From the
Department of Cardiovascular Medicine and
the
Department of Clinical Bioinformatics,
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8655, Japan, the ¶Laboratory of
Developmental Biology, Institute of Molecular and Cellular Biosciences, The
University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, and the
**Horikoshi Gene Selector Project, Exploratory
Research for Advanced Technology (ERATO), Japan Science and Technology
Corporation (JST), 5-9-6 Tokodai, Tsukuba, Ibaraki 300-2635, Japan
Received for publication, March 4, 2003 , and in revised form, May 13, 2003.
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ABSTRACT |
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INTRODUCTION |
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The DNA-binding regulatory transcription factor that plays a central role in dictating specific transcription (e.g. cell cycle, cell differentiation) consists of an activation/regulatory domain, which interacts with the basal transcription machinery and other protein-protein interactors (e.g. transcriptional cofactors), and the DNA-binding domain (DBD),1 which specifies the target promoter (1016). The DBD has been generally thought to possess a passive role to tether the activation/regulatory domain to the transcription machinery on the promoter, and functions other than its DNA binding activity have received little attention (17).
We have, however, focused on the role of the DBD as a target of regulation, and have shown in the past for Sp1 (18), the best studied and founding factor of the Sp/KLF (Sp1 and Krüppel-like factor) family (1921) of C2H2-type zinc finger transcription factors, to show differential regulation by protein-protein interaction and chemical modification through the DBD (22). Much research on the activation mechanisms through Sp1 has focused on interaction through the activation domain (e.g. transcription machinery, chromatin factors) (8, 23). Recently, we have shown that the Sp1 DBD interacts with acetyltransferase (22), and others have shown interaction of the Sp1 DBD with other factors (e.g. cell cycle regulatory factor E2F, Refs. 24 and 25; deacetylase HDAC1, Ref. 26; ATP-dependent nucleosomal remodeling enzyme SWI/SNF, Ref. 27; as well as other zinc finger transcription factors including Krüppel-like factors and nuclear receptors, Ref. 28). The DBD of Sp1, therefore, mediates protein-protein regulation important for transcription. To further understand functional regulation of Sp1 through its DBD, we have in the present study affinity-purified interacting factors and analyzed their functional effects on Sp1.
Here we show interaction of Sp1 through its DBD with the histone chaperone TAF-I. Interaction between these proteins is specific. This interaction inhibits Sp1 DNA binding, and also likely as a result of such, inhibits promoter activation by Sp1; thus TAF-I functions as a negative regulator of Sp1. This novel regulatory interaction between DNA-binding regulatory transcription factor and histone chaperone adds to our understanding of the mechanisms of how DNA-binding regulatory transcription factors are regulated by protein-protein interactions.
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EXPERIMENTAL PROCEDURES |
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TAF-I,
, and mutant constructs (gifts of Dr. Kyosuke Nagata)
were expressed in bacteria and purified using Probond resin (Invitrogen) with
buffer C (20 mM Tris-HCl, pH 7.4, 10% glycerol, 0.5 M
KCl, 50 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 1 µg/ml pepstatin
A). Washes were done with buffer C containing 20 mM imidazole, and
then eluted with buffer C containing 0.2 M imidazole. All protein
procedures were done at 4 °C.
Preparation of Nuclear ExtractNuclear extract from HeLa S3 cells was prepared as described (29). Briefly, cells were lysed by glass Dounce homogenizer in Dignam's buffer A, then centrifuged for 10 min at 700 x g to pellet nuclei. The pellet was suspended in Dignam's buffer C, rotated for 60 min to extract nuclear protein, and then centrifuged for 15 min at 18,000 x g. Buffer B was used for final dialysis. Dialysate was centrifuged for 15 min at 18,000 x g, and the supernatant was used as nuclear extract.
Isolation of Factors Interacting with Sp1 DNA-binding Domain50 µg of hexahistidine-tagged Sp1 DBD was bound to 50 µl of Probond resin (Invitrogen), and after washing, incubated with 725 µg of HeLa S3 nuclear extract. Proteins were eluted with buffer B containing 0.5 M imidazole. Samples were resolved by SDS-PAGE analysis and stained with Coomassie Brilliant Blue.
Protein Identification by MALDI/TOF Mass
SpectrometryProtein bands were excised, dehydrated with
acetonitrile, and after removing acetonitrile, dried, and then in-gel digested
with trypsin in 25 mM ammonium bicarbonate, pH 8. After soaking in
50% acetonitrile/5% tetrahydrofuran, the supernatant was collected, dried, and
then reconstituted by adding 50% acetonitrile, 0.1% tetrahydrofuran and mixed
with -cyano-4-hydroxycinnamic acid. Mass spectrometry (MALDI-TOF MS;
Voyager-DE STR, Applied Biosystems) was used to analyze proteins. Masses were
calibrated internally with peptides derived from trypsin autolysis, and
accuracy was within 10 ppm. Data base searches were done against a
nonredundant protein sequence data base of NCBI using the Protein Prospector
program V.3.2.1 (UCSF mass spectrometry facility).
Protein-Protein Interaction AssayGST fusion proteins were
immobilized to glutathione-Sepharose 4B resin (Amersham Biosciences) and
incubated with hexahistidine-tagged proteins in buffer of 20 mM
HEPES (pH 7.6 at 4 °C), 20% glycerol, 0.2 mM EDTA, 0.1% Triton
X-100, 100 mM NaCl, and 100 µM ZnSO4.
Reactions were carried out at 4 °C for 2 h and washed twice with the same
buffer. Bound proteins were resolved on a 10% SDS-PAGE gel, transferred to
nitrocellulose membrane, immunoblotted with anti-HIS-probe (G-18) antibody
(Santa Cruz Biotechnology), and then visualized by chemiluminescence (ECL,
Amersham Biosciences). Commercially available recombinant proteins were used
for p53, MyoD, and NFB (Santa Cruz Biotechnology).
Co-immunoprecipitation Assay1 µg of anti-Sp1 antibody
(PEP-2, Santa Cruz Biotechnology) or control IgG (sc-2027, Santa Cruz
Biotechnology) was bound to protein G-Sepharose (Amersham Biosciences)
followed by incubation with 1 mg of HeLa S3 cell extract. After washing with
radioimmune precipitation assay buffer (50 mM Tris-HCl, pH 7.9, 150
mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1
mM phenylmethylsulfonyl fluoride, 0.5 µg/ml leupeptin, and 1
µg/ml pepstatin), immunoprecipitates were subjected to SDS-PAGE, and then
immunoblotted using TAF-I (KM1715) and TAF-I
(KM1720) specific
antibodies (gifts from Dr. Kyosuke Nagata).
Gel-shift DNA Binding AssayGel-shift DNA binding assays
were done essentially as described
(22). Briefly, the Sp1
consensus binding sequence (top
5'-ATTCGATCGGGGCGGGGCGAGC-3') was used as the probe
(underlined nucleotides GG were substituted by TT for mutant analysis).
Annealed double-strand probe was gel-purified, and then kinase-labeled by
using [-32P]ATP (222 TBq(6000 Ci)/mmol, PerkinElmer Life
Sciences) and T4 polynucleotide kinase (Stratagene). Unincorporated
radiolabeled ATP was separated by NucTrap purification column (Stratagene).
Specific activity of the radiolabeled probe was adjusted by adding cold
double-stranded probe. Binding reactions were done in binding buffer of 20
mM HEPES (pH 7.6 at 4 °C), 20% glycerol, 0.2 mM
EDTA, 0.1% Triton X-100, 100 mM NaCl, and 100 µM
ZnSO4. Recombinant proteins were incubated at 30 °C for 15 min
in binding buffer prior to addition of 1.0 x 104 cpm (1 ng)
of labeled probe, followed by further incubation at 30 °C for 15 min
before separation on nondenaturing polyacrylamide gels. Gels were dried and
analyzed using BAS 1500 (Fuji Photo Film).
Co-transfection Reporter Assay25,000 HeLa cells were seeded
and transfected 24 h later with the SV40 early promoter reporter (100 ng) and
effector expression vectors by liposome-mediated transfer (Tfx-20; Promega).
Full-length human Sp1 cDNA (a gift of Dr. James Kadonaga) was inserted into
the expression vector pcDNA3 (Invitrogen). pCHA-TAF-I and
were
gifts of Dr. Kyosuke Nagata. p53-Luc, which contains 15 copies of a
p53-binding sequence upstream of the luciferease reporter and the expression
vector pFC-p53, were purchased from Stratagene. The total effector DNA amount
in transfection reactions was corrected to 1 µg by addition of empty
vector. Cells were harvested after 48 h and assayed for luciferase activity
(luciferase assay system, Promega). Luciferase activity was normalized against
protein concentration of cell lysates. Protein expression was examined by
Western blot using anti-HA antibody (Roche Diagnostics) for TAF-I, and
anti-Sp1 antibody (PEP2, Santa Cruz Biotechnology) for Sp1. Error bars denote
S.E.
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RESULTS |
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Coomassie Brilliant Blue (CBB) staining of the bound proteins as resolved by SDS-gel electrophoresis revealed several bands spanning molecular mass from 30 to 200 kDa, which were seen only when the binding reaction between the Sp1 DBD and HeLa nuclear extract was done (Fig. 1C, lane 2) and not when Sp1 DBD alone (lane 1) or HeLa nuclear extract alone (lane 3) was reacted with resin. Two major bands, which were the most abundant on CBB staining were of apparent molecular masses of 41 and 39 kDa and are hereafter referred to as p41 and p39, respectively.
To identify the proteins, the p41 and p39 bands were excised, trypsinized,
and then subjected to MALDI-TOF mass spectrometry. The proteins were
identified by peptide mass finger-printing with a computer search of the
nonredundant protein sequence NCBI data base as available for the mammalian
proteome, and then further subjected to post-source decay peptide sequencing.
Surprisingly, mass spectra of p41 and p39 identified them to be the products
of a single gene, template-activating factor-I (TAF-I)
(Fig. 2, AC)
(30,
31). p41 and p39 were
TAF-I and
, respectively. TAF-I
and
are the
alternative splicing products of a single gene and differ only in a short
region of their amino-terminal ends (Fig.
2C)
(31).
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TAF-I was originally identified as a cellular factor which stimulates
adenovirus core DNA replication
(30,
31), and has been shown to be
a histone chaperone which is a factor which can displace and/or assemble
nucleosomal histones in an ATP-independent manner
(3235).
TAF-I is identical to the SET oncogene whose translocation has been
implicated in leukemia
(36).
Sp1 and TAF-I Interact in Vitro and in VivoTo examine
whether Sp1 DBD directly binds the TAF-I proteins, GST pull-down binding
assays were done with recombinant TAF-I,
, and Sp1 DBD
(Fig. 3A). Under the
described binding conditions, both bacterially expressed hexahistidine-tagged
TAF-I
and
(lanes 1 and 2) bound to GST fusion
Sp1-DBD (lanes 4 and 6) but not to GST alone (lanes
3 and 5) showing that Sp1 DBD directly binds both TAF-I
and
. We have reproducibly seen that TAF-I
binds Sp1 DBD with a
slightly higher affinity than TAF-I
(lanes 4 and
6).
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To next see if TAF-I also binds full-length Sp1, GST pull-down binding
assays were done with recombinant TAF-I and full-length Sp1
(Fig. 3B). Under the
described binding conditions, bacterially expressed hexahistidine-tagged
TAF-I
(lane 1) bound to GST fusion full-length Sp1 (lane
3) but not to GST alone (lane 2) showing that full-length Sp1
directly binds TAF-I. TAF-I can therefore directly bind Sp1.
To further see whether these proteins interact in the cell,
immunoprecipitation was done using specific antibodies against TAF-I,
, and Sp1 (Fig.
3C). Sp1 was immunoprecipitated from HeLa cells followed
by immunoblotting using TAF-I
- and
-specific antibodies.
TAF-I
and
(lane 1) both bound Sp1 (lane 3) as
compared with the control immunoprecipitation using normal IgG antibody
(lane 2) confirming that these proteins do indeed interact in the
cell. Collectively, TAF-I interacts with Sp1 in vitro and in
vivo.
Specific Interaction between Sp1 and TAF-ITo see whether
interaction of Sp1 DBD with TAF-I is specific or common for histone
chaperones, a GST pull-down binding assay of Sp1 DBD was done with the histone
chaperone CIA (also known as ASF1 in Saccharomyces cerevisiae and
RCAF in Drosophila complexed with histones)
(3739)
(Fig. 4A). Under
conditions in which Sp1 DBD bound TAF-I (lanes 4 and
5), Sp1 DBD did not bind CIA (lanes 13) showing that
interaction between Sp1 DBD and TAF-I is specific. Therefore, interaction
between Sp1 DBD and TAF-I is a specific property of these factors, and not a
property common for histone chaperones.
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To next see whether interaction with TAF-I is specific for Sp1 or common
for DNA-binding factors, a GST pull-down binding assay with the DNA-binding
transcription factors p53, MyoD, and NFB was performed
(Fig. 4B). Under
conditions in which Sp1 DBD bound TAF-I
(lanes 13),
TAF-I
did not bind p53, MyoD, or NF
B (lanes 46)
thus showing that interaction between Sp1 DBD and TAF-I is specific. A CBB
stain of the GST fusion proteins is shown
(Fig. 4C). Therefore,
interaction between Sp1 DBD and TAF-I is a specific property of these two
factors.
Effects of the Acidic Carboxyl-terminal Regions of TAF-I in Interaction
with Sp1Histone chaperones including TAF-I have in common an
acidic region (32,
34,
38,
40), but as Sp1 DBD did not
bind CIA (Fig. 4A), we
thought that the acidic region may not mediate interaction between TAF-I and
Sp1 DBD. To address the effects of the acidic region of TAF-I on interaction
with Sp1 DBD, GST pull-down binding assays using mutants of TAF-I and
, which lack the common acidic carboxyl-terminal end,
TAF-I
C and TAF-I
C, respectively, were done
(Fig. 5A). We have
reproducibly seen that TAF-I
C binds Sp1 DBD with less affinity
than TAF-I
C (Fig.
5B, lanes 3 and 6), which may suggest
that the acidic carboxyl-terminal end participates in regulation of binding of
TAF-I
with Sp1 DBD greater than for TAF-I
. The acidic region of
TAF-I may therefore be involved in modulation of binding affinity.
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TAF-I Inhibits DNA Binding Activity of Sp1As TAF-I binds
the DNA-binding domain of Sp1, we examined the effects of TAF-I proteins on
the DNA binding activity of Sp1 by gel mobility shift analysis
(Fig. 6A). Under
conditions in which TAF-I and
did not show DNA binding activity
(lanes 2 and 3), incubation of TAF-I with Sp1 DBD resulted
in inhibition of specific DNA binding activity of Sp1 DBD to its cognate
binding sites (lanes 46) as shown by the dose-dependent
decrease in intensity of the shifted DNA-protein complex for TAF-I
(lanes 7 and 8) and TAF-I
(lanes 9 and
10). TAF-I
inhibited the DNA binding activity of Sp1 DBD to a
slightly greater extent than TAF-I
(lanes 7 and 8 versus
9 and 10). Under identical conditions, the acetyltransferase
p300 stimulates DNA binding activity of Sp1 DBD and thus this is not a
nonspecific effect of TAF-I (data not shown and Ref.
22). Therefore, TAF-I
inhibited the DNA binding activity of Sp1 DBD.
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TAF-I Inhibits Sp1-dependent Promoter ActivationAs
interaction of TAF-I proteins and Sp1 DBD inhibited the DNA binding activity
of Sp1 DBD, we examined whether TAF-I would inhibit Sp1-dependent promoter
activation as would be expected as a secondary result. Co-transfection
analysis was performed with a luciferase-reporter construct harboring the SV40
early promoter which contains six Sp1 binding sites
(Fig. 7A). As
expected, under conditions in which transfection of an expression plasmid
harboring full-length Sp1 showed dose-dependent activation of the SV40 early
promoter reporter construct in HeLa cells (lanes 13) and in
which TAF-I did not show activation of this reporter (TAF-I, lanes
1012; TAF-I
, lanes 1315), co-transfection of
TAF-I with Sp1 resulted in inhibition of Sp1-dependent promoter activation
(TAF-I
, lanes 46; TAF-I
, lanes
79). Protein expression from the expression vectors was confirmed
by Western blot. Both TAF-I
and
showed increasing amounts of
expression. Sp1 also showed increased expression, which shows that the effects
of TAF-I on Sp1 are not due to inhibition of Sp1 expression from its
expression vector. Further, as a control, we tested effects of TAF-I on p53
promoter activation as our binding studies showed that p53 does not associate
with TAF-I. Under our tested conditions, TAF-I did not inhibit p53 promoter
activation (Fig. 7B).
TAF-I therefore negatively regulates Sp1-mediated promoter activation likely
as a result of inhibition of DNA-binding.
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DISCUSSION |
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Novel Activity of Histone ChaperoneHistone chaperones are a class of factors that possess activity to mediate assembly and/or disassembly of nucleosomal histones in an ATP-independent manner and include the factors TAF-I, CIA, nucleoplasmin, and NAP-1 among others (30, 31, 3843). While interaction with histones and their activity to assemble/disassemble nucleosomal histones has been well addressed, their interaction with DNA-binding transcription factors has not been explored. The present study shows that the histone chaperone TAF-I functionally interacts with the DNA-binding transcription factor Sp1 (Figs. 3, 6, and 7). This interaction is specific as another histone chaperone examined did not bind Sp1, and as other tested DNA-binding transcription factors did not bind TAF-I (Fig. 4). TAF-I acts to negatively regulate the DNA binding and likely as a result of such also promoter activation by Sp1 (Figs. 6, 7, 8). TAF-I has been similarly shown to negatively regulate promoter activation by the retinoic acid receptor using cell co-transfection studies (43). Importantly, TAF-I may act to negatively regulate a subset of DNA-binding transcription factors that includes at least the zinc finger-type factors, which both Sp1 and nuclear receptors are.
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We note that TAF-I has been shown to stimulate transcription from in vitro chromatin template (35). The cell co-transfection assay used by us and others differs from the in vitro transcription reaction as the former is a cellular experiment in which a transfected episomal plasmid reporter is activated by forced expression of a transcription factor likely in the cytoplasm, in contrast to the latter biochemical study, which uses reconstituted components. The latter better reflects the involved fundamental reactions and allows for dissection of mechanisms of action; however, the former in contrast better reflects the collective surrounding regulatory reactions as seen in the cell albeit possible inherent limitations associated with compartmentation (e.g. cytoplasmic reaction) and concentration (e.g. effect of forced expression in contrast to basal endogenous levels). The apparent discrepancy in results between in vitro and in vivo experiments will be a topic needed to be addressed in further studies.
The mechanism of how histone chaperones are involved in reactions associated with specific promoters has also remained elusive. Functional interaction between DNA-binding transcription factor and histone chaperone may play a role in specifying the site of the reaction as dictated by the gene- and site-specific targeting properties of the DNA-binding factor. In reference, the centromeric proteins (CENP-A,B,C) through its DNA-binding component (CENP-B) shows centromere sequence-specific binding and localization allowing the CENP complex to modulate centromeric nucleosomes (44). This is one example in which concerted action between sequence-specific DNA-binding factor with histone-associated catalytic protein(s) results in specific and localized chromosomal/nucleosomal processes. Functional sequelae of the interaction between DNA-binding transcription factor and histone chaperone may be dictation of site-specificity by DNA-binding transcription factors for catalytic events to be mediated by the histone chaperone (e.g. nucleosome assembly/disassembly).
Regulatory Role of the DNA-binding DomainOf additional importance, interaction of Sp1 with TAF-I was mediated through the DBD. Much focus on regulation of Sp1 through protein-protein interaction has focused on the role of the activation domain (e.g. interaction with the basal machinery dTAF110/hTAF130 and transcriptional complex ARC) (8, 23) in contrast to the role of the DNA-binding domain, which has been poorly addressed. However, past studies by ourselves and others have shown that the DBD of Sp1 mediates important regulatory interactions such as with the cell cycle regulator E2F (24, 25), the acetyltransferase p300 (22), the histone deacetylase HDAC1 (26), the ATP-dependent nucleosomal remodeling enzyme SWI/SNF (27) as well as other zinc finger transcription factors including Krüppel-like factors (28).
Interestingly, the Sp1 DBD interacts with all three major chromatin-related factors consisting of chemical modification enzymes (e.g. acetyltransferase p300), ATP-dependent nucleosome assembly factor (e.g. SWI/SNF) and histone chaperone (e.g. TAF-I), which is a finding which has only been shown for histones. This finding is of particular interest because it implicates the DBD to play a likely role in mediating transcriptional regulatory processes in eukaryotes at the chromatin level. Combined regulation of the transcription factor by interaction with chromatin-related complexes through its activation domain (e.g. ARC, DRIP, TRAP) and the three factors through the DBD likely results in coordinated transcriptional regulation at the chromatin level. Importantly, as the DBD specifies the target gene or DNA sequence, selective and ordered interaction of chromatin-related factor with the DBD of the transcription factor may play a role in gene- and factor-selective regulation. Selective interaction between histone chaperones with DNA-binding factors such as interaction of TAF-I with zinc-finger type transcription factors is further suggestive of a specific regulatory role in transcription.
Cooperative Interaction of Histone Chaperone and DNA-binding Transcription FactorFunctional interaction between DNA-binding factor and histone chaperone is the most note-worthy new molecular mechanism, which results from our present study. As the interaction is specific, and as the histone chaperone negatively regulates activities of the DNA-binding factor, it is tempting to envision that TAF-I plays an important role to negatively regulate subsets of DNA-binding factors to affect selective gene expression. We, however, do not rule out the possibility that TAF-I may also participate in activation processes under certain regulatory conditions in consideration of the fact that TAF-I has been shown to possess stimulatory effects on transcription in vitro (35).
The next important questions which need to be answered are whether TAF-I contributes to continuous regulation/inactivation or if this is a triggered event, and how histones which also bind histone chaperones contribute to this process. It is note-worthy that the chaperone proteins including the Hsp90-co-chaperone p23, Hsp90, and Hsp70 modulate assembly as well as disassembly of transcriptional complexes as shown for nuclear receptors (4547). It is tempting to envision that histone chaperones also contribute to DNA-binding transcription factor regulation by mediating inactivation processes. Although the mechanisms of interaction of histone chaperone on DNA-binding factor are yet unclear, TAF-I may inhibit the activities of Sp1 by competitive interaction with the DNA-binding surface, but alternatively binding to the non-DNA-binding surface of Sp1 DBD may induce an allosteric/conformational change to Sp1 DBD making it transcriptionally incompetent/competent for further regulatory interactions (e.g. DNA binding, transcriptional activation).
Based on our data centered on Sp1 DBD, we have shown in the past that p300 acetyltransferase facilitates promoter access (22), therefore TAF-I as a negative regulator may act in concert with the acetyltransferase to mediate a balance of promoter activation and inactivation. Given that acetyltransferase and TAF-I have been shown to regulate acetylation and its inhibition on histones (43), respectively, this may be one of the signal modifications regulated by this concerted interaction (22, 43). As we have shown that Sp1 DBD is acetylated (22), further experiments to investigate whether TAF-I regulates inhibition of acetylation of DNA-binding transcription factor will also add to our understanding of transcriptional regulation. Further, although it would seem that inactivation/activation is an energy-consuming process, as histone chaperones are essentially non-ATP-dependent factors, their contribution would likely facilitate this process and allow for efficient transcriptional regulation.
Collectively, we have shown that the histone chaperone TAF-I negatively regulates a DNA-binding transcription factor. Our results provide an initial step in understanding the role of histone chaperones in the regulation of DNA-binding transcription factors.
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FOOTNOTES |
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|| These authors contributed equally to this work.
To whom requests for reprints should be addressed: Dept. of Cardiovascular
Medicine, Dept. of Clinical Bioinformatics, Graduate School of Medicine, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Tel.:
81-3-3815-5411 (ext. 33117); Fax: 81-3-5800-8824; E-mail:
torusuzu-tky{at}umin.ac.jp.
To whom correspondence should be addressed: Dept. of Cardiovascular Medicine,
Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-8655, Japan. Tel.: 81-3-5800-6526; Fax: 81-3-3815-2087; E-mail:
nagai-tky{at}umin.ac.jp.
1 The abbreviations used are: DBD, DNA-binding domain; GST; glutathione
S-transferase; CBB, Coomassie Brilliant Blue; HA, hemagglutinin;
TOF-MS, time-of-flight mass spectrometry; MALDI-TOF, matrix-assisted laser
desorption/ionization time-of-flight.
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ACKNOWLEDGMENTS |
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REFERENCES |
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