The role of activators in assembly of RNA polymerase II transcription complexes

Hori, Roderick T.; Carey, Michael

doi:10.1016/s0959-437x(05)80050-4

Cited by 89 publications

(49 citation statements)

References 58 publications

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“…Similar protein-protein interactions mediated by the progesterone receptor bound to distal hormone responsive elements have been reported for the uteroglobin gene (33). Interaction between distal regulatory loci and the basic transcription complex requires looping which depends on protein/ protein interactions between transcription factors bound, respectively, to the enhancer and to the proximal promoter (34). The fact that the intervening sequence between the t-PA enhancer and promoter can be deleted without reducing their response to dexamethasone and RA is suggestive for such a mechanism.…”

Section: Discussionsupporting

confidence: 55%

Identification of a Multihormone Responsive Enhancer Far Upstream from the Human Tissue-type Plasminogen Activator Gene

Bulens

Merchiers²,

Ibañez–Tallon³

et al. 1997

Journal of Biological Chemistry

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A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PAchloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp ؊7,501 and ؊7,974. The region from bp ؊7,145 to ؊9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from ؊7.1 to ؊8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at ؊7. Vascular patency is the result of a dynamic equilibrium between blood coagulation and fibrinolysis. Injury of the vessel wall can initiate the blood clotting cascade resulting in the formation of a hemostatic clot. To counterbalance fibrin deposition, blood contains the fibrinolytic system, one main function of which is to dissolve blood clots in the circulation. It is composed of the inactive precursor plasminogen, which can be converted into the proteolytic enzyme plasmin by the plasminogen activators (PAs) 1 tissue-type and urokinase-type PA (t-PA and u-PA, respectively) leading to the degradation of fibrin. Fibrinolytic activity can be inhibited both at the level of the PAs and plasmin by PA inhibitors (PAI-1 and -2) and ␣ 2 -antiplasmin, respectively (for a review, see ref. 1). Phenotypic analysis of mice deficient for either t-PA, u-PA, or both suggested that t-PA and u-PA were complementary, not only in the prevention of uncontrolled fibrin deposition in vivo but also in distinct processes which require local extracellular proteolytic activity, such as wound healing and gonadotropin-induced ovulation, as reviewed elsewhere (2).Dexamethasone, a synthetic glucocorticoid, and androgens increase t-PA synthesis in vitro (3-5) and in vivo (6, 7). The level of t-PA expression in breast carcinoma cells correlates with the endogenous level of progesterone receptor (8), and progesterone induces t-PA-related antigen secretion in primary human endometrial cells (9). Vitamin A, retinoic acid (RA), and some of its (synthetic) analogues induce t-PA-related antigen secretion by human umbilical vein endothelial cells in vitro (10 -12) and in the plasma as well as in specific tissues of vitamin A-deficient rats in vivo (11,13,14), suggesting that circulating RA might regulate t-PA expression in the vessel wall. The induction of t-PA expression by glucocorticoids and RA can be reproduced in HT1080 human fibrosarcoma cells, and interestingly, both agents induce t-PA mRNA steady state levels and t-PA-related antigen secretion in a cooperative manner.2 Both steroid hormones and RA are t...

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Section: Discussionsupporting

confidence: 55%

Identification of a Multihormone Responsive Enhancer Far Upstream from the Human Tissue-type Plasminogen Activator Gene

Bulens

Merchiers²,

Ibañez–Tallon³

et al. 1997

Journal of Biological Chemistry

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“…Alternatively, the receptors could be complexing a repressor. Transcription activation functions in general have been proposed to act as inhibitors (50), and reduced concentrations of SPT6, which is active in yeast and mammalian cells, caused a left shift in the estrogen dose-response curve (51). Genetic deletion of the yeast repressor SSN6 also caused increased percentages of agonist activity for antiestrogens, but not antiprogestins, and a Ն10-fold left shift in the dose-response curve for agonists bound to estrogen or progesterone receptors in yeast (52).…”

Section: Discussionmentioning

confidence: 99%

Induction Properties of a Transiently Transfected Glucocorticoid-responsive Gene Vary with Glucocorticoid Receptor Concentration

Szapary

Simons

1996

Journal of Biological Chemistry

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Transient transfections of steroid receptors have yielded much of the data used to construct the current models of steroid hormone action. These experiments invariably examine the ability of receptors to regulate transcription when occupied by saturating concentrations of steroid. We now report that other induction properties of a transiently transfected gene are not constant but vary with the concentration of transiently transfected glucocorticoid receptors. Thus, the percentage of maximal induction seen with subsaturating concentrations of glucocorticoid could be dramatically increased, and an antiglucocorticoid could be converted into a partial glucocorticoid, simply by increasing the concentration of glucocorticoid receptors. This behavior was observed in HeLa cells, containing endogenous receptors, or in CV-1 cells, containing almost no endogenous receptor, with either homologous or heterologous receptors. These increases were relatively insensitive to the concentration of reporter gene, suggesting the titration of some transcription factor(s) involved in regulating the position of the glucocorticoid dose-response curve and the agonist activity of an antiglucocorticoid. This property of transfected glucocorticoid receptors required a full-length, functionally active receptor but was retained, albeit reduced in magnitude, in the absence of binding to a glucocorticoid response element. Furthermore, this phenomenon was specific in that the A form of the human progesterone receptor had no effect under the same conditions. These variations in induction properties of antiglucocorticoids and of subsaturating concentrations of glucocorticoid, in a manner that was proportional to the amount of transfected receptor, reveal processes that are not operative with saturating concentrations of glucocorticoid. These variations also demonstrate that caution should be exercised in making mechanistic conclusions based solely on experiments conducted with saturating concentrations of glucocorticoid.The overriding experimental advantage of transient transfections is time. Thus, the biological consequences of altered nucleotide compositions in the cDNAs encoding active proteins, and in genomic sequences, can be examined in a fraction of the time required to establish cell lines with the same sequences stably integrated into the cellular genome. In the field of steroid receptors, most of the recent advances have emerged from transient transfection experiments, including the contributions of cis-acting elements (1, 2), of different nucleotides in receptor binding to the hormone-responsive element (3), of various regions of receptors in steroid binding and biological activity (reviewed in Ref. 4), of promoter structure and cell type as determinants for the activity of antisteroid activity (5), and of overlapping signaling systems such as dopamine (6), epidermal growth factor (7), and protein kinase A inducers (8, 9). The utility of transient transfections has been further enhanced by the development of the "two-hybrid" (10, 11) and...

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“…One of the key issues in the field is to understand how an upstream activator interacts with these factors to stimulate preinitiation complex assembly and transcription (2)(3)(4)(5)(6)(7). The chimeric activator GAL4-VP16 (8)(9)(10)) is a good model for studying this problem because of its potency in mammalian cell transfection assays (8) and in cell-free transcription systems (11)(12)(13).…”

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confidence: 99%

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

Hori

Pyo

Carey

1995

Proc. Natl. Acad. Sci. U.S.A.

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Transcriptional stimulation by the model activator GAL4-VP16 (a chimeric protein consisting of the DNA-binding domain of the yeast activator GAL4 and the acidic activation domain of the herpes simplex virus protein VP16) involves a series of poorly understood protein-protein interactions between the VP16 activation domain and components of the RNA polymerase II general transcription machinery. One of these interactions is the VP16-mediated binding and recruitment of transcription factor TFIIB. However, TATA box-binding protein (TBP)-associated factors (TAFs), or coactivators, are required for this interaction to culminate in productive transcription complex assembly, and one such TAF, Drosophila TAF40, reportedly forms a ternary complex with VP16 and TFIIB. Due to TFIIB's central role in gene activation, we sought to directly visualize the surfaces of this protein that mediate formation of the ternary complex. We developed an approach called protease footprinting in which the broad-specificity proteases chymotrypsin and alkaline protease were used to probe binding of 32P-end-labeled TFIIB to GAL4-VP16 or TAF40. Analysis of the cleavage products revealed two regions of TFIIB protected by VP16 from protease attack, one of which overlapped with a region protected by TAF40. The close proximity of the VP16 and TAF40 binding sites on the surface ofTFIIB suggests that this region could act as a regulatory interface mediating the effects of activators and coactivators on transcription complex assembly.The RNA polymerase II general transcription machinery is composed of six or more general factors called TFIIA through TFIIH (1). One of the key issues in the field is to understand how an upstream activator interacts with these factors to stimulate preinitiation complex assembly and transcription (2-7). The chimeric activator GAL4-VP16 (8-10) is a good model for studying this problem because of its potency in mammalian cell transfection assays (8) and in cell-free transcription systems (11-13). VP16 binds several potential targets in affinity chromatography studies (3-7, 14, 15), but the interaction with TFIIB is of particular interest because of its avidity (4, 5) and because VP16 recruits TFIIB to a promoter in assays of transcription complex assembly (4, 16). The interaction has also been validated by genetic studies: a point mutation in VP16, for example, simultaneously abolishes its interaction with TFIIB and its ability to stimulate transcription (4,17). Similarly, deletion mutations at the carboxyl ends of the two 77-aa repeats in TFIIB eliminate binding to VP16 (18)(19)(20), and point mutations in one of these regions, a putative basic amphipathic a-helix at the end of the first repeat, weaken TFIIB's interaction with VP16 and eliminate GAL4-VP16-responsive transcription in vitro (18 MATERIALS AND METHODSPlasmid Constructs. pETlld-C-HMK was constructed by inserting an oligonucleotide encoding a heart muscle kinase phosphorylation site (HMK) at the BamHI site of pETlid (Novagen). Nco I-BamHI fragments encod...

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The role of activators in assembly of RNA polymerase II transcription complexes

Cited by 89 publications

References 58 publications

Identification of a Multihormone Responsive Enhancer Far Upstream from the Human Tissue-type Plasminogen Activator Gene

Identification of a Multihormone Responsive Enhancer Far Upstream from the Human Tissue-type Plasminogen Activator Gene

Induction Properties of a Transiently Transfected Glucocorticoid-responsive Gene Vary with Glucocorticoid Receptor Concentration

Protease footprinting reveals a surface on transcription factor TFIIB that serves as an interface for activators and coactivators.

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