HSP-CBF is an NF-Y-dependent coactivator of the heat shock promoters CCAAT boxes.
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.
Imbriano
C
C
Dipartimento di Biologia Animale, U. di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy.
Bolognese
F
F
Gurtner
A
A
Piaggio
G
G
Mantovani
R
R
D000818:Animals; D023081:CCAAT-Binding Factor / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D050660:Core Binding Factors; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics*; D009363:Neoplasm Proteins; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D023081:CCAAT-Binding Factor; D050660:Core Binding Factors; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D014157:Transcription Factors