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INTRODUCTION |
The organization of chromatin structure is a fundamental, yet
extremely critical, component of gene regulation in all eukaryotic cells. Whether chromatin is transcriptionally active or repressed is
determined, at least in part, by the modification of nucleosomal histones. Of the many possible post-translational modifications of
histones, the most common are acetylation and deacetylation. In humans,
about 20 histone deacetylase
(HDAC)1 enzymes have been
identified whose functions are primarily to regulate the acetylation
status of histones and maintain regions of chromatin in
transcriptionally inactive states.
The first human histone deacetylase, HDAC1, was cloned by Schreiber and
colleagues (1) using a deacetylase inhibitor affinity matrix. The
second human histone deacetylase, HDAC2, was identified in our
laboratory in a yeast two-hybrid experiment with the YY1 transcription
factor as bait (2). HDAC1 and HDAC2 bear a high degree of sequence
homology to the yeast protein RPD3, and both enzymes typically coexist
in the same protein complexes.
The third human histone deacetylase, HDAC3, was cloned based on
sequence similarities to HDAC1 and HDAC2 (3-5). Unlike HDAC1/2, HDAC3
is present in a unique large multisubunit protein complex that contains
proteins different from those identified in HDAC1/2 complexes (6-9).
Also, unlike HDAC1 or HDAC2, HDAC3 is essential for cell viability and
is localized in both the nucleus and cytoplasm of cells (10-12).
Although HDAC3 clearly plays a crucial role in the regulation of gene
expression, the challenge has been to understand the exact mechanisms
by which HDAC3 modulates gene expression and affects the functions of
the transcriptional machinery. More importantly, there is a need to
address the functional differences between HDAC3 and HDAC1/2 and,
possibly, other HDACs.
The identification and characterization of HDAC1/2-binding proteins has
been tremendously useful in elucidating the mechanisms of action and
functions of these two HDACs. Following similar strategies, we isolated
an endogenous HDAC3-containing multisubunit complex using anti-HDAC3
immunoaffinity chromatography (9). Besides HDAC3, this complex also
contains NCoR/SMRT co-repressors plus several unidentified proteins.
In parallel, using anti-NCoR/SMRT combined with conventional
chromatography, four laboratories independently isolated complexes
containing NCoR/SMRT, HDAC3, the WD-40 repeat-containing protein TBL1,
SWI/SNF-related proteins, the co-repressor KAP-1, and multiple not-yet
characterized proteins (6-8, 13). We found that transcriptional
repression by NCoR is mediated by its interaction with HDAC3 and that
NCoR augments the enzymatic activity of HDAC3. Although these earlier
findings confirm and extend the concept that HDAC3 is a major player in
transcriptional repression, they raise several important questions.
What other proteins are present in the HDAC3 complex? Do these proteins
increase or antagonize HDAC3 activity? Does HDAC3 reciprocally modify
the function of these HDAC3-binding proteins?
We have now identified an additional component of the HDAC3 complex. We
show that the protein originally designated p130 (9) is the
transcription factor TFII-I (14, 15). TFII-I was earlier shown to be a
basal transcription factor that binds to and activates transcription
from the initiator element (16). Recent evidence suggests that TFII-I
is also an inducible multifunctional factor that selectively regulates
gene expression when activated by a variety of extracellular signals
(17).
In this report, we present evidence that TFII-I interacts specifically
with HDAC3, suggesting that HDAC3 may be a natural mediator of TFII-I
functions. Consistent with the ability of these two proteins to
interact, HDAC3 can also alter the transcriptional activity of
TFII-I.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The following plasmids have previously been
described: pGal4-VP16 (2), pGST-HDAC3 (3), expression plasmids for
HDAC3 deletion mutants (12), expression plasmids for various FLAG-HDACs (9, 18), pEDG and pEDG-TFII-I (19), pGal4TK-Luc (9), c-fos-
and V
-luciferase reporters (20), and pMcsrc (21).
pcDNA3-HA-TFII-I was constructed by subcloning TFII-I cDNA
downstream of the cytomegalovirus promoter and in-frame with the HA
sequence in pcDNA3.1 (Invitrogen). Plasmids that express HA-TFII-I deletions were constructed by digestion of pcDNA3-HA-TFII-I with various restriction enzymes, followed by religation. The plasmid expressing an enzymatic-deficient HDAC3 mutant (H134Q/H135A/A136S) was
generated using the Kunkel mutagenesis procedure as outlined in the
Muta-Gene system (Bio-Rad). All constructs were verified by DNA sequencing.
Immunochemical Reagents and Techniques--
Polyclonal
anti-TFII-I antibody has been described (22). Monoclonal anti-FLAG M2,
monoclonal anti-GST, and polyclonal anti-HA antibodies were obtained
from Sigma.
Immunoprecipitations were performed in a solution of PBS containing
0.1% Nonidet P-40 and protease inhibitors as previously described
(23). Immunocomplexes were washed six times with the same buffer, and
immunoprecipitated proteins were removed from protein A beads by either
boiling in gel loading buffer or by elution with excess peptide
antigens. For Western blot analyses, proteins were resolved on
SDS-polyacrylamide gels and transferred to polyvinylidene difluoride
membranes. After blocking with nonfat dried milk, the membranes were
treated with diluted primary antibodies, followed by diluted alkaline
phosphatase-conjugated secondary IgG. The blots were then developed
using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.
For intracellular localization of HDAC3 and TFII-I, COS1 cells were
grown on acid-etched coverslips in 100-mm tissue culture plates for
about 24 h and transfected with 0.5 µg of pFLAG-HDAC plasmid as
described below. Two days after transfection, the cells were washed
with ice-cold PBS, fixed in 4% paraformaldehyde for 15 min, and
permeabilized overnight in PBS containing 1% glycine and 0.1% Triton
X-100. Cells were incubated for 30 min at room temperature with
anti-FLAG coupled with fluorescein isothiocyanate (Upstate) and
anti-TFII-I antibodies. After washing with PBS, the cells were
incubated with diluted goat anti-rabbit IgG conjugated to tetramethyl
rhodamine (Sigma). The cells were then washed extensively with PBS, and
coverslips were applied with 40% glycerol before analyzing under a
Carl Zeiss confocal microscope.
GST Pull-down Assays--
GST and GST-HDAC3 were expressed and
purified as described (3). 35S-TFII-I was prepared using
the coupled transcription-translation rabbit reticulocyte lysate system
(Promega). Equal molar quantities of GST and GST-HDAC3, both conjugated
to glutathione-Sepharose beads, were incubated with radiolabeled
TFII-I. Binding reactions, washing conditions, and analysis by
electrophoresis and subsequent autoradiography were performed as
previously described (3).
Histone Deacetylase Assay--
Deacetylase activity was
determined using hyperacetylated core histones purified from HeLa cells
(24, 25). Briefly, each immunoprecipitated sample was mixed with 5000 cpm of [3H]acetate-labeled core histones. After
incubation at room temperature overnight, the reactions were quenched
with 1 M HCl and 0.16 M acetic acid (50 µl in
each sample). Released [3H]acetic acid was extracted with
600 µl of ethyl acetate by vortexing and centrifugation (5 min at
14,000 rpm). The ethyl acetate supernatants (250 µl from each sample)
were quantified by scintillation counting.
Transfection and Luciferase Assay--
COS1 cells were
cotransfected with plasmids directing the synthesis of various effector
proteins and a luciferase reporter using the FuGene 6 transfection
reagent (Roche). Each transfection contained 0.5 µg each of effector
and reporter DNAs, and all transfections were normalized to equal
amounts of DNA with parental expression vectors. For the
c-fos-luciferase assay, cells were refed with medium
containing 10% serum 24 h after transfection. Twelve hours later,
the cells were stimulated with 25 ng/ml recombinant human epidermal
growth factor (Sigma). Epidermal growth factor treatment was not
necessary for the V
-luciferase assay. Cells were collected and
luciferase activity was determined using the dual luciferase reporter
assay system (Promega).
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RESULTS |
Physical Interaction of TFII-I and HDAC3--
We previously
purified an endogenous HDAC3 complex from a total extract prepared from
HeLa cells using an anti-HDAC3 immunoaffinity column (9). We found that
in addition to HDAC3, eight proteins (p215, p205, p195, p130, p125,
p54, p52, and p35) specifically co-eluted with histone deacetylase
activity. When the 215K, 205K, and 195K polypeptides were
subjected to in-gel tryptic digestion followed by sequencing by
microcapillary HPLC ion trap mass spectrometry, the resulting peptide
sequences were shown to be derivatives of the nuclear receptor
co-repressors, NCoR and SMRT (9). We have now shown that protein
microsequencing of p130 has identified this HDAC3-associated
polypeptide as the transcription factor TFII-I (Table
I).
To ensure that the co-purification of TFII-I with anti-HDAC3 was not a
result of antibody cross-reactivity, we transfected HeLa cells with
plasmids expressing either HA-TFII-I or GST-TFII-I and FLAG-HDAC fusion
proteins. Extracts prepared from transfected cells were then
immunoprecipitated with an anti-FLAG or anti-GST antibody and
immunoblotted with an anti-HA antibody. As shown in Fig.
1A, top panel,
FLAG-HDAC3 interacted with HA-TFII-I and GST-TFII-I (lanes 3 and 8), whereas FLAG alone, FLAG-HDAC4, FLAG-HDAC5, and
FLAG-HDAC6 did not (lanes 1, 4-6,
9-11). Interestingly, HA-TFII-I or GST-TFII-I also bound,
to a limited extent, both FLAG-HDAC1 and FLAG-HDAC2 (lanes 2 and 7).

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Fig. 1.
Physical interaction between HDAC3 and
TFII-I. A, HeLa cells were transfected with plasmids
encoding the indicated proteins. Cell extracts were immunoprecipitated
with an anti-FLAG antibody and immunoblotted with an anti-HA antibody
(top left panel), an anti-GST antibody (top right
panel), or an anti-FLAG antibody (middle panel). Total
extracts were immunoblotted with an anti-HA antibody (bottom
panel). B, autoradiogram of in
vitro-translated TFII-I protein captured by a GST-HDAC3 fusion
protein (top panel). The input lane was loaded with
one-tenth the amount of 35S-labeled protein used in the
binding reactions. The gel was stained with Coomassie Blue before
autoradiography to ensure that GST-HDAC3 was not overloaded compared
with GST (bottom panel).
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To support the observation that HDAC3 and TFII-I interact, we tested
whether a GST-HDAC3 affinity matrix would capture TFII-I. Bacterially
expressed GST-HDAC3 was bound to glutathione-Sepharose beads and
incubated with 35S-labeled TFII-I produced by in
vitro translation in a reticulocyte lysate. The beads were then
washed and boiled in sample buffer, and the proteins released from the
beads were analyzed by electrophoresis in a SDS-polyacrylamide gel.
TFII-I was captured by the GST-HDAC3 fusion protein (Fig.
1B, lane 3) but not by the GST polypeptide alone
(lane 2).
Colocalization studies were performed to confirm the HDAC3-TFII-I
interaction in mammalian cells. COS1 cells were transiently transfected
with plasmids expressing FLAG-HDAC3, fixed with paraformaldehyde, and
immunostained with an anti-FLAG antibody. Consistent with our previous
findings, images obtained with a confocal laser scanning system showed
that HDAC3 was present in both the nucleus and cytoplasm (Fig.
2). TFII-I was regionally dispersed
throughout the nuclei of COS1 cells. Importantly, numerous distinct
nuclear regions in which TFII-I and HDAC3 colocalized were identified
(middle panel), in agreement with the observation that the
two proteins physically interact in vivo. As expected,
TFII-I also colocalized with HDAC1 (left panel). In
contrast, HDAC5 did not colocalize with TFII-I, acting as a negative
control (right panel).

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Fig. 2.
Colocalization of TFII-I with HDAC3.
Representative pictures of COS1 cells transfected with a FLAG-HDAC1,
-HDAC3, or -HDAC5 expression construct, fixed, stained with antibodies,
and analyzed by confocal microscopy. White dots indicate
areas where two signals are within 0.02 µm.
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Residues 373-401 of HDAC3 Are Necessary for the HDAC3-TFII-I
Interaction--
To identify the domain of HDAC3 that interacts with
TFII-I, we coprecipitated HeLa cells transfected with a plasmid
expressing HA-TFII-I with plasmids expressing FLAG fused to full-length
HDAC3 or various C-terminal truncated forms of HDAC3. As shown in Fig. 3, A and B,
full-length HDAC3 (1-428) and HDAC3 (1-401) clearly bound TFII-I
(Fig. 3B, lanes 1 and 2), whereas
fragments corresponding to HDAC3 residues 1-373, 1-313, 1-265, and
1-180 did not bind TFII-I (lanes 3-6). These data suggest
that residues 401-428 of HDAC3 are not necessary for HDAC3-TFII-I
association and that the minimal interaction region is located in
residues 373-401.

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Fig. 3.
Mapping of the TFII-I-interacting domain on
HDAC3. A, schematic diagram of full-length (1-428) and
various truncated FLAG-HDAC3 fusion proteins.
Oligomerization indicates the N-terminal region of HDAC3
that is required for self-association. NES denotes nuclear
export sequences. Unique represents a region within HDAC3
that has no similarity to any known proteins. For simplicity, the FLAG
portions of the fusion proteins are not shown here. The ability of each
FLAG-HDAC3 fusion protein to bind HA-TFII-I is indicated (+ or ).
B, anti-FLAG immunoprecipitates (top and
middle panels) and total extracts (bottom panel)
prepared from transfected cells were immunoblotted with the indicated
antibodies.
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Residues 363-606 of TFII-I Are Required for the HDAC3-TFII-I
Interaction--
In a reciprocal experiment, we identified the region
of TFII-I that interacts with HDAC3. C-terminal deletions of TFII-I
(1-781 and 1-606) bound HDAC3 (Fig.
4A; Fig. 4B,
lanes 1 and 2), suggesting that residues 606-958
of TFII-I are not important for HDAC3-TFII-I interaction. In contrast,
TFII-I (1-363) did not associate with HDAC3 (lane 3).
Further deletion analyses show that TFII-I (133-958 and 133-781)
interacted with HDAC3, whereas TFII-I (133-363) did not (lanes
4-6). Taken together, these results suggest that the minimal
HDAC3-binding domain is located between residues 363-606 of TFII-I but
that the interaction domain may extend from residues 133 to 606.

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Fig. 4.
Mapping of the HDAC3-interacting domain on
TFII-I. A, schematic diagram of various HA-TFII-I
deletion fusion proteins. The shaded boxes represent the
I-repeats (denoted as R1-R6). LZ represents the
leucine zipper motif. The nuclear localization sequence and the basic
region are marked as NLS and BR, respectively.
For simplicity, the HA portions of the fusion proteins are not shown
here. The ability of each HA-TFII-I fusion protein to bind FLAG-HDAC3
is indicated (+ or ). B, anti-FLAG immunoprecipitates
(top and middle panels) and total extracts
(bottom panel) prepared from transfected cells were
immunoblotted with the indicated antibodies.
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TFII-I Associates with Histone Deacetylase Activity--
To
determine whether the HDAC3-TFII-I interaction results in the
recruitment of HDAC enzymatic activity by TFII-I, we expressed GST-TFII-I in COS1 cells, prepared immunoprecipitates from extracts using an anti-GST antibody, and assayed for HDAC activity. On average,
HDAC activity was nearly 50-fold higher in immunocomplexes containing
GST-TFII-I when compared with GST alone (Fig.
5). The TFII-I-associated HDAC activity
was greatly reduced by Trichostatin A (TSA), a specific inhibitor of
deacetylases. Although we cannot exclude the possibility that HDAC1/2
(in addition to HDAC3) may contribute to the HDAC activity associated
with TFII-I, this result unequivocally confirms that TFII-I interacts
with HDACs.

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Fig. 5.
Association of HDAC enzymatic activity with
TFII-I. Histone deacetylase activity was assayed in anti-GST
immunoprecipitates prepared from COS1 cells transfected with either
pEDG or pEDG-TFII-I. + TSA indicates that the
immunoprecipitated proteins were treated with 400 nM TSA
prior to being assayed for HDAC activity. Each assay was performed in
duplicate from three independent samples, and the values shown are the
averages ± S.D.
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HDAC3 Regulates the Transcriptional Activity of TFII-I--
To
determine whether HDAC3 affects the transcriptional activity of TFII-I,
we examined its effect on the activity of the c-fos promoter, which is regulated by TFII-I (20). As predicted,
overexpression of TFII-I significantly activated the c-fos
promoter (Fig. 6A). More
importantly, overexpression of HDAC3 substantially inhibited the
activation of the c-fos promoter by TFII-I, consistent with the premise that HDAC3 can modulate the activity of TFII-I. The expression of TFII-I was not affected by HDAC3 (Fig. 6A,
right panel), ruling out the possibility that HDAC3
down-regulates the human EF-1
promoter used to express TFII-I.
Activation of the c-fos promoter by TFII-I was not affected
by an HDAC3 mutant (H134Q/H135A/A136S) lacking deacetylase activity or
by HDAC3 mutants that do not bind TFII-I (1-373, 1-313, 1-265,
1-180) (Fig. 6, A and B).

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Fig. 6.
HDAC3 inhibits the activity of TFII-I.
Expression plasmids for TFII-I, HDAC3, various HDAC3 mutants, c-Src,
HDAC6, or Gal4-VP16 were transfected into COS1 cells together with a
reporter construct as indicated. Luciferase activities are the
averages ± S.D. from three separate experiments. An anti-TFII-I
Western blot was performed to ensure that HDAC3 did not significantly
reduce the expression of TFII-I (A, right
panel).
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Recently, it was found that TFII-I can activate the c-fos
promoter through a Src-dependent mechanism (26). To
determine whether HDAC3 represses Src-dependent TFII-I
activation, we expressed TFII-I and c-Src, together with HDAC3, in the
presence of the c-fos reporter. As shown in Fig.
6C, activation of c-fos by TFII-I plus c-Src was
completely abolished by HDAC3.
In addition to c-fos, TFII-I is required for the
transcription of the naturally TATA-less but initiator-containing V
promoter (19, 27). In separate experiments, HDAC3 inhibited the ability of TFII-I to activate the V
promoter (Fig. 6D). However,
neither the HDAC3 mutant (H134Q/H135A/A136S) nor HDAC6 had an effect on the activation of the V
promoter by TFII-I. Taken together, these results unambiguously show that the transcriptional activity of TFII-I
is modulated by interaction with the HDAC3 protein.
To confirm that the inhibition of activation by the TFII-I protein is
not due to a general inhibitory effect of HDAC3 overexpression, we
transfected cells with pGal4-VP16 and pGal4-TKLuc. As shown, overexpression of HDAC3 did not affect transcriptional activation of
the Gal4-TK promoter by the Gal4-VP16 protein (Fig. 6E).
Thus, our data strongly suggest that HDAC3 is not a general cytotoxic protein but rather is a specific cellular inhibitor of TFII-I.
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DISCUSSION |
The two best studied human histone deacetylases, HDAC1 and HDAC2,
regulate gene expression, at least in part, by forming complexes with
other cellular factors. To obtain a greater mechanistic and functional
understanding of HDAC3, we previously purified and attempted to
identify proteins capable of forming heterologous complexes with HDAC3
(9). Here, we identify one of the previously isolated HDAC3-binding
proteins as the transcription factor, TFII-I.
Three lines of evidence confirmed that TFII-I is a genuine
HDAC3-associated protein. First, coimmunoprecipitation experiments showed that TFII-I interacts with HDAC3 but not HDAC-4, -5 or -6. Second, using a GST-HDAC3 fusion protein, biochemical evidence was
obtained indicating that HDAC3 and TFII-I interact in vitro. Finally, immunocytochemical methods revealed a similar subcellular distribution of HDAC3 and TFII-I in mammalian cells. Although the
present experiments rely on overexpression of HDAC3 or TFII-I, we
believe that they accurately reflect true in vivo
interactions, in part because TFII-I copurifies with HDAC3 in an
anti-HDAC3 immunoaffinity column using a whole cell extract without
overexpression of either protein (9).
In addition to its interaction with HDAC3, TFII-I also interacts very
weakly with HDAC1 and HDAC2 in coprecipitation experiments. Although
this observation is not surprising considering that several proteins
that bind HDAC1/2 also partner with HDAC3 (3, 9, 28-31), it is
conceivable that there are multiple affinity levels of interaction,
with HDAC3-TFII-I forming the most stable complex. Also, at least four
isoforms of TFII-I exist (15), and at this time, our protein
microsequencing data do not exclude the possibility that HDAC3 binds
more than one isoform of TFII-I.
Analysis of HDAC3 deletion mutants revealed that residues 373-401 of
HDAC3 are required for the HDAC3-TFII-I interaction. Although the
importance of this region with respect to mechanisms and functions of
HDAC3 is unknown at this time, this area is known to be outside both
the HDAC3 nuclear export sequence and the oligomerization domain and
contributes to the deacetylase and transcriptional activity of HDAC3
(12). In future studies, it will be important to determine whether this
TFII-I-interacting domain binds other cellular factors or associates
with TFII-I exclusively.
An unusual feature of the TFII-I protein is the presence of six highly
conserved 90-residue repeats (R1-R6), and a striking feature of these
repeats is the presence of HLH motifs, which have been implicated in
protein-protein interactions. Our deletion mutational studies indicate
that residues 363-606 of TFII-I are essential for the HDAC3-TFII-I
interaction. This region encompasses a portion of R2 and nearly the
entire R3 and R4 I-repeats. Although hetero- and homo-dimerization
mediated through HLH motifs is a well known phenomenon, it is presently
unclear how the HLH domain of TFII-I forms a complex with HDAC3, which
does not appear to contain an HLH domain. It is possible that HDAC3 and
TFII-I interact via a third protein that contains both an HLH motif and
an HDAC3 interaction domain. Further experiments using highly purified HDAC3 and TFII-I will resolve this issue.
The biological and functional significance of the HDAC3-TFII-I
interaction remains in question. One possibility is that, in addition
to its many other functions, TFII-I also recruits HDAC activity to
promoters containing TFII-I binding sites. By doing so, it may help
modulate the overall transcriptional activity from TFII-I-regulated
promoters. If so, TFII-I then joins a rapidly growing list of
sequence-specific DNA-binding transcription factors that regulate
transcription by recruitment of HDAC activity. In support of this
model, we show that TFII-I immunoprecipitates contain HDAC activity and
that HDAC3 inhibits the activity of the c-fos and V
promoters. The crucial question that needs to be addressed now is
whether the recruitment of HDAC3 by TFII-I is a regulated process and,
if so, how to identify the signal involved. It is also conceivable that
HDAC3 blocks the ability of TFII-I to activate transcription without
tethering itself to the transcription complex. Finally, the
possibility that TFII-I may be a substrate for HDAC3, and possibly for
HDAC1/2 as well, cannot yet be ruled out. Experiments designed to
explore each of these possibilities are now in progress.