From the 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
Received for publication, February 19, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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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.
Protection from cellular stress is a fundamental function that
enables all living organisms to counteract noxious environmental stimuli such as heat or toxic agents. A crucial aspect of the heat
shock response is the rapid and massive production of distinct classes
of related proteins conserved in evolution; based on their molecular
weight, three major families of heat shock proteins (HSPs)1 are catalogued:
middle HSPs, 64/74 kDa HSPs; large HSPs, 90/110 kDa; and small HSPs,
27/40 kDa (1). The whole process is controlled by increasing the
expression of the respective set of genes at the transcriptional level
(2, 3). This phenomenon is mediated by the heat shock transcription
factors (HSFs), which are present in a monomeric, non-DNA binding form
in the cytoplasm of unstressed cells. Upon activation, HSFs rapidly
form homotrimers, which translocate into the nucleus and bind to
discrete palyndromic sites (heat shock elements (HSEs)) present
in the promoters. In all cases tested so far, HSEs work in
conjunction with, and indeed require, nearby elements such as GC and
CCAAT boxes. Among heat shock promoters, HSP70s are perhaps the most
thoroughly studied. In essentially all species HSP70 promoters contain
CCAAT boxes (4-13). When multiple HSP70 genes are present in one
species, such as in mouse and man, they all harbor one or multiple
CCAAT sequences (4-6). Whenever tested in such diverse systems as
mammalian cells, Xenopus oocytes, transgenic tobacco or
zebrafish microinjection, the functional importance of CCAAT sequences
has been clearly established (14-20). The exception is represented by
Drosophila, which has no CCAAT boxes in their HSP70
promoters, a fact that is mirrored by the conspicuous absence of this
element in any type of promoter. In addition to the HSP70 family, human
HSP105 and HSP40 also contain CCAAT sequences, and as far as the
HSP40 is concerned, they have also been shown to be important
for expression (21, 22).
The CCAAT box is present not only in heat shock genes, but it is indeed
one of the most widespread cis-acting elements, being found in 30% of
eukaryotic promoters (23). In many DNA binding activators the acronym
CCAAT is present: CTF/NF1 (CCAAT transcription factor), C/EBP
(CCAAT/enhancer binding protein) and CDP (CCAAT displacement protein).
However, in the HSP system two polypeptides have been directly
implicated in CCAAT box function: NF-Y and CBF, hereafter renamed
HSP-CBF to avoid confusion with CBF, which is another acronym of NF-Y
(24, 25). NF-Y is a complex composed of three subunits: NF-YA, NF-YB,
and NF-YC, all required for DNA binding. A large body of evidence
indicates that NF-Y binds and activates many if not most CCAAT boxes in
diverse promoters in different kingdoms: yeast, plants, and mammals
(24, 25). Two of the subunits, NF-YB and NF-YC, contain histone-like
domains required for dimerization, association of NF-YA and CCAAT
binding. Two large Q-rich domains are present in NF-YA and NF-YC and
function in activation assays. At first sight, the CCAAT boxes found in the HSP70 promoters across species match very well the NF-Y consensus obtained by the alignment of a compilation of >300 sites
(26)2 and experimentally
derived with site-selection analysis (27). Analyzing the
Xenopus HSP70, the group of A. Wollfe came to the conclusion
that the CCAAT box is instrumental in maintaining an open chromatin
configuration of the promoter so that HSF could rapidly activate (20).
Evidence that Xenopus HSP70 activation is elicited through
interactions of NF-Y with the p300 coactivator has been presented (28).
In vivo footprinting experiments in mouse cells are
supportive of this idea because CCAAT boxes are protected
constitutively prior of the heat shock, whereas the HSEs become bound
by HSFs activators only following the thermal stimulation (29,
30). In another report, NF-Y was shown to be the target of negative
regulation by Myc to a On the other hand, work on the human HSP70 promoter pointed to a
different protein as crucial for CCAAT box activation. Screening of an
expression library with multimerized HSP70 CCAAT-containing oligonucleotides lead to the identification of HSP-CBF, a cDNA coding for a 999-amino acid protein (32, 33). HSP-CBF is ubiquitous and
highly conserved in many species; the Saccharomyces
cerevisiae equivalent, Mak21p, is an essential gene (34). It
contains a highly acidic N-terminal domain, usually a hallmark for
transcriptional activators, and indeed expression of HSP-CBF in COS
cells increased the activity of the human HSP70 promoter in a
CCAAT-dependent way. GAL4 fusion experiments further
pinpointed two regions important for transcriptional activation: the
acidic N-terminal domain and a central part highly conserved across
species (35). Interestingly, the N-terminal part of HSP-CBF is
contacted by the viral oncogene E1A (36) and by the anti-oncogene p53
(37); E1A and p53 have opposite effects on HSP-CBF; the former
increases, while the latter represses HSP-CBF-mediated activation (37,
38). To solve the issue of which protein is the bona
fide activator of heat shock genes CCAAT boxes, we employed
recombinant proteins in EMSA and immunoprecipitation assays in
vitro and expression vectors and antibodies for chromatin
immunoprecipitations in vivo on two promoters representative
of the HSP70 and HSP40 families.
Protein Production and Purification--
HSP-CBF cDNAs (wt
and mutants) were cloned into the His- and thioredoxin tagged
PET32 expression vector using the NcoI site; mutants 1-533,
1-366, and 1-100 were derived by cutting with EcoRI, XhoI, and HindIII, respectively, and religating.
Productions of the fusion proteins were as described in Refs. 39 and
40. The His-less NF-YB and NF-YA proteins were produced from PET3b, and
NF-YC from the His-tagged PET32 was modified to eliminate the
thioredoxin tag by cutting with NdeI and religating.
NF-YA and NF-YB subunits were found in inclusion bodies and were
resuspended in 6 M GnCl. Equimolar amounts of the
recombinant proteins were renatured by slowly dialyzing away the
denaturing agent, and the resulting material was centrifuged to remove
precipitates, loaded on an NTA-agarose column, and purified
according to standard procedures. HSP-CBF proteins were purified from
soluble fractions. For production of HSP-CBF in vitro, the
transcription/translation TnT system (Promega) was employed.
Immunoprecipitations--
For protein-protein interaction
studies, 50-100 ng of recombinant proteins were incubated in 200 µl
of 300 mM KCl, 20 mM HEPES, pH 7.9, 0.05%
Nonidet P-40, 0.5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, rotated for 2 h at 4 °C, and
then added to 25 µl of protein G-Sepharose to which 5 µg of the
anti-NF-YA Mab7 monoclonal antibody had been previously bound.
Incubation was pursued for 2 h at 4 °C, unbound material was
recovered after centrifugation, and the beads were washed with NDB100
with the addition of 0.1% Nonidet P-40. SDS buffer was added, and the
samples were boiled at 90 °C for 5 min and loaded on SDS gels.
Western blots were performed according to standard procedures with the
indicated primary antibody and revealed with a Pierce peroxidase
secondary antibody. The anti-HSP-CBF antibody was a kind gift of D. Linzer, Northwestern Univ., Evanston, IL.
EMSA--
For electrophoretic mobility shift assays
32P-labeled fragments, 10000 cpm, are incubated in NF-Y
buffer (20 mM HEPES, pH 7.9, 50 mM NaCl, 5%
glycerol, 5 mM MgCl2, 5 mM
Transfections--
The eukaryotic expression vector for HSP-CBF
has been described (32). 2.5 × 104 COS or SAOS2 cells
were transfected with 0.5 µl of LipofectAMINE (Life Technologies,
Inc.) in a 24-well plate using different doses of the activating
vectors, 0.1 µg of Xenopus HSP70-CAT (gift of N. Landsberger, Univ. Insubria, Varese, Italy) or HSP40-luciferase DNA (gift of K. Ohtsuka, Aichi Cancer Center, Nagoya, Japan), 50 ng of
N Chromatin Immunoprecipitations--
Formaldehyde cross-linking
and chromatin immunoprecipitation was performed as described in Ref.
41. Exponentially growing HeLa cells were washed in phosphate-buffered
solution and incubated for 10 min with the cross-linking solution,
containing 1% formaldehyde; after quenching the reaction with glycine
0.1 M cross-linked material was broken by sonication into
chromatin fragments of an average length of 500/1000 base pairs.
Immunoprecipitation was performed with ProtG-Sepharose. The chromatin
solution was precleared by adding ProtG-Sepharose for 15 min at
4 °C, aliquoted, and incubated with 2 µg of affinity-purified
rabbit polyclonal antibodies for 3 h at 4 °C with mild shaking.
Before use, ProtG-Sepharose was blocked with 1 µg/µl sheared
herring sperm DNA and 1 µg/µl bovine serum albumin for 4 h at
4 °C and then incubated with chromatin and antibody overnight.
Immunoprecipitates were eluted, and ethanol was precipitated. Recovered
material was treated with proteinase K, extracted with
phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated. The
pellets were resuspended in 30 µl of H2O and analyzed by
using polymerase chain reaction with the following primers specific for
the human HSP70 promoter: coding 5'-GGCGAAACCCCTGGAATATTCCCGA-3', non-coding 5'-AGCCTTGGGACAACGGGAG-3'; for the DHFR promoter: coding 5'-GGCCTCGCCTGCACAAATAGGG-3', non-coding 5'-GGGCAGAAATCAGCAACTGGGC-3'. The input sample was resuspended in 100 µl of H2O and
diluted 1:100.
Yeast Two-hybrid Assays--
The yeast strain used was PJ69-4A,
which contains three Gal4 inducible promoter elements fused to
selection and reporter genes (42). This strain was transformed with
plasmid pAS2-1 in which HSP-CBF-1-566 was cloned with
NcoI-EcoRI in frame with the GAL4 DNA binding
domain, AS2.1-HSP-CBFN, generating the strain PJ69-AS2.1-HSP-CBFN. This
strain was then transformed with the pACT2 plasmid
(CLONTECH) containing the NF-YB gene, cloned
BglII-EcoRI in frame with the GAL4 activation
domain. Transformants were plated onto synthetic media plates lacking
histidine, adenine, tryptophan, and leucine and containing 2% dextrose
as a carbon source and 1 mM 3-aminotriazole. His+ colonies were assayed for NF-Y Not HSP-CBF Binds to HSP70 and HSP40 CCAAT Boxes with High
Affinity--
CCAAT boxes are conserved in the HSP70 promoters across
species and are found, among others, in the human HSP40 promoter. To
characterize the proteins binding to them, we performed EMSA analysis
with nuclear extracts of mouse CH27 cells and two probes representative
of these families: the human HSP70 proximal
In an initial attempt to verify the relative affinity of NF-Y and
HSP-CBF for the CCAAT boxes of the HSP genes, we cloned HSP-CBF into
the PET32 E. coli expression vector. Recombinant HSP-CBF was
produced and purified using the His-tag of the protein on NTA
nickel columns; most of the resulting HSP-CBF is proteolytically cut to
a size of 55 kDa. We employed this material in EMSA analysis; no
binding of the recombinant HSP-CBF was observed on either targets even
at very high dosages (1 µg, not shown). Because of the poor yield of
intact bacterial HSP-CBF, we turned to a TnT transcription/translation system; HSP-CBF was abundantly produced in an intact form as checked by
[35S]methionine labeling of the protein (Fig.
1C). This material was used for EMSA as above, and again no
binding was observed. We systematically modified the concentrations of
salts, divalent cations, and detergents; we were incapable of observing
specific CCAAT binding of HSP-CBF on the HSP70 or HSP40 CCAAT probes
(Fig. 1D, lanes 1-4). EMSAs with HSP70 and HSP40
CCAAT oligonucleotides were then performed with both recombinant
proteins. Addition of the two proteins in the buffer conditions
described in Ref. 32 yielded a faint complex migrating more slowly than
the prevalent NF-Y complex (Fig. 1D, lanes 5-8);
this upper complex was NF-Y-dependent because it was
competed by cold CCAAT oligonucleotides and supershifted by anti-NF-Y
antibodies (not shown). In no other buffer conditions was this upper
complex detectable with the HSP40 or the HSP70 probe (Fig.
1E). The presence of HSP-CBF in the higher complexes was confirmed by supershift experiments with purified rabbit antibodies against HSP-CBF (Fig. 1F). Although these data do not rule
out completely the possibility that HSP-CBF binds DNA, they establish (i) that a clear difference in affinities between recombinant NF-Y and
HSP-CBF exists under standard EMSA conditions, and (ii) that NF-Y can
associate HSP-CBF, albeit with apparent modest affinity and strict
buffer requirements, when bound to DNA in vitro.
Interactions of NF-Y with the HSP70 CCAAT Box in Vivo--
To
verify the ability of NF-Y to interact with the HSP70 promoter in
vivo, we employed ChIP on growing HeLa cells. The cross-linked chromatin was immunoprecipitated using a polyclonal antibody against NF-YB. As negative controls, we included a reaction lacking a primary
antibody and one that contained an antibody against E2F1, an abundant
nuclear transcription factor that does not have a binding site on the
HSP70 promoter. After immunoprecipitation and reversal of the
cross-links, enrichment of the endogenous HSP70 promoter fragment in
each sample was monitored by polymerase chain reaction amplification
using primers amplifying the human HSP70 promoter region from HSP-CBF Activates HSP70 and HSP40 in an NF-Y-dependent
Manner--
Functional assays have indicated that the human HSP70
promoter is activated in a CCAAT-dependent way by HSP-CBF
(32, 35). Given the results obtained in our in vitro EMSA
analysis, we felt it important to verify this point. To this aim, we
cotransfected a CAT reporter gene driven by the Xenopus
HSP70 promoter that shows identical architecture and similar sequence
to the human counterpart with a HSP-CBF expression plasmid in COS and
SAOS2 cells. COS cells were chosen because, unlike CHO cells, they were shown to be permissive to HSP-CBF activity; the p53
In another set of conceptually similar experiments, we employed the
human HSP40 promoter, from position HSP-CBF Is Not a General Coactivator--
The results shown above
raised questions about the specificity of the transcriptional activity
of HSP-CBF for NF-Y and/or for heat shock promoters. To verify this, we
used other promoters in the same cotransfection systems. HSP70 genes
are regulated during the cell cycle, being activated at the
G1/S boundary (45, 46), thus it could be conceivable that
HSP-CBF activity might be involved in the large family of
CCAAT-containing cell cycle-regulated promoters (26). We employed
several cell cycle-regulated promoters either containing bona
fide NF-Y binding sites, such as TK, or lacking CCAAT boxes, such
as DHFR and Cyclin E. As a further control, we used the tissue-specific
MHC class II Ea that is highly dependent upon NF-Y binding.
Cotransfections of increasing doses of HSP-CBF with all these reporters
yielded levels of transcriptional activities that were not augmented
and, in fact to various extents, decreased when compared with
transfections in the absence of HSP-CBF (Fig. 4). These results rule out that HSP-CBF
is a general cofactor activating all types of promoters and further
support the idea that not all CCAAT-boxes are activated because TK and
Ea are clearly unaffected.
NF-Y and HSP-CBF Binds in Vitro and in Vivo--
A possible
explanation for the experiments shown above is that NF-Y and HSP-CBF
can bind to each other, and thus NF-Y could recruit HSP-CBF onto the
promoter. We started to evaluate this possibility by performing
immunoprecipitations with the recombinant proteins and the anti-NF-YA
monoclonal antibody Mab7. Using the wt HSP-CBF and NF-Y trimer, we
observed specific interactions between the two proteins because Western
blots revealed HSP-CBF in the bound fraction of the material
immunoprecipitated with Mab7 but not with an irrelevant anti-Gata1
antibody (Fig. 5, upper panel). As expected, NF-Y subunits were also found in the same bound fractions as exemplified for NF-YB (Fig. 5 and data not shown).
Deletion mutants of HSP-CBF were produced and assayed; HSP-CBF-1-533
and HSP-CBF-1-366 were still capable to bind NF-Y, although
HSP-CBF-1-100 was not. We then checked the protein-protein interactions of HSP-CBF-1-533 separately with NF-YA and with the NF-YB-NF-YC dimer; as shown in the lower panel of Fig. 5,
HSP-CBF is incapable of associating the separate NF-Y subunits. We
conclude that the NF-Y trimer, but not the single subunits, is able to interact with a domain of HSP-CBF located between amino acids 100 and
366.
To verify the interactions in an in vivo system, we employed
the two hybrid assay in yeast. We cloned the HSP-CBF-1-533 mutant that
interacts with NF-Y in vitro into the pAS2.1 vector
containing the DNA-binding domain of GAL4 deriving pAS2.1-CBFN and
NF-YB in the pACT vector, in frame with the GAL4 activation domain; these constructs were introduced in the PJ69-4A yeast strain and selected for Leu and Trp auxotrophy. This strain contains Ade and His selectable markers and a LacZ reporter gene that are under the
control of three different GAL4-dependent promoters, Gal2, Gal1, and Gal7, respectively (42). On Leu The CCAAT-boxes are extremely conserved in the promoters of heat
shock genes; in the HSP70 family, they have been found in mammals,
chicken, Xenopus, algae, oomycetes, fish, and
parasites (4-14). In this report, we clarified an important point for
our understanding of the molecular mechanisms leading to the induction of these genes, namely the identity of the proteins that activate such
sequence. Our data obtained with EMSA in vitro, with ChIP assays in vivo, and with transfections with an NF-YA
dominant negative vector conclusively establish NF-Y as the DNA-binding protein that recognizes HSP CCAAT boxes. However, our transfection results revealed that HSP-CBF is a gene-specific coactivator of heat
shock promoters, at least of those, the vast majority, containing this element.
It has long been known that the CCAAT boxes play a crucial role in HSP
transcription. Previous work performed in Xenopus oocytes on
the HSP70 promoter indicated that they are essential for induction after heat treatment. Most importantly, Wollfe's laboratory found that
their elimination brought profound alterations to the "open" chromatin configuration that normally allows HSFs to bind and activate
transcription shortly after the stimulus. Indeed in CCAAT-less constructs the whole promoter remains in a tight, closed nucleosomal configuration, inaccessible to HSFs and to the factors binding to the
core promoter sequences (20). In a later study, the same authors
investigated the NF-YB relationship with the coactivator p300, which
possesses a histone acetyltransferase activity important for
transcriptional activation. p300 acetylates the NF-YB subunit of NF-Y,
but the function of this modification, if any, remained unclear (30).
Formal proof that NF-Y was the protein involved in vivo was,
however, was lacking. These results lead to the hypothesis that
NF-Y presets chromatin configuration for activators to bind nearby and
for coactivators to be recruited, which was in full agreement with our
own data on the MHC class II Ea promoter performed with
nucleosomal and chromatin reconstitution systems in vitro. We found that NF-Y has a high intrinsic affinity for nucleosomal structures thanks to the NF-YB-NF-YC histone-like subunits (39, 40).
Consistent with this picture, previous in vivo
footprinting studies in mammalian cells showed that the CCAAT box of
the HSP70 promoter was protected constitutively even in unshocked cells (29, 30).
However, in the human system a different protein was shown to activate
the CCAAT-sequence; HSP-CBF, isolated thanks to library screenings with
multimerized human HSP70 CCAAT boxes, showed typical features of
activators and indeed behaved so (32). Although clear-cut evidence of
specific CCAAT binding activity by HSP-CBF was circumstantial,
additional features of HSP-CBF matched quite well the overall
regulation of HSP70 promoters; binding to the CR3 region of the 13SE1A,
a positive coactivator of heat shock genes (38), correlated with strong
superactivation of GAL4-HSP-CBF fusions (36). On the other hand,
binding to p53, a repressor of HSP transcription, lead to inhibition of
HSP-CBF activity (37). The crucial role of HSP-CBF in cellular
functions is further supported by the following observations: (i)
HSP-CBF genes can be retrieved from expressed sequence tags and genomic
data banks of a number of species including zebrafish,
Caenorhabditis elegans, Drosophila melanogaster, S. cerevisiae, and
Schizosaccharomyces pombe in addition to mouse, rat, and
man. (ii) Mak21p, the yeast homolog of HSP-CBF, was isolated in a
screening to complement mutations in genes allowing propagation of M1
double-stranded RNA and was shown to be essential for cell growth,
apparently being required for 60 S ribosomal biogenesis. The lack of
complementation of the Mak21-1 phenotype by the human gene, suggesting
notable interspecies differences and the absence of yeast conditional
mutants limit inferences from the yeast studies.
We were unable to detect any sequence-specific DNA binding capacity of
HSP-CBF on CCAAT oligonucleotides. On the other hand, clear-cut results
in vitro and in vivo implicate NF-Y as the
DNA binding factor. It is unclear to us how the original screenings yielded such polypeptide, but the likeliest explanation might be that
HSP-CBF has affinity for multimerized CCAAT boxes or for random longer
pieces of DNA in general irrespective of the sequence. Preliminary
evidence of this has been obtained in our
laboratory.4 These
discrepancies notwithstanding, it is clear that our data are entirely
consistent with the previous functional indications, but point to a
fundamentally different role of HSP-CBF in HSP activation, not direct
sequence-specific DNA binding but association with a CCAAT-bound NF-Y.
Our protein-protein interaction analysis identified the region of
HSP-CBF-NF-Y interactions between amino acids 100 and 366 of HSP-CBF.
Interestingly, this conserved region partially overlaps with the acidic
N-terminal region contacted by E1A and p53 (36, 37) and is distinct
from another domain, also evolutionarily conserved, that is important
for HSP transactivation (35). It is tempting to speculate that HSP-CBF
could be a protein scaffold that integrates interactions with different
transcription factors, both upstream and in the core promoter.
What would be the functional relationships between NF-Y and the HSP-CBF
coactivator? Coactivators are a large and heterogenous family of
molecules that do not bind DNA directly but act on prebound sequence-specific factors. Many coactivators, such as CBP/p300, PCAF,
GCN5, and PC4 (reviewed in Ref. 49) are considered as general cofactors
that act on many if not most genes. Another large family of
coactivators are factor-specific, that is, they recognize specific
classes of DNA binding activators. Upon hormonal induction, for
example, nuclear receptors bind coactivators that displace corepressor
molecules. Interestingly, many of these factor-specific coactivators
possess intrinsic HAT activities, to some extent similar to
those of p300/CBP (50). Our data on the cell cycle promoters, as well
as previous findings on the Our model predicting that HSP-CBF is a gene-specific coactivator brings
an important corollary: HSP-CBF makes contacts with NF-Y and,
consistent with this, it activates basal transcription provided that an
intact CCAAT box is present. However, it also activates heat shocked
transcription under conditions in which it is conceivable that it could
make important connections with HSFs. The apparent weak affinity of a
CCAAT-bound NF-Y for HSP-CBF, as detected in the experiments shown in
Fig. 1, suggests that additional factors, such as HSFs, and longer
fragments of DNA are required for stable interactions. GC boxes,
presumably binding to members of the Sp1 family, are also believed to
play a role, at least in human HSP70, and could be important in the
recruitment mechanism.
The mechanistic details of activation cannot be distinguished by our
experiments, but three possibilities can be envisaged. (i) HSP-CBF
might intervene early during the establishment of an open chromatin
configuration within the heat shock regulatory regions following NF-Y
binding (Fig. 7A). (ii)
Alternatively, it could be recruited only when HSFs binds to the HSEs
and increase the stability of the nucleoprotein complex (Fig.
7B). The highly acidic domain, which is required but not
sufficient for activation, would make contacts with proteins of the
basal machinery, such as some of the TAFIIs. In
these two schemes HSP-CBF might be important for chromatin remodeling
and would therefore play a role similar to p300/CBP. (iii) It could
help the release of the stalled polymerase by weakening the contacts
between the basal factors and the upstream activators (Fig.
7C, see Ref. 52). In Drosophila, the elongation factor P-TEFb is required for productive transcriptional elongation of
HSP70 mRNA, and HSF is required but not sufficient for P-TEFb recruitment (53). In this scenario, HSP-CBF might play a role in the
post-HSF recruitment phase of transcriptional activation. It is worth
reminding that the CIITA coactivator has been shown to be contacting
DNA-bound activators, proteins of the basal machinery, as well as the
elongation factor P-TEFb (51).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
155 upstream site in the human HSP70 promoter
(31), whose function is, however, unclear (15-17).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol) with 5 µg of CH27 nuclear extract or with
the recombinant proteins in a total volume of 10 µl; after incubation
for 15 min at 20 °C, we added 2 µl of 1× NF-Y buffer containing
bromphenol blue and samples loaded on a 4.5% polyacrylamide in 0.5×
Tris borate EDTA. The oligonucleotides used were the following: HSP70,
5'-CTCATCGAGCTCGGTGATTGGCTCAGAAGGGAAAA-3'and 5'-TTTTCCCTTCTGAGCCAATCACCGAGCTCGATGAG-3'; and HSP40,
5'-AGGGCGGCGGCGATTGGCCGGCGCCGCGGG-3' and
5'-CCCGCGGCGCCGGCCAATCGCCGCCGCCCT-3'. For supershift experiments, we used 300 ng of anti-NF-YA Mab7, anti-NF-YB, anti-NF-YC and anti-Gata1, and 100-300 ng of anti-HSP-CBF.
-galactopyranoside as an internal control and various amounts of
carrier pGEM plasmid to keep the total DNA concentration constant at 1 µg. Cells were recovered 24/36 h after transfection, washed in
phosphate-buffered solution (150 mM NaCl and 10 mM sodium phosphate, pH 7.4) and resuspended in TEN (150 mM NaCl and 40 mM Tris HCl, pH 7.4) for
measurements of
-galactosidase and CAT or luciferase activities.
Heat shocks were performed 30 h post-transfection, by placing the
plates for 90 min at 42 °C; following a change of medium, cells were
recovered after 5 h. A minimum of three independent transfections
in duplicate were performed. Standard deviations represented <20% of
the values.
-galactosidase activity
by a filter assay; colonies grown on selective plates were
replica-plated on nitrocellulose filters, dipped in liquid nitrogen to
permeabilize cells, thawed, and placed in a Petri dish containing
Whatman No. 3MM paper saturated with Z buffer containing
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 base pairs and
human HSP40 proximal
100 base pairs CCAAT boxes, both shown to be
essential for the full activities of the two promoters (15-17, 21). A
single complex was observed with both oligonucleotides (Fig.
1, A and B,
lanes 2). In parallel, we incubated Escherichia
coli, produced NF-Y in the same assay, and promptly observed complexes (Fig. 1, A and B, lanes
1); it was immediately apparent that the complexes generated with
nuclear extracts and with recombinant NF-Y had similar electrophoretic
mobilities. Both the HSP70 and HSP40 complexes were specifically self-
and cross-competed by unlabeled oligonucleotides containing the two CCAAT boxes but not by an unrelated oligonucleotide harboring the
MHC class II Ea X box (Fig. 1, A and B,
lanes 3-8). These complexes were also supershifted by
anti-NF-YB and anti-NF-YC antibodies and inhibited by the anti-NF-YA
Mab7 monoclonal (Fig. 1, A and B, lanes
9-13). These results establish conclusively that the more
readably seen DNA-binding protein in EMSA assays is NF-Y and represents
the first demonstration that NF-Y is the HSP40 proximal CCAAT-binding
protein.
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Fig. 1.
NF-Y is the HSP40 and HSP70
CCAAT-binding entity in nuclear extracts. EMSA of NF-Y and HSP-CBF
bound to the HSP70 (A) and HSP40 (B) proximal
CCAAT box regions. Lane 1, 0.5 ng of recombinant NF-Y;
lanes 2-13, CH27 nuclear extracts. Lanes 2 and
9, no competitor; lanes 3 and 4 in
A and lanes 5 and 6 in
B, 20- and 100-fold excess of cold competitor HSP70
oligonucleotide; lanes 5 and 6 in A
and lanes 3 and 4 in B, 20- and
100-fold excess of cold competitor HSP40 oligonucleotide. Lane
10, anti-NF-YA Mab7, lane 11, anti NF-YB, lane
12, anti-NF-YC, lane 13, anti-Gata1. C, TnT
expression of the control (Luciferase) and HSP-CBF labeled with
[35S]methionine. D, dose-response of in
vitro translated HSP-CBF (0.5, 1, and 2 µl in lanes
2-4 and 6-8) without (lanes 2-4) and with
0.5 ng of recombinant NF-Y (lanes 6-8). E, NF-Y
alone (lanes 1-5), or with 1 µl of HSP-CBF (lanes
6-10) on the HSP70 (upper panels) or HSP40
(lower panels) in different buffers. Buffer A: 50 mM NaCl, 5 mM MgCl2, 20 mM Tris HCl, pH 7.8, 0.5 mM EDTA, 5% glycerol.
Buffer B: 50 mM NaCl, 10 mM Tris HCl, pH 7.8, 1 mM EDTA, 5% glycerol. Buffer C: 50 mM NaCl, 5 mM MgCl2, 10 mM Tris HCl, pH 7.8, 5% glycerol. Buffer D: 5 mM MgCl2, 10 mM Tris HCl, pH 7.8, 0.5 mM EDTA, 5% glycerol.
Buffer E: 1 mM MgCl2, 10 mM Tris
HCl, pH 7.8, 0.5 mM EDTA, 5% glycerol. F,
antibody supershift of the NF-Y-HSP-CBF complex with anti-HSP-CBF
antibodies on the HSP70 CCAAT box. Lane 1, HSP-CBF with 600 ng of purified anti-HSP-CBF purified antibody; lane 2, same
with 600 ng of anti-Gata1 antibody; lanes 3 and 4, 200 and 600 ng of anti-HSP-CBF antibody on NF-Y and HSP-CBF; lane
5, same as lanes 3 and 4 except that the
control anti-Gata1 antibody was used.
117 to
+75 base pairs. The results show that only the anti-NF-Y antibody
immunoprecipitates chromatin containing the HSP70 promoter (Fig.
2A). This result mirrors the binding of NF-Y to other promoters of cell cycle-regulated
genes3. To rule out
unspecific effects of the ChIP assays performed with the anti-NF-YB
antibody, the same immunoprecipitate was also used to amplify DHFR, a
CCAAT-less cell cycle-regulated promoter; no amplification was observed
(Fig. 2B), which is consistent with the lack of NF-Y binding
sites on this promoter. These results provide in vivo
evidence for a specific retention of NF-Y to the HSP70 promoter,
indicating that this transcription factor plays a critical role in the
activation of the HSP70 gene.
View larger version (24K):
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Fig. 2.
NF-Y binds the HSP70 promoter in
vivo. Chromatin immunoprecipitation analysis of NF-Y
binding to HSP70 CCAAT box region in vivo. From the same
HeLa immunoprecipitates corresponding to the anti-NF-YB and anti-E2F1
(Santa Cruz) antibodies, DNA corresponding to the HSP70 (A)
and DHFR (B) were polymerase chain reaction amplified with
the indicated oligonucleotides.
/
SAOS2 were chosen because of the possible interfering role of p53 on HSP-CBF activation (37). As shown in Fig.
3A, HSP-CBF overexpression
leads to a 4-5-fold increase in promoter activity in COS cells. The
effect is strictly dependent upon the integrity of the CCAAT boxes
because a promoter containing mutations in the two CCAAT boxes,
HSP70mut in Fig. 3A, does not respond to HSP-CBF
overexpression. To establish the specific role of NF-Y in such system,
we cotransfected a highly diagnostic vector coding for the DNA
binding-defective NF-YAm29 mutant; the protein produced by this
construct acts in a dominant negative fashion associating the
NF-YB-NF-YC dimer and preventing the endogenous trimer to bind CCAAT
boxes (43). Under these conditions, CAT activity was not enhanced by
HSP-CBF. Next, we heat shocked COS cells cotransfected as above and
found that (i) the levels of CAT activity are, as expected, increased
upon heat treatment, an effect that requires intact CCAAT boxes (20,
44); (ii) HSP-CBF further increased overall activity; and (iii)
cotransfections with the dominant negative vector abolished the
positive effect of the heat shock and of HSP-CBF overexpression (Fig.
3B).
View larger version (18K):
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Fig. 3.
HSP-CBF transactivation of HSP70 and HSP40
promoters requires NF-Y. Transfections of COS cells A,
Xenopus wt or CCAAT-mutated HSP70 promoters fused to
the CAT reporter gene (250 ng) were assayed with 300 ng of HSP-CBF,
with or without 100 ng of NF-YAm29 dominant negative vector under basal
or heat shocked conditions. B, same as A except
that 200 ng of HSP40 Luciferase gene (21) was used. The data refer to
the average fold induction (similar data were obtained in SAOS2
cells). Standard deviations are indicated by error
bars.
227 to +30, which contains two
CCAAT boxes at
100 and
220 and an HSE at -70. This promoter is
inducible upon heat shock at 42 °C in HeLa cells (21). Again, we
observed a robust effect of HSP-CBF, both under basal and after heat
treatment, and a negative effect of cotransfecting the dominant
negative vector (Fig. 3B). The same experiments were performed in SAOS2 cells, and we obtained equivalent results on both
promoters (data not shown). From this set of experiments, we conclude
that (i) HSP-CBF is an activator of HSP transcription beyond the HSP70
system and (ii) that the integrity of the CCAAT box and a functional
DNA binding NF-Y trimer are required.
View larger version (15K):
[in a new window]
Fig. 4.
HSP-CBF is not a general coactivator.
The DHFR, Ea, TK, and CyclinE reporters were transiently transfected in
COS cells alone or together with 100 ng, 300 ng, 1 µg of HSP-CBF. The
average activities are shown and standard deviations were <20%.
View larger version (53K):
[in a new window]
Fig. 5.
NF-Y-HSP-CBF protein-protein interactions
in vitro. Immunoprecipitations of recombinant
proteins with the anti-NF-YA Mab7 monoclonal antibody or with a control
anti-Gata1 are shown. L, load material;
FT, follow-through, unbound proteins; B, bound
material. A, recombinant NF-Y trimer and the wt or the
indicated HSP-CBF mutants were used. Immunoblots were performed with
anti-HSP-CBF and anti-NF-YB antibodies. B, we used the
separated NF-YA (upper panel) or NF-YB-NF-YC dimer
(lower panel) with Mab7 and anti-NF-YB antibodies,
respectively.
Trp
Ade
His
plates,
robust growth was observed of the double transformants containing
pACT-NF-YB and pAS2.1-CBFN but not of the transformants containing
either plasmids with the empty counterpart or of the single plasmids
(Fig. 6). The resulting colonies are
white, an indication of adenine auxotrophy, and positive in
-galactopyranoside assays (data not shown). This proves that a
functional interaction occurs between NF-YB and HSP-CBF. It should be
noted that the yeast S. cerevisiae does contain NF-Y
homologs, the HAP2/3/5 complex involved in activation of cytochrome
genes (Ref. 47 and references therein), and that the yeast subunits can
associate the mammalian homologs (48). Thus we cannot exclude the
possibility that the overexpressed mouse NF-YB is in reality complexed
with the yeast HAP2/5 and present as a trimeric complex; in this case
HSP-CBF would interact with a hybrid trimer in yeast. Overall, these
data are in agreement with the in vitro
immunoprecipitations, and we can conclude that HSP-CBF and NF-Y can
form complexes in solution both in vitro and in
vivo.
View larger version (27K):
[in a new window]
Fig. 6.
NF-YB interacts with HSP-CBF in yeast.
Yeast strain PJ69-4A was transformed with the indicated plasmids and
grown in a Ade-Leu-Trp-His- minimal plate in the presence of 1 mM 3-AT.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin, clearly rule out that HSP-CBF
is an NF-Y-specific coactivator because many NF-Y-dependent
promoters are not responsive to HSP-CBF. A third class of coactivators
is gene-specific; a prototypical example is represented by CIITA, the
master switch regulator of genes involved in antigen presentation (51).
CIITA does not bind DNA directly, but it recognizes a nucleoprotein
platform composed of RFX, CREB, and NF-Y that binds to the conserved
S-X-X2-Y boxes in all genes concerned. Upon overexpression in cells, it
has some affinity for the single factors, NF-Y and RFX, but it is
incapable of associating either factor in DNA-binding assays. It
contains a highly acidic N-terminal part that is essential for
transcriptional activation. All these features are highly reminiscent
of the HSP-CBF activities described here and in previous studies.
View larger version (26K):
[in a new window]
Fig. 7.
Possible mechanisms of HSP-CBF-mediated
activation.
Finally, results presented here have wider implications that
potentially apply to other systems; HSPs, in fact, are but one category
of genes that are up-regulated by an increase in temperature. MDR1 and
Erp72/Grp-78 families are also activated, but in a HSE-independent manner. Grp78 is evolutionarily related to HSPs and belongs to the
chaperone endoplasmic reticulum proteins involved in the unfolded protein response (54-56). Genes coding for such proteins, which include Grp94, calreticulin, protein disulfide isomerase, and Herp are
activated through a conserved composite promoter sequence termed ERSE
(54-60). Molecular dissection of ERSEs have detailed that they contain
a CCAAT box, and indeed heavily rely on NF-Y for their function
(52-54), and also contain an ATF6 binding sequence spaced by a linker
of variable sequence and conserved distance (58, 60). It has been
proposed that ATF6, a DNA-binding transcription factor normally
residing in the endoplasmic reticulum, is cleaved upon endoplasmic
reticulum stress; the resulting product unmasks a hidden
activation domain that upon binding of ATF6 close to NF-Y strongly
activates transcription. Cooperative binding between the two activators
on DNA is essential (58). An intriguing possibility is that HSP-CBF
might serve as a more general coactivator of a cellular "stress
response" by functioning also on the ATF6-NF-Y couple. The reagents
and assays developed here will help elucidate these points.
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ACKNOWLEDGEMENTS |
---|
We thank the following scientists for the gifts of reporter plasmids: K. Ohtsuka (Aichi Cancer Center, Nagoya, Japan) for HSP40-Luciferase; N. Landsberger (Univ. Insubria, Varese, Italy) for HSP70-CAT; P. Farnham (Univ. Wisconsin, WI) for DHFR-luciferase; and E. Wintersberger (Univ. Vienna, Austria) for TK-luciferase. We particularly thank D. Linzer (Northwestern Univ., Evanston, IL) for the gift of HSP-CBF vectors and antisera.
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FOOTNOTES |
---|
* 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.
Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M101553200
2 R. Mantovani, unpublished data.
3 A. Gurtner and G. Piaggio, submitted for publication.
4 C. Imbriano and R. Mantovai, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HSP, heat shock protein; HSF, heat shock transcription factor; HSE, heat shock element; wt, wild type; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; TnT, troponin T; ChIP, chromatin immunoprecipitation assay.
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