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Oct 26, 1992 - Irwin Davidson1, Yves Lutz, Jun Wu,. Jean-Marc Egly, Laszlo ... Ptashne, 1988; Mitchell and Tjian, 1989; Ptashne and Gann,. 1990). One of the ...

The EMBO Journal vol.12 no.2 pp.489-499, 1993

Distinct TFIID complexes mediate the effect of different transcriptional activators

Christel Broul, Sunita Chaudhary1, Irwin Davidson1, Yves Lutz, Jun Wu, Jean-Marc Egly, Laszlo Tora and Pierre Chambon Laboratoire de Genetique Mol6culaire des Eucaryotes du CNRS, Unitd 184 de Biologie Moleculaire et de G6nie Gen6tique de l'INSERM, Institut de Chimie Biologique, Faculte de Medecine, 11 rue Humann, 67085 Strasbourg Cedex, France IC.B., S.C. and I.D. should be considered as equal first authors. Communicated by P.Chambon

Multiple chromatographically separable complexes containing the TATA binding protein (TBP), which exhibit different functional properties, exist in HeLa cells. At least three distinct subpopulations of such complexes can be functionally defined as TFUD since they function with RNA polymerase II. Using a partially reconstituted HeLa cell in vitro transcription system and immunoprecipitation with a monoclonal antibody directed against TBP, we show that stimulation of transcription by the chimeric activators GAL-VP16, GAL-TEF-1 and GAL-ER(EF) requires the presence of factors which are tightly associated with these TFHD complexes. Moreover, the activity of GAL-TEF-1 appears to be mediated by at least two chromatographically distinct populations of TFIID. The factor(s) associated with one of these populations is also required for the activity of GAL-ER (EF) and GAL-VP16, while the factor(s) associated with the other population functions selectively with GALTEF-1. These two TFLlD populations are composed of both common and unique TBP associated factors (TAFs). Key words: activation domain/coactivators/immunoprecipitation/in vitro transcription/TBP associated factors

Introduction Initiation of transcription by RNA polymerase II (B) requires the ordered assembly of a multiprotein preinitiation complex (Davison et al., 1983; Buratowski et al., 1989; Maldonado et al., 1990; and references therein). In addition to RNA polymerase II at least eight other HeLa cell factors are involved in basal transcription initiated at the adenovirus-2 major late promoter (AdMLP), TFIIA (J), TFIIB, TFIID, TFIIE, TFIIF, TFIIG, TFJIH (also called BTF-2) and TFII-I (Sopta et al., 1989; Summimoto et al., 1990, 1992; Gerard etal., 1991; Malik etal., 1991; Moncollin etal., 1991; Ohkuma et al., 1991; Peterson et al., 1991; Roy et al., 1991; Usuda et al., 1991; Aso et al., 1992; Cortes et al., 1992; Finkelstein et al., 1992; Flores et al., 1992; Ha et al., 1992; and references therein; reviewed in Saltzman and Weinman, 1989; Sawadogo and Sentenac, 1990; Zawel and Reinberg, 1992). One of these transcription factors, TFIIE, may be dispensable for transcription from the immuno(©) Oxford University Press

globulin heavy chain gene promoter, suggesting that the requirement for all of these factors may not be general (Parvin et al., 1992). The formation of preinitiation complexes can be modulated both in vivo and in vitro by activator proteins which bind specifically to sequences located either upstream or downstream of the transcription start site (for reviews see Ptashne, 1988; Mitchell and Tjian, 1989; Ptashne and Gann, 1990). One of the main components of the preinitiation complex is the TATA box binding protein (TBP) (Buratowski et al., 1988; Cavallini et al., 1989; Hahn et al., 1989; Horikoshi et al., 1989; Hoffman et al., 1990; Kao et al., 1990; Peterson et al., 1990, and references therein; for review see Pugh and Tjian, 1992; Sharp, 1992), which in higher eukaryotes is found tightly associated with other proteins [TBP associated factors (TAFs), for review see Gill and Tjian, 1992] in the TFIID and SLL complexes (Dynlacht et al., 1991; Pugh and Tjian, 1991; Tanese et al., 1991; Comai et al., 1992). The TFIID complex appears to be one of the targets for both positive (Stringer et al., 1990; Dynlacht et al., 1991; Horikoshi et al., 1991; Ingles et al., 1991; Lee et al., 1991; Liebermann and Berk, 1991; Tanese et al., 1991) and negative (Meisterernst and Roeder, 1991; Meisterernst et al., 1991; Inostroza et al., 1992) regulators of promoter activity. Site-specific activators of polymerase II transcription usually contain separable domains involved in DNA binding and transcriptional activation. The various activation domains (AD), have no apparent common structure but some of them are characterized by the presence of a high content of certain amino acids (for review see Mitchell and Tjian, 1989). One of the first identified classes of AD, which is characterized by its richness in acidic amino acids (Giniger and Ptashne, 1987), is exemplified by the acidic AD (AAD) of the Herpes simplex virus VP16 protein (O'Hare and Goding, 1988; Preston et al., 1988; Triezenberg et al., 1988). When fused to a heterologous DNA binding domain, the VP16 AAD can activate transcription both in vivo (Sadowski et al., 1988; Tora et al., 1989) and in vitro (Chasman et al., 1989; Berger et al., 1990; Carey et al., 1990; Kelleher et al., 1990; White et al., 1991). In vitro in HeLa cell extracts, the VP16 AAD acts after the formation of template-committed complexes to increase the number of active transcription complexes without notably increasing the rate of their formation (White et al., 1992a). This effect may involve the increased recruitment of TFIIB or stabilization of its interaction with the preinitiation complex (Lin and Green, 1991). However, the action of the AAD requires the presence of a component(s) present in crude TFIID fractions which cannot simply be replaced by the recombinant human or yeast TBP or by the recombinant human TFIIB (White et al., 1991, 1992a). The AB and EF regions of the human estrogen receptor (ER) contain non-acidic activation functions (AF-1 and AF-2, formerly called TAF- I and TAF-2) which exhibit properties 489

C.Brou et al.



IPhosphocellulose P1l

0 0.1 0.3 0.5 1.0M KCI

1.0M KCI Fig. 1. The chromatography protocol used to isolate fractions containing factors required for the activity of the chimeric activators GAL-VP16, GAL-TEF-1 and GAL-ER(EF) is outlined. All procedures were performed at 4°C as described in Materials and methods. The concentrations of KCI and ammonium sulfate (AS) used in column elutions are indicated. Arrows indicate the use of a linear gradient between the concentrations indicated at each end of the arrow.

distinct from those of the AADs in vivo (Tora et al., 1989). While AADs can homosynergize and heterosynergize with the AdMLP upstream element factor (UEF also called MLTF or USF) and ER AF-1, ER AF-1 itself cannot homosynergize. On the other hand ER AF-2 homosynergizes and heterosynergizes with UEF but not with AADs (Tora et al., 1989). Thus, these three activation functions each have distinct properties. Furthermore in transcriptional interference or 'squelching' experiments (Ptashne, 1988; Meyer et al., 1989), ER AF-1 and ER AF-2 interfere in vivo with activation by AADs, but AADs do not interfere with the activation by ER AF-1 or ER AF-2 suggesting the The transcriptional enhancer factor 1 (TEF-1) is a third transcriptional activator which has been studied in our laboratory and exhibits properties different from those of the above activators. This factor was originally identified as a HeLa cell protein binding to two distinct enhansons of the SV40 enhancer (Xiao et al., 1987; Davidson et al., 1988). Interestingly transfection of vectors expressing recombinant TEF-1 in HeLa cells does not result in an increase in the level of transcription from TEF- 1 reporters, over that generated by the endogenous HeLa cell TEF-1 itself, but rather represses the transactivation activity of the endogenous TEF- 1 by an interference/squelching phenomenon (Xiao et al., 1991). Moreover, expression of recombinant TEF-1 in lymphoid cells does not activate expression from TEF-1 reporter plasmids (Xiao et al., 1991; Ishiji etal., 1992). These results strongly suggest that activation of transcription by TEF-1 requires the action of a TIF(s) which is highly limiting in HeLa cells and may be cell specific. Taken altogether, these studies suggest that the activation functions of each of the -transactivators mentioned above may require different TIFs in order to stimulate the initiation of transcription. In this study we have used an in vitro transcription assay in order to identify such factors. We show that HeLa cell extracts contain chromatographically separable 490

activities which are required for the stimulation of transcription by the different activators. One of these activities is required by all three recombinant chimeric GAL -TEF-1, GAL-ER(EF) and GAL -VP16 activators, while another is specific for GAL -TEF-1. Immunoprecipitation experiments indicate that these activities are tightly associated with the TBP. Thus, activation by TEF-1 can be mediated by two chromatographically separable populations of TFIID complexes, one of which functions specifically with this activator.

Results Chromatographic isolation of HeLa cell factor(s) required for the activity of the VP16, TEF-1 and ER(EF) activation domains in vitro HeLa cell nuclear proteins were first chromatographed on a heparin-ultrogel column (Figure 1 and Materials and methods) to identify proteins required for the stimulation of transcription by the purified recombinant chimeric activators GAL-VP16, GAL-ER(EF) and GAL-TEF-1 (see Introduction for references). The pooled heparin 0.6 M KCl fractions (HO.6), when supplemented with partially purified TFHA, generated basal transcription which could be stimulated by addition of each of the three chimeric activators (data not shown). We have previously reported that following chromatography of HeLa whole cell extracts on phosphocellulose P11 (PC) a combination of the general transcription factors, purified from the PCO.5 M KCl fraction (PCO.5) and partially purified TFIIA allowed basal transcription which was only weakly stimulated by GAL -VP 16 (White et al., 1992a). Addition of a DEAE 0.25 M KCl fraction, derived from the PC 1.0 M KCl fraction (PC1.0), to the PCO.5-derived basal system resulted in an efficient stimulation by GAL -VP16 (White et al., 1992a), which suggested that the PCO.5 fraction contained the factors necessary for basal transcription, but lacked at

Transcriptional activation by distinct TFIID complexes

..0 PC Fractions GAL-TEFI



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