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PubMed2006

Inhibition and down-regulation of gene transcription and guanylyl cyclase activity of NPRA by angiotensin II involving protein kinase C.

The objective of this study was to investigate the role of protein kinase C (PKC) in the angiotensin II (Ang II)-dependent repression of Npr1 (coding for natriuretic peptide receptor-A, NPRA) gene transcription. Mouse mesangial cells (MMCs) were transfected with Npr1 gene promoter-luciferase construct and treated with Ang II and PKC agonist or antagonist. The results showed that the treatment of MMCs with 10 nM Ang II produced a 60% reduction in the promoter activity of Npr1 gene. MMCs treated with 10nM Ang II exhibited 55% reduction in NPRA mRNA levels, and subsequent stimulation with 100 nM ANP resulted in 50% reduction in guanylyl cyclase (GC) activity. Furthermore, the treatment of MMCs with Ang II in the presence of PKC agonist phorbol ester (100 nM) produced an almost 75% reduction in NPRA mRNA and 70% reduction in the intracellular accumulation of cGMP levels. PKC antagonist staurosporine completely reversed the effect of Ang II and phorbol ester. This is the first report to demonstrate that ANG II-dependent transcriptional repression of Npr1 gene promoter activity and down-regulation of GC activity of translated protein, NPRA is regulated by PKC pathways.

Angiotensin II↗
PubMed2004

Does looping and clustering in the nucleus regulate gene expression?

There has been considerable interest in the way that chromatin is spatially organised within the cell nucleus and how that may relate to gene expression and its control. New molecular techniques have identified looped chromatin domains at the mammalian beta-globin and the Drosophila hsp70 loci. Looped domains may insulate chromatin from the influence of neighbouring domains, and the bases of loops may also act to concentrate proteins locally within the nucleus. The spatial clustering of sequences from the Drosophila bithorax complex, located in trans, has also been demonstrated. An emerging theme is that bringing DNA and proteins together within a defined sub-region of the nuclear volume facilitates both the activation and the repression of gene expression. Nuclear compartments may also be involved in the post-translational modification of proteins by sumoylation and ubiquitylation.

Animals↗
PubMed1993

Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro.

Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate or NADPH, or with an inhibitor, methotrexate, repressed its ability to interact with DHFR mRNA. An in vitro rabbit reticulocyte lysate translation system was used to show that the addition of exogenous human recombinant DHFR protein to in vitro translation reactions specifically inhibited DHFR mRNA translation. These studies suggest that the direct interaction between DHFR protein and its mRNA may be a mechanism for regulation of DHFR synthesis.

Base Sequence↗
PubMed1979

Superinduction of alpha 2u globulin by actinomycin D: evidence for drug-mediated increase in alpha 2u mRNA.

Actinomycin D, an inhibitor of DNA-dependent RNA synthesis, increased the hepatic concentration of alpha 2u globulin, an androgen-inducible protein in the rat. Spayed female rats with a marginally induced state of alpha 2u synthesis showed an approximately 5-fold increase in hepatic alpha 2u globulin within 3-6 hr after treatment with actinomycin D. Initial treatment of these animals with 5 alpha-dihydrotestosterone, followed by actinomycin D, resulted within 2-3 hr in a more than 2-fold increase in hepatic alpha 2u globulin compared to animals treated with the androgen alone. In spite of inhibition of hepatic synthesis of poly(A)-containing RNA to less than 25% of control, superinduction with actinomycin D resulted in a parallel increase in the translatable mRNA for alpha 2u globulin. These results showing increase in both alpha 2u globulin and its translatable mRNA after superinduction with actinomycin D support the concept of post-transcriptional repression of alpha2u synthesis.

Alpha-Globulins↗
PubMed1998

Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA.

The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Up-regulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1beta (IL-1beta) increases prostaglandin E2 (PGE2) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25-40% by dexamethasone, while PGE2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1beta-induced PGE2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1beta treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3'-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3'-UTR for specificity.

Cycloheximide↗
PubMed2006

Ribosomal stress couples the unfolded protein response to p53-dependent cell cycle arrest.

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Cell Cycle↗
PubMed1987

Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals.

The nar operon, which encodes the three subunits of nitrate reductase in Escherichia coli, is fully induced under anaerobic conditions with nitrate. Two distinct regulatory domains have been delineated in the 5' region of the operon which respond respectively to positive induction by the fnr gene product under anaerobic conditions and to positive induction by the narL gene product in the presence of nitrate (S.F. Li, T. Rabi, and J.A. DeMoss, J. Bacteriol. 164:25-32). To characterize these two regulatory regions, we determined the DNA sequence for a 500-base-pair (bp) region extending upstream from the first structural gene of the nar operon. Analysis of subsequent subclones of the operon established that the 5' limit of the nar operon lies between 215 and 260 bp upstream from the translational start site of the first structural gene. The region required for induction by the fnr gene product is located within 160 bp from the translation start site, while the region responding to induction by nitrate extends an additional 100 bp upstream. Protein fusions of lacZ with the N-terminal sequence of the narG gene were constructed so that beta-galactosidase formation was under the control of the nar promoter and one or both regulatory domains. Analysis of strains bearing these fusion plasmids indicated that the expression of the hybrid proteins paralleled that of nitrate reductase by the parent plasmids, demonstrating that the regulatory signals did not extend significantly into the first structural gene. The transcriptional start site and the level of the transcription were determined by the S1 mapping procedure. One major transcript was identified which initiated -50 bp from the translational start site of the first structural gene. The synthesis of the transcript was repressed aerobically, was fully induced by nitrate anaerobically, and was greatly reduced in an Fnr- mutant. Possible regulatory sequences were identified in the 200-bp regulatory region extending upstream from the transcription start site.

Aerobiosis↗
PubMed1989

La Crosse virus nucleocapsid protein controls its own synthesis in mosquito cells by encapsidating its mRNA.

Within 24 to 48 h of La Crosse virus infection of mosquito cells, greater than 75% of the S mRNA was found to band in CsCl density gradients at the position of genome or antigenome nucleocapsids. The encapsidation of the S mRNA correlates with the repression of N protein synthesis in vivo, and the encapsidated S mRNA cannot be translated in vitro. Unlike genome and antigenome assembly, S mRNA assembly is a relatively slow process, which is not coupled to its synthesis. Within the encapsidated S mRNA population, three forms could be distinguished, those with intact primers which were or were not also assembled with N protein and those in which the primer and up to 3 template bases had been lost. We suggest that genome replication, but not transcription, is down regulated with time in mosquito cells for reasons that are unclear. The pool of unassembled N protein then increased to the point at which it began to interact with its own mRNA, as this mRNA also contains what is considered to be the assembly site, i.e., the conserved sequences at the 5' ends of all genome and antigenome chains. This lead to the assembly of the entire mRNA, except for the nontemplate primer. Some of the primers were then also assembled with N protein, whereas others were digested to produce truncated mRNAs.

Animals↗
PubMed2005

The Polycomb group gene Ezh2 prevents hematopoietic stem cell exhaustion.

The molecular mechanism responsible for a decline of stem cell functioning after replicative stress remains unknown. We used mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) to identify genes involved in the process of cellular aging. In proliferating and senescent MEFs one of the most differentially expressed transcripts was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG) involved in histone methylation and deacetylation. Retroviral overexpression of Ezh2 in MEFs resulted in bypassing of the senescence program. More importantly, whereas normal HSCs were rapidly exhausted after serial transplantations, overexpression of Ezh2 completely conserved long-term repopulating potential. Animals that were reconstituted with 3 times serially transplanted control bone marrow cells all died due to hematopoietic failure. In contrast, similarly transplanted Ezh2-overexpressing stem cells restored stem cell quality to normal levels. In a "genetic genomics" screen, we identified novel putative Ezh2 target or partner stem cell genes that are associated with chromatin modification. Our data suggest that stabilization of the chromatin structure preserves HSC potential after replicative stress.

Animals↗
PubMed1994

Acidic-phospholipid deficiency represses the flagellar master operon through a novel regulatory region in Escherichia coli.

The Escherichia coli pgsA3 mutation, which causes acidic-phospholipid deficiency, was found to repress the flagellar master operon, as assessed by the beta-galactosidase activities of flhD-lacZ fusions. This explained the impairment of flagellar formation and motility by the mutation. A series of deletion analysis indicated that a 40-bp region, at the 5' end of the flhD locus examined, was responsible for the repression of a downstream transcription initiation that was catabolite-repression sensitive. This novel regulatory region was 200 bp upstream of the first possible translation initiation site.

Bacterial Proteins↗
PubMed2000

A splice variant of the neuron-restrictive silencer factor repressor is expressed in small cell lung cancer: a potential role in derepression of neuroendocrine genes and a useful clinical marker.

The neuron-restrictive silencer factor [NRSF (RE-1 silencing transcription factor/X box repressor)] is a transcriptional silencer, which we have previously implicated in deregulation of the vasopressin promoter in small cell lung cancer (SCLC). Here we describe a novel splice variant of the NRSF transcript, which is highly expressed in SCLCs. The variant was detected in both established cell lines and primary SCLC cultures as well as in some primitive neuroectodermal tumor biopsies. It was present at very low levels in human brain tissue, non-SCLC tumors, and normal bronchial epithelium. This human splice variant, which is massively overexpressed in SCLCs, incorporates a 50-bp insert between exons 5 and 6, introducing a stop codon and predicting translation of a truncated NRSF isoform. We propose that the encoded isoform may antagonize repression of the vasopressin promoter and other "neuronal" genes with neuron-restrictive silencer elements in SCLCs. Thus, up-regulated expression of this NRSF isoform may be a key early factor in defining the neuroendocrine phenotype of these tumors. The NRSF splice variant represents a specific clinical marker that could prove useful in detection of the majority of SCLCs.

Alternative Splicing↗
PubMed1993

Repression of hepatic delta-aminolevulinate synthase by heme and metalloporphyrins: relationship to inhibition of heme oxygenase.

Heme- and tin-chelated metalloporphyrins are known to decrease the activity of hepatic delta-amino-levulinate synthase, the rate-controlling enzyme of heme synthesis. We performed experiments in primary chick embryo liver cells with tin-, zinc- and copper-chelated porphyrins to assess their effects on activities of delta-aminolevulinate synthase induced by prior treatment of cells with glutethimide and ferric nitrilotriacetate. These different metalloporphyrins were tested to form the experimental foundation for eventual studies in patients with acute porphyrias, in which uncontrolled induction of hepatic delta-amino-levulinate synthase, which plays a key role in pathogenesis of disease. Zinc and tin porphyrins reduced delta-aminolevulinate synthase activities, whereas copper-chelated porphyrins did not. When heme (iron protoporphyrin) was added with zinc or tin porphyrins, delta-aminolevulinate synthase activity was further reduced. Effects of the nonheme metalloporphyrins on delta-aminolevulinate synthase were closely correlated with their abilities to inhibit heme oxygenase (r = 0.78). The largest decrease of delta-aminolevulinate synthase (67%) was obtained with zinc mesoporphyrin and heme. Dose-response data indicated that only nanomolar concentrations of zinc mesoporphyrin and heme are required to obtain this effect. We found no effect of exposure to heme (10 mumol/L) or heme (200 nmol/L) plus zinc mesoporphyrin (50 nmol/L) on the half-life of activity of delta-aminolevulinate synthase (1.9 to 2.1 hr, regardless of treatment). This result suggests that the repressive effect of heme is directed toward decreasing synthesis, increasing breakdown or decreasing the translation of the messenger RNA of delta-aminolevulinate synthase.(ABSTRACT TRUNCATED AT 250 WORDS)

5-Aminolevulinate Synthetase↗
PubMed1991

Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli.

When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion. Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.

Base Sequence↗
PubMed1993

[N-acetylglutamate-5-phosphotransferase of the thermophilic bacterium Bacillus stearothermophilus: nucleotide sequence of the gene and enzyme characterization].

A nucleotide sequence of the argB gene of strain Bacillus stearothermophilus NCIB 8224 was determined. The argB gene codes for N-acetylglutamate-5-phosphotransferase of 258 amino acids with a molecular weight of 26918 D. This value is in good agreement with the SDS-PAG electrophoresis gata for identification of the heat stable B. stearothermophilus argB product synthesized in mesophilic Escherichia coli host cells. The substrates MgATP and N-acetyl-L-glutamate efficiently protect the enzyme against temperature denaturation. Amino acid sequences of bacterial (B. stearothermophilus and E. coli) and yeast (Saccharomyces cerevisiae and S. pombe) N-acetylglutamate-5-phosphotransferases share homologous conservative sites which can be responsible for MgATP binding and other structural and functional features of the enzymes of evolutionary distant microorganisms. Gel-filtration followed by K-phosphate buffer/N-acetyl-L-glutamate elution points out that the enzyme should have a molecular weight of 55,000 D. This predicts a dimeric form of the enzyme in physiological conditions. N-acetylglutamate-5-phosphotransferase activity is not inhibited by arginine-the end product of the biosynthetic pathway. The enzyme synthesis is repressed 4-fold in B. stearothermophilus by adding arginine to a growth medium. On the contrary, in E. coli hosts independent of their argR status, bacillary enzyme synthesis is not influenced by arginine. The plasmid-cloned B. stearothermophilus argB gene is well expressed in heterologous host cells (N-acetylglutamate-5-phosphotransferase activity was more than 150 and 600-fold higher in comparison with the plasmidless E. coli and B. stearothermophilus hosts, respectively). This is a result of efficient utilization of bacillary transcriptional and translational signals, convenient codon usage of the argB gene in E. coli and the absence of any repressive action of arginine and E. coli ArgR repressor on mRNA synthesis.

Amino Acid Sequence↗
PubMed1999

Identification of a cis-acting regulatory sequence responsible for the repression of brnQ in Salmonella typhimurium.

brnQ is the gene encoding the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium. The expression of the gene is transcriptionally repressed by an excess of glycyl-l-leucine added to the bacterial culture. To investigate the mechanism of regulation, we constructed brnQ-lacZ translational fusions with various deletions upstream from the promoter of brnQ, and examined the effects of the deletions on the regulation. We found a cis-acting region, 5'-GTGTTTTA-3', for the repression of brnQ expression, which was located 94 base pairs upstream from the transcription start site. Removal of the sequence resulted in derepression of brnQ. Two homologous sequences were found 45 base pairs downstream and 42 base pairs upstream from the sequence. We designated these sequences as O1, O2, and O3, in the order from the sequence proximal to the promoter to that distal to the promoter, respectively. The gleR1 mutation, which we reported previously to be a regulatory mutation enhancing transcription of brnQ, was a G-to-T transversion in the O1 sequence 50 base pairs upstream from the transcription start site. Insertion of five nucleotides between O1 and O2 resulted in derepression of brnQ. Further insertion of five nucleotides did not restore the original regulation of brnQ, indicating the importance of the proper spacing of these sequences. We also showed that the protein product of livS, the gene responsible for regulation of the LIV-I transport system, may bind to the O2 sequence. Furthermore, LivS was shown to be an allele of Lrp based on complementation experiments.

Artificial Gene Fusion↗
PubMed1988

Mutations in the leader sequence and initiation codon of the gene for ribosomal protein S20 (rpsT) affect both translational efficiency and autoregulation.

We have transferred the complete structural gene and part of the leader for ribosomal protein S20 of Escherichia coli to a controllable expression vector and have used oligonucleotide-directed mutagenesis to create mutations in the untranslated leader of the plasmid-borne gene. We have assayed for posttranscriptional regulation of the synthesis of S20 after inducing transcription of the mutant S20 mRNA from the expression vector. We found that two mutations lead to loss of feedback control of S20 synthesis: (i) a change of the initiation codon from UUG to AUG and (ii) a replacement of part of the S20 leader with a nonhomologous sequence including an AUG initiation codon. These mutations also lead to increases in both the intrinsic translational efficiency of the plasmid-encoded S20 mRNA in vitro and its half-life in vivo. A double mutation (GA to CT) at residues -3 and -4 relative to the initiation codon does not result in overproduction of S20. Rather, it reduces translational efficiency in vitro and mRNA stability in vivo. Our results demonstrate the fundamental importance of the UUG initiation codon in mediating autogenous repression of S20 synthesis.

Base Sequence↗
PubMed2006

Variable coordination of cotranscribed genes in Escherichia coli following antisense repression.

BACKGROUND: A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. RESULTS: To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA) targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A) and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. CONCLUSION: The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes. Consequently, no simple and specific methods for expression control of a single gene within polycistronic operons are available, and a thorough understanding of mRNA regulation and stability is required to understand the results from both knock-down and knock-out methods used in bacteria.

Codon, Initiator↗