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PubMed2005

The H-NS protein represses transcription of the eltAB operon, which encodes heat-labile enterotoxin in enterotoxigenic Escherichia coli, by binding to regions downstream of the promoter.

Heat-labile enterotoxin, a major virulence determinant of enterotoxigenic Escherichia coli, is encoded by the eltAB operon. To elucidate the molecular mechanism by which the heat-stable nucleoid-structural (H-NS) protein controls transcription of eltAB, the authors constructed an eltAB-lacZ transcriptional fusion and performed beta-galactosidase analysis. The results showed that H-NS protein exerts fivefold repression on transcription from the eltAB promoter at 37 degrees C and 10-fold repression at 22 degrees C. Two silencer regions that were required for H-NS-mediated repression of eltAB expression were identified, both of which were located downstream of the start site of transcription. One silencer was located between +31 and +110, the other between +460 and +556, relative to the start site of transcription, and they worked cooperatively in repression. DNA sequences containing the silencers were predicted to be curved by in silico analysis and bound H-NS protein directly in vitro. Repression of eltAB transcription by H-NS was independent of promoter strength, and the presence of H-NS protein did not affect promoter opening in vitro, indicating that repression was achieved by inhibiting promoter clearance or blocking transcription elongation, probably via DNA looping between the two silencers.

Artificial Gene Fusion↗
PubMed1996

smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo.

nanos mRNA, which encodes the localized component of the Drosophila posterior body patterning determinant, is normally translated only at the posterior pole of the embryo, where the mRNA is concentrated. Here we identify two similar cis-acting sequences in the nanos mRNA 3' untranslated region that mediate translational repression. These sequences bind an embryonic protein of 135 kD, smaug, and we refer to them as smaug recognition elements (SREs). Analysis of point mutations in the SREs reveals a strong correlation between smaug binding and translational repression; mutants unable to bind smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind smaug remain repressed translationally. These results strongly suggest that smaug acts in translational repression of unlocalized nanos mRNA. Translational repression is essential, as embryos expressing a nanos mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of the RNA.

Animals↗
PubMed1996

Silencers are required for inheritance of the repressed state in yeast.

Transcriptional silencers in the yeast Saccharomyces induce position-specific, sequence-independent repression by promoting formation of a heterochromatin-like structure across sequences adjacent to them. We have examined the role of silencers in maintenance and inheritance of repression at the silent mating-type cassettes in yeast by monitoring the expression state of one of these cassettes following in vivo deletion of the adjacent silencer. Our experiments indicate that although silencer sequences are dispensable for the maintenance of repression in the absence of cell-cycle progression, silencers are required for the stable inheritance of a repressed state. That is, silenced loci from which the silencer is deleted most often become derepressed within one generation of losing the silencer. Thus, the heritability of a repressed state is not intrinsic to a silenced locus or to the chromatin encompassing it; rather, heritability of repression appears to be a property of the silencer itself.

DNA Nucleotidyltransferases↗
PubMed1998

Histone deacetylase activity of Rpd3 is important for transcriptional repression in vivo.

Eukaryotic organisms from yeast to human contain a multiprotein complex that includes Rpd3 histone deacetylase and Sin3 corepressor. The Sin3-Rpd3 complex, when recruited to promoters by specific DNA-binding proteins, can direct transcriptional repression of specific classes of target genes. It has been proposed that the histone deacetylase activity of Rpd3 is important for repression, but direct evidence is lacking. Here, we describe four Rpd3 derivatives with mutations in evolutionarily invariant histidine residues in a putative deacetylation motif. These Rpd3 mutants lack detectable histone deacetylase activity in vitro, but interact normally with Sin3 in vivo. In yeast cells, these catalytically inactive mutants are defective for transcriptional repression. They retain some residual Rpd3 function in vivo, however, suggesting that repression by the Sin3-Rpd3 complex may not be attributable exclusively to its intrinsic histone deacetylase activity. Finally, we show that a human Rpd3 homolog can interact with yeast Sin3 and repress transcription when artificially recruited to a promoter. These results suggest that the histone deacetylase activity of Rpd3 is important, but perhaps not absolutely required, for transcriptional repression in vivo.

Acetylation↗
PubMed1990

General repression of enhanson activity by the adenovirus-2 E1A proteins.

It has been shown previously that the adenovirus 2 (Ad2) E1A proteins repress activation of transcription by the SV40, polyomavirus and immunoglobulin gene enhancers. Here, we demonstrate that the repression of the SV40 enhancer is not specifically mediated by one of its constituent enhansons and/or proto-enhancers, but that each is subject to repression individually. This inhibitory effect of the E1A proteins is also observed with the AP-1 factor-binding enhansons from the polyomavirus and human metallothionein enhancers, and the MHC class I gene H-2Kb enhanson, which binds the KBF1/H2TF1/TC-IIB protein. Repression by the E1A gene products may, in fact, extend to all enhancer trans-activators, because the transcriptional activities of nuclear receptors (e.g., the estrogen and glucocorticoid receptors), of the yeast enhancer factor GAL4 expressed in HeLa cells, and of chimeric trans-activators (such as GAL-VP16) are all similarly inhibited. The E1A protein domains 2 and 3, including the acidic amino acid stretch that has been shown previously to be necessary for E1A-mediated trans-activation, are not required for repression. These results indicate that the amino-terminal region of the protein, which contains domain 1, plays a crucial role in repression, possibly by interfering in the transcriptional activation process at a step common to all trans-acting enhancer factors.

Adenovirus Early Proteins↗
PubMed1993

Repression by the yeast meiotic inhibitor RME1.

The RME1 gene product, a negative regulator of meiosis with three zinc finger motifs, acts by preventing transcript accumulation from IME1, whose product is required for meiotic gene expression. We have isolated a 404-bp segment from a region 2 kb upstream of IME1 that is sufficient for RME1-dependent repression of a heterologous promoter. This DNA contains an RME1-response element (RRE) and another region called the modulation region. The modulation region is required for repression because DNA containing the RRE alone did not repress but was able to confer RME1-dependent transcriptional activation of a reporter gene. In gel mobility retardation assays, RME1 formed a specific complex with the RRE, and RRE point mutations that reduced the affinity for RME1 also blocked repression and activation. Footprinting of the RME1-RRE complex revealed a 21-bp protected region that included the positions of these RRE mutations. We conclude that RME1 binding to this RRE is required for repression. Thus, the mechanism of meiotic inhibition by RME1 is direct transcriptional repression of IME1.

Base Sequence↗
PubMed2001

Active repression and E2F inhibition by pRB are biochemically distinguishable.

To understand mechanistically how pRB represses transcription, we used a reconstituted transcription assay and compared pRB activity on naked versus chromatin templates. Surprisingly, when pRB was directly recruited to a naked template, no transcriptional repression was observed. However, we observed active repression when the same promoter was assembled into chromatin. Histone deacetylases do not appear to play a role in this observed repression. Further experiments showed repression could occur after preinitiation complex assembly, in contrast with pRB inhibition of E2F, suggesting discrete mechanisms by which pRB directly inhibits an activator such as E2F or actively represses proximally bound transcription factors.

Carrier Proteins↗
PubMed2002

A molecular link between gene-specific and chromosome-wide transcriptional repression.

Gene-specific and chromosome-wide mechanisms of transcriptional regulation control development in multicellular organisms. SDC-2, the determinant of hermaphrodite fate in Caenorhabditis elegans, is a paradigm for both modes of regulation. SDC-2 represses transcription of X chromosomes to achieve dosage compensation, and it also represses the male sex-determination gene her-1 to elicit hermaphrodite differentiation. We show here that SDC-2 recruits the entire dosage compensation complex to her-1, directing this X-chromosome repression machinery to silence an individual, autosomal gene. Functional dissection of her-1 in vivo revealed DNA recognition elements required for SDC-2 binding, recruitment of the dosage compensation complex, and transcriptional repression. Elements within her-1 differed in location, sequence, and strength of repression, implying that the dosage compensation complex may regulate transcription along the X chromosome using diverse recognition elements that play distinct roles in repression.

Animals↗
PubMed2001

Role of mRNA secondary structure in translational repression of the maize transcriptional activator Lc(1,2).

Lc, a member of the maize (Zea mays) R/B gene family, encodes a basic helix-loop-helix transcriptional activator of the anthocyanin biosynthetic pathway. It was previously shown that translation of the Lc mRNA is repressed by a 38-codon upstream open reading frame (uORF) in the 5' leader. In this study, we report that a potential hairpin structure near the 5'end of the Lc mRNA also represses downstream translation in the rabbit reticulocyte in vitro translation system and in transient transformation assays. Base pairing of the hairpin is important for repression because its destabilization increases translation of the uORF and the downstream ORF. However, translation of the uORF is not required for the hairpin-mediated repression. Instead, the uORF and the 5'-proximal hairpin mediate two independent levels of repression. Although the uORF represses downstream translation due to inefficient reinitiation of ribosomes that translate uORF, the hairpin inhibits ribosome loading at the 5' end of the mRNA.

Base Sequence↗
PubMed2004

Aux/IAA proteins contain a potent transcriptional repression domain.

Aux/IAA proteins are short-lived nuclear proteins that repress expression of primary/early auxin response genes in protoplast transfection assays. Repression is thought to result from Aux/IAA proteins dimerizing with auxin response factor (ARF) transcriptional activators that reside on auxin-responsive promoter elements, referred to as AuxREs. Most Aux/IAA proteins contain four conserved domains, designated domains I, II, III, and IV. Domain II and domains III and IV play roles in protein stability and dimerization, respectively. A clear function for domain I had not been established. Results reported here indicate that domain I in Aux/IAA proteins is an active repression domain that is transferable and dominant over activation domains. An LxLxL motif within domain I is important for conferring repression. The dominance of Aux/IAA repression domains over activation domains in ARF transcriptional activators provides a plausible explanation for the repression of auxin response genes via ARF-Aux/IAA dimerization on auxin-responsive promoters.

Amino Acid Sequence↗
PubMed1992

Carbon catabolite repression can account for the temporal pattern of expression of a penicillin biosynthetic gene in Aspergillus nidulans.

Aspergillus nidulans synthesizes penicillins as secondary metabolites when grown under certain culture conditions. Broths containing carbon (C) sources that give rise to carbon catabolite repression (CCR) support a much lower antibiotic yield than broths with non-repressing C sources. Steady-state levels of the isopenicillin N synthetase (IPNS) gene transcript are considerably reduced in mycelia grown with repressing C sources and are depressed in mycelia grown with sugars which do not cause CCR, indicating that penicillin biosynthesis is regulated by CCR through transcriptional control of structural genes. CCR is sufficient to explain the temporal window of expression of the IPNS gene during the growth cycle since (i) the transcript becomes derepressed as soon as the concentration of a repressing C source drops to non-repressing levels and (ii) derepressing C sources sustain derepressed IPNS transcription at all tested moments of the growth cycle. Several tested hypofunctional mutations in creA (the negatively acting regulatory gene which mediates CCR in A. nidulans) do not cause full derepression of IPNS transcript in the presence of a repressing C source. The slight degree of IPNS derepression caused by some creAd (derepressed) alleles parallels the strength of the mutation (as determined by the morphological effect they elicit).

Aspergillus nidulans↗
PubMed1995

Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine-mediated repression, but still able to activate at sigma 70 promoters.

In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates sigma 70, not sigma 54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine-mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine- and phenylalanine-mediated repression of aroP. In contrast, it was able to effect wild-type levels of phenylalanine-mediated repression of aroG, tryptophan-mediated repression of aroP and transcriptional activation of mtr and tyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine-dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine-mediated repression by TyrR but not for tyrosine- or phenylalanine-mediated activation. E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP-dependent aromatic amino acid-binding site.

Adenosine Triphosphatases↗
PubMed2001

Catabolite repression mediated by the CcpA protein in Bacillus subtilis: novel modes of regulation revealed by whole-genome analyses.

Previous studies have shown that the CcpA protein of Bacillus subtilis is a major transcription factor mediating catabolite repression. We report here whole-transcriptome analyses that characterize CcpA-dependent, glucose-dependent gene expression and correlate the results with full-genome computer analyses of DNA binding (CRE) sites for CcpA. The data obtained using traditional approaches show good agreement with those obtained using the transcriptome approach. About 10% of all genes in B. subtilis are regulated > 3x by glucose, with repressed genes outnumbering activated genes three to one. Eighty per cent of these genes depend on CcpA for regulation. Classical approaches have provided only evidence for CcpA-mediated, glucose-dependent activation or repression. We show here that CcpA also mediates glucose-independent activation or repression, and that glucose may alter either the direction or the intensity of either effect. Computer analyses revealed the presence of CRE sites in most operons subject to CcpA-mediated glucose repression, but not in those subject to glucose activation, suggesting that either secondary transcription factors regulate the latter genes or activation by CcpA involves a dissimilar binding site. Operons encoding the constituents of ABC-type transporters that are subject to CcpA-mediated glucose regulation show two distinct patterns: either all genes in the operon are regulated in parallel (the minor class) or the gene encoding the extracytoplasmic solute-binding receptor is preferentially regulated (the major class). Genes subject to CcpA-independent catabolite repression are primarily concerned with sporulation. Several transcription factors were identified that are themselves regulated by CcpA at the transcriptional level. Representative data with functionally characterized genes are presented to illustrate the novel findings. The comprehensive transcriptome data are available on our website: www.biology.uesd.edu/~MSAIER/regulation/ and also on http://www.blackwell-science.com/ products/journals/suppmat/MMI/MMI2328/MMI2328sm.htm

ATP-Binding Cassette Transporters↗
PubMed2006

Catabolite repression of the citST two-component system in Bacillus subtilis.

In Bacillus subtilis, expression of the citrate transporter CitM is under strict control. Transcription of the citM gene is induced by citrate in the medium mediated by the CitS-CitT two-component system and repressed by rapidly degraded carbon sources mediated by carbon catabolite repression (CCR). In this study, we demonstrate that citST genes are part of a bicistronic operon. The promoter region was localized in a stretch of 58 base pairs upstream of the citS gene by deletion experiments. Transcription of the operon was repressed in the presence of glucose by the general transcription factor CcpA. A distal consensus cre site in the citS-coding sequence was implicated in the mechanism of repression. Furthermore, this repression was relieved in Bacillus subtilis mutants deficient in CcpA or Hpr/Crh, components essential to CCR. Thus, we demonstrate that CCR represses the expression of the citST operon, which is responsible for the induction of citM, through the cre site located 1326 bp from transcriptional start site of citST.

Bacillus subtilis↗
PubMed2002

trans-Activation and repression properties of the novel nonsteroid glucocorticoid receptor ligand 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four stereoisomers.

Glucocorticoids are potent anti-inflammatory and immunosuppressant agents. However, they also produce serious side effects that limit their usage. It has been proposed that anti-inflammatory properties of glucocorticoids are caused mostly by repression of activator protein 1- and nuclear factor kappabeta-stimulated synthesis of inflammatory mediators, whereas most of their adverse effects are associated with trans-activation of genes involved with metabolic processes. Our laboratories have sought to discover novel glucocorticoid receptor (GR) ligands that have high repression but low trans-activation activities. We describe here cellular properties of 2,5-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5-(1-methylcyclohexen-3-y1)-1H-[1]benzopyrano[3,4-f]quinoline (A276575) and its four enantiomers. Similar to dexamethasone, A276575 exhibited high affinity for GR and potently repressed interleukin (IL) 1beta-stimulated IL-6 production in human skin fibroblasts, prostaglandin (PG) E(2) production in A549 human lung epithelial cells, and concanavalin A-induced monocyte proliferation. In contrast to dexamethasone, A276575 caused smaller induction of aromatase activity in human skin fibroblasts and antagonized dexamethasone-induced activation of an mouse mammary tumor virus-glucocorticoid-response element (GRE) reporter gene construct. Among the four enantiomers of A276575, the two (-)-enantiomers showed 10- to 30-fold higher affinities for GR than their respective (+)-enantiomers. Both (-)-Syn and (-)-Anti enantiomers of A276575 were potent inhibitors of IL-1beta-stimulated PGE2 production in A549 lung epithelial cells; unexpectedly, however, only the (-)-Anti enantiomer inhibited regulated on T-cell activation, normal T-cell expressed and secreted (RANTES) production in A549 cells. In summary, A276575 is a novel, nonsteroidal GR ligand that possesses high repression activities against inflammatory mediator production but has lower GRE trans-activation activities than traditional steroids. Differential repression of RANTES and PGE2 production in a cell by the two (-)-enantiomers of A276575 illustrates the complexity of repression by GR.

Aromatase↗
PubMed1996

Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWW0 under various conditions of nutrient limitation in chemostat culture.

In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWW0) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer. This report describes catabolite repression of the TOL pathway by succinate in chemostat cultures run at a low dilution rate (D = 0.05 h-1) under different conditions of inorganic-nutrient limitation. The activity of benzylalcohol dehydrogenase (BADH) in cell extracts was used as a measure of the expression of the TOL upper pathway. When cells were grown in the presence of 10 to 15 mM succinate under conditions of phosphate or sulfate limitation, the BADH activity in response to the nonmetabolizable inducer o-xylene was less than 2% of that of cells grown under conditions of succinate limitation. Less repression was found under conditions of ammonium or oxygen limitation (2 to 10% and 20 to 35%, respectively, of the BADH levels under succinate limitation). The BADH expression levels determined under the different growth conditions appeared to correlate well with the mRNA transcript levels from the upper pathway promoter (Pu), which indicates that repression was due to a blockage at the transcriptional level. The meta-cleavage pathway was found to be less susceptible to catabolite repression. The results obtained suggest that the occurrence of catabolite repression is related to a high-energy status of the cells rather than to a high growth rate or directly to the presence of growth-saturating concentrations of a primary carbon and energy source.

Alcohol Oxidoreductases↗
PubMed2005

Integration of signals through Crc and PtsN in catabolite repression of Pseudomonas putida TOL plasmid pWW0.

Toluene degradation in Pseudomonas putida KT2440 pWW0 plasmid is subjected to catabolite repression. Pu and P(S1) promoters of the pWW0 TOL plasmid are down-regulated in vivo during exponential growth in rich medium. In cells growing on minimal medium, yeast extract (YE) addition mimics exponential-phase rich medium repression of these promoters. We have constructed and tested mutants in a series of global regulators described in Pseudomonas. We describe that a mutant in crc (catabolite repression control) partially relieves YE repression. Macroarray experiments show that crc transcription is strongly increased in the presence of YE, inversely correlated with TOL pathway expression. On the other hand, we have found that induced levels of expression from Pu and P(S) in the presence of YE are partially derepressed in a ptsN mutant of P. putida. PtsN but not Crc seems to directly interfere with XylR activation at target promoters. The effect of the double mutation in ptsN and crc is not the sum of the effects of each independent mutation and suggests that both regulators are elements of a common regulatory pathway. Basal expression levels from these promoters in the absence of inducer are still XylR dependent and are also repressed in the presence of yeast extract. Neither crc nor ptsN could relieve this repression.

Bacterial Proteins↗
PubMed2006

Effect of sequence-directed nucleosome disruption on cell-type-specific repression by alpha2/Mcm1 in the yeast genome.

In Saccharomyces cerevisiae, a-cell-specific genes are repressed in MATalpha cells by alpha2/Mcm1, acting in concert with the Ssn6-Tup1 corepressors and the Isw2 chromatin remodeling complex, and nucleosome positioning has been proposed as one mechanism of repression. However, prior studies showed that nucleosome positioning is not essential for repression by alpha2/Mcm1 in artificial reporter plasmids, and the importance of the nucleosome positioning remains questionable. We have tested the function of positioned nucleosomes through alteration of genomic chromatin at the a-cell-specific gene BAR1. We report here that a positioned nucleosome in the BAR1 promoter is disrupted in cis by the insertion of diverse DNA sequences such as poly(dA) . poly(dT) and poly(dC-dG) . poly(dC-dG), leading to inappropriate partial derepression of BAR1. Also, we show that isw2 mutation causes loss of nucleosome positioning in BAR1 in MATalpha cells as well as partial disruption of repression. Thus, nucleosome positioning is required for full repression, but loss of nucleosome positioning is not sufficient to relieve repression completely. Even though disruption of nucleosome positioning by the cis- and trans-acting modulators of chromatin has a modest effect on the level of transcription, it causes significant degradation of the alpha-mating pheromone in MATalpha cells, thereby affecting its cell type identity. Our results illustrate a useful paradigm for analysis of chromatin structural effects at genomic loci.

Adenosine Triphosphatases↗