Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 26332-26339 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 276 |
| Issue number | 28 |
| DOIs | |
| Publication status | Published - Jul 13 2001 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
HSP-CBF is an NF-Y-dependent Coactivator of the Heat Shock Promoters CCAAT Boxes. / Imbriano, Carol; Bolognese, Fabrizio; Gurtner, Aymone; Piaggio, Giulia; Mantovani, Roberto.
In: Journal of Biological Chemistry, Vol. 276, No. 28, 13.07.2001, p. 26332-26339.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - HSP-CBF is an NF-Y-dependent Coactivator of the Heat Shock Promoters CCAAT Boxes
AU - Imbriano, Carol
AU - Bolognese, Fabrizio
AU - Gurtner, Aymone
AU - Piaggio, Giulia
AU - Mantovani, Roberto
PY - 2001/7/13
Y1 - 2001/7/13
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0035854652&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0035854652&partnerID=8YFLogxK
U2 - 10.1074/jbc.M101553200
DO - 10.1074/jbc.M101553200
M3 - Article
C2 - 11306579
AN - SCOPUS:0035854652
VL - 276
SP - 26332
EP - 26339
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 28
ER -