From the Institute of Molecular Medicine and Genetics and Department of Radiology, Medical College of Georgia, Augusta, Georgia 30912
Received for publication, October 10, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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The heat shock transcription factors (HSFs)
regulate the expression of heat shock proteins (hsps), which are
critical for normal cellular proliferation and differentiation. One of
the HSFs, HSF-4, contains two alternative splice variants, one of which
possesses transcriptional repressor properties in vivo. This repressor isoform inhibits basal transcription of hsps 27 and 90 in tissue culture cells. The molecular mechanisms of HSF-4a isoform-mediated transcriptional repression is unknown. Here, we
present evidence that HSF-4a inhibits basal transcription in vivo when it is artificially targeted to basal promoters via the DNA-binding domain of the yeast transcription factor, GAL4. By using a
highly purified, reconstituted in vitro transcription system, we show that HSF-4a represses basal transcription at an early
step during preinitiation complex assembly, as pre-assembled preinitiation complexes are refractory to the inhibitory effect on
transcription. This repression occurs by the HSF-4a isoform, but not by
the HSF-4b isoform, which we show is capable of activating transcription from a heat shock element-driven promoter in
vitro. The repression of basal transcription by HSF-4a occurs
through interaction with the basal transcription factor TFIIF. TFIIF
interacts with a segment of HSF-4a that is required for the
trimerization of HSF-4a, and deletion of this segment no longer
inhibits basal transcription. These studies suggest that HSF-4a
inhibits basal transcription both in vivo and in
vitro. Furthermore, this is the first report identifying an
interaction between a transcriptional repressor with the basal
transcription factor TFIIF.
In mammalian cells, three heat shock transcription factors, HSF-1,
-2, and -4, have been isolated. These factors share high levels of
sequence homology in their DNA-binding domains and hydrophobic heptad
repeats (1-5). Various HSF1
family members are expressed as at least two isoforms, and the precise
function of the different HSF isoforms is unclear; however, they may
play a regulatory role in heat shock element (HSE)-driven transcription
(1, 6). The recently cloned human HSF-4 is structurally different from
other members of the HSF family. Alternative splicing between the two
isoforms, namely HSF-4a and -b introduces a frameshift leading to a
smaller HSF-4a protein (463 versus 493 amino acid residues)
(7). The HSF-4a contains a DNA-binding domain and the N-terminal
hydrophobic heptad repeats, but it lacks a transcriptional activation
domain (2, 7). This suggests that this HSF-4 isoform may act as a
repressor of other HSFs through its ability to bind either directly to
the HSE or to oligomerize with other members of the family. Cells
expressing exogenous HSF-4a protein exhibit lower levels of basal and
inducible heat shock protein expression
(2).2 The HSF-4b protein
appears to be a relatively weak transcriptional activator after heat
shock, when compared with other members of the HSF family, such as
HSF-1 (7). Interestingly, HSF-4b contains putative mitogen-activated
protein kinase phosphorylation sites, which are absent from the HSF-4a
isoform. The HSF-4 is expressed in many tissues in human and mouse, but
as with other HSF family members, the molecular function, as well as
the ratio of expression of the different isoforms in different tissues
and at different times, has not been clearly determined.
Transcriptional repressors play an important role in regulation of gene
expression, particularly during development, differentiation, and cell
growth (8, 9). Of several major classes of repressors, one class
consists of DNA-binding proteins that repress transcription via active
repression. The targets of such repressors can be activator or
co-activator proteins, co-repressors, or proteins that interact with
basal transcription factors or GTFs. The repressors that repress
activators or co-activators can only inhibit a limited number of
promoters (9). The YY1 protein is an example of one such repressor; it
represses the fos promoter and requires a DNA-binding site for YY1 as well as a proximal cyclic AMP response element (10).
Those repressors that interact with co-repressors exert their effect
via interaction of the co-repressor with the transcriptional machinery.
Examples of such co-repressors are mSin3A and mSin3B, which interact
with Mad and MxiI, which in turn are transcriptional repressors when
they dimerize with other members of the c-Myc family (11, 12). The Mad
proteins contain a motif that interacts with a paired amphipathic helix
3 present in Sin3 proteins. Recruitment of Sin3, the transcriptional
corepressor N-Cor, and a histone deacetylase is how Mad proteins are
thought to suppress transcription (13, 14). Finally, the repressors
that interact with basal transcription factors repress a minimal RNA
polymerase II promoter containing an initiator element or a TATA box
(9, 15-17). The Drosophila homeodomain protein Eve and the
DNA-binding protein Kruppel (KR) are capable of repressing
basal transcription by binding to general transcription factors TBP and
TFIIE, respectively (18, 19). Other repressors such as the
Drosophila Dorsal Switch Protein (DSP1) act as a
transcriptional repressor for several activator families in
vitro. DSP1 binds directly to the TATA-binding protein (TBP)
complexes containing TFIIA and displaces TFIIA from binding to TBP
(20). Another repressor that has been shown to interact with basal
factors is the retinoblastoma tumor suppressor protein, which represses
the activation of transcription mediated by E2F by preventing
TFIIA/TFIID from entering the preinitiation complex (21).
Transcriptional initiation by RNA polymerase II in an in
vitro transcription system occurs in the presence of five general transcription factors, namely TFIIB, TFIID, TFIIE, TFIIF, and TFIIH.
The initiation of transcription occurs following the assembly of the
preinitiation complex (PIC), which consists of RNA polymerase II and
general transcription factors binding to a promoter (22-25). For those
genes that contain a consensus TATA element, the assembly of factors
begins with the binding of TBP, which is the TATA box-binding protein
and is a subunit of TFIID. Complex formation is completed by the
assembly of other general transcription factors and RNA polymerase II
(22-26).
The basal transcription factor TFIIF is a heterotetramer of RNA
polymerase II-associating protein RAP74 and RAP30 subunits (27). TFIIF
is involved in both the initiation and elongation stages of
transcription and has been shown to be essential for transcription of
all RNA polymerase II promoters that have been examined (28). The RAP30
subunit of TFIIF is involved in the recruitment of RNA polymerase II to
promoter-bound TBP and TFIIB. TFIIF has been shown to bind directly to
other basal factors, TFIID, TFIIE, and TFIIB as well as RNA polymerase
II. TFIIB and RNA polymerase II binding occurs at conserved region III
of RAP74. The binding of TAF250 and RAP30 to RAP74 occurs at conserved
region I (29). Transcriptional activators, such as serum response
factor (SRF), have been shown to bind RAP74 in the middle of the
molecule, which encodes conserved region II (30). On promoters such as adenovirus major late promoter, the RAP74 subunit helps to wrap the DNA
approximately one turn around the general transcription factors and RNA
polymerase II. The TFIIF transcription factor may have a role in
isomerization of the preinitiation complex, resulting in helix
untwisting before the open complex is formed (31). TFIIF is proposed to
play a role in recruitment, isomerization, initiation, and stimulation
of elongation by RNA polymerase II. The RAP30 component of TFIIF can
enhance the assembly of RNA polymerase II into the initiation complex,
and RAP74 binding to the initiation complex will allow RNA polymerase
II to make promoter contact (29, 31, 32).
We report here studies on the molecular mechanisms of transcriptional
repression by the HSF-4a isoform, using both in vivo and
cell-free transcription assays. Our findings indicate that the target
of HSF-4a repression is the basal transcription machinery, and the
repression occurs through inhibition of the early step in PIC assembly.
We further show that the basal transcription factor TFIIF is the
specific target of HSF-4a-mediated repression. This repression occurs
through the interaction of TFIIF with HSF-4a, and deletion of amino
acid residues 124-194 from HSF-4a renders the protein incapable of
inhibiting basal transcription.
Cell Culture--
H1299 and HeLa cells are derived from human
lung and cervical carcinomas, respectively, and were purchased from
American Type Culture Collection (ATCC). These cells were maintained in
Dulbecco's minimal essential medium, supplemented with 10% fetal calf serum.
Plasmids--
For in vitro transcription assays,
three different G-less cassette constructs were used as follows: one
promoter was 2× HSEs upstream of the core heat shock promoter, another
had the HSEs removed, and the other contained the complete adenovirus
major late promoter (MLP). These plasmids were prepared as follows. For
the G-less cassette containing two HSEs, the previously reported plasmids pHSP
GAL4-HSF-4a deletion mutants were constructed as follows: using the
HSF-4a cDNA as a template, different fragments were amplified using
primers that incorporated an EcoRI site at the 5'-terminal end and a HindIII site at the 3'-terminal end of the HSF-4a
cDNA. The fragments were subcloned in-frame into pSG424 downstream
of the GAL4-(1-147) DNA-binding domain (34, 35). The sequences of the primers are as follows: HSF-4 R1373HindIII EcoRI,
5'-GGAATTCCCA-AGCTTAGGGGGAGGGACTGGCTTCCGG-3'; HSF4F582EcoRI,
5'-GGAATTCTTT-GGGCCACTTCAGGCGGGGCCG-3'; HSF4F811 EcoRI, 5'-GGAATTCACATCCCAGA-AGACTCTCCATCCC-3';
HSF4F1110EcoRI, 5'-GGAATTCCTAGATGTGC-TGGGCCCCAGT-3; and HSF4
1242EcoRI, 5'-GGAATTCAAGGACCCCACGCTCGGGGCC-3'.
The N-terminal His6-tagged HSF-4a deletion mutants
were constructed with the same primers as above, except that the
restriction enzyme recognition sites incorporated were NheI
and HindIII at the 5'- and 3'-ends of HSF-4a cDNA,
respectively. The fragments were inserted into pET28b vector (Novagen,
Madison, WI), and their nucleotide sequences were confirmed by
automated DNA sequencing. The human HSF-4a cDNA was the gift of Dr.
A. Nakai (Kyoto University, Japan). The human HSF-4b isoform was
amplified by polymerase chain reaction using cDNA obtained from
normal human skeletal muscle cells (BioWhittaker, Inc.). The reporter
plasmids, GAL4-TK-CAT and GAL4-MLP-CAT, were obtained from Dr. D. Dean
(Washington University, St. Louis) (36).
Transient Transfection Assays--
Transient transfections were
performed by calcium phosphate precipitation technique or by
LipofectAMINE (Life Technologies, Inc.). Transfected DNA mixes included
2 µg of expression plasmid DNA and, where indicated, 1.5 µg of
GAL4-TK-CAT or GAL4-MLP-CAT DNA, and 0.1 µg of firefly luciferase DNA
with pBluescript carrier DNA added to a total of 4 µg. The DNA mixes
were added to 0.5 to 1 × 106 cells. Forty eight hours
after transfection, cells were lysed, and firefly luciferase activity
was determined according to the manufacturer's instruction (Promega,
Madison, WI). CAT activity was determined by chromatography or
enzyme-linked immunosorbent assay (Roche Molecular Biochemicals). The
activity of firefly luciferase in which a cytomegalovirus promoter
drives its constitutive expression was used as an indicator of
transfection frequency (37, 38). All transient transfection experiments
were performed at least 3 times with both H1299 and HeLa cells, and
results were consistent.
Electrophoretic Mobility Shift Assays--
Electrophoretic
mobility shift assays using whole cell extracts have been described in
detail previously (38-40). Briefly, after each treatment, cells were
rinsed with phosphate-buffered saline and lysed in 100 µl of
extraction buffer (10 mM HEPES (pH 7.9), 0.4 mM
NaCl, 0.1 mM EDTA, 0.5 mM dithiothreitol, 5%
glycerol, 0.5 mM phenylmethylsulfonyl fluoride). The
protein concentration of samples was estimated by the bicinchoninic
acid method (Pierce). Equal amounts of protein (10 µg) in extraction
buffer (volume not exceeding 15 µl) were added to the reaction
mixture, which contained 4 µl of binding buffer (37.5 mM
NaCl, 15 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol), 10 µg of poly(dI-dC),
and 1 ng of 32P-labeled GAL4 oligonucleotide (see below)).
The mixture was incubated for 15 min at 25 °C and resolved on a
4.5% nondenaturing polyacrylamide gel. After electrophoresis, gels
were fixed in 7% (v/v) acetic acid for 5 min, rinsed once in distilled
water, dried under vacuum, and exposed to x-ray film. The
double-stranded oligonucleotide containing the GAL4-binding site was as
follow: 5'-GGGATCTCGGA GTACTGTCCTCCGA-3'and
5'-GGTCGGAGGACAGTACTCCGAGAT-3' (41). The oligonucleotide was labeled
using Klenow fragment of DNA polymerase I, deoxynucleotide
triphosphates, and [ Purification of Components of the in Vitro Transcription
System--
All proteins required for in vitro
transcription were purified in HE buffer (25 mM HEPES (pH
7.9), 1 mM EDTA, 0.1% Nonidet P-40, 15% glycerol, 4 mM 2-mercaptoethanol, 10 µg of phenylmethylsulfonyl fluoride with the appropriate amount of KCl) and have been described previously (42). Recombinant full-length and HSF-4a deletion mutants
were His6-tagged and were purified using
Ni2+-chelate nitrilotriacetic acid chromatography using
denaturing or non-denaturing conditions and extensively dialyzed
against 0.1× HE buffer (37).
In Vitro Transcription Assays--
In vitro
transcription assays were carried out using methods described
previously with several modifications (42). For each 50-µl assay,
purified factors were added in the following approximate amounts: 15 ng
of TFIIA, 7 ng of TFIIB, 50 ng of 56-kDa TFIIE, 30 ng of 34-kDa TFIIE,
50 ng of the complex of 30- and 74-kDa TFIIF, 1-2 ng of TBP in eTFIID,
and 50-100 ng of RNA polymerase II, with additional factors added as
indicated in figure legends. Reactions were carried out in 0.5× HE
buffer (0.05 M KCl, 12.5 mM HEPES (pH 7.9), 0.5 mM EDTA, 0.05% Nonidet P-40, 7.5% glycerol, 2 mM 2-mercaptoethanol, 5 µg of phenylmethylsulfonyl
fluoride/ml) supplemented with 3.4 mM MgCl2.
250 ng of supercoiled, G-less cassette promoter construct was then
added for 30 min to allow formation of PICs. Nucleotide triphosphates,
ATP, UTP, CTP (and [ Protein-Protein Interactions, Immunoprecipitation, and
Immunoblotting--
To detect the interactions of HSF-4a and TFIIF
in vitro, the recombinant His6-tagged HSF-4a
protein was purified and dialyzed extensively against the 0.1× HE
buffer. Purification of TFIIF has been described previously (42). 150 ng of the purified recombinant HSF-4a and 125 ng of recombinant TFIIF
were mixed and incubated at 30 °C for 30 min. The complexes were
then immunoprecipitated using standard protocols (37). Antibody used in
the immunoprecipitation experiments was against the large subunit of
TFIIF, RAP74 (Santa Cruz Biotechnology). The immunoprecipitated
materials were immunoblotted as described previously (37).
HSF-4a Inhibits Transcription via Basal Promoter Elements in
Vivo--
The HSF-4a isoform has been shown to inhibit basal
expression of heat shock protein 90 and 27 in tissue culture cell lines (7). This isoform of HSF-4 also inhibits the heat inducibility of
reporter plasmids containing an HSE.2 These results and the
fact that this HSF-4 isoform has a DNA-binding domain, but lacks a
transcriptional activation domain, suggests that it could be an active
transcriptional repressor (9). To determine whether HSF-4a inhibits
transcription from basal promoters as other active repressors do, we
constructed a series of chimeric proteins in which the HSF-4a
DNA-binding domain was replaced by a yeast GAL-4 transcription factor.
In addition, the remainder of the HSF-4a protein was truncated as shown
in Fig. 1A. These constructs
included the remaining regions of HSF-4a, which included amino acid
residues 124-463 (construct 2 in Fig. 1A);
residues 194-463 (construct 3 in Fig. 1A);
residues 270-463 (construct 4 in Fig. 1A);
366-463, and residues 414-463 (constructs 5 and 6, respectively, in Fig. 1A). The ability of each
construct to bind to the GAL4-binding site was determined using
electrophoretic mobility shift assays following the transient
transfection of each construct into H1299 cells. All constructs were
capable of binding to an oligonucleotide containing a GAL-4-binding
site (Fig. 1B). The expression constructs shown in Fig.
1A were co-transfected with reporter plasmids GAL4-MLP-CAT
or GAL4-TK-CAT into H1299 cells and 48 h following transfection,
and CAT activity for each construct was determined. Results indicate
that the GAL4-HSF-4a construct containing amino acid residues 124-463
repressed the expression of both thymidine kinase and MLP basal
promoters by more than 5-fold when targeted to these promoters (Fig. 1,
C-E). HSF-4a repression of transcription of these basal
promoters was less pronounced in the more severe GAL4-HSF-4a deletion
mutants. There was no reduction in CAT expression when cells were
co-transfected with plasmid construct pSG424, which contained the
GAL4-(1-147) DNA-binding domain (construct 1), when
compared with transfection of cells with reporter genes alone.
The results of these experiments suggest that sequences encoding the
amino acid residues 124-194 of HSF-4a protein are required for
repression of basal transcription. This region encodes the N-terminal
hydrophobic heptad repeats (HHR in Fig. 1A).
To ensure that the inhibition of basal transcription that was observed
in the presence of GAL4-HSF-4a (Fig. 1) was not due to nonspecific
squelching, the following experiment was performed. Reporter plasmids
GAL4-TK-CAT or GAL4-MLP-CAT containing GAL4-binding sites were
co-transfected with or without an expression construct containing
full-length (hemagglutinin)-tagged HSF-4a. This HSF-4a protein contains
a DNA-binding domain that requires heat shock element for binding and,
therefore, is unable to bind to the GAL4-binding sites present in the
reporter constructs. However, HSF-4a would be able to bind to other
factors present in the cell and cause nonspecific squelching of
transcription of these reporter constructs. 48 h after
transfection, CAT expression was determined. The results indicate that
there was no inhibition of basal transcription driving GAL4-MLP-CAT or
GAL4-TK-CAT by HSF-4a if the protein was not targeted specifically to
these promoters (Fig. 2).
HSF-4a Inhibits Basal Transcription in an in Vitro Model System
Prior to the Formation of the Preinitiation Complex--
Both the
thymidine kinase and adenovirus major late promoters used in the
previous experiments are minimal basal promoters containing a consensus
TATA element. For transcription, these promoters have been shown to
require RNA polymerase II and the so-called "basal" transcription
factors (also known as general transcription factors, or GTFs), which
include the TBP (TATA-binding polypeptide) subunit of TFIID and TFIIA,
TFIIB, TFIIE, TFIIF, and TFIIH (24). To explore the mechanisms of
HSF-4a repression of basal transcription, we used a highly purified,
reconstituted in vitro transcription system (42). These
reactions contain affinity-purified RNA polymerase II, recombinant
TFIIB, and other factors such as TFIIA, TFIID, TFIIE, TFIIF, and TFIIH
purified from HeLa cell nuclear fractions. GTFs and a G-less cassette
containing an hsp 70 promoter with two HSEs were incubated for 30 min
at 30 °C with increasing concentrations of purified HSF-4a protein. This incubation step allows the formation of the PICs (42). Nucleotides
were then added (see Fig. 3A
for a schematic), and reactions were allowed to proceed for an
additional 45 min at 30 °C and then terminated. As shown in Fig.
3B, additions of 30, 60, or 120 ng of purified
recombinant HSF-4a protein to the transcription reaction inhibited
basal transcription in a concentration-dependent manner. The
addition of 120 ng of purified HSF-4a protein inhibited transcription
by more than 5-fold.
We then asked whether HSF-4a could inhibit basal transcription if it
was added after the assembly of the preinitiation complex on the DNA.
For this, GTFs were allowed to incubate with the DNA template for 30 min at 30 °C in the absence of nucleotides. Following this
incubation period, HSF-4a and nucleotide triphosphates were added;
transcription was allowed to proceed for 45 min at 30 °C, and
samples were analyzed. No inhibition of basal transcription by HSF-4a
was detected under these conditions (Fig. 3C). The results of these experiments suggest that HSF-4a inhibits basal transcription at an early step during the PIC assembly.
HSF-4a Inhibits in Vitro Transcription from Promoters with No
HSE--
We and others (Ref. 2 and data not shown) have shown that
recombinant or in vitro translated HSF-4a protein can bind
constitutively to the HSE. To test whether HSF-4a binding to the HSE
was required for its inhibition of basal transcription, two additional
constructs lacking HSEs were tested. One was identical to the HSE
construct used in Fig. 3 except the HSEs were removed, and the other
was a construct with the adenovirus major late core promoter. For these
experiments, GTFs were allowed to incubate with DNA, with or without
120 ng of purified HSF-4a protein, for 30 min at 30 °C before the
transcription reaction was allowed to proceed at 30 °C for 45 min
(see Fig. 4A for schematic).
The results indicate that HSF-4a inhibited transcription from all
promoters used (compare lanes 1 and 2, Fig. 4,
B-D). As with the HSE-containing promoter, no effect was
seen when 120 ng of HSF-4a was added after the preinitiation complex
had already formed (lane 3, Fig. 4, B-D).
Although we consistently obtained a greater inhibition of the basal
transcription when the template DNA contained HSEs (Fig.
4B), HSF-4a could still inhibit transcription of the non-HSE
containing DNA templates (Fig. 4, C and D). One
explanation is that HSF-4a binds directly to one or more components of
the PIC and prevents the proper assembly of the complex on the TATA
element. Thus, the presence of the HSE is not an absolute
requirement.
Promoter-bound HSF-4a Inhibits Transcription--
The fact that
HSF-4a could potentially bind to a component of the basal transcription
machinery and inhibit in vitro transcription would be
reasonable if we assumed that the factors are more easily accessible to
each other while in a test tube, but that in vivo the HSE is
critical for targeting HSF-4a to the promoter. To explore if the
fraction of HSF-4a that is bound to the HSE is indeed inhibitory to
in vitro basal transcription, we performed the following
experiment. A biotinylated DNA fragment (1.2 µg or 0.7 pmol final
concentration) containing 2 HSEs bound to streptavidin-coated beads was
incubated with full-length HSF-4a (400 ng or 2.6 pmol final
concentration) or with the same amounts of truncated HSF-4a lacking the
DNA-binding domain at 25 °C for 20 min. The mixture was then rinsed
to remove excess HSF-4a, and basal transcription was performed as
above. The results show that only the full-length HSF-4a bound to the HSE is able to inhibit in vitro basal transcription (Fig.
4E, lanes 5 and 6 and 4F), whereas the
construct HSF-4a (residues 124-463), which does not bind to the HSE,
does not inhibit basal transcription (Fig. 4E, lanes
3 and 4, and F). The fraction of the
full-length and truncated HSF-4a that bound to the DNA beads is shown
in the immunoblot analysis in Fig. 4G. Our previous
experiments using gel mobility shift analysis to measure HSF-4a DNA
binding ability to HSE indicate that purified HSF-4a binds specifically to HSE maximally when it is preincubated at 4 or 25 °C, and its DNA-binding ability is reduced when it is preincubated at elevated temperatures.2
HSF-4a Interacts with the TFIIF Transcription Factor, Leading to
Inhibition of Basal Transcription--
Two observations suggested to
us that HSF-4a might be interacting with one of the GTFs and thus
inhibit transcription: 1) HSF-4a exerted its inhibitory effect
independent of the HSE, and 2) HSF-4a lost this inhibitory effect if
added after the PIC had already been formed. To address this question,
we performed in vitro transcription assays as outlined in
Fig. 5A. These assays were
designed to determine the minimal set of GTFs that were needed to make
the HSF-4a repression-resistant complex seen in Figs. 3 and 4. In this
experiment, selected GTFs were left out of the initial preincubation
step, allowing formation of the partial PICs. The "deleted" GTFs
were then added to the reaction, in the presence or absence of HSF-4a,
along with nucleotides, and transcription was allowed to proceed.
TFIIA, -B, and -D can form a stable complex on DNA (24). Preinitiation
complexes containing these factors were still susceptible to repression
by HSF-4a (Fig. 5, A and B). Addition of RNA
polymerase II and TFIIA, -B, -D did not significantly alter these
results (Fig. 5C). Significantly, when TFIIF was included (TFIIA, -B, and -D), RNA polymerase II, and TFIIF (Fig. 5D)
or TFIIA, -B, -D, -E, and -F and RNA polymerase II (Fig.
5E), transcription could no longer be repressed.
To test further TFIIF in HSF-4a-mediated repression, we determined
whether the presence of TFIIF was critical for the promoter that was
used in this in vitro transcription model. For this
experiment, the preincubation reaction included RNA polymerase II and
all the GTFs except for TFIIF. A 5-fold reduction in basal
transcription occurred when recombinant TFIIF was left out (Fig.
6, A-C, compare lane
2 to 1). We also asked whether addition of TFIIF to the
transcription reaction after a 30-min preincubation period would have
allowed transcription to take place, because the order of PIC assembly requires TFIIE and TFIIH to enter the complex after TFIIF. The results
show that the level of transcription was not affected by the late
addition of TFIIF to the reaction (Fig. 6, B and
C, compare lanes 3 to 1), and HSF-4a
was inhibitory to the transcription reaction (compare lanes
4 to 3).
From the data shown in Fig. 6, it therefore appeared that HSF-4a
inhibited basal transcription by interfering with TFIIF function. One
possible mechanism for the interference of TFIIF function is through a
direct interaction of HSF-4a with TFIIF. We therefore tested for an
interaction in pull-down experiments. Purified HSF-4a and TFIIF were
incubated at 30 °C for 30 min followed by immunoprecipitation using
antibody to RAP74, a component of TFIIF. Complexes were immunoblotted
with antibody to the His6 to detect the
co-immunoprecipitated His6-tagged HSF-4a protein. As shown
in Fig. 6D, HSF-4a specifically co-immunoprecipitated with
TFIIF (Fig. 6D, lane 3), suggesting that HSF-4a
interacts with a component of the TFIIF complex.
Another transcriptional activator that has been shown to interact with
TFIIF is SRF. Addition of SRF to the in vitro basal transcription system enhances transcription. However, increasing amounts of SRF leads to inhibition, possibly through a mechanism called
squelching (30). "Squelching" by SRF could be overcome by the
addition of TFIIF. To investigate whether adding TFIIF back to the
transcription reaction would rescue the inhibitory effect observed
following addition of HSF-4a to the basal transcription reaction,
experiments were performed where 0.26 pmol (40 ng) of HSF-4a trimers
was incubated with 0.36 pmol (75 ng) of TFIIF tetramers for 20 min at
25 °C. This mixture was then added to the standard GTF mixture which
also included TFIIF, and these factors were allowed to assemble on DNA
for 30 min before the onset of transcription reaction. The results
indicate that if sufficient amounts of TFIIF are prebound to HSF-4a,
HSF-4a no longer inhibits transcription (Fig. 6, E and
F).
The Domain of HSF-4a That Inhibits TFIIF Function Is Encoded by
Amino Acid Residues 124-194--
To determine the region of HSF-4a
that is responsible for interacting with TFIIF during transcription, a
series of His6-tagged HSF-4a deletion mutants were
constructed (Fig. 7A) and were
purified using Ni2+-chelate nitrilotriacetic acid
chromatography (Fig. 7B). These mutant proteins were then
added to the preincubation stage of the transcription reaction together
with GTFs (Fig. 7C). Only the full-length HSF-4a protein or
the HSF-4a deletion mutant containing amino acid residues 124-463 was
able to inhibit basal transcription 3-4-fold (Fig. 7, D and
E, constructs 1 and 2). Construct 3, containing amino acid residues 194-463, was slightly inhibitory (reduced transcription by 20-30%), but constructs containing amino acid residues 270-463 or 366-463 (constructs 4 and 5) did not inhibit the
in vitro transcription reaction (Fig. 7, D and
E). These results suggest that the region of HSF-4a that
interferes with transcription is encoded by amino acid residues 124 to
a few residues beyond amino acid residue 194, which includes the
N-terminal hydrophobic heptad repeat or leucine zippers 1-3
(N-terminal HHR in Fig. 7A). These findings are
consistent with the regions of HSF-4a required for repression of basal
transcription in vivo (Fig. 1).
The Transcriptional Activator HSF-4b Isoform Can Activate
HSE-driven Transcription in Vitro--
To determine if HSF-4a and
HSF-4b, which differ in part of their amino acid sequence, have the
opposite effect on HSE-driven transcription, we titrated HSF-4a or
HSF-4b to the in vitro transcription reactions (Fig.
8A). We observed that HSF-4a
was not able to activate transcription at any of the concentrations
tested (5-120 ng). Rather transcription was inhibited in the
30-120-ng range (Fig. 8B). In sharp contrast, HSF-4b
stimulated transcription at all the concentrations tested except at the
highest (120 ng per reaction). Under the same conditions of in
vitro transcription, addition of another HSF family member, HSF-1,
activates an HSE-driven transcription (Fig. 8C). These
results are consistent with our conclusion that HSF-4a is a
transcriptional inhibitor, and HSF-4b is a transcriptional activator.
HSF-4 has been detected in two isoforms. One isoform (HSF-4a)
appears to be a transcriptional repressor, because it contains a
conserved DNA-binding domain but lacks an activation domain. This
suggests that this isoform can bind an HSE without being able to
activate transcription. The second isoform, HSF-4b, which is predicted
to have differences in residues in the central domain of the protein
due to alternative splicing, can activate transcription (7). The HSF-4
transcription factor is expressed in several tissues in both mouse and
human as determined by Northern blots as well as polymerase chain
reaction analysis of cDNA obtained from human tissues
(7).2 HSF-4 binds an HSE constitutively but loses its DNA
binding activity upon a mild heat shock when it is synthesized in an
in vitro transcription/translation-coupled reaction
(7).2 However, the exact role of HSF-4 in transcriptional
regulation of heat shock proteins in vivo is not known.
In these studies, we analyzed the molecular mechanisms underlying
HSF-4a-mediated transcriptional repression. We found that HSF-4a
represses basal transcription when it is targeted artificially, through
fusion with a GAL4 DNA-binding domain, to basal promoters containing
GAL4-binding sites fused to the thymidine kinase or the adenovirus
major late promoters in vivo. Interestingly, other transcriptional repressors, such as Rb, inhibit specific basal promoters when targeted to them (36). Rb repression of basal promoters
occurs via two different mechanisms. One mechanism is dependent on
histone deacetylase activity. This is used for Rb repression of the
adenovirus MLP (36). The second mechanism, which is used with the SV-40
enhancer, is independent of histone deacetylase activity and occurs by
direct inhibition of transcription factors at the promoter (36).
Further studies using in vitro transcription models to
understand the molecular mechanism of Rb-mediated repression indicate
that Rb prevents the TFIID-TFIIA complex from contacting the consensus
TATA element (21). To understand in more detail how HSF-4a-mediated
repression of basal transcription occurs, we performed experiments
using purified HSF-4a and a purified in vitro transcription
system. We demonstrate here that HSF-4a can inhibit basal transcription
in this model system. HSF-4a inhibited transcription from core
promoters with a consensus TATA element without HSE, although greater
inhibitory activity was seen for promoters that contained HSE. This
suggests that HSF-4a repression of basal transcription occurs through
its interference with one of the factors involved in basal
transcription. By using order-of-addition experiments, we demonstrate
binding of HSF-4a to TFIIF. TFIIF is a complex of 74- and 30-kDa
subunits, and our preliminary results suggest that the larger RAP74
subunit directly interacts with HSF-4a (data not shown). The RAP74
subunit of TFIIF has been shown to interact with transcriptional
activators such as SRF and GTFs such as TAFII250, RAP30, TFIIB, and RNA
polymerase II (24, 30, 43). The activation domain of SRF, for example, associates with amino acid residues 172-357 of RAP74 (30). It has
therefore been suggested that the SRF interaction with TFIIF is
required for SRF-activated transcription. Furthermore, since TFIIF
binds RNA polymerase II through the RAP30 subunit, SRF could facilitate
the recruitment of RNA polymerase II to the promoter or alter the
conformation of the initiation complex (30). By using amino acid
deletion studies, we were able to identify the region of interaction of
TFIIF with HSF-4a. Deletion of N-terminal amino acid residues on the
C-terminal side of residue 194 exerts weak to no inhibition of
transcription. This suggests that the region that spans leucine zippers
1-3 of HSF-4a is the segment that interacts with TFIIF and prevents
its proper role in transcription. Because HSF-4a possesses a
DNA-binding domain, it is presumably an active repressor, that is it
could inhibit transcription by binding to specific sites on the DNA and
prevent other members of the HSF family to bind to the same sites. We
therefore hypothesize that HSF-4a may constitutively bind certain HSEs
under physiological conditions and prevent transcription via those
sites, perhaps by preventing the complete assembly of the preinitiation
complex. Alternatively, HSF-4a may bind certain HSEs after stress
stimuli and therefore prevent other HSFs from binding and activating
transcription. The fact that in the in vitro transcription
model HSF-4a inhibits TFIIF from entering the preinitiation complex
whether it is bound to HSE or not could be explained by two
possibilities. The first is that in vivo, HSF-4a can
function only as a repressor and prevent TFIIF from entering PIC when
HSF-4a is bound to HSE. Therefore, HSF-4a is targeted to specific
promoters. The second possibility is that HSF-4a is not abundantly
expressed in cells, but when it is expressed, it could bind and tie up
TFIIF and perhaps cause a general repression of transcription. HSF-4a
could conceivably have a short half-life, as it is not expressed
abundantly in many cell lines that we have tested (data not shown).
The HSF-4a interaction with TFIIF could inhibit the ability of TFIIF to
interact with other factors such as RNA polymerase II. HSF4a could
function in preventing the proper assembly of preinitiation complexes
as indicated by its inability to inhibit transcription from PICs that
already contain TFIIF. Since the HSF-4b isoform contains the leucine
zipper 1-3 region but also contains amino acid residues that differ
from that of the HSF-4a isoform, HSF-4b could potentially generate a
weaker interaction with TFIIF. Conversely, these differences in amino
acid residues between HSF-4a and HSF-4b may be critical for the
function of HSF-4b in its ability to activate transcription.
Interestingly, addition of HSF-4b to an in vitro
transcription reaction is capable of activating transcription in the
range of concentration for HSF-4a that was found to be repressive to
basal transcription. Since other members of the HSF family, namely
HSF-1 and HSF-2, also contain leucine zippers 1-3 and have high
homology to HSF-4b, it is conceivable that all HSF family members
potentially interact with TFIIF to exert their positive effect on
transcription. HSF-1 and HSF-2 also have other isoforms as well, but
their isoforms lack a whole exon, and in all cases the amino acid
sequence encoding the activation domain remains intact. More studies
are needed to investigate the role of these various HSF isoforms in the
regulation of heat shock protein gene expression in
vivo.
Extensive studies indicate that the PIC assembly pathway differs from
promoter to promoter and is dependent on both the specific elements
involved as well as the promoter context. The TFIIF transcription factor, however, has been shown to be required for all transcriptional activators so far tested (24). Nevertheless, the requirements for the
general transcription factors and the dependence of the HSE promoter
used was tested to ensure the requirement for TFIIF of such promoters.
We found that TFIIF was required for transcription from basal
promoters, since elimination of recombinant TFIIF from PIC assembly
significantly decreased basal transcription. From this and other
results presented, a hypothetical model is presented in Fig.
9 to describe our findings. Under the
conditions whereby HSF-4a binds an HSE, proper assembly of the
preinitiation complex is hindered or prevented, perhaps by interfering
with the recruitment of RNA polymerase II and other factors to the
complex through its association with TFIIF. HSF-4b, on the other hand,
could also potentially interact with TFIIF, but this interaction has a
positive, rather than a negative, regulatory role on transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
50HSE2, which contains two tandem heat shock elements (HSEs) followed by a TATA element, was used (33). The G-less cassette
with no HSE was similar to pHSP
50HSE2 but with no HSE. Both G-less
cassettes generated 190-bp sized transcripts. The MLP-containing G-less
cassette generated a 170-bp sized transcript (gift of Dr. D. Peterson,
Texas A & M University, Houston).
32P]dCTP. Antibody to GAL4
DNA-binding domain used in supershift experiments (10 µg of whole
cell extract incubated with 0.1 µg of antibody for 20 min at 25 °C
prior to further analysis) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA).
-32P]CTP), and
3'-O-methyl-GTP, were then added, and the reaction was
incubated for 45 min to allow transcription to proceed only through the
170- or 190-bp G-less region depending on the template. All reactions
were terminated by the addition of NaCl (133 mM final
concentration), SDS (to 0.5%), EDTA (10.5 mM final
concentration), Tris-Cl (pH 7.9) (3.3 mM final
concentration), and carrier tRNA (100 µg/ml). Reactions were then
extracted with phenol/chloroform, ethanol-precipitated, and separated
by urea, 5% polyacrylamide gel electrophoresis. Gels were dried,
quantitated, and analyzed by PhosphorImager using a linear scale IQMac
version 1.2 (42). In experiments where immobilized templates were used,
template consisted of the hsp 70 promoter fused to a 190-base pair
G-less cassette and was the gift of Dr. W. Dynan. Briefly, the
linearized DNA (XbaI digest) was biotinylated in a fill-in
reaction containing 1 unit/µg of Klenow fragment of Escherichia
coli DNA polymerase 1 and 0.025 mM of deoxynucleotides
and biotin-21-dUTP (CLONTECH). After 30 min at
30 °C, the product was digested with SphI to remove biotinylated end upstream of the hsp 70 promoter followed by spin column chromatography to remove any released and unincorporated materials. The biotinylated DNA was then incubated with
streptavidin-coated paramagnetic beads (Dynal, Dynabeads M-280) at 5 pmol of DNA/mg beads for 1 h at 43 °C in buffer containing 5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 M NaCl. At the end of the incubation period, the
supernatant was removed, and the DNA content was quantitated. The beads
were resuspended in water at a concentration of 40 µg of DNA/ml after
several washes in the above buffer as well as water.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HSF-4a inhibits transcription via basal
promoters. A, constructs showing deletion mutants of
HSF-4a that were fused to the DNA-binding domain of yeast transcription
factor GAL4-(1-147). Constructs are presented according to the amino
acid residues of HSF-4a present in the construct. DBD,
DNA-binding domain; HHR, hydrophobic heptad repeats (leucine
zippers 1-3). B, electrophoretic mobility shift assays of
constructs shown in A. 48 h after transfection, equal
amounts of cleared cell lysates were analyzed by gel mobility shift
assays. +Ab samples are lysates incubated in the presence of
antibody to GAL4 (1:20 dilution) for 20 min at 25 °C before
analysis. Panels 1-6 are constructs shown in A. Panel 7 is untransfected cell lysate. Panel 8 is
the cell lysate as in panel 3 but with 200× excess cold
oligonucleotide added. Nonspecific and specific indicate the presence
of a nonspecific band that is commonly seen with some mammalian cell
lysates using oligonucleotides containing GAL4-binding sites (41).
However, the GAL4 DNA-binding domain fragment encoded by plasmid PSG424
as well as the other small mutant proteins also run with the same
mobility (see Ab in lanes 5 and 6).
Note that larger fragments such as those shown in lanes
2-4 appear above the nonspecific band in the
Ab lanes. Antibody to GAL4 in +Ab
lanes are able to supershift all fusion proteins. The presence of
multiple bands in
Ab lanes most likely represent
multimers. C, CAT assays. Constructs 1-6 shown
in A were co-transfected into H1299 cells with the reporter
construct GAL4-MLP-CAT or GAL4-TK-CAT and firefly luciferase as an
indicator of transcriptional frequency. 48 h post-transfection,
cells were lysed, and CAT and luciferase activity was determined from
80 or 20 µg of protein, respectively. D and E,
quantitation of the data shown in C as well as data from
other experiments. Data are presented as % conversion of
[14C]chloramphenicol acetyltransferase to major
acetylated forms relative to internal control.
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Fig. 2.
HSF-4a inhibition of basal transcription
in vivo requires targeting to the promoter. H1299
cells were transiently transfected with GAL4-TK-CAT (TK) or
GAL4-MLP-CAT (MLP) with or without a full-length HSF-4a
cDNA subcloned into pcDNA3 expression vector.
A, 48 h after transfection, CAT expression was
determined in equal amounts of protein (150 µg) from cell lysates
using enzyme-linked immunosorbent assay. B, immunoblot
analysis of cell lysates showing the expression of HSF-4a in
transiently transfected cells. Primary antibody was to hemagglutinin
present in pcDNA3-HA-HSF-4a expression construct.
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Fig. 3.
HSF-4 inhibits basal transcription in
vitro at an early step during preinitiation complex
assembly. A, schematic of transcription reaction. As
indicated, GTFs and RNA polymerase II were allowed to incubate with an
HSE-containing promoter for 30 min at 30 °C. During this incubation
time, different concentrations of purified HSF-4a were added to
appropriate reactions as indicated. Nucleotides were then added, and
reactions were allowed to proceed for 45 min and then stopped. Samples
were processed as described under "Materials and Methods" and
analyzed by gel electrophoresis and quantitated by PhosphorImager.
B, top panel is a PhosphorImager analysis of a
representative transcription experiment. Lanes 1-4 indicate
the concentration of HSF-4a protein in each reaction being 0, 30, 60, and 120 ng of protein, respectively. The graph shows
quantitation by PhosphorImager of the experiment shown, as well as data
from other experiments. C, similar to B but GTFs
were allowed to incubate with DNA template at 30 °C for 30 min
before nucleotides and 120 ng of HSF-4a were added to the reaction.
Lanes 1 and 2 indicate reactions without or with
the addition of 120 ng of HSF-4a protein, respectively.
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Fig. 4.
HSF-4a inhibits basal transcription with
several promoters. A, schematic of transcription
reaction. Indicated GTFs and template DNA with or without HSF-4a were
incubated at 30 °C for 30 min. Nucleotides and HSF-4a were then
added, and the reaction was allowed to proceed for 45 min. Reactions
were then stopped, and samples were processed as described under
"Materials and Methods," analyzed by gel electrophoresis, and
quantitated by PhosphorImager. B, template DNA contained 2 HSEs. C, template DNA contained no HSEs. D,
template DNA was MLP. Lanes 1-3 are as follows:
1, GTFs and nucleotides, without HSF-4a; 2, GTFs
and nucleotides with the addition of 120 ng of HSF-4a; 3, HSF-4a added to the reaction after the formation of the preinitiation
complex assembly. E, DNA beads were incubated with HSF-4a
protein in 70 µl reaction. After 20 min at 25 °C, beads were
washed 4 times with 200 µl of 0.5× HE buffer. Transcription was then
carried out in duplicate by the addition of various components of
in vitro transcription. Products were analyzed as above.
Lanes 1 and 2 indicate DNA beads only;
lanes 3 and 4, DNA beads plus HSF-4a (residues
124-463); lanes 5 and 6, DNA beads plus HSF-4a
(residues 1-463). F, quantitation of the data shown in
E as well as in other experiments. G, immunoblot
analysis showing the fraction of HSF-4a that bound to HSE beads after
HSF-4a was incubated with HSE containing DNA beads. Lanes 1 and 2, input full-length HSF-4a (residues 1-453) and
truncated HSF-4a (residues 124-463) that lacks DNA-binding domain,
respectively, to show the position of the two proteins. Lanes
3 and 4, fraction of full-length and truncated HSF-4a
bound to DNA, respectively. Lanes 5 and 6,
fraction of full-length and truncated HSF-4a that were not bound to DNA
and were present in the first wash, respectively.
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Fig. 5.
Order of addition transcription reactions
eliminates TFIIA, -B, -D, -E, -H and polymerase II as factors that may
be targeted by HSF-4a. A, schematic of transcription
reaction. Indicated GTFs and template DNA were incubated at 30 °C
for 30 min. Nucleotides and the remaining GTFs in the absence of 120 ng
of HSF-4a (lane 1 in B-E) or presence of 120 ng
of HSF-4a (lane 2 in B-E) were added to the
reactions, and the reactions were allowed to proceed for 45 min.
Reactions were then stopped, and samples were processed as described
under "Materials and Methods" and analyzed by gel electrophoresis
and quantitated by PhosphorImager. B, TFIIE/F/H/polymerase
II were not present during PIC assembly. C, TFIIE/F/H were
not present during PIC assembly. D, TFIIE/H were not present
during PIC assembly. E, TFIIH was not present during the PIC
assembly.
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Fig. 6.
TFIIF is the target of HSF-4-mediated
transcriptional repression. A, schematic of
transcription reaction. Indicated GTFs and template DNA were incubated
at 30 °C for 30 min. Nucleotides with or without HSF-4a were then
added to the reaction, which was allowed to proceed for 45 min. TFIIF
was added with or without HSF-4a during PIC assembly or after 30 min of
preincubation as indicated. Reactions were then stopped, and samples
were processed as in Fig. 4. B, transcription reactions.
Lane 1, GTFs with TFIIF added during PIC assembly.
Lane 2, GTFs without TFIIF during PIC assembly. Lane
3, GTFs were incubated for 30 min to assemble PICs without TFIIF,
which was added at 30 min at the onset of transcription reaction.
Lane 4, GTFs were incubated for 30 min to assemble PICs
without TFIIF which was then added at 30 min with HSF-4a at the onset
of transcription. C, quantitation of the data shown in
B and other experiments using PhosphorImager. D,
pull-down experiments showing HSF-4a interaction with TFIIF. TFIIF was
preincubated with purified histidine-tagged HSF-4a protein (30 min at
30 °C). Anti-RAP74 (large subunit of TFIIF) was then added to the
mixture, and following further incubation, protein A beads were added.
The co-immunoprecipitated materials were analyzed by SDS-polyacrylamide
gel electrophoresis and detected using antibody to His6 to
detect presence of recombinant HSF-4a. Lane 1, purified
HSF-4a protein in the input reaction. Lane 2, HSF-4a unbound
to protein A beads. Lane 3, HSF-4a/TFIIF
co-immunoprecipitates. Lane 4, immunoprecipitation
(IP) of HSF-4a without the addition of TFIIF to the
reaction. Lane 5, control reaction mixture containing
HSF-4a, TFIIF, and protein A but not using antibody to RAP-74 for
immunoprecipitation. E, HSF-4a bound to TFIIF added prior to
the assembly of the transcription components does not inhibit basal
transcription. HSF-4a was incubated for 20 min with TFIIF, and the
mixture was added to the components of the transcription that contained
TFIIF. Lane 1, transcription reaction containing all the
components. Lane 2, HSF-4a added to the transcription
reaction. Lane 3, same as lane 2 but preincubated
HSF-4a-TFIIF was added before the onset of the transcription.
F, quantitation of the data shown in E. Lane numbers correspond to that for E.
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Fig. 7.
TFIIF targets HSF-4a at a region encoding the
N-terminal hydrophobic heptad repeats. A, constructs
showing various deletion mutants of HSF-4a that were fused to the
His6 tag. Constructs are indicated according to the amino
acids of HSF-4a present in the construct. DBD, DNA-binding
domain; HHR, hydrophobic heptad repeats (N-terminal leucine
zippers). B, Coomassie Blue staining of HSF-4a deletion
mutants (constructs 1-5 shown in A). Molecular
weight markers are shown on the right. C,
schematic of transcription reaction. D, transcription
reaction contained no HSF-4a (lane indicated as ) or with HSF-4a wild
type (lane 1), HSF-4a with amino acid residues 124-463
(lane 2), HSF-4a with amino acid residues 194-463
(lane 3), HSF-4a with amino acid residues 270-463
(lane 4), HSF-4a with amino acid residues 366-463
(lane 5). E, quantitation of the results in
D as well as other experiments using PhosphorImager.
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Fig. 8.
HSF-4b isoform activates transcription.
A, schematic of transcription reaction. As indicated GTFs
and RNA polymerase II were allowed to incubate with HSE-containing
promoter for 30 min at 30 °C. During this incubation time, 5-120 ng
of purified HSF-4a or HSF-4b was added to appropriate reactions as
indicated. Nucleotides were then added, and reactions were allowed to
proceed for 45 min and then stopped. Samples were processed as
described under "Materials and Methods," analyzed by gel
electrophoresis, and quantitated by PhosphorImager. B, top
panel, representative gel showing transcription reactions.
0 indicates control without HSF-4a or HSF-4b. Bottom
panel, the gel shown here as well as data from other experiments
were quantitated using PhosphorImager. C, transcription was
performed as in A but with the addition of 100 ng of HSF-1.
0, indicates transcription without the addition of HSF-1 to
the reaction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
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Fig. 9.
Hypothetical model of HSF-4a repression of
HSE-driven transcription. A, HSF-4b is an activator of
transcription. B, HSF-4a binds and ties up TFIIF or HSF-4a
binds HSE and via interaction with TFIIF prevents the proper assembly
of the preinitiation complex.
In conclusion, we have presented evidence of an interaction of the
transcriptional repressor HSF-4a with TFIIF, leading to inhibition of
transcription. Future experiments will help to determine better the
details of the HSF-4a-TFIIF interaction and the mechanism by which
HSF-4a functions as transcriptional repressor.
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ACKNOWLEDGEMENTS |
---|
We thank the following investigators for providing many valuable materials: Dr. D. Dean for GAL4-MLP-CAT and GAL4-TK-CAT; Dr. Nakai for HSF-4a cDNA; Dr. Rhea-Beth Markowitz for critical reading of the manuscript.
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FOOTNOTES |
---|
* This work was supported by NCI Grants CA85947 and CA62130 from the National Institutes of Health.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.
To whom correspondence should be addressed: Institute of Molecular
Medicine and Genetics and Dept. of Radiology, Medical College of
Georgia, 1120, 15th St., CB2803, Augusta, GA 30912. Tel.: 706-721-8759; Fax: 706-721-8752; E-mail: mivechi@immag.mcg.edu.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M009224200
2 N. H. Mivechi, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: HSFs, heat shock transcription factors; hsps, heat shock proteins; HSE, heat shock element; TBP, TATA-binding protein; bp, base pair; PIC, preinitiation complex; GTF, general transcription factors; MLP, major late promoter; Ab, antibody; SRF, serum response factor; CAT, chloramphenicol acetyltransferase; Rb, retinoblastoma.
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