Synergistic activation and repression of transcription by Drosophila homeobox proteins

Han, Kyuhyung; Levine, Michael; Manley, James L.

doi:10.1016/0092-8674(89)90580-1

Cited by 344 publications

(280 citation statements)

References 51 publications

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“…1A). Each plasmid was then cotransfected into Drosophila Schneider cells as described previously (16,28), and the expression level and transcriptional activity of each derivative were compared with those of wild-type GAL4-ftzQ.…”

Section: Resultsmentioning

confidence: 99%

“…Variations in transfection efficiencies within a given experiment were almost always less than twofold. CAT assays were also performed as previously described, and promoter activities were calculated as CATto-␤-galactosidase activity ratios (28). All CAT values reported were within the previously determined linear range of the assay.…”

Section: Methodsmentioning

confidence: 99%

“…Whole-cell extracts were prepared by thawing and then sonicating for 2 min in an ultrasonic Sonifier (Branson); this step was followed by centrifugation at full speed for 10 min in a microcentrifuge. Transfection efficiencies were determined by assaying for ␤-galactosidase activity as previously described (28). Variations in transfection efficiencies within a given experiment were almost always less than twofold.…”

Section: Methodsmentioning

confidence: 99%

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A Direct Interaction between a Glutamine-Rich Activator and the N Terminus of TFIIB Can Mediate Transcriptional Activation In Vivo

Colgan

Ashali

Manley

1995

Molecular and Cellular Biology

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Studies examining the mechanism by which transcriptional activators function have suggested that the general transcription factor IIB (TFIIB) can be a target for certain regulatory proteins. For example, we showed previously that expression of a mutant form of TFIIB can specifically inhibit activation in vivo mediated by the strong, glutamine-rich activator protein GAL4-ftzQ. Using transient cotransfection assays, we have defined the regions in both GAL4-ftzQ and TFIIB that are required for activity in vivo and provide evidence that a potential zinc finger structure at the N terminus of TFIIB is necessary for the observed functional interaction between the two proteins. Using a protein binding assay, we have demonstrated that GAL4-ftzQ can specifically interact with TFIIB in vitro. This interaction requires the same regions in both molecules necessary for function in vivo and is reduced or eliminated by mutations predicted to disrupt the zinc finger in TFIIB. These results support the idea that a direct interaction between a regulatory protein and TFIIB can be important for transcriptional activation in vivo and, combined with previous data of others, suggest that different activators can function by contacting distinct regions of TFIIB.Initiation of transcription by RNA polymerase II (RNAP II) occurs through a complex set of interactions involving promoter DNA, a set of general transcription factors, and genespecific regulatory proteins. Promoters recognized by RNAP II usually consist of two classes of sequence elements in order to accomodate these interactions. Core, or basal, promoter elements include the TATA box and the initiator, which, individually or in tandem, can support the assembly of the general transcription factors into functional preinitiation complexes (for reviews, see references 5, 56, and 64). Core promoter elements are usually surrounded by members of the second class of promoter sequences, which consists of elements recognized by gene-specific factors that function to regulate the assembly and/or activity of the general transcription machinery (35,49,58).Principally through fractionation and reconstitution of transcription in vitro, the factors constituting the RNAP II general transcription machinery have been isolated and characterized (for reviews see references 6, 17, and 70). These general, or basal, transcription factors, designated TFIIA, -B, -D, -E, -F, and -H, were once thought to all be necessary in addition to the polymerase for transcription, but studies using a variety of promoters and/or more purified systems have suggested that several of these factors may be dispensible under certain conditions (19,25,39,51,59,60). The general factors can form preinitiation complexes on core promoter elements in vitro by way of an ordered assembly pathway involving protein-DNA and protein-protein contacts (7, 61). For TATA-containing promoters, this assembly process begins with the binding of TFIID to the TATA element, which may be facilitated by TFIIA, and is followed by the association of T...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Direct Interaction between a Glutamine-Rich Activator and the N Terminus of TFIIB Can Mediate Transcriptional Activation In Vivo

Colgan

Ashali

Manley

1995

Molecular and Cellular Biology

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“…All DNA mixtures also contained 2 l of copia-lacZ plasmid to monitor transcription efficiencies and 2 g of chloramphenicol acetyltransferase (CAT) reporter plasmids. A modified 2ϫ HEBS solution (280 mM NaCl, 7 mM KCl, 1.25 mM Na 2 HPO 4 , 0.2% dextrose, 40 mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid [HEPES; pH 7.1]) was used instead of the one previously described (25). All solutions were used at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Transfection of Schneider cells was performed essentially as described previously (10,25). The total actin 5C expression plasmid (pACTPPA) in each tube was adjusted to a final amount of 7 g by the addition of the actin 5C expression plasmid without any insert.…”

Section: Methodsmentioning

confidence: 99%

Functional Interaction between p53, the TATA-Binding Protein (TBP), and TBP-Associated Factors In Vivo

Farmer

Colgan

Nakatani

et al. 1996

Molecular and Cellular Biology

102

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The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAF II 230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAF II 40 and TAF II 60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAF II 110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53.Exposure to DNA-damaging agents causes cellular levels of the p53 tumor suppressor protein to dramatically increase, resulting in a G 1 /S arrest in the cell cycle. This arrest is presumably due to the direct activation by p53 of genes involved in DNA repair, cell cycle regulation, and apoptosis (for reviews, see references 12 and 23). The role of p53 as a DNA-bindingdependent transcriptional activator is an important one since mutations that alter this ability lead to unregulated cellular growth and tumorigenesis (for a review, see reference 13). Therefore, it is of great interest to understand the mechanism of transcriptional activation by p53.Proper transcriptional activation of class II genes requires specific interactions between activators bound to regulatory elements and the general transcription factors that assemble on the TATA box and/or the initiator element (reviewed in reference 59). Besides TFIID, the factor that directly binds to the TATA box, the general factors include TFIIA, -B, -E, -F, and -H and RNA polymerase II (Pol II) (3, 11, 65), many of which have been shown to interact directly with transcriptional activators in vitro (8,31,40,46,64). The TATA-binding protein (TBP), the TFIID subunit that directly binds the TATA motif (for a review, see reference 26), has also been shown to interact directly with transcriptional activators in vit...

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Expression of homeobox gene-GBX2 in human prostatic cancer cells

Gao

Isaacs

1996

Prostate

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Synergistic activation and repression of transcription by Drosophila homeobox proteins

Cited by 344 publications

References 51 publications

A Direct Interaction between a Glutamine-Rich Activator and the N Terminus of TFIIB Can Mediate Transcriptional Activation In Vivo

A Direct Interaction between a Glutamine-Rich Activator and the N Terminus of TFIIB Can Mediate Transcriptional Activation In Vivo

Functional Interaction between p53, the TATA-Binding Protein (TBP), and TBP-Associated Factors In Vivo

Expression of homeobox gene-GBX2 in human prostatic cancer cells

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