A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor.

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

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Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2832-2839.1990 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2832-2839

Author(s):

A S Ponticelli ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Initiation Site ◽

Activator Proteins ◽

Nuclear Extracts ◽

Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

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Analysis of Saccharomyces cerevisiae his3 transcription in vitro: biochemical support for multiple mechanisms of transcription.

Molecular and Cellular Biology ◽

10.1128/mcb.10.6.2832 ◽

1990 ◽

Vol 10 (6) ◽

pp. 2832-2839 ◽

Cited By ~ 45

Author(s):

A S Ponticelli ◽

K Struhl

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Initiation Site ◽

Activator Proteins ◽

Nuclear Extracts ◽

Transcription In Vitro

The promoter region of the Saccharomyces cerevisiae his3 gene contains two TATA elements, TC and TR, that direct transcription initiation to two sites designated +1 and +13. On the basis of differences between their nucleotide sequences and their responsiveness to upstream promoter elements, it has previously been proposed that TC and TR promote transcription by different molecular mechanisms. To begin a study of his3 transcription in vitro, we used S. cerevisiae nuclear extracts together with various DNA templates and transcriptional activator proteins that have been characterized in vivo. We demonstrated accurate transcription initiation in vitro at the sites used in vivo, transcriptional activation by GCN4, and activation by a GAL4 derivative on various gal-his3 hybrid promoters. In all cases, transcription stimulation was dependent on the presence of an acidic activation region in the activator protein. In addition, analysis of promoters containing a variety of TR derivatives indicated that the level of transcription in vitro was directly related to the level achieved in vivo. The results demonstrated that the in vitro system accurately reproduced all known aspects of in vivo his3 transcription that depend on the TR element. However, in striking contrast to his3 transcription in vivo, transcription in vitro yielded approximately 20 times more of the +13 transcript than the +1 transcript. This result was not due to inability of the +1 initiation site to be efficiently utilized in vitro, but rather it reflects the lack of TC function in vitro. The results support the idea that TC and TR mediate transcription from the wild-type promoter by distinct mechanisms.

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Multiple Roles of the τ131 Subunit of Yeast Transcription Factor IIIC (TFIIIC) in TFIIIB Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.298-308.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 298-308 ◽

Cited By ~ 23

Author(s):

Hélène Dumay-Odelot ◽

Joël Acker ◽

Rosalia Arrebola ◽

André Sentenac ◽

Christian Marck

Keyword(s):

Transcription Factor ◽

Transcription Initiation ◽

In Vitro Transcription ◽

Initiation Factor ◽

Physical Interaction ◽

Restrictive Temperature ◽

Class Iii ◽

High Concentrations

ABSTRACT Yeast transcription factor IIIC (TFIIIC) plays a key role in assembling the transcription initiation factor TFIIIB on class III genes after TFIIIC-DNA binding. The second largest subunit of TFIIIC, τ131, is thought to initiate TFIIIB assembly by interacting with Brf1/TFIIIB70. In this work, we have analyzed a TFIIIC mutant (τ131-ΔTPR2) harboring a deletion in τ131 removing the second of its 11 tetratricopeptide repeats. Remarkably, this thermosensitive mutation was selectively suppressed in vivo by overexpression of B”/TFIIIB90, but not Brf1 or TATA-binding protein. In vitro, the mutant factor preincubated at restrictive temperature bound DNA efficiently but lost transcription factor activity. The in vitro transcription defect was abolished at high concentrations of B” but not Brf1. Copurification experiments of baculovirus-expressed proteins confirmed a direct physical interaction between τ131 and B”. τ131, therefore, appears to be involved in the recruitment of both Brf1 and B”.

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Regulation of endothelial thrombomodulin expression by inflammatory cytokines is mediated by activation of nuclear factor-kappa B

Blood ◽

10.1182/blood-2004-03-0928 ◽

2005 ◽

Vol 105 (10) ◽

pp. 3910-3917 ◽

Cited By ~ 79

Author(s):

Richard H. Sohn ◽

Clayton B. Deming ◽

David C. Johns ◽

Hunter C. Champion ◽

Ce Bian ◽

...

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Inflammatory Cytokines ◽

Nuclear Factor Kappa B ◽

Nuclear Factor ◽

Mobility Shift ◽

Factor Binding ◽

Nuclear Factor Kappa ◽

Tnf Α

AbstractInflammation and thrombosis are increasingly recognized as interrelated biologic processes. Endothelial cell expression of thrombomodulin (TM), a key component of the anticoagulant protein C pathway, is potently inhibited by inflammatory cytokines. Because the mechanism underlying this effect is largely unknown, we investigated a potential role for the inflammatory transcription factor nuclear factor-kappa B (NF-κB). Blocking NF-κB activation effectively prevented cytokine-induced down-regulation of TM, both in vitro and in a mouse model of tumor necrosis factor-α (TNF-α)–mediated lung injury. Although the TM promoter lacks a classic NF-κB consensus site, it does contain tandem Ets transcription factor binding sites previously shown to be important for both constitutive TM gene expression and cytokine-induced repression. Using electrophoretic mobility shift assay and chromatin immunoprecipitation, we found that multiple Ets species bind to the TNF-α response element within the TM promoter. Although cytokine exposure did not alter Ets factor binding, it did reduce binding of p300, a coactivator required by Ets for full transcriptional activity. Overexpression of p300 also prevented TM repression by cytokines. We conclude that NF-κB is a critical mediator of TM repression by cytokines. Further evidence suggests a mechanism involving competition by NF-κB for limited pools of the transcriptional coactivator p300 necessary for TM gene expression.

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Methanobacterium thermoautotrophicum RNA Polymerase and Transcription In Vitro

Journal of Bacteriology ◽

10.1128/jb.181.14.4424-4429.1999 ◽

1999 ◽

Vol 181 (14) ◽

pp. 4424-4429 ◽

Cited By ~ 42

Author(s):

Trevor J. Darcy ◽

Winfried Hausner ◽

Donald E. Awery ◽

Aled M. Edwards ◽

Michael Thomm ◽

...

Keyword(s):

Transcription Factor ◽

Complex Formation ◽

Rna Polymerase ◽

Transcription Initiation ◽

Methanobacterium Thermoautotrophicum ◽

Tata Binding Protein ◽

Gene Promoters ◽

Transcription In Vitro ◽

Archaeal Histone

ABSTRACT RNA polymerase (RNAP) purified from Methanobacterium thermoautotrophicum ΔH has been shown to initiate transcription accurately in vitro from the hmtB archaeal histone promoter with either native or recombinant forms of the M. thermoautotrophicum TATA-binding protein and transcription factor TFB. Efforts to obtain transcription initiation from hydrogen-regulated methane gene promoters were, however, unsuccessful. Two previously unrecognized archaeal RNAP subunits have been identified, and complex formation by the M. thermoautotrophicum RNAP and TFB has been demonstrated.

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Transcription factor/DNA interactions visualized by electron spectroscopic imaging

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122654 ◽

1992 ◽

Vol 50 (1) ◽

pp. 450-451

Author(s):

David P. Bazett-Jones ◽

Mark L. Brown

Keyword(s):

Transcription Factor ◽

Dna Sequences ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Phosphorus Content ◽

Spectroscopic Imaging ◽

Electron Spectroscopic Imaging ◽

Dna Backbone ◽

Two Parameters ◽

High Level

A multisubunit RNA polymerase enzyme is ultimately responsible for transcription initiation and elongation of RNA, but recognition of the proper start site by the enzyme is regulated by general, temporal and gene-specific trans-factors interacting at promoter and enhancer DNA sequences. To understand the molecular mechanisms which precisely regulate the transcription initiation event, it is crucial to elucidate the structure of the transcription factor/DNA complexes involved. Electron spectroscopic imaging (ESI) provides the opportunity to visualize individual DNA molecules. Enhancement of DNA contrast with ESI is accomplished by imaging with electrons that have interacted with inner shell electrons of phosphorus in the DNA backbone. Phosphorus detection at this intermediately high level of resolution (≈lnm) permits selective imaging of the DNA, to determine whether the protein factors compact, bend or wrap the DNA. Simultaneously, mass analysis and phosphorus content can be measured quantitatively, using adjacent DNA or tobacco mosaic virus (TMV) as mass and phosphorus standards. These two parameters provide stoichiometric information relating the ratios of protein:DNA content.

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High resolution mapping of nucleic acid containing complexes in vitro and in situ

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162703 ◽

1996 ◽

Vol 54 ◽

pp. 48-49

Author(s):

D. P. Bazett-Jones ◽

M. J. Hendzel

Keyword(s):

Nucleic Acid ◽

High Resolution ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Heavy Atom ◽

Regulation Of Transcription ◽

High Exposure ◽

Resolution Mapping ◽

Tata Sequence

Structural analysis of combinations of nucleosomes and transcription factors on promoter and enhancer elements is necessary in order to understand the molecular mechanisms responsible for the regulation of transcription initiation. Such complexes are often not amenable to study by high resolution crystallographic techniques. We have been applying electron spectroscopic imaging (ESI) to specific problems in molecular biology related to transcription regulation. There are several advantages that this technique offers in studies of nucleoprotein complexes. First, an intermediate level of spatial resolution can be achieved because heavy atom contrast agents are not necessary. Second, mass and stoichiometric relationships of protein and nucleic acid can be estimated by phosphorus detection, an element in much higher proportions in nucleic acid than protein. Third, wrapping or bending of the DNA by the protein constituents can be observed by phosphorus mapping of the complexes. Even when ESI is used with high exposure of electrons to the specimen, important macromolecular information may be provided. For example, an image of the TATA binding protein (TBP) bound to DNA is shown in the Figure (top panel). It can be seen that the protein distorts the DNA away from itself and much of its mass sits off the DNA helix axis. Moreover, phosphorus and mass estimates demonstrate whether one or two TBP molecules interact with this particular promoter TATA sequence.

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Desethylamiodarone antagonizes the effect of thyroid hormone at the molecular level

Acta Endocrinologica ◽

10.1530/eje.0.1450059 ◽

2001 ◽

pp. 59-64 ◽

Cited By ~ 18

Author(s):

F Bogazzi ◽

L Bartalena ◽

S Brogioni ◽

A Burelli ◽

F Raggi ◽

...

Keyword(s):

Thyroid Hormone ◽

Molecular Mechanisms ◽

Thyroid Hormone Receptor ◽

Molecular Level ◽

Inhibitory Effect ◽

Mobility Shift ◽

Down Regulation ◽

Nih3t3 Cells ◽

Peripheral Tissues

OBJECTIVE: To evaluate the molecular mechanisms of the inhibitory effects of amiodarone and its active metabolite, desethylamiodarone (DEA) on thyroid hormone action. MATERIALS AND METHODS: The reporter construct ME-TRE-TK-CAT or TSHbeta-TRE-TK-CAT, containing the nucleotide sequence of the thyroid hormone response element (TRE) of either malic enzyme (ME) or TSHbeta genes, thymidine kinase (TK) and chloramphenicol acetyltransferase (CAT) was transiently transfected with RSV-TRbeta into NIH3T3 cells. Gel mobility shift assay (EMSA) was performed using labelled synthetic oligonucleotides containing the ME-TRE and in vitro translated thyroid hormone receptor (TR)beta. RESULTS: Addition of 1 micromol/l T4 or T3 to the culture medium increased the basal level of ME-TRE-TK-CAT by 4.5- and 12.5-fold respectively. Amiodarone or DEA (1 micromol/l) increased CAT activity by 1.4- and 3.4-fold respectively. Combination of DEA with T4 or T3 increased CAT activity by 9.4- and 18.9-fold respectively. These data suggested that DEA, but not amiodarone, had a synergistic effect with thyroid hormone on ME-TRE, rather than the postulated inhibitory action; we supposed that this was due to overexpression of the transfected TR into the cells. When the amount of RSV-TRbeta was reduced until it was present in a limited amount, allowing competition between thyroid hormone and the drug, addition of 1 micromol/l DEA decreased the T3-dependent expression of the reporter gene by 50%. The inhibitory effect of DEA was partially due to a reduced binding of TR to ME-TRE, as assessed by EMSA. DEA activated the TR-dependent down-regulation by the negative TSH-TRE, although at low level (35% of the down-regulation produced by T3), whereas amiodarone was ineffective. Addition of 1 micromol/l DEA to T3-containing medium reduced the T3-TR-mediated down-regulation of TSH-TRE to 55%. CONCLUSIONS: Our results demonstrate that DEA, but not amiodarone, exerts a direct, although weak, effect on genes that are regulated by thyroid hormone. High concentrations of DEA antagonize the action of T3 at the molecular level, interacting with TR and reducing its binding to TREs. This effect may contribute to the hypothyroid-like effect observed in peripheral tissues of patients receiving amiodarone treatment.

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Transcriptional properties of BmX, a moderately repetitive silkworm gene that is an RNA polymerase III template

Molecular and Cellular Biology ◽

10.1128/mcb.8.2.624-631.1988 ◽

1988 ◽

Vol 8 (2) ◽

pp. 624-631

Author(s):

E T Wilson ◽

D P Condliffe ◽

K U Sprague

Keyword(s):

Control Region ◽

Transcription Initiation ◽

Rna Polymerase Iii ◽

Sequence Element ◽

Direct Transcription ◽

Factor Binding ◽

Large Gene ◽

Polymerase Iii ◽

Downstream Control

We analyzed the transcriptional properties of a repetitive sequence element, BmX, that belongs to a large gene family (approximately 2 x 10(4) copies) in the genome of the Bombyx mori silkworm. We discovered BmX elements because of their ability to direct transcription by polymerase III in vitro and used them to test the generality of the properties of previously identified silkworm polymerase III control elements. We found that the signals that act in cis to control BmX transcription strongly resemble those that direct transcription of other silkworm polymerase III templates. As with silkworm tRNA and 5S RNA genes, transcription of BmX requires sequence signals located both upstream and downstream from the site of transcription initiation. The critical upstream sequences are structurally as well as functionally similar in the three kinds of templates. The downstream control region of BmX resembles the corresponding part of a silkworm alanine tRNA gene in that it provides a large (greater than 100 base pairs) region that influences transcription factor binding. Moreover, the factor-binding regions of both tRNA(Ala) and BmX genes are remarkable in that under certain conditions, key elements within them (the B boxes, for example) appear dispensable. This behavior can be understood if, in both of these templates, the downstream control region acts as a large target for interaction with a multifactor complex.

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