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At least 19 recordsLinked to original sources
PubMed2000

Repression of the Escherichia coli melR promoter by MelR: evidence that efficient repression requires the formation of a repression loop.

The Escherichia coli MelR protein is a transcription activator that, in the presence of melibiose, activates expression of the melAB operon by binding to four sites located just upstream of the melAB promoter. MelR is encoded by the melR gene, which is expressed from a divergent transcript that starts 237 bp upstream of the melAB promoter transcript start point. In a recent study, we have identified a fifth DNA site for MelR that overlaps the melR promoter transcript start and -10 region. Here we show that MelR binding to this site can downregulate expression from the melR promoter; thus, MelR autoregulates its own expression. Optimal repression of the melR promoter is observed in the absence of melibiose and requires one of the four other DNA sites for MelR at the melAB promoter. The two MelR binding sites required for this optimal repression are separated by 177 bp. We suggest that, in the absence of melibiose, MelR forms a loop between these two sites. We argue that, in the presence of melibiose, this loop is broken as the melAB promoter is activated. However, in the presence of melibiose, the melR promoter can still be partially repressed by MelR binding to the site that overlaps the transcript start and -10 region. Parallels with the Escherichia coli araC-araBAD regulatory region are discussed.

Binding Sites↗
PubMed1999

Role of HU and DNA supercoiling in transcription repression: specialized nucleoprotein repression complex at gal promoters in Escherichia coli.

Efficient repression of the two promoters P1 and P2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA looping-mediated repression involves binding of the Gal repressor (GalR) to two gal operators (OE and OI) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between OE and OI, and supercoiled DNA. Without DNA looping, GalR binding to OE partially represses P1 and stimulates P2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P2 was entirely dependent upon DNA supercoiling in vivo. The P2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P2, but not P1, was also totally derepressed by the absence of HU or the OI operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNA-multiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P1 but not P2 is still repressed by GalR binding to OE. The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.

Aminocoumarins↗
PubMed2001

YY1 represses vitamin D receptor-mediated 25-hydroxyvitamin D(3)24-hydroxylase transcription: relief of repression by CREB-binding protein.

Ying Yang transcription factor (YY1) can repress or activate transcription. 25-Hydroxyvitamin D(3)-24-hydroxylase [24(OH)ase], an enzyme involved in the catabolism of 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], is up-regulated at the transcriptional level by 1,25-(OH)(2)D(3) to self-induce its deactivation. Here we report that YY1 can repress 1,25-(OH)(2)D(3)-induced 24(OH)ase transcription in CV1 cells transfected with vitamin D receptor (VDR) expression vector or in LLCPK(1) cells that contain VDR endogenously. With increasing amounts of YY1 DNA transfected (500 ng to 2 microg), ligand-dependent VDR activation of 24(OH)ase transcription was steadily repressed (maximum repression was 10-fold). Thus, YY1 may be a key modulator preventing activation at times that do not require the enzyme to be expressed. Relief of YY1 repression was observed in the presence of TFIIB or CBP (CREB binding protein) suggesting that YY1 may exert repression, in part, by sequestering TFIIB/CBP. Glutathione-S-transferase (GST) pull-down assays identified regions in the N and C termini of CBP that can bind YY1. In addition, the N-terminal region of CBP that interacts with YY1 can inhibit YY1 from binding to TFIIB. Thus, CBP may alleviate YY1-mediated repression, in part, by preventing YY1 from binding to TFIIB, which is required for VDR-mediated transcription. In summary, our results suggest that YY1 represses 24(OH)ase transcription, at least in part, by sequestering activator proteins involved in VDR-mediated transcription. In addition, our findings demonstrate a role for CBP in relief of repression of VDR-mediated transcription.

Animals↗
PubMed2001

Drosophila single-minded represses gene transcription by activating the expression of repressive factors.

The Drosophila single-minded gene controls CNS midline cell development by both activating midline gene expression and repressing lateral CNS gene expression in the midline cells. The mechanism by which Single-minded represses transcription was examined using the ventral nervous system defective gene as a target gene. Transgenic-lacZ analysis of constructs containing fragments of the ventral nervous system defective regulatory region identified sequences required for lateral CNS transcription and midline repression. Elimination of Single-minded:Tango binding sites within the ventral nervous system defective gene did not affect midline repression. Mutants of Single-minded that removed the DNA binding and transcriptional activation regions abolished ventral nervous system defective repression, as well as transcriptional activation of other genes. The replacement of the Single-minded transcriptional activation region with a heterologous VP16 transcriptional activation region restored the ability of Single-minded to both activate and repress transcription. These results indicate that Single-minded indirectly represses transcription by activating the expression of repressive factors. Single-minded provides a model system for how regulatory proteins that act only as transcriptional activators can control lineage-specific transcription in both positive and negative modes.

Animals↗
PubMed1999

A novel mutation, of the Bacillus subtilis hut operon that relieves both catabolite repression and amino acid repression.

A mutation, designated hutCR11, which resulted in high expression of the hut operon and release of the catabolite repression and amino-acid repression of hut expression, was isolated and determined to be a T-to-G transversion at position +30 (+1 indicates the transcription-initiation site). In the hutCR11 mutant, levels of hutP mRNA were 5-fold higher than those in wild-type cells under conditions of non-induction and induction and 11-fold higher under conditions of catabolite repression and amino-acid repression. Mutation analysis showed that two types of base change (T-->A and T-->C) at position +30 did not cause high expression of the hut operon, indicating that this was specifically caused by the single base substitution (T-->G) at position +30. The base substitution of A for T at position +30 also led to partial relief of both catabolite repression and amino-acid repression. These results indicate that the nucleotide sequence at +30 is important for regulation of both catabolite repression and amino-acid repression of the hut operon.

Bacillus subtilis↗
PubMed2001

Repression domains of class II ERF transcriptional repressors share an essential motif for active repression.

We reported previously that three ERF transcription factors, tobacco ERF3 (NtERF3) and Arabidopsis AtERF3 and AtERF4, which are categorized as class II ERFs, are active repressors of transcription. To clarify the roles of these repressors in transcriptional regulation in plants, we attempted to identify the functional domains of the ERF repressor that mediates the repression of transcription. Analysis of the results of a series of deletions revealed that the C-terminal 35 amino acids of NtERF3 are sufficient to confer the capacity for repression of transcription on a heterologous DNA binding domain. This repression domain suppressed the intermolecular activities of other transcriptional activators. In addition, fusion of this repression domain to the VP16 activation domain completely inhibited the transactivation function of VP16. Comparison of amino acid sequences of class II ERF repressors revealed the conservation of the sequence motif (L)/(F)DLN(L)/(F)(x)P. This motif was essential for repression because mutations within the motif eliminated the capacity for repression. We designated this motif the ERF-associated amphiphilic repression (EAR) motif, and we identified this motif in a number of zinc-finger proteins from wheat, Arabidopsis, and petunia plants. These zinc finger proteins functioned as repressors, and their repression domains were identified as regions that contained an EAR motif.

Amino Acid Motifs↗
PubMed1995

A domain of the even-skipped protein represses transcription by preventing TFIID binding to a promoter: repression by cooperative blocking.

We examined the mechanism by which the C-terminal 236 amino acids of the even-skipped protein (region CD) repress transcription. A fusion protein, CDGB, was created that contains region CD fused to the glucocorticoid receptor DNA binding domain. This protein repressed transcription in an in vitro system containing purified fractions of the RNA polymerase II general transcription factors, and repression was dependent upon the presence of high-affinity glucocorticoid receptor binding sites in the promoter. Repression by CDGB was prevented when the promoter DNA was preincubated with TFIID or TBP, whereas preincubation of the template DNA with CDGB prevented TFIID binding. Together, these results strongly imply that CDGB represses transcription by inhibiting TFIID binding, and further experiments suggested a mechanism by which this may occur. Region CD can mediate cooperative interactions between repressor molecules such that molecules bound at the glucocorticoid receptor binding sites stabilize binding of additional CDGB molecules to low-affinity binding sites throughout the basal promoter. Binding to some of these low-affinity sites was shown to contribute to repression. Further experiments suggested that the full-length eve protein also represses transcription by the same mechanism. We speculate that occupancy of secondary sites within the basal promoter by CDGB or the eve protein inhibits subsequent TFIID binding to repress transcription, a mechanism we term cooperative blocking.

Animals↗
PubMed1969

Catabolite repression of the lac operon. The contribution f trascriptional repression.

1. Experiments were carried out to distinguish the contributions of transcriptional and translational repression to catabolite repression of the lac operon. 2. In strain EZ16-3-G of Escherichia coli the synthesis of thiogalactoside transacetylase is directed by a gene situated on an episome, and the operator, promotor and regulator genes that lay cis to this gene have been deleted, so that the normal mechanism for controlling transcription is abolished. The extent of catabolite repression in this strain was much less than that in wild-type strains. 3. The same episome is responsible for the synthesis of thiogalactoside transacetylase in strain RM32/F'd25, and in this strain a second lac operon directs the synthesis of beta-galactosidase under the control of a wild-type operator-promotor-regulator system. The extent of catabolite repression of thiogalactoside transacetylase in strain RM32/F'd25 was substantially more than in strain EZ16-3-G, but less than that of beta-galactosidase in strain RM32/F'd25. 4. Since the synthesis of thiogalactoside transacetylase in these organisms is presumably subject to translational repression only, it is concluded that in strain RM32/F'd25 the synthesis of beta-galactosidase is subject to both transcriptional and translational repression. It is also concluded that the extent of translational repression varies between strains.

Acyltransferases↗
PubMed1994

Transcriptional repression by the human bZIP factor E4BP4: definition of a minimal repression domain.

The bZIP factor E4BP4 overlaps in DNA binding site specificity with the transcriptional activator CREB and members of the ATF family of transcription factors, but is an active transcriptional repressor. In this study we have mapped the repressing activity of E4BP4 to a small 'domain' of 65 amino acids that retains its ability to repress transcription when transferred to the heterologous DNA binding domain of the yeast transcriptional activator GAL4. This segment of the E4BP4 polypeptide contains a high proportion of charged amino acids and does not resemble the repression domains that have been characterized so far from other active transcriptional repressors such as the Drosophila Krüppel, Engrailed or Even-skipped proteins. A mutation which changes the charge configuration of this repression module resulted in a complete loss of repressor activity. The E4BP4-GAL4 fusion protein is able to repress the residual transcription from minimal promoters containing the adenovirus E4 or E1b TATA box. This is consistent with a mechanism of action whereby E4BP4 interacts with some component of the general transcription machinery to cause repression of basal and activated transcription. Although a number of nuclear proteins are able to interact with the E4BP4 repression domain in vitro, these proteins do not appear to include the general transcription factors TFIIB or TBP.

Amino Acid Sequence↗
PubMed2004

Transforming growth factor beta-mediated transcriptional repression of c-myc is dependent on direct binding of Smad3 to a novel repressive Smad binding element.

Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor beta (TGF-beta) signal. The ability of the Smads to act as transcriptional activators via TGF-beta-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-beta target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-beta-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-beta, and this repression is required for the manifestation of the TGF-beta cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-beta-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-beta inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter.

Animals↗
PubMed1994

Repression of transforming growth factor beta 1 promoter by the adenovirus oncogene E1A. Identification of a unique GC-rich sequence as a target for E1A repression.

The transforming growth factor beta 1 (TGF-beta 1) is a key regulator of proliferation and differentiation in a wide variety of cell types. It is a potent growth inhibitor for most epithelial, endothelial, lymphoid, and myeloid cells. In the present study, we showed that a DNA virus oncoprotein, E1A, strongly repressed the activity of the TGF-beta 1 promoter in a variety of cell lines. Interestingly, this repression was specific for 12 S E1A because 13 S E1A was much less active in this assay. Analysis of a series of E1A mutants showed that the repression was dependent on the amino terminus and the conserved region 1 of the E1A protein. To identify the target sequence for E1A repression in the TGF-beta 1 promoter, a series of mutant promoters were analyzed and a 10-base pair GC-rich sequence between -91 and -82 was found to be the major target for E1A repression of the promoter. Using chimeric reporter constructs, we provide evidence that the 10-base pair GC-rich sequence is sufficient to impart sequence-specific E1A repression to a heterologous promoter. Additionally, we suggest that the mechanism of E1A repression through this GC-rich element does not involve abrogation of the retinoblastoma control of the TGF-beta 1 promoter.

Adenovirus E1A Proteins↗
PubMed2004

Propagation of silencing; recruitment and repression of naive chromatin in trans by polycomb repressed chromatin.

The Polycomb group (PcG) proteins maintain stable and heritable repression of homeotic genes. Typically, Polycomb response elements (PRE) that direct PcG repression are located at great distances (10s of kb) from the promoters of PcG-repressed genes, and it is not known how these PREs can communicate with promoters over such distances. Using Class II mouse PRC core complexes (mPCCs) assembled from recombinant subunits, we investigated how PcG complexes might bridge distant chromosomal regions. Like native and recombinant Drosophila Class II complexes, mPCC represses chromatin remodeling and transcription. Interestingly, mPCC bound to one polynucleosome template can recruit a second template from solution and renders it refractory to transcription and chromatin remodeling. A Drosophila PRC core complex (dPCC) also is able to recruit a second template. Posterior sex combs (PSC), a subunit of dPCC, inhibits chromatin remodeling and transcription efficiently but requires assembly with dRING1 to recruit chromatin. Thus, repression and template bridging require different subunits of PcG complexes, suggesting that long-range effects may be mechanistically distinct from repression.

Animals↗
PubMed1970

Repression of acetyl-coenzyme A carboxylase by unsaturated fatty acids: relationship to coenzyme repression.

It has been reported that the level of d-biotin in the growth medium of Lactobacillus plantarum regulates the synthesis of apoacetyl-coenzyme A (CoA) carboxylase; high levels cause repression, and deficient levels effect derepression. In this study, evidence has been obtained which suggests that coenzyme repression by biotin is an indirect effect; i.e., biotin regulates the synthesis of unsaturated fatty acids which are the true repressors of the acetyl-CoA carboxylase. This was observed in an experiment in which long-chain unsaturated fatty acids were added to media containing deficient, sufficient, or excess levels of d-biotin. In every case, independently of the biotin concentration for growth, the unsaturated fatty acids caused a severe repression of the carboxylase. Saturated fatty acids were without effect. The level of oleic acid required to give maximal repression was 50 mug/ml. The free fatty acids had no adverse effect on the activity of the cell-free extracts nor on the permeation of d-biotin into the cell. Saturated and unsaturated fatty acids decreased the rate of holocarboxylase formation from d-biotin and the apoacetyl-CoA carboxylase in the extracts. It is concluded that there are at least three mechanisms that control the acetyl-CoA carboxylase in this organism: (i) indirect coenzyme repression by d-biotin, (ii) repression by unsaturated fatty acids, and (iii) regulation of the activity of the holocarboxylase synthetase by both saturated and unsaturated fatty acids.

Biotin↗
PubMed1973

Regulation of argE-argH expression with arginine derivatives in Escherichia coli: extreme non-uniformity of repression and conditional repressive action.

In regulatory studies of the arginine biosynthetic system of Escherichia coli, alpha-N-acetyl-l-arginine (AcA) is a useful restrictive arginine source. In strain 39A-23R3 (argA(-)), at 25 mug/ml, AcA gives suboptimal growth rates and is fully derepressive for acetylornithinase (specified by argE) and approximately 50% derepressive for argininosuccinase (specified by argH). At 10 mug/ml, the growth rate decreases, whereas the extent of derepression is unchanged; at 500 mug/ml, full repression results. In strain 3670 (argB(-)argG(-)), AcA (25 mug/ml) leads to partial derepression of acetylornithinase but full repression of argininosuccinase. Thus, the repression patterns for both strains, although not identical, are nonuniform. AcA utilization is antagonized by alpha-N-acetyl-l-ornithine (AcO). In strain 3670 (blocked before and after acetylornithinase), the growth rate on AcA (25 mug/ml) is lowered by AcO (500 mug/ml); acetylornithinase is completely derepressed, whereas argininosuccinase is fully repressed. This difference in regulatory behavior represents extreme nonuniform repression. Unexpectedly, the effect of AcO is attributable to the conversion of AcO to citrulline (Cit). In strain 3670, mixtures of AcA (25 mug/ml) and Cit (300 mug/ml) permit complete derepression of acetylornithinase; there is evidence that Cit enters the cell. In contrast, in the arginine-limited chemostat, Cit represses acetylornithinase. These opposite regulatory effects of Cit appear to stem from the difference in arginine restriction. AcA enters the cell via AcO permease and is deacylated by acetylornithinase (K(m), 5.0 mM). AcA competitively inhibits AcO cleavage (K(i), 2.4 mM), but Cit is not inhibitory. The antagonism of AcA utilization by AcO or Cit is thought to be exerted at the AcO permease.

Acetylation↗
PubMed1990

Determination of the cis sequence involved in catabolite repression of the Bacillus subtilis gnt operon; implication of a consensus sequence in catabolite repression in the genus Bacillus.

The mechanism underlying catabolite repression in Bacillus species remains unsolved. The gluconate (gnt) operon of Bacillus subtilis is one of the catabolic operons which is under catabolite repression. To identify the cis sequence involved in catabolite repression of the gnt operon, we performed deletion analysis of a DNA fragment carrying the gnt promoter and the gntR gene, which had been cloned into the promoter probe vector, pWP19. Deletion of the region upstream of the gnt promoter did not affect catabolite repression. Further deletion analysis of the gnt promoter and gntR coding region was carried out after restoration of promoter activity through the insertion of internal constitutive promoters of the gnt operon before the gntR gene (P2 and P3). These deletions revealed that the cis sequence involved in catabolite repression of the gnt operon is located between nucleotide positions +137 and +148. This DNA segment contains a sequence, ATTGAAAG, which may be implicated as a consensus sequence involved in catabolite repression in the genus Bacillus.

Bacillus subtilis↗
PubMed1993

Transcriptional repression by the Drosophila even-skipped protein: definition of a minimal repression domain.

We have used a transient expression assay employing Drosophila tissue culture cells to study the transcriptional repression activity of the homeo domain protein Even-skipped (Eve). Eve was found to repress all promoters that contained Eve-binding sites, including both TATA-containing and TATA-lacking minimal promoters, as well as promoters activated by several different classes of activator proteins. These findings suggest that the general transcription machinery can be a target of Eve. By analyzing properties of a variety of Eve mutants and chimeric fusion proteins, we have identified several features important for efficient repression. In addition to the DNA-binding domain, a potent repressor requires a repression domain, which can be as small as 27 residues. The minimal 57-residue Eve repression domain, as well as several others studied here, were all found to be proline rich and to contain a high percentage of hydrophobic residues. An intriguing feature of the strong repressors was that their DNA-binding activities, measured by gel retention assays with nuclear extracts, were significantly less than those of derivatives inactive in repression.

Amino Acid Sequence↗
PubMed1996

The human cut homeodomain protein can repress gene expression by two distinct mechanisms: active repression and competition for binding site occupancy.

By analogy with other homeodomain proteins conserved in evolution, mammalian Cut proteins are believed, as in Drosophila melanogaster, to play an important role in determining cell type specificity in several tissues. At the molecular level, Cut proteins appear to serve as transcriptional repressors. In this study, we have examined the mechanism by which the human Cut (hCut) protein down-regulates gene expression. The homeodomain and the three regions called Cut repeats are evolutionarily conserved and were previously shown to function as DNA binding domains. The carboxy-terminal region, although it does not show amino acid sequence homology per se, in all cases is enriched in alanine and proline residues, a distinctive feature of some transcriptional repression domains. Our results reveal two distinct modes of repression: competition for binding site occupancy and active repression. On one hand, the composite DNA binding domain formed by Cut repeat 3 and the Cut homeodomain was shown to bind to CCAAT and Sp1 sites within the tk gene promoter and to reduce gene expression, presumably by preventing activation by the corresponding transcription factors. On the other hand, the carboxy-terminal region of mammalian Cut proteins was found to function as an active repression domain in a distance-independent manner. We have further narrowed this activity to two subdomains that can independently repress activated transcription. Finally, we present a model to illustrate the two mechanisms by which Cut proteins repress gene expression.

Amino Acid Sequence↗