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PubMed1997

Evidence for inhibition of MyEF-2 binding to MBP promoter by MEF-1/Pur alpha.

Myelin basic protein (MBP) is a major component of the myelin sheath whose production is developmentally controlled during myelinogenesis. Earlier studies have indicated that programmed expression of the MBP gene is regulated at the level of transcription. Evidently, the MB1 regulatory motif located between nucleotides -14 to -50 plays an important role in transcription of the MBP promoter in both in vivo systems. The MB1 element contains binding sites for the activator protein MEF-1/Pur alpha and the repressor protein MyEF-2. In this study we use bandshift assays with purified MEF-1/Pur alpha and MyEF-2 and demonstrate that binding of MyEF-2 to its target sequence is inhibited by MEF-1/Pur alpha. Under similar conditions, MyEF-2 enhances the association of MEF-1/Pur alpha with MB1 DNA. MEF-1/Pur alpha binds to MB1 in mono- and dimeric forms. Inclusion of MyEF-2 in the binding reaction increases the dimeric association of MEF-1/Pur alpha with the MB1 sequence. The use of MEF-1/Pur alpha variants in the bandshift assay suggests that two distinct regions of this protein may be involved in its binding to the MB1 sequences, and its ability to block MyEF-2 interaction with the MB1 sequence. Based on previous studies on the programmed expression of MEF-1/Pur alpha and MyEF-2 during myelination and the current findings on their interplay for binding to the MB1 motif, a model is proposed for their involvement in transcriptional regulation of the MBP gene during the course of brain development.

Adenosine Triphosphate↗

PubMed2003

Adenovirus 2 E1B-55K protein relieves p53-mediated transcriptional repression of the survivin and MAP4 promoters.

It is well established that adenovirus E1B-55K protein functions as an inhibitor of the tumor suppressor protein p53 by binding and inactivating p53 as a transcriptional activator protein. Here we show that the adenovirus 2 E1B-55K protein also blocks p53 as a transcriptional repressor protein of the survivin and the MAP4 promoters. The repression is dependent on the ability of E1B-55K to bind to p53 and is enhanced by coexpression of the adenovirus E4orf6 protein. Overexpression of the transcriptional corepressor protein Sin3A partially relieves the inhibitory effect of E1B-55K, suggesting that E1B-55K blocks p53 functions by interfering with the Sin3 complex.

Adenovirus E1B Proteins↗

PubMed1994

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

Base Sequence↗

PubMed1994

Mutational analysis of the arginine repressor of Escherichia coli.

Arginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild-type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N-terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by L-arginine at the same concentration as the wild-type repressor. The other two mutant repressors map in the C-terminal half of the protein. They are poorly precipitated by L-arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super-repressors. Such mutants behave as arginine auxotrophs as a result of hyper-repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super-repressor proteins were purified. In comparison with the wild-type repressor, two of them were shown to have a higher DNA-binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA-binding affinity when bound to arginine.

Arginine↗

PubMed2002

Kinetic analysis of the interaction between the QutA and QutR transcription-regulating proteins.

The QutR protein is a multidomain repressor protein that interacts with the QutA activator protein. Both proteins are active in the signal transduction pathway that regulates transcription of the quinic acid utilization (qut) gene cluster of the microbial eukaryote Aspergillus nidulans. In the presence of quinate, production of mRNA from the eight genes of the qut pathway is stimulated by the QutA activator protein. The QutR protein plays a key role in signal recognition and transduction, and a deletion analysis has shown that the N-terminal 88 amino acids are sufficient to inactivate QutA function in vivo. Using surface plasmon resonance we show here that the N-terminal 88 amino acids of QutR are able to bind in vitro to a region of QutA that genetic analysis has previously implicated in transcription activation. We further show that increasing the concentration of a full-length (missense) mutant QutR protein in the original mutant strain can restore its repressing function. This is interpreted to mean that the qutR mutation in this strain increases the equilibrium dissociation constant for the interaction between QutA and QutR. We propose a model in which the QutA and QutR proteins are in dynamic equilibrium between bound (transcriptionally inactive) and unbound (transcriptionally active) states.

Aspergillus nidulans↗

PubMed1997

Alpha2p controls donor preference during mating type interconversion in yeast by inactivating a recombinational enhancer of chromosome III.

Homothallic strains of Saccharomyces cerevisiae can change mating type as often as every generation by replacing the allele at the MAT locus with a copy of mating type information present at one of two storage loci, HML and HMR, located on either end of chromosome III. Selection of the appropriate donor locus is dictated by a mating type-specific repressor protein, alpha2p: Cells containing alpha2p select HMR, whereas those lacking alpha2p select HML. As a repressor protein, alpha2p binds to DNA cooperatively with the transcriptional activator Mcm1p. Here we show that two alpha2p/Mcm1p-binding sites, DPS1 and DPS2, control donor selection. DPS1 and DPS2 are located approximately 30 kb from the left arm of chromosome III, well removed from HML, HMR, and MAT. Precise deletion of only DPS1 and DPS2 results in random selection of donor loci and in a cells without affecting selection in alpha cells. Reciprocally, deletion of only the alpha2p binding segments in each of these two sites results in selection of the wrong donor loci in alpha cells without affecting preference in a cells. These results suggest that Mcm1p, bound to these two sites in the absence of alpha2p, activates HML as donor. Binding of alpha2p blocks the ability of Mcm1p bound to DPS1 and DPS2 to activate HML, resulting in default selection of HMR as donor. DPS1 and DPS2 also regulate expression of several noncoding RNAs, although deletion of at least one of these RNA loci does not affect donor preference. This suggests that transcriptional activation, rather than transcription of a specific product, is the initiating event in activating the left arm of chromosome III for donor selection.

Binding Sites↗

PubMed1995

The molecular cloning and characterization of BM1P1 and BM1P2 proteins, putative positive transcription factors involved in barbiturate-mediated induction of the genes encoding cytochrome P450BM-1 of Bacillus megaterium.

Analysis of a 1.3-kilobase segment of 5'-flanking DNA from the barbiturate-inducible P450BM-1 gene (CYP106) of Bacillus megaterium revealed two open reading frames. One, BM1P1, encodes 98 amino acids and is located 267 base pairs upstream from the sequence encoding cytochrome P450BM-1 but in the opposite orientation. The second, BM1P2 (88 amino acids), is 892 base pairs upstream from the P450BM-1 coding sequence and in the same coding strand. The expression of BM1P1 and BM1P2 was strongly stimulated in cells grown in the presence of pentobarbital, and the BM1P1 gene product exerted positive control on expression of P450BM-1. When a 177-base pair fragment encompassing the overlapping promoter regions of the P450BM-1 and BM1P1 genes was used as a probe in DNA binding assays, the BM1P1 and BM1P2 gene products and Bm3R1 (the repressor protein regulating the barbiturate-mediated expression of P450BM-3) could bind individually, but the addition of BM1P1 or BM1P2 to a binding mixture containing Bm3R1 completely prevented the appearance of a Bm3R1 binding band. When a 208-base pair fragment containing a Barbie box sequence and located upstream of the 177-base pair fragment was used as a probe, only a Bm3R1 binding band was detected. Although neither BM1P1 and BM1P2 appeared to bind to this 208-base pair fragment, their presence strongly inhibited the binding of Bm3R1 to the same probe. The evidence suggests that BM1P1 and BM1P2 may, in part, act as positive regulatory proteins involved in the expression of the P450BM-1 gene by interfering with the binding of the repressor protein, Bm3R1, to the regulatory regions of P450BM-1.

Amino Acid Sequence↗

PubMed2005

Steady-state analysis of glucose repression reveals hierarchical expression of proteins under Mig1p control in Saccharomyces cerevisiae.

Glucose repression is a global transcriptional regulatory mechanism commonly observed in micro-organisms for the repression of enzymes that are not essential for glucose metabolism. In Saccharomyces cerevisiae, Mig1p, a homologue of Wilms' tumour protein, is a global repressor protein dedicated to glucose repression. Mig1p represses genes either by binding directly to the upstream repression sequence of structural genes or by indirectly repressing a transcriptional activator, such as Gal4p. In addition, some genes are repressed by both of the above mechanisms. This raises a fundamental question regarding the physiological relevance of the varied mechanisms of repression that exist involving Mig1p. We address this issue by comparing two well-known glucose-repression systems, that is, SUC2 and GAL gene expression systems, which encompass all the above three mechanisms. We demonstrate using steady-state analysis that these mechanisms lead to a hierarchical glucose repression profile of different family of genes. This switch over from one carbon source to another is well-calibrated as a function of glucose concentration through this hierarchical transcriptional response. The mechanisms prevailing in this repression system can achieve amplification and sensitivity, as observed in the well-characterized MAPK (mitogen-activated protein kinase) cascade system, albeit through a different structure. A critical feature of repression predicted by our steady-state model for the mutant strain of S. cerevisiae lacking Gal80p agrees well with the data reported here as well as that available in the literature.

DNA-Binding Proteins↗

PubMed1997

Dimerization specificity of P22 and 434 repressors is determined by multiple polypeptide segments.

The repressor protein of bacteriophage P22 binds to DNA as a homodimer. This dimerization is absolutely required for DNA binding. Dimerization is mediated by interactions between amino acids in the carboxyl (C)-terminal domain. We have constructed a plasmid, p22CT-1, which directs the overproduction of just the C-terminal domain of the P22 repressor (P22CT-1). Addition of P22CT-1 to DNA-bound P22 repressor causes the dissociation of the complex. Cross-linking experiments show that P22CT-1 forms specific heterodimers with the intact P22 repressor protein, indicating that inhibition of P22 repressor DNA binding by P22CT-1 is mediated by the formation of DNA binding-inactive P22 repressor:P22CT-1 heterodimers. We have taken advantage of the highly conserved amino acid sequences within the C-terminal domains of the P22 and 434 repressors and have created chimeric proteins to help identify amino acid regions required for dimerization specificity. Our results indicate that the dimerization specificity region of these proteins is concentrated in three segments of amino acid sequence that are spread across the C-terminal domain of each of the two phage repressors. We also show that the set of amino acids that forms the cooperativity interface of the P22 repressor may be distinct from those that form its dimer interface. Furthermore, cooperativity studies of the wild-type and chimeric proteins suggest that the location of cooperativity interface in the 434 repressor may also be distinct from that of its dimerization interface. Interestingly, changes in the dimer interface decreases the ability of the 434 repressor to discriminate between its wild-type binding sites, O(R)1, O(R)2, and O(R)3. Since 434 repressor discrimination between these sites depends in large part on the ability of this protein to recognize sequence-specific differences in DNA structure and flexibility, this result indicates that the C-terminal domain is intimately involved in the recognition of sequence-dependent differences in DNA structure and flexibility.

Amino Acid Sequence↗

PubMed2004

CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island.

Vascular endothelial growth factor (VEGF) is produced by several cell types in the kidney, and its expression is tightly regulated for the maintenance of normal renal physiology. Increases or decreases in its expression are associated with proteinuria and renal disease. Recently, we found that the expression of VEGF is markedly induced following interactions between CD40 ligand (CD40L) and CD40. Here, endothelial cells (EC) or Jurkat T cell lines were transiently transfected with luciferase reporter constructs under the control of the human VEGF promoter and were treated with human soluble CD40L (sCD40L). We identified a CD40-responsive 68-bp region (bp -50 to +18) of the promoter and 43 bp within this region (bp -25 to +18) that have 97% homology to a sequence of CpG dinucleotides. A computerized search revealed that the CpG region has putative binding domains for the transcriptional repressor protein methyl CpG binding protein-2 (MeCP2). In EMSA, we found that the 43-bp methylated sequence formed four complex(es) with nuclear extracts from untreated EC and reduced binding of at least one complex when nuclear lysates from sCD40L-activated EC (30 min) were used. Supershift analysis using anti-MeCP2 demonstrated that most of the complex(es) in both untreated and sCD40L-activated EC involved interactions between the 43-bp DNA and MeCP2. In addition, we found that other CpG binding proteins may also interact with this region of the promoter. Taken together, this is the first demonstration that CpG binding transcriptional repressor proteins including MeCP2 may be of importance in VEGF biology.

CD40 Antigens↗

PubMed2006

Human SLUG does not directly bind to CtBP1.

SLUG is a transcriptional repressor protein implicated to have major role in the oncogenesis and metastasis of human breast cells. We previously have shown by chromatin immunoprecipitation assay that human SLUG (hSLUG) is co-localized with the co-repressor protein CtBP1 as bound to the BRCA2 gene silencer [M.K. Tripathi, S. Misra, S.V. Khedkar, N. Hamilton, C. Irvin-Wilson,, C. Sharan, L. Sealy, G. Chaudhuri, J. Biol. Chem. 280 (2005) 17163-17171]. hSLUG was predicted to be binding directly to CtBP1 because of an apparent presence of CtBP1 binding site in its amino acid sequences. Here, we provide evidence through yeast two-hybrid and in vitro co-immunoprecipitation analyses that hSLUG does not directly interacts with hCtBP1. This observation will help in the study of the mode of action of hSLUG in human cells.

Alcohol Oxidoreductases↗

PubMed1987

Base sequence-specific interactions of operator DNA fragments with the lambda-cro repressor coupled with changes in their conformations.

The mechanism of interaction of the operator DNA with the lambda-cro repressor protein was investigated using proton n.m.r. and photo CIDNP. Three kinds of DNA duplexes, the lambda-OR3 17-mer, phi80-OR2 19-mer and CRP binding site 22-mer, were prepared, and all of their imino proton resonances of the complexes with lambda-cro were assigned to individual base pairs. By monitoring the assigned signals of the DNA fragments and lambda-cro, it was found that in the complex of lambda-cro with lambda-OR3, two subunits of the cro dimer bind to the right and left halves of the OR3, respectively, and the bidentate binding induces a structural distortion in the middle of the 17-mer. lambda-cro itself also undergoes a conformational change including loosening of the dimeric form. In the complex of lambda-cro with phi 80-OR2, which has a 6-bp sequence common to that of lambda-OR3, one subunit of the cro dimer seems to bind specifically to the common part. However, there is only a slight conformational change in the cro dimer. In the mixture of the CRP binding site 22-mer and lambda-cro, soft contact without any conformational change was observed between them.

Base Sequence↗

PubMed1994

Prokaryotic test system for evaluation of oligonucleotide-affected antisense inhibition.

A new test system is introduced for the estimation of the antisense effect of various types of oligodeoxy-nucleotides. This system is based upon the inhibition of translation of P2 phage repressor protein. Evidence for a direct correlation between phage yield and antisense inhibition was obtained by comparing the concentration-dependent effectiveness of an antisense phosphorothioate 18-mer, its mutated version, and the unmodified oligonucleotide. Oligonucleotides of complementary sequences were synthesized against the ribosome binding site and different sites of the coding region. In our test system only small differences were found based on the location of the target sequence. Rather, the degree of inhibition was related to the calculated ability to hybridize based on Tm values. In this way the sensitivity of the test system could also be demonstrated.

Bacteriophage P2↗

PubMed1996

CytR/cAMP-CRP nucleoprotein formation in E. coli: the CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP.

The CytR repressor protein relies on protein-protein interactions to the cAMP-CRP complex to bind its operators with sufficiently high affinity to repress transcription. Here, the quaternary structure of CytR and the mechanism underlying the cooperative binding of CytR and cAMP-CRP have been analyzed. Using a modified Ferguson analysis in which protein-DNA complexes are separated in a non-denaturing gel system, we show that CytR binds its operators as a dimer alone as well as in a ternary complex with cAMP-CRP. Analyses of DNA binding of CytR at low protein concentrations indicate that CytR is a dimer in solution at physiological concentrations. Moreover, the CytR inducer cytidine was found not to have any effect on the oligomerization of free CytR or DNA bound CytR. Thus, these data rule out the possibility that the cooperative DNA binding of CytR and cAMP-CRP involves induced dimerization of CytR, and they suggest that cytidine interrupts the cooperative binding of CytR and cAMP-CRP solely by perturbing the protein-protein interactions between the two proteins.

Bacterial Proteins↗

PubMed2002

Neuron restrictive silencer factor as a modulator of neuropeptide gene expression.

We hypothesize that the transcription factor neuron restrictive silencer factor (NRSF) is an important determinant of the expression of the preprotachykinin (PPTA) gene (encoding substance P and Neurokinin A) and arginine vasopressin (AVP) both in neuronal and nonneuronal cells. NRSF, a zinc finger repressor protein, binds the NRSE motif found in many neuronal specific genes at a variety of promoter locations. However, it is found in a similar location at the major transcriptional start site, within both PPTA and AVP peptide promoters. We have correlated modulation of NRSF activity with expression of AVP and PPTA in a variety of cell types, indicating the general mechanism by which this protein may regulate expression. Specifically, they are as follows:(1). Expression of NRSF dramatically represses PPTA promoter activity in reporter gene constructs in primary cultures of DRG neurons.(2). The PPTA promoter activity is regulated differentially in osteoarthritic compared to normal chondrocytes. This regulation correlates with the region containing the NRSE site.(3). We have correlated a splice variant of NRSF with the establishment and progression of small cell lung carcinoma (SCLC) and demonstrated that NRSF variants can directly affect the activity of the AVP promoter in reporter gene constructs. If the deregulated expression of peptides in these diseases point to the mechanism determining the pathology, then perhaps targeting protocols that correct this deregulation may also reverse the specific disease phenotypes. Our data would indicate that modulation of NRSF activity would be a target for such intervention.

Animals↗

PubMed2002

Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems.

In mammalian cells, proper gene regulation is achieved by the complex interplay of transcription factors that activate or repress gene expression by binding to the regulatory regions of target promoters. While transcriptional activators have been extensively characterised and classified into functional groups, relatively little is known about the comparative strength and cell type-specificity of transcriptional repressors. Here, we have compared the ability of a series of eukaryotic repression domains to silence basal and activated transcription. A series of the most potent repression domains was further tested in the context of a gene therapy gene-switch system in various cell types. The results indicate that the analysed repression domains exert varying silencing activities in different promoter contexts. Furthermore, their potential for gene silencing varies also depending on the cellular context. When multimerised within one chimeric repressor protein, particular combinations of repressor domains were found to display synergistic repressing effects and efficient repression in a panel of cell lines. This approach thus allowed the identification of transcriptional repressors that are both potent and versatile in terms of cellular specificity as a basis for gene switch systems.

Cell Line↗

PubMed2004

Quantification of plasmid loss in Escherichia coli cells by use of flow cytometry.

A method was developed to study plasmid stability in Escherichia coli cells, which utilised the high speed analysis properties of flow cytometry. To discriminate between plasmid-harbouring cells and plasmid-free cells a plasmid-encoded Lac repressor protein was used to regulate the expression of a chromosomally inserted green fluorescent protein gene in the host cells. Flow cytometric analysis enabled detection and quantification of plasmid-free cells due to their green fluorescent phenotype. The reported system offers real-time analysis in combination with a very low detection level of plasmid loss in bacterial populations. This could be useful in future investigations of plasmid stability and population selection in bacterial communities.

Colony Count, Microbial↗

PubMed1995

Large activation energy barriers to chaperone-peptide complex formation and dissociation.

To probe the mechanism of chaperone substrate selection, we have investigated the kinetics of complex formation and dissociation between the molecular chaperone DnaK and a short peptide (Cro, representing amino acids 1-12 of the cro repressor protein). The Cro protein was N-terminally labeled with the environmentally sensitive fluorophore dansyl chloride (Cro*), and steady-state and stopped-flow fluorescence spectroscopies and fluorescence-detected high-performance size exclusion chromatography (HPSEC) were used to monitor complex formation and dissociation over a range of temperatures in the absence of ATP. The results are summarized as follows: (i) Cro* binds to DnaK with a second-order rate constant, k(on), which varies from 8 to 200 M-1 s-1 between 15 and 37 degrees C. The slow on-rate is a consequence of a large activation energy barrier. The activation enthalpy (delta H*) and the prefactor [omega exp delta S*/R)] are 26 kcal mol-1 and 7 x 10(20) M-1 s-1, respectively. (ii) Once formed, DnaK-Cro* complexes are long-lived, especially at low temperatures (T < 15 degrees C). The off-rate is unusually temperature-sensitive, for example, there is a 478-fold increase in k(off) from 2.3 x 10(6) to 1.1 x 10(-3) s-1 over a range of only 30 degrees C (5-35 degrees C). The steep temperature-dependence of the off-rate is a consequence of a very large activation energy barrier to DnaK-Cro* complex dissociation [delta H* = 34.6 kcal mol-1 and omega exp (delta S*/R) = 2 x 10(21) s-1]. The relatively low affinity of the Cro* peptide for DnaK is due to a large kinetic barrier to binding. We discuss possible causes for these large kinetic barriers.

Amino Acid Sequence↗