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PubMed1991

Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor.

Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or beta-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes.

Actins↗

PubMed1996

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

Animals↗

PubMed1997

Expansion of a CUG trinucleotide repeat in the 3' untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts.

Expansion of a CTG trinucleotide repeat in the 3' untranslated region (UTR) of DMPK, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.

Cell Line↗

PubMed1998

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 alphaP3A2.TKO expression cassette causes exactly the same ectopic oral expression of a coinjected wild-type CyIIIa construct. Furthermore, the alphaP3A2.TKO cassette derepresses the endogenous CyIIIa gene in the oral ectoderm and in the endoderm. alphaP3A2.TKO thus abrogates the function of the endogenous SpP3A2 transcription factor with respect to spatial repression of the CyIIIa gene. Widespread expression of alphaP3A2.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.

Actins↗

PubMed1998

Enhancement of serum-response factor-dependent transcription and DNA binding by the architectural transcription factor HMG-I(Y).

The mechanisms by which HMG-I proteins regulate cell growth are unknown, and their effects on gene expression have only been partially elucidated. We explored the potential interaction between HMG-I proteins and serum-response factor (SRF), a member of the MADS-box family of transcription factors. In cotransfection experiments, HMG-I(Y) potentiated SRF-dependent activation (by more than 5-fold) of two distinct SRF-responsive promoters, c-fos and the smooth muscle-specific gene SM22alpha. This effect was also observed with a heterologous promoter containing multiple copies of the CC(A/T)6GG (CArG) box. HMG-I proteins bound specifically to the CArG boxes of c-fos and SM22alpha in gel mobility shift analysis and enhanced binding of SRF to these CArG boxes. By chelating peptide-immobilized metal affinity chromatography, we mapped the domain of HMG-I(Y) that interacts with SRF to amino acids 50-81, a region that does not bind specifically to DNA in electrophoretic mobility shift assays even though it includes the third AT-hook DNA-binding domain. Surprisingly, HMG-I(Y) mutants that failed to bind DNA still enhanced SRF binding to DNA and SRF-dependent transcription. In contrast, deletion of the HMG-I(Y) 50-81 domain that bound SRF prevented enhancement of transcription. To our knowledge, this is the first report of an HMG-I protein interacting with a MADS-box transcription factor. Our observations suggest that members of the HMG-I family play an important role in SRF-dependent transcription and that their effect is mediated primarily by a protein-protein interaction.

Animals↗

PubMed1998

A transcriptional coactivator, steroid receptor coactivator-3, selectively augments steroid receptor transcriptional activity.

Estrogen receptors ERalpha and ERbeta are members of the family of nuclear hormone receptors and act as ligand-inducible transcriptional factors, which regulate the expression of target genes on binding to cognate response elements. We report here the characterization of steroid receptor coactivator-3 (SRC-3), a coactivator of nuclear receptor transcription that is a member of a family of steroid receptor coactivators that includes SRC-1 and transcription intermediate factor-2. SRC-3 enhanced ERalpha and progesterone receptor-stimulated gene transcription in a ligand-dependent manner, but stimulation of ERbeta-mediated transcription was not observed. Protein-protein interaction assays, including real-time interaction analyses with BIAcore, demonstrated that the affinity of the ERalpha interaction with SRC-3 was much higher than that observed for the ERbeta interaction with SRC-3. Mutational analysis suggests a potential interplay between the transactivation function-1 and -2 domains of ERalpha and SRC-3. Furthermore, an intrinsic transactivation function was observed in the C-terminal half of SRC-3. Finally, SRC-3 was differentially expressed in various tissues and, among several tumor cells examined, was most abundant in the nuclear fraction of MCF-7 breast cancer cells. Therefore, SRC-3, a third member of a family of steroid receptor coactivators, has a distinct tissue distribution and intriguing selectivity between ERalpha and ERbeta.

Acetyltransferases↗

PubMed2004

Analysis of mRNA transcripts from the NAD(P)H oxidase 1 (Nox1) gene. Evidence against production of the NADPH oxidase homolog-1 short (NOH-1S) transcript variant.

Recent reports indicate that NAD(P)H oxidase 1 (Nox1) mRNA undergoes alternative splicing, producing a short transcript (NOH-1S) encoding a novel H+ channel. Although the H+ transport properties of NOH-1S-transfected cells resemble those of many cells, the production of a NOH-1S protein was never documented. We characterized Nox1 transcripts in colon-derived cells and present evidence that mRNA splicing does not produce NOH-1S; rather, NOH-1S appears to be an artifact of template switching during cDNA synthesis. The NOH-1S transcript was not observed by Northern blotting, despite claims of its abundance based on RNase protection assays. The shortened cDNA was generated by avian myeloblastosis virus reverse transcriptase, but not by thermally stable reverse transcriptase under conditions that produce full-length Nox1. Analysis of shortened cDNAs detected NOH-1S sequence and other variants that differ at the alleged splice junction site. Although no appropriate RNA splicing sites were found within Nox1 to account for NOH-1S formation, we found repetitive sequence elements bordering the deleted region, which could promote intramolecular template switching during cDNA synthesis. Template switching was confirmed in vitro, where the deleted cDNA was generated by avian myeloblastosis virus reverse transcriptase from a synthetic, full-length Nox1 RNA template. A survey of the expressed sequence tags database suggests that similar switching phenomena occur between repetitive elements in other Nox family transcripts, indicating such cloning artifacts are common. In contrast, genuine RNA splicing does account for another Nox1 transcript lacking the entire exon 11, which is abundant in colon cells but encodes a protein incapable of supporting superoxide production.

Alternative Splicing↗

PubMed2005

The CGL2612 protein from Corynebacterium glutamicum is a drug resistance-related transcriptional repressor: structural and functional analysis of a newly identified transcription factor from genomic DNA analysis.

The emergence of antibiotic-resistant bacteria often causes serious clinical problems. The TetR family is one of the major transcription factor families that regulate expression of genes involved in bacterial antimicrobial resistance systems. CGL2612 protein is a transcription factor newly identified by genomic DNA analysis on Corynebacterium glutamicum, which belongs to the mycolic acid-containing Actinomycetales, including the well known pathogens Corynebacterium diphtheriae and Mycobacterium tuberculosis. Crystal structure analysis showed that the CGL2612 protein exhibits significant structural similarity to the multidrug resistance (MDR)-related transcription factor QacR from Staphylococcus aureus, despite poor amino acid sequence similarity between these proteins. Binding DNA sequence analysis of CGL2612 protein using the systematic evolution of ligands by the exponential enrichment (systematic evolution of ligands by exponential enrichment, or SELEX) method revealed that this protein is a new member of the TetR family, which regulates expression of the immediately upstream gene, cgl2611, probably encoding a major facilitator superfamily permease. Subsequent functional analyses confirmed a function of the CGL2612 as a transcriptional repressor responsible for the antimicrobial resistance system in C. glutamicum. The strategy used in the present study is one of the most convenient and powerful methods to analyze functionally unknown transcription factors, and the results obtained here will contribute to our understanding of the drug resistance mechanism not only in C. glutamicum but also in the related bacteria, C. diphtheriae and M. tuberculosis.

Amino Acid Sequence↗

PubMed2006

The anti-rheumatic gold salt aurothiomalate suppresses interleukin-1beta-induced hyaluronan accumulation by blocking HAS1 transcription and by acting as a COX-2 transcriptional repressor.

Gold compounds are among the oldest disease-modifying drugs and are still widely used today for treating rheumatoid arthritis. Despite decades of use, little is known about the mode of action of this class of drugs. Here we have demonstrated that aurothiomalate (AuTM) suppresses hyaluronan accumulation by blocking interleukin (IL)-1beta-induced hyaluronan synthase-1 transcription. We have further demonstrated that, in fibroblast-like synoviocytes (FLSs), AuTM acts as a specific COX-2 transcriptional repressor in that IL-1beta-induced COX-2 transcription is blocked, whereas COX-1 transcription and translation is unaffected. As a consequence, PGE2 levels released by FLS are dose-dependently reduced in cells exposed to AuTM. Of similar importance is the demonstration that AuTM does block NFkappaB-DNA interaction. In addition, two other transcription factors implicated in inflammatory events, namely AP-1 and STAT3, are blocked as well. The effect on NFkappaB likely explains the inhibition of COX-2 as well as that of HAS1, as both are genes that depend on the activation of NFkappaB. Interestingly, AuTM does not interfere with IL-1beta-induced IkappaB alpha degradation, in most cases a prerequisite for subsequent NFkappaB activation. Furthermore, evidence is presented that, in FLS, AuTM blocks NFkappaB-DNA interaction neither by binding to NFkappaB binding sites nor by interacting with activated NFkappaB proteins. Taken together, AuTM treatment of FLS blocks two of the most important proinflammatory events that are associated with rheumatoid arthritis. AuTM blocks the release of PGE2 and prevents the activation of NFkappaB, therefore blocking IL-1beta-induced hyaluronan accumulation and likely a series of other pro-inflammatory NFkappaB-dependent genes.

Antirheumatic Agents↗

PubMed1995

Regulation of the balance of cytokine production and the signal transducer and activator of transcription (STAT) transcription factor activity by cytokines and inflammatory synovial fluids.

The balance between type 1 and 2 T helper cell cytokine production plays an important role in several animal models of autoimmunity, and skewed patterns of cytokine expression have been described in human inflammatory diseases. Many cytokines activate signal transducer and activation of transcription (STAT) transcription factors, which, in turn, activate transcription of inflammatory effector genes. We used mononuclear cell priming cultures and inflammatory synovial fluids (SFs) derived from arthritis patients to examine the regulation of cytokine production and STAT activity by an inflammatory synovial microenvironment. Exposure to SFs during priming resulted in an 81% inhibition of interferon (IFN)-gamma, but not interleukin (IL) 4, production by effector cells generated in priming cultures. SF suppression was mediated by IL-4 and IL-10 and inhibition of IL-12 expression, and it was reversed in a dominant fashion by exogenous IL-12. SFs blocked the sustained activity of transcription factor Stat1, but not Stat3, during the priming period, and Stat1 activity was differentially regulated by cytokines in parallel with their positive or negative regulation of IFN-gamma production. Active Stat3, but not Stat1, was detected in cells from inflamed joints. These results suggest a role for altered balance of Stat1 and Stat3 transcriptional activity in the regulation of T cell differentiation and in the pathogenesis of inflammatory synovitis.

Arthritis, Rheumatoid↗

PubMed2002

Downstream E-box-mediated regulation of the human telomerase reverse transcriptase (hTERT) gene transcription: evidence for an endogenous mechanism of transcriptional repression.

Regulation of the hTERT gene encoding the telomerase catalytic subunit plays an important role in human cell senescence, immortalization, and carcinogenesis. By examining the activity of various deleted or mutated hTERT promoter fragments, we show that an E-box element downstream of the transcription initiation site is critical to differential hTERT transcription between the telomerase/hTERT-positive renal cell carcinoma cell line (RCC23) and its telomerase/hTERT-negative counterpart containing a transferred, normal chromosome 3 (RCC23+3). This E-box element mediated repression of hTERT transcription in RCC23+3 but not in RCC23. A copy number-dependent enhancement of the repression suggested active repression, rather than loss of activation, in RCC23+3. Endogenous expression levels of c-Myc or Mad1, which could activate or repress hTERT transcription when overexpressed, did not account for the differential hTERT transcription. Gel mobility shift assays identified the upstream stimulatory factors (USFs) as a major E-box-binding protein complex in both RCC23 and RCC23+3 and, importantly, detected an RCC23+3-specific, E-box-binding factor that was distinct from the USF and Myc/Mad families. The E-box-mediated repression was also active in normal human fibroblasts and epithelial cells and inactive in some, but not all, telomerase/hTERT-positive cancer cells. These findings provide evidence for an endogenous, repressive mechanism that actively functions in telomerase/hTERT-negative normal cells and becomes defective during carcinogenic processes, e.g., by an inactivation of the telomerase repressor gene on chromosome 3.

Cell Cycle Proteins↗

PubMed2001

The defect in transcription-coupled repair displayed by a Saccharomyces cerevisiae rad26 mutant is dependent on carbon source and is not associated with a lack of transcription.

Nucleotide excision repair (NER) is an evolutionarily conserved pathway that removes DNA damage induced by ultraviolet irradiation and various chemical agents that cause bulky adducts. Two subpathways within NER remove damage from the genome overall or the transcribed strands of transcribing genes (TCR). TCR is a faster repair process than overall genomic repair and has been thought to require the RAD26 gene in Saccharomyces cerevisiae. Rad26 is a member of the SWI/SNF family of proteins that either disrupt chromatin or facilitate interactions between the RNA Pol II and transcription activators. SWI/SNF proteins are required for the expression or repression of a diverse set of genes, many of which are differentially transcribed in response to particular carbon sources. The remodeling of chromatin by Rad26 could affect transcription and/or TCR following formation of DNA damage and other stress-inducing conditions. We speculate that another factor(s) can substitute for Rad26 under particular growth conditions. We therefore measured the level of repair and transcription in two different carbon sources and found that the defect in the rad26 mutant for TCR was dependent on the type of carbon source. Furthermore, TCR did not correlate with transcription rate, suggesting that disruption of RAD26 leads to a specific defect in DNA repair and not transcription.

Carbon↗

PubMed2006

Transcriptional regulation of the human GD3 synthase gene expression in Fas-induced Jurkat T cells: a critical role of transcription factor NF-kappaB in regulated expression.

The transcriptional regulation mechanisms involved in the up-regulation of Fas-induced GD3 synthase gene have not yet been elucidated. 5'-Rapid amplification of cDNA end (5'-RACE) using mRNA prepared from Fas-induced Jurkat T cells revealed the presence of multiple transcription start sites of human GD3 synthase gene, and the 5'-end analysis of the longest of its product showed that transcription started from 650 nucleotides upstream of the translational initiation site. Promoter analyses of the 5'-flanking region of the human GD3 synthase gene using luciferase gene reporter system showed strong promoter activity in Fas-induced Jurkat T cells. Deletion study revealed that the region from -1146 to -646 (A of the translational start ATG as position +1) was indispensable for the Fas response. This region lacks apparent TATA and CAAT boxes but contains putative binding sites for transcription factors c-Ets-1, cAMP-responsive element-binding (CREB) protein, activating protein 1 (AP-1), and NF-kappaB. Base-substitution experiment showed that only the NF-kappaB-binding site of putative binding sites is required for the maximal expression induced by Fas. Both DNase I footprint and electrophoretic mobility shift assays with the nuclear extract of Fas-induced Jurkat T cells revealed that NF-kappaB was bound specifically to the probe being mediated by its binding site in the promoter sequence. Taken together, these results indicate that NF-kappaB plays an essential role in the transcriptional activity of human GD3 synthase gene in Fas-induced Jurkat T cells. In addition, the translocation of NF-kappaB-binding protein to nucleus by Fas activation is also crucial for the increased expression of the GD3 synthase gene in Fas-activated Jurkat T cells.

Base Sequence↗

PubMed1984

A potential stem-loop structure and the sequence CAAUCAA in the transcript are insufficient to signal rho-dependent transcription termination at lambda tR1.

It has been suggested that a sequence in the RNA transcript that can form a stem and loop structure, followed by the sequence CAAUCAA, is the signal for rho-dependent transcription termination. We tested this hypothesis by synthesizing a DNA duplex whose sequence corresponds to a region of the lambda tR1 terminator that contains these structural features. We cloned this synthetic DNA fragment under the control of the lacUV5 promoter, and showed that it does not cause rho-dependent termination in vitro. RNA polymerase pauses during in vitro transcription across the synthetic sequence, although less efficiently than at the corresponding sequence on the lambda template. No rho-mediated termination was detected even under conditions that prolonged transcriptional pausing at the synthetic site, indicating that the synthetic sequence is defective as a transcript release site. We suggest that unlike rho-independent terminators, rho-dependent terminators require sequences in addition to those immediately before the sites of termination.

Base Sequence↗

PubMed1984

Use of complementary DNA oligomers to probe trp leader transcript secondary structures involved in transcription pausing and termination.

DNA oligomers were synthesized that are perfectly complementary to different segments of the tryptophan (trp) operon leader transcript. These 15 nucleotide long oligomers were used as probes of the involvement of transcript secondary structures in two processes: transcription pausing at the pause site located near base pair 90 in the leader region, and transcription termination at the attenuator. The 15-mers were complementary to the four segments of the trp leader transcript which have been shown to form the alternative secondary structures that are believed to be responsible for pausing, termination, and antitermination. Oligomers complementary to RNA segments 1 and 3 relieved termination while the 15-mer complementary to RNA segment 1 relieved pausing. 15-mers complementary to segment 2 had no effect on pausing and the oligomer complementary to segment 4 had virtually no effect on termination.

Base Sequence↗

PubMed1985

Eukaryotic ternary transcription complexes: transcription complexes of RNA polymerase II are associated with histone-containing, nucleosome-like particles in vivo.

Using a psoralen crosslinking, radioactive labelling technique, we have previously been able to study ternary transcription complexes containing DNA-dependent RNA polymerases I and II which are released from rat liver nuclei by endogenous nuclease digestion [Sargan and Butterworth, refs 1 and 2]. Although the DNA component of these complexes was found to have a 'nucleosome-like' size profile and although the experimental conditions for autodigestion were designed to minimise histone rearrangement, it is necessary to provide further evidence that the periodicity of nuclease cutting around these transcription complexes is conferred by histones. Studies using secondary nuclease digestion of the released transcription complexes now show a digestion barrier characteristic of that conferred by nucleosomal histones which is lost if histones are removed from the complexes. Furthermore, antibodies raised against histones are effective in precipitating transcription complexes of RNA polymerase II and, to a lesser extent, of RNA polymerase I. The data suggest that, in rat hepatic tissue, transcription complexes are in very close proximity (within a few hundred base pairs) of histone-containing, nucleosome-like particles in vivo.

Animals↗

PubMed1985

A transcription map of a yeast centromere plasmid: unexpected transcripts and altered gene expression.

YCp19 is a yeast centromere plasmid capable of autonomous replication in both yeast and E. coli (J. Mol. Biol., 158: 157-179, 1982). It is stably maintained as a single copy in the yeast cell and is therefore a model yeast "minichromosome" and cloning vector. We have located the positions and measured the abundance of the in vivo yeast transcripts from YCp19. Transcripts from the selectable marker genes TRP1 and URA3 were present at increased levels relative to chromosomal copies of the genes. Unanticipated transcripts from the yeast CEN4 and E. coli pBR322 sequences were also found. Although much of the plasmid vector is actively transcribed in vivo, the regions around the most useful cloning sites (BamHI, EcoRI, SalI) are free of transcripts. We have analyzed transcription of BamHI inserts containing promoter variants of the HIS3 gene and determined that although initiation events are accurate, plasmid context may alter levels of gene expression.

Centromere↗

PubMed1985

Transcription of a Drosophila tRNAArg gene in yeast extract: 5'-flanking sequence dependence for transcription in a heterologous system.

The Drosophila tRNA gene encoded on pArg is efficiently transcribed in extracts of Saccharomyces cerevisiae, but the efficiency is 5'-flanking sequence dependent: deletion to between positions -21 and -17 (relative to position +1 of the mature coding sequence) reduces transcription to a very low level. This demonstrates that requirement for wild-type 5'-flanking sequence exists in the case of a heterologous combination of a tRNA gene and transcription extract. Expression of pArg in vivo in S. cerevisiae is also dependent on the wild-type 5'-flanking sequence, but only with deletion to between -17 and -11 is the steady-state level of pArg transcripts reduced to near zero. The 5'-flanking sequence requirement in S. cerevisiae extract is similar to that found in Drosophila Kc cell extract. However, transcription kinetics distinguish S. cerevisiae extract from that of Drosophila Kc cells. tRNA genes added to S. cerevisiae extract exhibit a lag phase before initiation of active transcription, but this lag is much shorter and much less temperature dependent than is the lag phase in Drosophila Kc cell extract.

Animals↗