A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives

Carey, Michael; Lin, Yaojian; Green, Michael R.; Ptashne, Mark

doi:10.1038/345361a0

Cited by 389 publications

(265 citation statements)

References 15 publications

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“…The interaction of acidic activators with multiple general initiation factors (e.g., TFIID, TFIIB, and TFIIH) is compatible with the high degree of synergy that is observed in transcriptional activation (7,11) as discussed by Greenblatt (32) and Herchlag and Johnson (41). Formation of at least a partial open complex and transcriptional initiation on linear DNA do not require TFIIE, TFIIH, or ATP, since RNA polymerase II can synthesize a promoter-specific trinucleotide in the absence of these factors (31).…”

Section: Discussionmentioning

confidence: 79%

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Xiao¹,

Pearson²,

Coulombe³

et al. 1994

Mol. Cell. Biol.

339

274

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Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIHH and its Saccharomyces cerevisuze counterpart, factor b. The VP16-and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.Accurate transcription in vitro by human RNA polymerase II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF (RAP30 and RAP74), TFIIH (also known as BTF2), and TFIIJ (reviewed in references 13 and 121). Beginning with TFIID, which recognizes the TATA boxes present in many promoters for RNA polymerase II, these factors and RNA polymerase II can be assembled in a defined order onto a promoter (5). TFIIH and TFIIJ are the last of these factors to bind to an assembling initiation complex (12,14,26), and TFIIH, also known as BTF2, is the only factor known to possess associated enzymatic activities. These include an ATP-dependent DNA helicase activity (95, 96) and a protein kinase activity that can phosphorylate the carboxyterminal heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase 11 (12, 21,68 herpes simplex virus transactivator VP16 (107) and in the N-terminal 73 amino acids of the mammalian tumor suppressor protein p53 (23). The p53 protein has a site-specific DNA-binding domain in its carboxy-terminal portion (101). VP16 does not, by itself, bind specifically to DNA, but instead its amino-terminal portion binds DNA in association with mammalian factors, including the POU homeodomain protein Oct-1 that recognizes octamer sequences in DNA (28,60,102). When fused to the DNA-binding domain of GAL4, the VP16 and p53 activation domains stimulate transcription in yeast and human cells of a gene bearing GAL4-binding sites (9,16,23,74,87,92). These observations imply that common mechanisms for transcriptional activation by acidic activators may exist in fungi and mammals.Many activation domains have been shown to interact with the TATA-box-binding protein (TBP) subunit of TFIID. These include the highly acidic activation domains in VP16 (50,103), p53 (10,67,72,84,97,108),118), and E2F-1 (37), as well as other kinds of activation domains found in the adenovirus activator ElA (48, 62), the Epstein-Barr virus proteins Zta (64) and R (70), the human T-cell leukemia virus type 1 (HTLV-1) activator Taxl (8), the transactivator Tat of human immunodeficiency virus type 1 (HIV-1) (53), and human c-Fos and c-Jun (85), PU-1 (38), and Spl (20). In certain cases, reduced binding of TBP by activation domains wit...

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Section: Discussionmentioning

confidence: 79%

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Xiao¹,

Pearson²,

Coulombe³

et al. 1994

Mol. Cell. Biol.

339

274

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“…pGal 5 -c-myc P2 luciferase was constructed by inserting the PvuII-SacI fragment of pGal 5 -P2 CAT (73) into the SmaI-SacI sites of pGL2 (Promega). pGal 5 -Sp 4 -HIV2 CAT was constructed from pGal 5 -HIV2 CAT (73) by insertion of an oligonucleotide [(GGGGCGGGAC) 4 ] into the XbaI site. pGal 5 -Sp 4 -E1b CAT was constructed by inserting the same oligonucleotide into the XbaI site of pGal 5 -E1b CAT (Gal4/E1bTATA) (41).…”

Section: Methodsmentioning

confidence: 99%

“…In many cases, it is not known why some combinations synergize while others do not. Three mechanisms of transcriptional synergism have been identified: (i) cooperative DNA binding (18), (ii) formation of multiple contacts between activators and GTFs (4,6,42), and (iii) derepression of chromatin templates by displacement of nucleosomes (5). It has also been suggested that synergy could result from the stimulation of different rate-limiting steps in transcription (25).…”

mentioning

confidence: 99%

“…Multiple copies of a single activator, as well as two different activators, can synergize (4,43). For example, Tat synergizes with Sp1, but not with VP16 (29,62).…”

mentioning

confidence: 99%

“…In many cases, it is not known why some combinations synergize while others do not. Three mechanisms of transcriptional synergism have been identified: (i) cooperative DNA binding (18), (ii) formation of multiple contacts between activators and GTFs (4,6,42), and (iii) derepression of chromatin * Corresponding author. Present address: Amgen Institute,…”

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Three Functional Classes of Transcriptional Activation Domains

Blau

Xiao

McCracken

et al. 1996

Molecular and Cellular Biology

258

263

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We have studied the abilities of different transactivation domains to stimulate the initiation and elongation (postinitiation) steps of RNA polymerase II transcription in vivo. Nuclear run-on and RNase protection analyses revealed three classes of activation domains: Sp1 and CTF stimulated initiation (type I); human immunodeficiency virus type 1 Tat fused to a DNA binding domain stimulated predominantly elongation (type IIA); and VP16, p53, and E2F1 stimulated both initiation and elongation (type IIB). A quadruple point mutation of VP16 converted it from a type IIB to a type I activator. Type I and type IIA activators synergized with one another but not with type IIB activators. This observation implies that synergy can result from the concerted action of factors stimulating two different steps in transcription: initiation and elongation. The functional differences between activators may be explained by the different contacts they make with general transcription factors. In support of this idea, we found a correlation between the abilities of activators, including Tat, to stimulate elongation and their abilities to bind TFIIH.Stimulation of eukaryotic gene expression requires sequence-specific factors with DNA binding domains and activation domains that interact with the general transcription factors (GTFs) and recruit RNA polymerase II (pol II) to the promoter (3, 65). In vivo the transcriptionally active form of pol II is probably a holoenzyme complex which contains a number of the GTFs as well as other polypeptides (36). Different activation domains interact with different GTFs, including TFIIB (44), TFIID (17,19,63), and TFIIF (76). These interactions are thought to recruit, stabilize, and/or modify the activity of the pol II holoenzyme.We recently demonstrated that the activation domains of p53, VP16, and E2F1 bind directly to TFIIH (50a, 72). TFIIH is a multisubunit factor, different forms of which are required for both transcription and nucleotide excision repair of DNA (9). TFIIH is the only GTF which has enzymatic activities: it has two helicase subunits and a cyclin-dependent protein kinase subunit which phosphorylates the pol II large-subunit C-terminal domain (CTD) (12,52,58,60). Both of these enzymatic activities are implicated in steps in transcription which occur shortly after initiation of the RNA chain. First, a helicase is required for efficient formation of open complexes and for promoter clearance on linear templates in vitro (20). Second, when paused polymerases resume elongation on several Drosophila genes in vivo, the CTD becomes phosphorylated, suggesting a possible role for TFIIH kinase in regulating elongation (50, 70). Furthermore, inhibitors of the TFIIH kinase inhibit elongation under activated (74), but not basal (57), transcription conditions.In vivo, rate-limiting steps after initiation have been well documented for a number of genes. For example, polymerases stall 20 to 40 bases downstream of the start sites in the Drosophila hsp70 and human c-myc genes (38, 51, 64) and terminate...

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Schaffner

2002

Encyclopedia of Molecular Biology

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A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives

Cited by 389 publications

References 15 publications

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

Three Functional Classes of Transcriptional Activation Domains

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