JBC Ideal method for primary cell transfection

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Originally published In Press as doi:10.1074/jbc.M205860200 on July 9, 2002

J. Biol. Chem., Vol. 277, Issue 36, 32875-32882, September 6, 2002
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TATA-binding Protein-free TAF-containing Complex (TFTC) and p300 Are Both Required for Efficient Transcriptional Activation*

Sara HardyDagger , Marjorie BrandDagger §, Gerhard Mittler, Jun Yanagisawa||, Shigeaki Kato||, Michael Meisterernst, and Làszlò Tora**

From the Institut de Génétique et de Biologie Moléculaire et Cellulaire, UMR 7104, Department of Transcriptional and Post-transcriptional Control of Gene Regulation, Communauté Urbaine de Strasbourg, France, boite postale 10142-67404 Illkirch Cedex, the  National Research Center for Environment and Health, Department of Gene Expression, Marchioninistrasse 25, D-81377 München, Germany, and the || Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Initiation of transcription of protein-encoding genes by RNA polymerase II was thought to require transcription factor TFIID, a complex comprising the TATA-binding protein (TBP) and TBP-associated factors (TAFs). In the presence of TBP-free TAF complex (TFTC), initiation of polymerase II transcription can occur in the absence of TFIID. TFTC contains several subunits that have been shown to play the role of transcriptional coactivators, including the GCN5 histone acetyltransferase (HAT), which acetylates histone H3 in a nucleosomal context. Here we analyze the coactivator function of TFTC. We show direct physical interactions between TFTC and the two distinct activation regions (H1 and H2) of the VP16 activation domain, whereas the HAT-containing coactivators, p300/CBP (CREB-binding protein), interact only with the H2 subdomain of VP16. Accordingly, cell transfection experiments demonstrate the requirement of both p300 and TFTC for maximal transcriptional activation by GAL-VP16. In agreement with this finding, we show that in vitro on a chromatinized template human TFTC mediates the transcriptional activity of the VP16 activation domain in concert with p300 and in an acetyl-CoA-dependent manner. Thus, our results suggest that these two HAT-containing co-activators, p300 and TFTC, have complementary rather than redundant roles during the transcriptional activation process.

* This work was supported by funds from INSERM, CNRS, Hôpital Universitaire de Strasbourg, Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, Ligue Nationale contre le Cancer, the Human Frontier Science Program (RG 196/98 to L. T. and M. M.), and by European Community Grants HPRN-CT-2000-00087 and HPRN-CT-2000-00088.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These are equal first authors; both were supported by a fellowship from the Ministère de l'Education Nationale, de l'Enseignement Supérieur, de la Recherche et de la Technologie.

§ Present address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Mail Stop A3-025, Seattle, WA 98112.

** To whom correspondence should be addressed. Tel.: 33-388-65-34-44; Fax: 33-388-65-32-01; E-mail: laszlo@igbmc.u-strasbg.fr.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.


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