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Originally published In Press as doi:10.1074/jbc.M101553200 on April 16, 2001

J. Biol. Chem., Vol. 276, Issue 28, 26332-26339, July 13, 2001
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HSP-CBF Is an NF-Y-dependent Coactivator of the Heat Shock Promoters CCAAT Boxes*

Carol ImbrianoDagger , Fabrizio Bolognese§, Aymone Gurtner||, Giulia Piaggio||, and Roberto MantovaniDagger **

From the Dagger  Dipartimento di Biologia Animale, U. di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy, the § Dipartimento di Genetica e di Biologia dei Microrganismi, U. di Milano, Via Celoria 26, 20133 Milano, Italy, and the || Istituto Regina Elena, Centro Ricerca Sperimentale, Via delle Messi d'oro 156, 00158 Roma, Italy

The cellular response to toxic stimuli is elicited through the expression of heat shock proteins, a transcriptional process that relies upon conserved DNA elements in the promoters: the Heat Shock Elements, activated by the heat shock factors, and the CCAAT boxes. The identity of the CCAAT activator(s) is unclear because two distinct entities, NF-Y and HSP-CBF, have been implicated in the HSP70 system. The former is a conserved ubiquitous trimer containing histone-like subunits, the latter a 110-kDa protein with an acidic N-terminal. We analyzed two CCAAT-containing promoters, HSP70 and HSP40, with recombinant NF-Y and HSP-CBF using electrophoretic mobility shift assay, protein-protein interactions, transfections and chromatin immunoprecipitation assays (ChIP) assays. Both recognize a common DNA-binding protein in nuclear extracts, identified in vitro and in vivo as NF-Y. Both CCAAT boxes show high affinity for recombinant NF-Y but not for HSP-CBF. However, HSP-CBF does activate HSP70 and HSP40 transcription under basal and heat shocked conditions; for doing so, it requires an intact NF-Y trimer as judged by cotransfections with a diagnostic NF-YA dominant negative vector. HSP-CBF interacts in solution and on DNA with the NF-Y trimer through an evolutionary conserved region. In yeast two-hybrid assays HSP-CBF interacts with NF-YB. These data implicate HSP-CBF as a non-DNA binding coactivator of heat shock genes that act on a DNA-bound NF-Y.

* This work was supported in part by grants from Ministero Universita `E Ricerca Scientifica (Progetti Rilevante Interesse Nazionale "Nucleic Acids-Protein Interactions") and Associazione Italiana Ricerca Sul Cancro (to R. M.).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.

Recipient of an Fondazione Italiana Ricerca Sul Cancro fellowship.

** To whom correspondence should be addressed. Tel.: 39-59-2055542; Fax: 39-59-2055548; E-mail: mantor@mail.unimo.it.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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