Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5′ strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3′ strand are located 280 nt downstream, immediately upstream of the Shine–Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

Transcriptional regulation of the Xlim-1 gene by activin is mediated by an element in intron I

The Xlim-1 gene is activated in the late blastula stage of Xenopus embryogenesis in the mesoderm, and its RNA product becomes concentrated in the Spemann organizer at early gastrula stage. A major regulator of early expression of Xlim-1 is activin or an activin-like signal. We report experiments aiming to identify the activin response element in the Xlim-1 gene. The 5′ flanking region of the gene contains a constitutive promoter that is not activin responsive, whereas sequences in the first intron mediate repression of basal promoter activity and stimulation by activin. An intron-derived fragment of 212 nt is the smallest element that could mediate activin responsiveness. Nodal and act-Vg1, factors with signaling properties similar to activin, also stimulated Xlim-1 reporter constructs, whereas BMP-4 did not stimulate or repress the constructs. The mechanism of activin regulation of Xlim-1 and the sequence of the response element are distinct from activin response elements of other genes studied so far.

Myc represses transcription of the growth arrest gene gas1

Cell proliferation is regulated by the induction of growth promoting genes and the suppression of growth inhibitory genes. Malignant growth can result from the altered balance of expression of these genes in favor of cell proliferation. Induction of the transcription factor, c-Myc, promotes cell proliferation and transformation by activating growth promoting genes, including the ODC and cdc25A genes. We show that c-Myc transcriptionally represses the expression of a growth arrest gene, gas1. A conserved Myc structure, Myc box 2, is required for repression of gas1, and for Myc induction of proliferation and transformation, but not for activation of ODC. Activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen was sufficient to repress gas1 gene transcription. These findings suggest that transcriptional repression of growth arrest genes, including gas1, is one step in promotion of cell growth by Myc.

Direct repeat sequences in the Streptomyces chitinase-63 promoter direct both glucose repression and chitin induction

The chi63 promoter directs glucose-sensitive, chitin-dependent transcription of a gene involved in the utilization of chitin as carbon source. Analysis of 5′ and 3′ deletions of the promoter region revealed that a 350-bp segment is sufficient for wild-type levels of expression and regulation. The analysis of single base changes throughout the promoter region, introduced by random and site-directed mutagenesis, identified several sequences to be important for activity and regulation. Single base changes at −10, −12, −32, −33, −35, and −37 upstream of the transcription start site resulted in loss of activity from the promoter, suggesting that bases in these positions are important for RNA polymerase interaction. The sequences centered around −10 (TATTCT) and −35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial promoters. Overlapping the RNA polymerase binding site is a perfect 12-bp direct repeat sequence. Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation. Other base changes resulted in loss of glucose repression while retaining the requirement for chitin induction, suggesting that this sequence is also involved in glucose repression. The fact that cis-acting mutations resulted in glucose resistance but not inducer independence rules out the possibility that glucose repression acts exclusively by inducer exclusion. The fact that mutations that affect glucose repression and chitin induction fall within the same direct repeat sequence module suggests that the direct repeat sequence facilitates both chitin induction and glucose repression.

Interference with gene regulation in living sea urchin embryos: Transcription factor Knock Out (TKO), a genetically controlled vector for blockade of specific transcription factors

“TKO” is an expression vector that knocks out the activity of a transcription factor in vivo under genetic control. We describe a successful test of this concept that used a sea urchin transcription factor of known function, P3A2, as the target. The TKO cassette employs modular cis-regulatory elements to express an encoded single-chain antibody that prevents the P3A2 protein from binding DNA in vivo. In normal development, one of the functions of the P3A2 transcription factor is to repress directly the expression of the CyIIIa cytoskeletal actin gene outside the aboral ectoderm of the embryo. Ectopic expression in oral ectoderm occurs if P3A2 sites are deleted from CyIIIa expression constructs, and we show here that introduction of an αP3A2⋅TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the αP3A2⋅TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. αP3A2⋅TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of αP3A2⋅TKO in the endoderm has the additional lethal effect of disrupting morphogenesis of the archenteron, revealing a previously unsuspected function of SpP3A2 in endoderm development. In principle, TKO technology could be utilized for spatially and temporally controlled blockade of any transcription factor in any biological system amenable to gene transfer.

Estradiol repression of tumor necrosis factor-α transcription requires estrogen receptor activation function-2 and is enhanced by coactivators

The tumor necrosis factor-α (TNF-α) promoter was used to explore the molecular mechanisms of estradiol (E2)-dependent repression of gene transcription. E2 inhibited basal activity and abolished TNF-α activation of the TNF-α promoter. The E2-inhibitory element was mapped to the −125 to −82 region of the TNF-α promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) β is more potent than ERα at repressing the −1044 TNF-α promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERβ, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E2-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.

Nrf1 and Nrf2 positively and c-Fos and Fra1 negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductase1 gene

Twenty-four base pairs of the human antioxidant response element (hARE) are required for high basal transcription of the NAD(P)H:quinone oxidoreductase1 (NQO1) gene and its induction in response to xenobiotics and antioxidants. hARE is a unique cis-element that contains one perfect and one imperfect AP1 element arranged as inverse repeats separated by 3 bp, followed by a “GC” box. We report here that Jun, Fos, Fra, and Nrf nuclear transcription factors bind to the hARE. Overexpression of cDNA derived combinations of the nuclear proteins Jun and Fos or Jun and Fra1 repressed hARE-mediated chloramphenicol acetyltransferase (CAT) gene expression in transfected human hepatoblastoma (Hep-G2) cells. Further experiments suggested that this repression was due to overexpression of c-Fos and Fra1, but not due to Jun proteins. The Jun (c-Jun, Jun-B, and Jun-D) proteins in all the possible combinations were more or less ineffective in repression or upregulation of hARE-mediated gene expression. Interestingly, overexpression of Nrf1 and Nrf2 individually in Hep-G2 and monkey kidney (COS1) cells significantly increased CAT gene expression from reporter plasmid hARE-thymidine kinase-CAT in transfected cells that were inducible by β-naphthoflavone and tert-butyl hydroquinone. These results indicated that hARE-mediated expression of the NQO1 gene and its induction by xenobiotics and antioxidants are mediated by Nrf1 and Nrf2. The hARE-mediated basal expression, however, is repressed by overexpression of c-Fos and Fra1.

Histone deacetylase inhibitor selectively induces p21WAF1 expression and gene-associated histone acetylation

Histone deacetylases (HDACs) catalyze the removal of acetyl groups on the amino-terminal lysine residues of core nucleosomal histones. This activity is associated generally with transcriptional repression. We have reported previously that inhibition of HDAC activity by hydroxamic acid-based hybrid polar compounds, such as suberoylanilide hydroxamic acid (SAHA), induces differentiation and/or apoptosis of transformed cells in vitro and inhibits tumor growth in vivo. SAHA is a potentially new therapeutic approach to cancer treatment and is in Phase I clinical trials. In several tumor cell lines examined, HDAC inhibitors alter the expression of less than 1% of expressed genes, including the cell cycle kinase inhibitor p21WAF1. In T24 bladder carcinoma cells, SAHA induces up to a 9-fold increase in p21WAF1 mRNA and protein, which is, at least in part, because of an increase in the rate of transcription of the gene. SAHA causes an accumulation of acetylated histones H3 and H4 in total cellular chromatin by 2 h, which is maintained through 24 h of culture. An increase in the accumulation of acetylated H3 and H4 was detected throughout the p21WAF1 promoter and the structural gene after culture with SAHA. The level of histone acetylation did not change in chromatin associated with the actin and p27 genes, and their mRNA expression was not altered during culture of T24 cells with SAHA. Thus, the present findings indicate that the induction of p21WAF1 by SAHA is regulated, at least in part, by the degree of acetylation of the gene-associated histones and that this induced increase in acetylation is gene selective.

The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant

In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of α-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of α-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The α-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of αAmy3 3′ untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire αAmy3 3′ UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire αAmy3 3′ UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the αAmy3 3′ UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the α-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.

Glucocorticoid-mediated repression of nuclear factor-κBdependent transcription involves direct interference with transactivation

Glucocorticoids exert multiple anti-inflammatory activities, one of which is the inhibition of transcription dependent on the nuclear factor (NF)-κB. It has been suggested that the effect of dexamethasone (DEX), a glucocorticoid analog, is attributed to an increased production of the inhibitory IκB molecule, which in turn would bind and remove activated, DNA-bound NF-κB complexes in the cell nucleus. Upon investigating DEX-mediated repression of interleukin-6 expression induced by tumor necrosis factor, DEX treatment was found to act directly on NF-κB-dependent transcription, without changing the expression level of IκB. Neither the mRNA of IκB nor the protein was significantly elevated by a combined treatment with tumor necrosis factor and DEX of murine endothelial or fibroblast cells. The DNA-binding activity of induced NF-κB also remained unchanged after stimulation of cells with DEX. Evidence for a direct nuclear mechanism of action was obtained by analysis of cell lines stably expressing a fusion protein between the DNA-binding domain of the yeast Gal4 protein and the transactivating p65 subunit of NF-κB. Expression of a Gal4-dependent luciferase reporter gene activated by this nuclear fusion protein was also strongly repressed after addition of DEX. Because the DNA-binding activity of the Gal4 fusion protein was not affected by DEX, it can be concluded that the reduction of gene activation was caused by interference of the activated glucocorticoid receptor with the transactivation potential of the NF-κB p65 subunit.

Insulin inhibits transcription of IRS-2 gene in rat liver through an insulin response element (IRE) that resembles IREs of other insulin-repressed genes

Recent data indicate that sustained elevations in plasma insulin suppress the mRNA for IRS-2, a component of the insulin signaling pathway in liver, and that this deficiency contributes to hepatic insulin resistance and inappropriate gluconeogenesis. Here, we use nuclear run-on assays to show that insulin inhibits transcription of the IRS-2 gene in the livers of intact rats. Insulin also inhibited transcription of a reporter gene driven by the human IRS-2 promoter that was transfected into freshly isolated rat hepatocytes. The human promoter contains a heptanucleotide sequence, TGTTTTG, that is identical to the insulin response element (IRE) identified previously in the promoters of insulin-repressed genes. Single base pair substitutions in this IRE decreased transcription of the IRS-2-driven reporter in the absence of insulin and abolished insulin-mediated repression. We conclude that insulin represses transcription of the IRS-2 gene by blocking the action of a positive factor that binds to the IRE. Sustained repression of IRS-2, as occurs in chronic hyperinsulinemia, contributes to hepatic insulin resistance and accelerates the development of the diabetic state.

Changes in Growth CO2 Result in Rapid Adjustments of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunit Gene Expression in Expanding and Mature Leaves of Rice1

The accumulation of soluble carbohydrates resulting from growth under elevated CO2 may potentially signal the repression of gene activity for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS). To test this hypothesis we grew rice (Oryza sativa L.) under ambient (350 μL L−1) and high (700 μL L−1) CO2 in outdoor, sunlit, environment-controlled chambers and performed a cross-switching of growth CO2 concentration at the late-vegetative phase. Within 24 h, plants switched to high CO2 showed a 15% and 23% decrease in rbcS mRNA, whereas plants switched to ambient CO2 increased 27% and 11% in expanding and mature leaves, respectively. Ribulose-1,5-bisphosphate carboxylase/oxygenase total activity and protein content 8 d after the switch increased up to 27% and 20%, respectively, in plants switched to ambient CO2, but changed very little in plants switched to high CO2. Plants maintained at high CO2 showed greater carbohydrate pool sizes and lower rbcS transcript levels than plants kept at ambient CO2. However, after switching growth CO2 concentration, there was not a simple correlation between carbohydrate and rbcS transcript levels. We conclude that although carbohydrates may be important in the regulation of rbcS expression, changes in total pool size alone could not predict the rapid changes in expression that we observed.

Estrogen and thyroid hormone interaction on regulation of gene expression.

Estrogen receptor (ER) and thyroid hormone receptors (TRs) are ligand-dependent nuclear transcription factors that can bind to an identical half-site, AGGTCA, of their cognate hormone response elements. By in vitro transfection analysis in CV-1 cells, we show that estrogen induction of chloramphenicol acetyltransferase (CAT) activity in a construct containing a CAT reporter gene under the control of a minimal thymidine kinase (tk) promoter and a copy of the consensus ER response element was attenuated by cotransfection of TR alpha 1 plus triiodothyronine treatment. This inhibitory effect of TR was ligand-dependent and isoform-specific. Neither TR beta 1 nor TR beta 2 cotransfection inhibited estrogen-induced CAT activity, although both TR alpha and TR beta can bind to a consensus ER response element. Furthermore, cotransfection of a mutated TR alpha 1 that lacks binding to the AGGTCA sequence also inhibited the estrogen effect. Thus, the repression of estrogen action by liganded TR alpha 1 may involve protein-protein interactions although competition of ER and TR at the DNA level cannot be excluded. A similar inhibitory effect of liganded TR alpha 1 on estrogen induction of CAT activity was observed in a construct containing the preproenkephalin (PPE) promoter. A study in hypophysectomized female rats demonstrated that the estrogen-induced increase in PPE mRNA levels in the ventromedial hypothalamus was diminished by coadministration of triiodothyronine. These results suggest that ER and TR may interact to modulate estrogen-sensitive gene expression, such as for PPE, in the hypothalamus.

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

Long-range repression in the Drosophila embryo.

Transcriptional repressors can be characterized by their range of action on promoters and enhancers. Short-range repressors interact over distances of 50-150 bp to inhibit, or quench, either upstream activators or the basal transcription complex. In contrast, long-range repressors act over several kilobases to silence basal promoters. We describe recent progress in characterizing the functional properties of one such long-range element in the Drosophila embryo and discuss the contrasting types of gene regulation that are made possible by short- and long-range repressors.