PU.1 phosphorylation and increased binding affinity.

The p47(phox) gene encodes a cytosolic component of the phagocytic NADPH oxidase complex. Expression of p47(phox) is both tissue-specific and developmentally regulated. Stable transfection of the myeloid cell lines PLB985 and HL60, with reporter gene constructs containing as little as 58 bp of proximal promoter sequence, was capable of directing significant reporter gene activity in myeloid cells, which increased significantly on induction of myeloid differentiation. EMSA analysis of a binding site for the Ets family member, PU.1, located at positions -39 to -44 revealed that the pattern of complex formation changed significantly on induction of myeloid differentiation. All EMSA complexes were competed by a functional PU.1 binding site and could be supershifted in the presence of polyclonal anti-PU.1 antibody. Reaction of EMSA complexes with anti-phosphoserine antibody, treatment with phosphatase, or Western blotting of proteins captured on the PU.1 binding site, was used to demonstrate that the changes in PU.1 complex formation dependent on myeloid differentiation were associated with increased levels of PU.1 phosphorylation. Furthermore, the more highly phosphorylated forms of PU.1 were shown to have a greater affinity for the p47(phox) PU.1 consensus binding site. Up-regulated transcriptional activity in response to myeloid differentiation can therefore be correlated with increased levels of PU.1 phosphorylation and a greater binding affinity.

Base Sequence↗

PubMed1999

Induction of inducible nitric oxide synthase gene expression by Pokeweed mitogen.

The present study has characterized the expression of iNOS gene in Pokeweed mitogen (PWM)-driven murine macrophage RAW 264.7 cells. PWM significantly induced nitric oxide production in a dose-dependent manner. Quantitative reverse transcription-polymerase chain reaction analysis demonstrated that the inducible nitric oxide synthase gene expression is increased by PWM treatment. Since iNOS transcription has recently been shown to be under the control of the nuclear factor (NF)-kappaB/Rel family of transcription factors, the effects of PWM on NF-kappaB/Rel activation were examined using a transient transfection assay and an electrophoretic mobility shift assay (EMSA). Transient expression assays with NF-kappaB/Rel binding sites linked to the chloramphenicol acetyltransferase gene suggest that the PWM-induced increase in transcription is mediated by the NF-kappaB/Rel transcription factor complex. Using DNA fragments containing the NF-kappaB/Rel binding sequence, PWM was shown to activate the protein/DNA binding of NF-KB/Rel to its cognate site as measured by EMSA. Supershift EMSA showed the presence of p50 and c-Rel protein in the complex at the kappaB site. Western blot analysis of isolated nuclear fractions, using p65 and c-Rel-specific antibodies, provided further evidence that c-Rel is increased by PWM treatment. N-Tosyl-1-phenylalanine chloromethyl ketone, a potent inhibitor of NF-kappaB/Rel activation, inhibited PWM-induced nitrite generation in a dose-dependent manner. Collectively, the results of these experiments indicate that c-Rel is positively regulated by PWM to assist in the initiation of iNOS gene expression.

Animals↗

PubMed1999

Immunoregulatory potential of urothelium: characterization of NF-kappaB signal transduction.

PURPOSE: To characterize the basal and activated form of nuclear factor-kappaB (NF-kappaB) complex in normal urothelial cell cultures after stimulation with tumor necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), and double-stranded ribonucleic acid (dsRNA). MATERIALS AND METHODS: Human urothelial cells cultured from normal bladder specimens underwent immunohistochemical staining and cellular extracts were prepared for Electrophoretic Mobility Shift Assays (EMSA), Western blot analyses, RNA isolation and Northern Blot analyses before and after stimulation with TNF-alpha, LPS, and dsRNA. RESULTS: In normal human urothelial cells, activation of the NF-kappaB complex in response to stimulation with TNF-alpha, LPS, and dsRNA was detected by immunohistochemical methods and EMSA. Depending on the stimulus, a specific NF-kappaB complex was activated as seen by supershift experiments in EMSA. By Western blot, the inhibitor of NF-kappaB complex, IkappaB-alpha, degraded in response to stimulation. Northern blot analysis from total RNA revealed subsequent inducible interleukin-8 (IL-8) mRNA expression of normal urothelial cells when treated with TNF-alpha, LPS, and dsRNA. CONCLUSIONS: Normal human urothelial cells contain basal NF-kappaB complexes in an inactivated state. When these cells are challenged by different agents such as TNF-alpha, LPS, and dsRNA, the cells respond by activation of the NF-kappaB signal transduction pathway, degradation of its inhibitor, IkappaB-alpha, and translocation of this primary factor into the nucleus to induce specific genetic responses such as IL-8 expression.

Cells, Cultured↗

PubMed2001

The transcription factors Sp1 and Sp3 are required for human angiotensin II type 1 receptor gene expression in H295-R cells.

The peptide hormone angiotensin II regulates a variety of physiological responses which are mediated by its interaction with high affinity G protein-coupled receptors localized on the surface of target cells. Our previous studies have demonstrated that a 145 bp sequence within the promoter region was required for basal level expression of the human angiotensin II type 1 receptor (hAT(1)R) gene. In the present study, deletional analysis of the hAT(1)R promoter localized the major regulatory sequence to two overlapping GC boxes harbored within the -105 to -85 bp region relative to the transcription start site in H295-R cells. Electrophoretic mobility shift assays (EMSAs) using a double-stranded (ds) oligonucleotide corresponding to this region and H295-R cell nuclear extract resulted in five specific DNA-protein complexes. EMSAs performed with competitive ds-oligonucleotides which harbored the consensus binding site for Sp1 prevented the formation of the DNA-protein complexes. Supershift EMSAs also demonstrated that Sp1 and Sp3 could bind to the GC boxes present within the -105 to -85 bp region of the hAT(1)R promoter. Transactivation experiments utilizing Drosophila SL2 cells, which lack endogenous Sp family transcription factors, demonstrated that Sp1 and Sp3 activated the hAT(1)R promoter and that maximal activation was only achieved when both GC boxes were present. Taken together, these findings suggest that Sp1 and Sp3 are necessary for the expression of the hAT(1)R gene in H295-R cells.

Acetaldehyde↗

PubMed2001

Molecular analysis of estrogen induction of preproenkephalin gene expression and its modulation by thyroid hormones.

Estrogen receptors (ER) and thyroid hormone receptors (TR) are ligand-dependent nuclear transcription factors. Estrogen-induced preproenkephalin (PPE) gene expression in the hypothalamus is directly related to estrogen-induced lordosis behavior in the rat. In the present study, we showed that the PPE mRNA level in the ventromedial hypothalamus of female rats was significantly decreased by ovariectomy. This decrease was reversed by estrogen replacement in a dose- and time-dependent manner. Using transient transfection and electrophoretic mobility shift assays (EMSA), functional estrogen response elements (ERE) were identified between -437 and -145 base pairs (bp) of the rat PPE gene promoter region. Two ERE-like elements are present between -405 and -364 of the rat PPE gene promoter, which bind ERalpha as demonstrated by EMSA. Estrogen produced a dose-dependent increase in CAT activity in cotransfection assays with ERalpha expression vector and a 437PPE-CAT reporter construct containing 437 bp of the rat PPE gene promoter and the CAT reporter gene. This estrogen-induced PPE promoter activity was inhibited by liganded-TR in transient cotransfection assays. Analysis of DNA-protein interactions by EMSA revealed that both ERalpha and TR (alpha1 and beta1) could bind to the EREs in the rat PPE gene promoter. Furthermore, estrogen induction of PPE mRNA in the ventromedial hypothalamus of the ovariectomized female rat was significantly attenuated by concomitant administration of triiodothyronine. These results suggest that estrogen regulation of the hypothalamic PPE gene expression is mediated through an estrogen-receptor complex directly interacting with the functional EREs in its promoter region; and that this estrogen effect can be modified by thyroid hormones.

Animals↗

PubMed1999

Promoter activity of the beta-amyloid precursor protein gene is negatively modulated by an upstream regulatory element.

Alzheimer's disease (AD) is characterized by the aggregation of the amyloid beta-peptide (Abeta) which is generated from a larger beta-amyloid precursor protein (betaAPP). An overexpression of the betaAPP gene in certain areas of the AD brain has been suggested to be an important factor in the neuropathology of AD. Here we have further characterized an upstream regulatory element (URE) located between -2257 and -2234 of the human betaAPP promoter. In addition to its location in the promoter, BLAST search reveals that URE is present in several introns of the betaAPP gene and is also detected in many other genes. For functional studies, two promoter regions were cloned upstream of the reporter gene, chloramphenicol acetyl transferase (CAT): (i) phbetaE-B - the plasmid that contains the human (h) promoter region (-2832 to +101) including URE, and (ii) prhbetaE-B - the plasmid that contains the rhesus (rh) promoter region excluding URE as it lacks a 270 bp region of the hbetaAPP promoter (-2435 to -2165). Transient transfection studies indicate that phbetaE-B displayed significantly less CAT-promoter activity than prhbetaE-B in C6, PC12 and SK-N-SH cells. To determine the role of URE in a heterologous promoter, a pbetaURE construct was made by subcloning URE in an enhancerless promoter vector pCATP. The pbetaURE-CAT construct displayed threefold to fourfold less promoter activity than pCATP when different cell lines were transfected with the plasmids. URE interacts with a novel protein(s) as determined by the electrophoretic mobility shift assay (EMSA). Although the core DNA region of URE resembles with the NF-kB element, URE-binding protein is not related to the NF-kB transcription factor. When EMSA was performed with specific competitors in different cell lines, the labeled URE probe was not competed by the oligonucleotides specific for either the AP3, NF-1 or NF-kB transcription factor. The migration of the URE-protein complex was different from the NF-kB-protein complex in the EMSA gel. A distinct URE-specific nuclear factor was also detected in frontal cortex of a normal human brain. These results suggest that the URE region acts as a repressor element, that the URE-binding protein is not related to the known transcription factors tested, and that the protein is present in astrocytic, neuroblastoma, PC12 cells and in the human brain.

Amyloid beta-Protein Precursor↗

PubMed1997

Hepatic nuclear proteins that bind cis-regulatory elements in the proximal promoter of the rat corticosteroid-binding globulin gene.

The plasma transport protein for glucocorticoids, corticosteroid-binding globulin (CBG), is produced by hepatocytes, and expression of the CBG gene (Cbg) in the liver is controlled by a variety of hormones, environmental stimuli, and developmental cues. The rat Cbg proximal promoter, including 145 base pairs (bp) from the transcription start site, contains two cis-regulatory elements (designated P1 and P2), and is as transcriptionally active as a much more extended region (approximately 1.2 kbp) of the promoter. We have now characterized the rat liver nuclear proteins that interact with P1 and P2. Several proteins interacted specifically with P1 during an electrophoretic mobility shift assay (EMSA), and based on ultraviolet (UV) cross-linking and Southwestern blot analyses, as well as an antibody-supershifting EMSA, one of these has been identified as hepatic nuclear factor (HNF)1 beta. The major band shift formed with P2 in an EMSA appears to comprise a protein that migrates as a doublet of 58 and 62 KDa on sodium dodecylsulfate-polyacrylamide gel ectrophoresis (SDS-PAGE) after UV cross-linking with an oligonucleotide containing P2, as well as during Southwestern blot analyses. Mutations of the CCAAT sequence within P2 also prevent binding to this protein, the physicochemical properties of which resemble the CCAAT-binding protein CP2. Functional analyses of this region of the rat Cbg proximal promoter fused to a luciferase reporter gene demonstrated that mutations within P1, which prevent its interaction with NHF1, do not influence adversely its transcriptional activity. Thus, although members of the HNF1 family of nuclear proteins play an essential role in the transcriptional activation of several other related genes (e.g., thyroxin-binding globulin and alphal-antitrypsin) in hepatocytes, HNF1 beta does not appear to be required for the basal activity of the rat Cbg promoter. In addition, deletion of P2 from the proximal promoter abolishes transcriptional activity and the CCAAT-binding protein that interacts with P2 probably represents an important determinant of Cbg expression under different physiological conditions.

Animals↗

PubMed2002

Effect of heat stress on promoter binding by transcription factors in the cytosol of the archaeon Methanosarcina mazeii.

Regulation of archaeal stress genes is not yet fully understood. This work is part of a research effort aimed at elucidating the molecular mechanisms of transcription initiation and regulation of the stress genes in the hsp70(dnaK) locus of the mesophilic, methanogenic archaeon Methanosarcina mazeii. The locus has the stress genes 5'-grpE-hsp70(dnaK)-hsp40(dnaJ)-3' encoding the chaperone machine components GrpE, Hsp70(DnaK), and Hsp40(DnaJ), respectively, flanked by non-heat shock inducible genes, orf16 and orf11-trkA. Thus, the M. mazeii hsp70(dnaK) locus offers the opportunity for studying heat shock and non-heat shock inducible genes side by side. The objectives of the work reported here were to develop procedures for studying basal transcription factors in the cytosol of M. mazeii and their interaction with these genes' promoters in stressed cells for comparison with unstressed counterparts. The preparation of non-radioactive DNA probes for electrophoretic mobility shift assay (EMSA), and the combination of EMSA with Western blotting for DNA-binding protein identification were standardized for this investigation. DNA probes bearing the genes' promoter regions were used for detecting and identifying DNA-binding proteins in the cytosol of unstressed and heat-shocked cells. Cytosolic TATA-binding protein (TBP) was found to bind the stress-gene promoters in both unstressed and heat-shocked cells but more strongly in the latter. Likewise, in stressed cells TBP-transcription factor B (TFB)(TFIIB) association was increased by comparison with unstressed controls. The level of cytosolic TBP assessed by its DNA-binding activity using EMSA remained unchanged during the various phases of culture growth in the absence of heat stress. The results indicate that heat stress of cells in culture modulates the level and/or the stress-gene promoter-binding activity of the M. mazeii TBP, and enhances TBP-TFB association in the cytosol and DNA binding.

Base Sequence↗

PubMed2002

Regulation of transcription of the human prepronociceptin gene by Sp1.

Nociceptin/orphanin FQ is a recently discovered neuropeptide and the endogenous ligand for opioid receptor-like-1. The promoter region of the precursor protein prepronociceptin (ppN/OFQ) has been cloned and sequenced. We have previously shown that a stretch of 110 bases immediately 5' to the first intron 23 bp upstream of the ATG start codon is responsible for significant enhancement of transcription of the human ppN/OFQ gene. We performed electromobility shift assays (EMSAs) using oligonucleotides spanning portions of the promoter region close to the intron to determine which DNA elements were important for transcriptional regulation. EMSAs using Sp1 antibody revealed a cis-acting regulatory element from bases 35-67 that appeared to bind Sp1 transcription factor and cause a shift to higher molecular weight. Deletion of this 30-bp region of DNA from the 1.2 kb promoter caused a significant loss of transcription as measured by luciferase reporter assays. Mutation of four bases at the Sp1 binding site also induced a significant loss of transcription compared to wildtype constructs. Finally, an Sp1- but not Etf-binding consensus oligonucleotide was able to compete with the interaction of the oligo with the NS20Y nuclear extract. Combined with the data from the supershift EMSAs, it appears that Sp1 is the transcription factor binding to the GC region close to the intron to regulate transcription of the human ppN/OFQ gene.

5' Flanking Region↗

PubMed2002

Multimerization of restriction fragments by magnesium-mediated stable base pairing between overhangs: a cause of electrophoretic mobility shift.

The electrophoretic mobility shift assay (EMSA) or simply the "gel shift assay" is one of the most sensitive methods for studying the ability of a protein to bind to DNA. EMSAs are also widely used to investigate protein- or sequence-dependent DNA bending. Here we report that electrophoresis using physiological concentrations of Mg(2+) can cause a mobility shift of restriction fragments in nondenaturing polyacrylamide gels as the overhangs form stable base pairs. This phenomenon was observed at even 37 degrees C. The retardation was, however, more pronounced at low temperatures, where a three-nucleotide overhang 5'-GAC also caused a mobility shift. The stability of the pairing was generally high when the overhangs of four nucleotides display high GC content, while the mobility shift caused by 5'-AATT was greater than those caused by 5'-GATC, 5'-TCGA, and 5'-CTAG. This observation should be taken into account to avoid misinterpretation of the data when the EMSA, especially the circular permutation gel mobility shift assay, is performed using a running buffer that contains Mg(2+) ions. The stable adhesion between short overhangs may present an important basis for genome stability and many genetic processes occurring in living cells.

Base Pairing↗