Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID

Stringer, Keith; Ingles, C J; Greenblatt, Jack

doi:10.1038/345783a0

Cited by 600 publications

(390 citation statements)

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“…These data indicate that additional interactions with components of the transcription machinery or transcription factors might be necessary for the high transactivation potential of EWS-Fli1. We know from virion protein VP16 that a single strongly activating domain can contact four di erent transcription factors (Xiao et al, 1994;Stringer et al, 1990;Lin et al, 1991;Goodrich et al, 1993). Since the EWS amino terminus is largely composed of a degenerate repeated peptide motif we cannot exclude that there are several interfaces contacting hsRPB7 downstream of the ®rst 82 amino acids.…”

Section: Discussionmentioning

confidence: 99%

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II

et al. 1998

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As a result of the t(11;22)(q24;q12) chromosomal translocation characterizing the Ewing family of tumors (ET), the amino terminal portion of EWS, an RNA binding protein of unknown function, is fused to the DNA-binding domain of the ets transcription factor Fli1. The hybrid EWS-Fli1 protein acts as a strong transcriptional activator and, in contrast to wildtype Fli1, is a potent transforming agent. Similar rearrangements involving EWS or the highly homologous TLS with various transcription factors have been found in several types of human tumors. Employing yeast twohybrid cloning we isolated the seventh largest subunit of human RNA polymerase II (hsRPB7) as a protein that speci®cally interacts with the amino terminus of EWS. This association was con®rmed by in vitro immunocoprecipitation. In nuclear extracts, hsRPB7 was found to copurify with EWS-Fli1 but not with Fli1. Overexpression of recombinant hsRPB7 speci®cally increased gene activation by EWS-chimeric transcription factors. Replacement of the EWS portion by hsRPB7 in the oncogenic fusion protein restored the transactivating potential of the chimera. Our results suggest that the interaction of the amino terminus of EWS with hsRPB7 contributes to the transactivation function of EWS-Fli1 and, since hsRPB7 has characteristics of a regulatory subunit of RNA polymerase II, may in¯uence promoter selectivity.

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Section: Discussionmentioning

confidence: 99%

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II

et al. 1998

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“…E2F1 contains an acidic activation domain and a number of such transactivators, including VP16, have been shown to interact with general transcription factors, including TBP (Stringer et al, 1990), TFIIB and TFIIH (Xiao et al, 1994). Therefore, we tested the GST-E2F1(342 ± 437) column eluates for the presence of these factors to compare the targets of the E2F1 activation domain with those of previously characterized activation domains.…”

Section: Identi®cation Of General Factors That Interact With the Actimentioning

confidence: 99%

“…TBP is a highly conserved molecule (reviewed by Greenblatt, 1991) and certain activation domains, including those of VP16 and p53, that have been shown to bind human TBP can also bind yeast TBP if they are able to function in S. cerevisiae (Stringer et al, 1990;Liu et al, 1993;Martin et al, 1993;Truant et al, 1993). We expected the same to be true of the E2F1 activation domain and tested whether it could bind the yeast TBP in a yeast whole cell extract.…”

Section: Interaction Of the E2f1 Activation Domain With Human And Yeamentioning

confidence: 99%

“…A series of a nity columns was prepared containing immobilized GST-E2F1(342 ± 437) (lanes 1,2 and 3) or, as a control, a GST-VP16(413 ± 490) fusion protein containing the activation domain of herpes simplex virus VP16 (lane 4). We demonstrated previously that the activation domain of VP16 also binds yeast TBP directly and selectively (Stringer et al, 1990;Ingles et al, 1991). An E. coli extract containing yeast TBP was applied to each column and the columns were subsequently washed with low salt bu er and then eluted with either GST or GST-Rb, as indicated in the Figure. The eluates were analysed by Western blotting with antibody against TBP.…”

Section: The E Ect Of Rb On the Interactions Of E2f1 With Tbp And P62mentioning

confidence: 99%

“…Numerous activator proteins, including the herpes simplex virus transactivator VP16, bind TBP (e.g. see Stringer et al, 1990), the DNA-binding subunit of the general transcription factor TFIID, and results from mutational studies with several activators, including VP16, have demonstrated a good correlation between the ability of an activator to bind TBP and its ability to activate transcription (Ingles et al, 1991;Seto et al, 1992;Truant et al, 1993;Melcher and Johnston, 1995). Interactions have also been reported between certain transcriptional activators and TFIIB Ing et al, 1992), with certain TBP-associated factors (TAFs) (Goodrich et al, 1993;Hoey et al, 1993;Lu and Levine, 1995;Thut et al, 1995), and also with TFIIF (Zhu et al, 1994;Joliot et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

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Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH

Pearson

Greenblatt

1997

Oncogene

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The transcriptional activator E2F1 regulates the expression of genes at the G1/S boundary. We have characterized interactions of the E2F1 activation domain with two general transcription factors, the TATA-box binding protein (TBP) and TFIIH. Two distinct binding sites on E2F1 were identi®ed for TBP (amino acids 386 ± 417 and 415 ± 437) each of which supported activation in mammalian cells when expressed as a fusion to a heterologous DNA-binding domain. Neither of these minimal activation domains independently bound TFIIH; rather, the TFIIH binding site of E2F1 overlaps both domains. Loss of TFIIH-binding by E2F1 resulted in a 60 ± 65% reduction in transactivation, suggesting that the E2F1/TFIIH interaction is important, but not essential, for transactivation. The retinoblastoma protein (Rb) binds directly to E2F1 and represses E2F1-mediated transactivation. We have demonstrated that recombinant Rb can compete with TBP and the p62 subunit of TFIIH for binding to immobilized E2F1. A tumorigenic form of Rb de®cient in repressing E2F1-mediated transactivation is likewise de®cient in displacing TBP from E2F1. We propose that competition between Rb and both TBP and TFIIH for binding to E2F1 is a mechanism by which Rb inhibits transactivation by E2F1.

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Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

2000

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The herpes simplex virus transactivator protein VP16 is frequently used to regulate gene expression in several experimental systems, including transgenic mice. It has been suggested that high levels of VP16 expression in mice may be lethal. In order to systematically address this issue, we linked the VP16 gene to promoters that are active early and in a variety of tissues throughout development, such as the human β‐actin promoter or the rat nestin gene enhancer. VP16 expression was assayed using a LacZ reporter gene linked to a VP16‐responsive immediate early gene promoter. We show here that expression of VP16 at high levels is detrimental to pre‐implantation development. By culturing embryos in vitro, we demonstrate that this effect is exerted at the transition from the 2‐cell to the 4‐cell stage, reducing survival to the blastocyst stage dramatically. On the other hand, transgenic mice expressing VP16 transgenes at postimplantation stages are viable. These results suggest a differential sensitivity to VP16 expression in different cell types and stages of development. The reduction of embryo survival by VP16 implicates herpes virus infection as a potential cause of infertility. Mol. Reprod. Dev. 55:37–46, 2000. © 2000 Wiley‐Liss, Inc.

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Direct and selective binding of an acidic transcriptional activation domain to the TATA-box factor TFIID

Cited by 600 publications

References 47 publications

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II

Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH

Herpes simplex virus transcriptional activator VP16 is detrimental to preimplantation development in mice

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