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At least 271 records · Page 15Linked to original sources
PubMed2004

The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter.

The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation. In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R'). At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac). Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.

Base Sequence↗
PubMed2001

The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters.

In human cells, telomerase activity is regulated by transcriptional control of the telomerase reverse transcriptase gene (hTERT) whose product is the catalytic subunit of the enzyme. The hTERT promoter is active in virtually all types of tumors and immortal cells, but is silent in most adult somatic tissues. In this study, we placed the herpes simplex virus thymidine kinase gene under the control of the hTERT promoter with the aim of restricting its expression to tumor cells. In transfection experiments, the hTERT promoter driven thymidine kinase gene (hTERTp/TK) conferred ganciclovir sensitivity to all tumor and immortal cell lines tested, whereas normal somatic cells remained largely unaffected. Human hTERTp/TK-positive cancer cells implanted in nude mice developed into tumors that could be eradicated by ganciclovir treatment. The hTERTp/TK cassette was inserted into an adenovirus vector and its efficacy in reducing tumor growth was compared with that of an adenovirus carrying the thymidine kinase gene under the control of the cytomegalovirus immediate-early promoter (CMVp/TK). In a xenograft model using the human 143B osteosarcoma cell line, a single injection of either virus resulted in equivalent tumor regression and survival upon ganciclovir treatment. In animals injected intratumorally with the CMVp/TK adenovirus, expression of the thymidine kinase gene was detected in tumors, as well as in liver samples. Expression of the suicide gene in combination with ganciclovir resulted in severe liver histopathology and in an elevation of hepatic enzymes. In sharp contrast, when the hTERT promoter controlled the thymidine kinase gene, transgene expression was observed in tumors, but not in liver samples. Normal liver function in these animals was confirmed by serum levels of hepatic enzymes that were indistinguishable from those of control healthy mice. These results indicate that by restricting thymidine kinase expression to tumor cells, the hTERT promoter allows the tumoricidal effect of the suicidal gene to be exerted without detrimental consequences on healthy tissues and vital organs. The tight specificity of expression imparted by the hTERT promoter will assist the development of novel approaches to the treatment of a broad array of cancer types.

Adenoviridae↗
PubMed1997

Identification and functional analysis of multiple murine myeloperoxidase (MPO) promoters and comparison with the human MPO promoter region.

Myeloperoxidase (MPO) is an important component of the oxidative antibacterial defense system of granulocytes. Mammalian MPO gene expression has been most extensively studied in human and murine cells. Transcription of the human MPO gene appears to begin at a single initiation site and we have recently described the isolation and characterization of the corresponding human MPO promoter. On the other hand, MPO transcripts in murine myeloid cells show several distinct 5'-termini, suggesting the existence of multiple murine MPO promoters. However, significant levels of endogenous murine MPO promoter activity have not been demonstrated heretofore, although several murine MPO enhancers have been described. We now report the identification and preliminary functional characterization of four distinct murine MPO promoters. Sequence comparison of the human and murine MPO promoter regions reveals homologues of three out of four of these murine promoters within the human MPO gene. However, only one of these sites appears to be functionally active in human myeloid cells, possibly because of the interposition of Alu sequences between the putative promoter sites in the human gene.

Animals↗
PubMed2002

The Escherichia coli cAMP receptor protein bound at a single target can activate transcription initiation at divergent promoters: a systematic study that exploits new promoter probe plasmids.

We report the first detailed quantitative study of divergent promoters dependent on the Escherichia coli cAMP receptor protein (CRP), a factor known to activate transcription initiation at target promoters by making direct interactions with the RNA polymerase holoenzyme. In this work, we show that CRP bound at a single target site is able to activate transcription at two divergently organized promoters. Experiments using promoter probe plasmids, designed to study divergent promoters in vivo and in vitro, show that the divergent promoters function independently. Further in vitro experiments show that two holo RNA polymerase molecules cannot be accommodated simultaneously at the divergent promoters.

Base Sequence↗
PubMed2001

DNA supercoiling-dependent transcriptional coupling between the divergently transcribed promoters of the ilvYC operon of Escherichia coli is proportional to promoter strengths and transcript lengths.

The twin-domain model of Liu and Wang suggested that high levels of DNA supercoiling generated in the region between closely spaced divergently transcribed promoters could serve to couple the activities of these promoters transcriptionally. In this report, we use topoisomer sets of defined superhelical densities as DNA templates in a purified in vitro transcription system to demonstrate transcriptional coupling between the divergently transcribed ilvY and ilvC promoters of the ilvYC operon of Escherichia coli. Current evidence for this type of DNA supercoiling-dependent transcriptional coupling, based largely on the in vivo activities of promoters contained in engineered DNA constructs, suggests that the transcription complex must be physically hindered to generate DNA supercoils and to prevent their diffusion throughout the DNA duplex. However, the in vitro results presented here demonstrate that (i) transcriptional coupling is observed between the divergent promoters of the ilvYC operon in the absence of transcript anchoring; (ii) the magnitude of the negative DNA supercoiling generated in the divergent promoter region is proportional to the sum of the global and transcription-induced superhelicity; and (iii) the magnitude of this transcription-induced superhelicity is proportional to promoter strengths and transcript lengths.

Alcohol Oxidoreductases↗
PubMed2001

Hormonal regulation of multiple promoters of the rat mitochondrial glycerol-3-phosphate dehydrogenase gene: identification of a complex hormone-response element in the ubiquitous promoter B.

Rat mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is regulated by multiple promoters in a tissue-specific manner. Here, we demonstrate that thyroid hormone (3,5,3'-tri-iodo-L-thyronine) and steroid hormone but not the peroxisome proliferator clofibrate and retinoic acid stimulate the activation of the ubiquitous promoter B in a receptor-dependent manner, whereas the more tissue-restricted promoters A and C are not inducible by these hormones. Thyroid hormone action is mediated by a direct repeat +4 (DR+4) hormone-response element as identified by deletion and mutation analyses of promoter B in transient transfection analyses. The DR+4 element was able to bind to an in vitro translated thyroid hormone receptor in band-shift and supershift experiments. The hormone-response element comaps with a recognition site for the transcription factor Sp1, suggesting complex regulation of this sequence element. Mutation of this Sp1-recognition site reduces the basal promoter B activity dramatically in HepG2 and HEK293 cells in transient transfection and abolishes the binding of Sp1 in band-shift experiments. As demonstrated by Western-blot experiments, administration of tri-iodothyronine to euthyroid rats increases hepatic mGPDH protein concentrations in vivo. As it has recently been reported that human mGPDH promoter B is not regulated by tri-iodothyronine, this is the first example of a differentially tri-iodothyronine-regulated orthologous gene promoter in man and rat.

Animals↗
PubMed2002

Expression of foreign genes in Mycobacterium bovis BCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovis BCG strains.

SETTING: Optimization of BCG as a vehicle for live recombinant vaccines requires improved strategies for stable antigen expression. OBJECTIVES: To investigate the effects of various combinations of post-translational signals and promoters on expression and stability in different BCG strains. DESIGN: Plasmids were constructed using mycobacterial promoters (hsp60, 19-kDa antigen, 85A antigen--from the Mycobacterium tuberculosis complex--and the 18-kDa antigen from Mycobacterium leprae) and post-translation signals (85A antigen secretion and 19-kDa antigen acylation signals), coupled with reporter genes. RESULTS: The 19-kDa acylation signal had little effect on expression, while the 85A secretion signal enhanced markedly the levels of cell-associated product. Inclusion of the hsp60 promoter caused plasmid instability; various deletions affecting the promoter region occurred during or soon after transformation, but not during subsequent growth of the transformants, nor with other promoters. BCG Moreau appeared to be more susceptible to deletions than other BCG strains. CONCLUSIONS: The 85A signal may prove useful in optimizing gene expression in BCG, irrespective of secretion of the product. Deletions associated with the hsp60 promoter may be due to a transient lethal induction of the hsp60 promoter associated with electroporation. With intact plasmid there was no marked difference in expression between BCG strains.

Animals↗
PubMed1994

Ribosomal gene promoter domains can function as artificial enhancers of RNA polymerase I transcription, supporting a promoter origin for natural enhancers in Xenopus.

Enhancers of RNA polymerase I transcription in higher eukaryotes are repetitive elements within the intergenic spacers of rRNA genes. In Xenopus and mouse, enhancers and the gene promoter bind the activator protein, upstream binding factor, and in Xenopus, enhancers also share sequence similarity with an upstream domain of the promoter. This upstream promoter domain can act as an efficient enhancer when polymerized and cloned adjacent to a ribosomal gene promoter injected into oocytes. A core promoter domain lacking similarity with spacer sequences in Xenopus laevis but analogous to a repeated sequence in Xenopus borealis can also function as an enhancer. These data demonstrate functional relatedness between the promoter and enhancers, supporting the hypothesis that enhancers could have evolved from duplicated promoter domains that bind essential transcription factors. The ability of upstream binding factor to bind enhancers inactivated by mutation suggests that upstream binding factor binding alone cannot explain enhancer function.

Animals↗
PubMed1996

Krüppel-associated box-mediated repression of RNA polymerase II promoters is influenced by the arrangement of basal promoter elements.

The evolutionarily conserved Krüppel-associated box (KRAB) is present in the N-terminal regions of more than one-third of all Krüppel-class zinc finger proteins. Recent experiments have demonstrated that the KRAB-A domain tethered to a promoter DNA by connecting to heterologous DNA-binding protein domain or targeted to a promoter-proximal RNA sequence acts as a transcriptional silencing of RNA polymerase II promoters. Here we show that expression of KRAB domain suppresses in vivo the activating function of various defined activating transcription factors, and we demonstrate that the KRAB domain specifically silences the activity of promoters whose initiation is dependent on the presence of a TATA box. Promoters whose accurate transcription initiation is directed by a pyrimidine-rich initiator element, however, are relatively unaffected. We also report in vitro transcription experiments indicating that the KRAB domain is able to repress both activated and basal promoter activity. Thus, the KRAB domain appears to repress the activity of certain promoters through direct communication with TATA box-dependent basal transcription machinery.

Base Sequence↗
PubMed1997

Silencer elements controlling the B29 (Igbeta) promoter are neither promoter- nor cell-type-specific.

The murine B29 (Igbeta) promoter is B cell specific and contains essential SP1, ETS, OCT, and Ikaros motifs. Flanking 5' DNA sequences inhibit B29 promoter activity, suggesting this region contains silencer elements. Two adjacent 5' DNA segments repress transcription by the murine B29 promoter in a position- and orientation-independent manner, analogous to known silencers. Both these 5' segments also inhibit transcription by several heterologous promoters in B cells, including mb-1, c-fos, and human B29. These 5' segments also inhibit transcription by the c-fos promoter in T cells suggesting they are not B cell-specific elements. DNase I footprint analyses show an approximately 70-bp protected region overlapping the boundary between the two negative regulatory DNA segments and corresponding to binding sites for at least two different DNA-binding proteins. Within this footprint, two unrelated 30-bp cis-acting DNA motifs (designated TOAD and FROG) function as position- and orientation-independent silencers when located directly 5' of the murine B29 promoter. These two silencer motifs act cooperatively to restrict the transcriptional activity of the B29 promoter. Neither of these motifs resembles any known silencers. Mutagenesis of the TOAD and FROG motifs in their respective 5' DNA segments eliminates the silencing activity of these upstream regions, indicating these two motifs as the principal B29 silencer elements within these regions.

Animals↗
PubMed1998

Promoter architecture, cofactors, and orphan receptors contribute to cell-specific activation of the retinoic acid receptor beta2 promoter.

Expression of retinoic acid receptor beta (RARbeta) is spatially and temporally restricted during embryonal development. Also during retinoic acid (RA)-dependent embryonal carcinoma (EC) cell differentiation, RARbeta expression is initially up-regulated, while in later phases of differentiation expression is down-regulated, by an unknown mechanism. To gain insight into the regulation of RARbeta, we studied the activity of the RARbeta2 promoter and mutants thereof in various cell lines. While the RARbeta2 promoter is activated by RA in a limited number of cell lines, synthetic RA-responsive reporters are activated in most cell types. We show that the expression levels of proteins that bind to the beta-retinoic acid response element (RAR/retinoid X receptors and orphan receptors) and also the differential expression of a number of coactivators modulate the RA response on both natural and synthetic reporters. We further show that cell type-specific activation of the RARbeta2 promoter is dependent on the promoter architecture including the spacing between retinoic acid response element and TATA-box and initiator sequence (betaINR). Mutation within these regions caused a decrease in the activity of this promoter in responsive EC cells, while an increase in activity in non-EC cell lines was observed. Cell-specific complexes were formed on the betaINR, suggesting that the betaINR contributes to cell-specific activation of the promoter. On this basis we propose that promoter context-dependent and more general RA response-determining mechanisms contribute to cell-specific RA-dependent activation of transcription.

Animals↗
PubMed1998

Abortive initiation of transcription at a hybrid promoter. An analysis of the sliding clamp activator of bacteriophage T4 late transcription, and a comparison of the sigma70 and T4 gp55 promoter recognition proteins.

Bacteriophage T4 late promoters are transcribed by an RNA polymerase holoenzyme comprising the Escherichia coli core, E, the phage gene 55-encoded promoter recognition subunit, gp55, and the gene 33-encoded co-activator, gp33. Transcriptional initiation is activated by the T4 gene 45-encoded sliding clamp, which is loaded on to DNA at enhancer-like sites by its clamp-loader. Correct initiation of transcription at late promoters in basal mode requires only RNA polymerase core and gp55 (E.gp55). Dinucleotide-primed abortive initiation of basal and activated T4 late transcription has been compared. Only the trinucleotide non-productive transcript is made at a high rate; all other short transcripts are made at rates of less than one molecule per productive transcript. Gp45 increases abortive trinucleotide synthesis along with productive transcription, although the proportion of productive transcripts is also elevated. Nevertheless, this increase accounts for only a small part of the activation of T4 late transcription that is generated by its activator and co-activator. The pattern of production of short transcripts differs subtly between basal and enhanced transcription, indicating that linking the RNA polymerase with its sliding clamp activator only generates minor changes in the transition from abortive to productive RNA chain elongation. The T4 late promoter is converted to a strong sigma70 promoter by inserting an appropriate -35 promoter element. A direct comparison at such a hybrid promoter shows sigma70 and gp55 generating qualitatively and quantitative different patterns of abortive initiation at the same start site.

Bacteriophage T4↗
PubMed2001

The mouse fetoprotein transcription factor (FTF) gene promoter is regulated by three GATA elements with tandem E box and Nkx motifs, and FTF in turn activates the Hnf3beta, Hnf4alpha, and Hnf1alpha gene promoters.

Fetoprotein transcription factor (FTF) is an orphan nuclear receptor that activates the alpha(1)-fetoprotein gene during early liver developmental growth. Here we sought to define better the position of FTF in transcriptional cascades leading to hepatic differentiation. The mouse FTF gene was isolated and assigned to chromosome 1 band E4 (one mFTF pseudogene was also found). Exon/intron mapping shows an mFTF gene structure similar to that of its close homologue SF1, with two more N-terminal exons in the mFTF gene; exon mapping also delimits several FTF mRNA 5'- and 3'-splice variants. The mFTF transcription initiation site was located in adult liver at 238 nucleotides from the first translation initiator codon, with six canonical GATA, E box, and Nkx motifs clustered between -50/-140 base pairs (bp) from the cap site; DNA/protein binding assays also pinpointed an HNF4-binding element at +36 bp and an FTF-binding element at -257 bp. Transfection assays and point mutations showed that the mFTF promoter is activated by GATA, HNF4alpha, FTF, Nkx, and basic helix-loop-helix factors, with marked cooperativity between GATA and HNF4alpha. A tandem GATA/E box activatory motif in the proximal mFTF promoter is strikingly similar to a composite motif coactivated by differentiation inducers in the hematopoietic lineage; a tandem GATA-Nkx motif in the distal mFTF promoter is also similar to a composite motif transducing differentiation signals from transforming growth factor-beta-like receptors in the cardiogenic lineage. Three genes encoding transcription factors critical to early hepatic differentiation, Hnf3beta, Hnf4alpha, and Hnf1alpha, each contain dual FTF-binding elements in their proximal promoters, and all three promoters are activated by FTF in transfection assays. Direct DNA binding action and cooperativity was demonstrated between FTF and HNF3beta on the Hnf3beta promoter and between FTF and HNF4alpha on the Hnf1alpha promoter. These combined results suggest that FTF is an early intermediary between endodermal specification signals and downstream genes that establish and amplify the hepatic phenotype.

Alternative Splicing↗
PubMed2001

Requirement for yeast TAF145 function in transcriptional activation of the RPS5 promoter that depends on both core promoter structure and upstream activating sequences.

The general transcription factor TFIID has been shown to be involved in both core promoter recognition and the transcriptional activation of eukaryotic genes. We recently isolated TAF145 (one of TFIID subunits) temperature-sensitive mutants in yeast, in which transcription of the TUB2 gene is impaired at restrictive temperatures due to a defect in core promoter recognition. Here, we show in these mutants that the transcription of the RPS5 gene is impaired, mostly due to a defect in transcriptional activation rather than to a defect in core promoter recognition, although the latter is slightly affected as well. Surprisingly, the RPS5 core promoter can be activated by various activation domains fused to a GAL4 DNA binding domain, but not by the original upstream activating sequence (UAS) of the RPS5 gene. In addition, a heterologous CYC1 core promoter can be activated by RPS5-UAS at normal levels even in these mutants. These observations indicate that a distinct combination of core promoters and activators may exploit alternative activation pathways that vary in their requirement for TAF145 function. In addition, a particular function of TAF145 that is deleted in our mutants appears to be involved in both core promoter recognition and transcriptional activation.

Gene Expression Regulation, Fungal↗
PubMed2001

Sodium butyrate induces transcription from the G alpha(i2) gene promoter through multiple Sp1 sites in the promoter and by activating the MEK-ERK signal transduction pathway.

Sodium butyrate, an erythroid differentiation inducer and a histone deacetylase inhibitor, increases G alpha(i2) levels in differentiating K562 cells. Here we show that sodium butyrate induces G alpha(i2) gene transcription via sequences at -50/-36 and -92/-85 in the G alpha(i2) gene promoter. Both sequences contain core sequence motif for Sp1 binding; electrophoretic mobility shift as well as supershift assays confirmed binding to Sp1. Transcription from the G alpha(i2) gene promoter was also activated by two other histone deacetylase inhibitors, trichostatin A and Helminthsporium carbonium toxin (HC toxin), which also induce erythroblastic differentiation in K562 cells. However, hydroxyurea, a potent erythroid differentiation inducer in these cells, did not activate transcription from this gene promoter, indicating that promoter activation is inducer-specific. Mutations within the Sp1 sites at -50/-36 and -92/-85 in the G alpha(i2) gene promoter substantially decreased transcriptional activation by sodium butyrate, trichostatin A, or HC toxin. Transfection with constitutively activated ERKs indicated that this promoter can be activated through the MEK-ERK signal transduction pathway. Inhibition of the MEK-ERK pathway with U0126 or reduction in the expression of endogenous ERK with an antisense oligonucleotide to ERK significantly inhibited sodium butyrate- and HC toxin-induced transcription but had no effect on trichostatin A-induced transcription. Inhibition of the JNK and p38 MAPKs, using selective inhibitors, had no effect on sodium butyrate-induced transcription. In cells in which sodium butyrate induction of promoter activation had been inhibited by various concentrations of U0126, constitutively activated ERK2 reversed this inhibition. These results show that the MEK-ERK signal transduction pathway is important in butyrate signaling, which eventually converges in the cell nucleus.

Butyrates↗
PubMed2004

Opposite Smad and chicken ovalbumin upstream promoter transcription factor inputs in the regulation of the collagen VII gene promoter by transforming growth factor-beta.

A critical component of the epidermal basement membrane, collagen type VII, is produced by keratinocytes and fibroblasts, and its production is stimulated by the cytokine transforming growth factor-beta (TGF-beta). The gene, COL7A1, is activated by TGF-beta via Smad transcription factors in cooperation with AP1. Here we report a previously unsuspected level of complexity in this regulatory process. We provide evidence that TGF-beta may activate the COL7A1 promoter by two distinct inputs operating through a common region of the promoter. One input is provided by TGF-beta-induced Smad complexes via two Smad binding elements that function redundantly depending on the cell type. The second input is provided by relieving the COL7A1 promoter from chicken ovalbumin upstream promoter transcription factor (COUP-TF)-mediated transcriptional repression. We identified COUP-TFI and -TFII as factors that bind to the TGF-beta-responsive region of the COL7A1 promoter in an expression library screening. COUP-TFs bind to a site between the two Smad binding elements independently of Smad or AP1 and repress the basal and TGF-beta-stimulated activities of this promoter. We provide evidence that endogenous COUP-TF activity represses the COL7A1 promoter. Furthermore, we show that TGF-beta addition causes a rapid and profound down-regulation of COUP-TF expression in keratinocytes and fibroblasts. The results suggest that TGF-beta signaling may exert tight control over COL7A1 by offsetting the balance between opposing Smad and COUP-TFs.

3T3 Cells↗
PubMed2006

Phosphorylation of the alpha 2(1) procollagen promoter binding proteins is required for promoter activity.

We have previously identified transcription factors binding to the proximal promoter region of the human COL1A2 gene that are essential for promoter activity. Two regions in the proximal promoter, the CCAAT-element and the CME bind the CCAAT-binding factor and an as yet uncharacterized DNA-binding protein, respectively. In this study we show that phosphorylation of both the CCAAT-binding and CME-binding proteins are essential for DNA-binding activity. While the binding of the CCAAT-binding proteins to the COL1A2 proximal promoter was inhibited after treatment of nuclear proteins with calf intestinal phosphatase, PP2A treatment had a significant inhibitory effect on the binding of the CME-binding proteins. The treatment of type I collagen producing fibroblasts, CT-1, with the kinase inhibitor, staurosporine, interfered with DNA-protein interactions on both elements on the COL1A2 proximal promoter. This inhibition was associated with a significant decrease in both endogenous COL1A2 gene expression as observed by northern blot analysis and proximal promoter activity as assayed in transient transfection assays. Our results suggest that the phosphorylation of transcription factors interacting with the COL1A2 proximal promoter is essential for both DNA binding and for the expression of this gene.

CCAAT-Enhancer-Binding Proteins↗
PubMed2004

Health-promotion behaviors that promote self-healing.

A large body of evidence has shown that health-promotion programs in smoking cessation, stress management, fitness, nutrition, weight control, and medical self care have been successful in helping people improve their health practices and related health conditions. However, the impact of these programs on promoting self-healing among people with acute and chronic diseases is mixed. The purpose of this paper is to identify research opportunities important to fostering a better understanding of health promotion behaviors that promote self healing. To provide context, the health-promotion concept is discussed, as is the literature on workplace health-promotion programs provided to overtly healthy people. Next, examples of the literature on health-promotion programs for people with chronic illness are provided. Finally, a research agenda and opportunities for research are presented.

Attitude to Health↗