From the Department of Molecular Biology, Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama 350-0495, Japan
Received for publication, December 26, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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Positive cofactor 4 (PC4), originally identified
as a transcriptional coactivator, possesses the ability to suppress
promoter-driven as well as nonspecific transcription via its DNA
binding activity. Previous studies showed that the repressive activity
of PC4 on promoter-driven transcription is alleviated by transcription
factor TFIIH, possibly through one of its enzymatic activities. Using recombinant TFIIH, we have analyzed the role of TFIIH for alleviating PC4-mediated transcriptional repression and determined that the excision repair cross complementing (ERCC3) helicase activity of TFIIH
is the enzymatic activity that alleviates PC4-mediated repression via
Positive cofactor 4 (PC4)1 was originally
identified in the upstream-factor stimulatory activity that augments
activator-dependent transcription in vitro (1,
2). PC4 stimulates transcription in vitro with
diverse kinds of activators, including VP16 (3, 4), thyroid hormone
receptor (5), octamer transcription factor-1 (6), and BRCA-1 (7),
presumably by facilitating assembly of the preinitiation complex
through bridging between activators and the general transcriptional
machinery (4, 8). Studies on the interaction of PC4 with activators and
TFIIA, as well as in vitro functional analyses, suggest that
interaction between TFIIA and PC4 plays a pivotal role for facilitating
the preinitiation complex (PIC) assembly (3, 4). Further studies also
demonstrated the importance of PC4 for transcriptional activation by
AP-2 (9) and HIV transactivator Tat (10) in vivo. In
addition, a yeast homologue of PC4, SUB1/TSP1 (11, 12), which is
essential for viability in the presence of TFIIB mutations (12), was
shown to function as a coactivator for GCN4 and HAP proteins.
The N-terminal region of PC4 contains a serine-rich portion termed the
SEAC domain, which exhibits similarity to viral immediate-early
proteins (3). Phosphorylation of the serine residues in the SEAC domain
negatively regulates the coactivator activity of PC4 (3, 13) possibly by a conformational change.
In addition to the role as coactivator, PC4 was subsequently shown to
repress promoter-driven transcription as well as nonspecific transcription in vitro (14, 15). The analyses of PC4 mutants demonstrated that the repressive activity is a separate function from
the coactivator activity (14); therefore, the repressive activity of
PC4 may play an as yet unknown function in regulating transcription
in vivo. In fact, the primary function of PC4 in vivo could possibly be to repress transcription rather than to enhance transcription because phosphorylated PC4, which is inactive as
a coactivator but retains repressive activity, is the predominant form
(~95%) within the cells (13). Transcriptional repression by PC4
correlates with the single-stranded (ss) DNA binding activity present
in its C-terminal region, which shows preferential binding to melted
double-stranded (ds) DNA and to heteroduplex DNA (14). The structural
studies show that PC4 forms a homodimer via its C-terminal region that
contains four-stranded Here we used the recombinant TFIIH mutants that lack one of the
enzymatic activities (cdk7 kinase, ERCC2 helicase, or ERCC3 helicase)
(18) and examined the mechanism by which TFIIH counteracts the
repressive effect of PC4. We have found that TFIIH counteracts PC4-mediated repression via ERCC3 helicase activity and that neither ERCC2 helicase nor cdk7 kinase activity is required for alleviating the
repression, an observation further supported by the fact that TFIIH
does not phosphorylate PC4. Our results suggest that PC4 and the ERCC3
helicase activity of TFIIH may act together to increase the specificity
of transcription and also to provide more intricate regulation of transcription.
Purification of Transcription Factors--
PC4 was expressed in
Escherichia coli, BL21(DE3)pLysS, harboring the plasmid
pET11c-PC4, and the extract was prepared by sonication in buffer A (20 mM Hepes-KOH, pH 7.9, 10% glycerol, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol containing 100 mM KCl). The extract was
applied onto a HiTrap SP column, and the bound proteins were eluted
with a 5-column volume of a linear gradient of 0.1-0.6 M KCl. The
eluted fractions were diluted to adjust the conductivity to that of 0.1 M KCl and then loaded onto a HiTrap heparin column. The
bound proteins were eluted with a 5-column volume of linear gradient of
0.1-0.6 M KCl. RNA polymerase II (RNAPII), TFIIB, TFIIE, TFIIF, and
FLAG-tagged TBP (f:TBP) were prepared essentially as described
(19).
Preparation of Recombinant TFIIH--
Recombinant TFIIH and its
mutants were reconstituted in High Five cells using three
baculoviruses, each of which expresses three subunits of TFIIH (18,
19). The purification of TFIIH was done essentially as described (19)
except that TALONTM metal affinity resin
(Clontech) was used in place of Ni-nitrilotriacetic acid (NTA) superflow (Qiagen). The amount of each TFIIH, whose cdk7
subunit is C-terminal-tagged with a FLAG epitope, was adjusted by using
silver-stained gels as well as quantitative immunoblots with anti-FLAG
M2 antibody.
In Vitro Transcription--
In vitro transcription
reactions were carried out in a 25-µl reaction containing 12 mM Hepes-KOH, pH 7.9, 6% glycerol, 60 mM KCl,
0.6 mM EDTA, 8 mM MgCl2, 5 mM dithiothreitol, 20 units of RNase inhibitor (TaKaRa),
0.2 mM ATP, 0.2 mM UTP, 0.1 mM
3'-O-methyl GTP, 12.5 µM CTP, 10 µCi
of [ Kinase Assays--
Phosphorylation of GST-CTD (carboxyl-terminal
domain) and PC4 by TFIIH was performed essentially as described (19).
Where indicated, casein kinase II (New England Biolabs) was used in place of TFIIH as indicated.
Requirement of
We next tested whether recombinant TFIIH could alleviate
transcriptional repression by PC4. As shown in Fig.
2A, even in the absence of
TFIIH, the negatively supercoiled template allowed production of the
specific 390-nt transcript (lane 1), which was suppressed to
less than 5% by the addition of PC4 (lane 2). Adding the
increasing amounts of TFIIH, however, gradually restored the levels of
transcription (lanes 3-6) to 40-60% of those seen in the
absence of both TFIIH and PC4 (lane 1), indicating that
recombinant TFIIH can reverse the repressive effect of PC4 in a
dose-dependent manner as does natural TFIIH (14, 15).
Using the highly purified reconstituted system, we then tested the
requirement for The ERCC3 Helicase Activity of TFIIH Is Essential for Alleviating
PC4-mediated Repression--
The requirement of TFIIH Does Not Phosphorylate PC4--
Because the previous result
showed that PC4 is released from the template upon phosphorylation by
TFIIH (21), the dispensability of the cdk7 kinase for PC4-mediated
repression was somewhat unexpected. Furthermore, lack of any consensus
phosphorylation site for cdks (S/TPXR/K)
in PC4 prompted us to re-address whether TFIIH is indeed able to
phosphorylate PC4 in vitro as previously reported (15, 21).
As shown in Fig. 4A, wild-type
TFIIH, K48A, and K346A phosphorylated CTD efficiently but K41A did not
phosphorylate CTD, indicating that the substitution of lysine with
alanine at the 41st residue of cdk7 eliminated the kinase activity to
an undetectable level. Phosphorylation of CTD by TFIIH produced the
hypophosphorylated form as well as the hyperphosphorylated form that
showed a slower migration on the SDS gel (Fig. 4A). Casein
kinase II also phosphorylated CTD, although phosphorylation did not
shift the migration of GST-CTD (Fig. 4A, lanes 1 and 2).
We next tested whether casein kinase II and the same set of TFIIH
mutants could phosphorylate PC4. Casein kinase II efficiently phosphorylated PC4 as previously reported (3, 13) and altered PC4 from
the faster migrating form (~15 kDa) to the slower migrating form
(~20 kDa) (Fig. 4B, bottom panel, lanes
1 and 2). In contrast, wild-type TFIIH, K346A, and
K48A, all of which retain cdk7 kinase activity (Fig. 4A,
lanes 4, 6, and 7), did not
phosphorylate PC4 (Fig. 4B, lanes 4,
6, and 7). The low levels of PC4 labeling
observed on a longer exposure of the gel (Fig. 4B,
middle panel, lanes 3-7) is not because of the
TFIIH kinase activity because the TFIIH mutant K41A, which lacks the
kinase activity (Fig. 4A, lane 5), showed the
same degree of labeling as wild-type TFIIH. Our results demonstrate
that TFIIH does not phosphorylate PC4 and argue against the involvement
of PC4 phosphorylation by TFIIH for alleviating PC4-mediated repression
of transcription.
Quantitative Analysis of PC4-mediated Repression in the Absence of
the ERCC3 Helicase Activity--
The in vivo concentration
of PC4 is estimated to be ~1 µM in HeLa cells, and
~95% of PC4 is phosphorylated in vivo, presumably by
casein kinase II (3, 13, 14). Therefore, we tested whether phosphorylated and non-phosphorylated PC4 can distinguish the presence
and absence of ERCC3 helicase activity within the general transcriptional machinery at the physiological PC4 concentration. PC4
was first phosphorylated by CKII as shown in Fig. 4B, and then increasing amounts of both phosphorylated and non-phosphorylated PC4 were added to the transcriptional reactions containing either wild-type TFIIH or K346A. As shown in Fig.
5, the levels of transcription were
reduced to less than 40% at 0.125 µM of PC4 and to
~10% at 1 µM of PC4 in the absence of ERCC3 helicase
activity. Non-phosphorylated PC4 repressed transcription slightly
better than phosphorylated PC4 in the absence of ERCC3 helicase
activity (Fig. 5). By contrast, in the presence of wild-type TFIIH,
transcription remained markedly more resistant to repression by PC4
(Fig. 5) (14). These results indicate that PC4 represses transcription
regardless of its phosphorylation status in the absence of ERCC3
helicase activity. In addition, because repression by PC4 occurs
similarly in the presence of K346A (Fig. 5) as in the absence of TFIIH
(data not shown) (14), mutual exclusion of PC4 and TFIIH on the
promoter is an unlikely mechanism for the antagonistic effect of PC4
and TFIIH.
Our results show that the ERCC3 helicase activity of TFIIH
counteracts PC4-mediated transcriptional repression and that neither the ERCC2 helicase nor the cdk7 kinase has any role in this process. The fact that the ERCC3 helicase, but not the cdk7 kinase, of TFIIH
relieves PC4-mediated repression provides a clue as to the mechanism by
which TFIIH and PC4 act antagonistically to regulate transcription.
Negatively supercoiled templates allow specific transcription by RNAPII
in the absence of TFIIH and ATP in vitro (22, 23),
presumably by the transfer of free energy stored on the negatively
supercoiled templates (24-26). This transfer of free energy appears to
be constrained by PC4, because the property of negatively supercoiled
DNA templates bound by PC4 is similar to that of linear DNA templates
with regard to the absolute requirement of TFIIH and ATP for specific
promoter-driven transcription (22, 23). This effect of PC4 transmitted
indirectly through DNA to the general transcriptional machinery is
consistent with the functional antagonism between TFIIH and PC4 that
does not involve the mutual exclusion of TFIIH and PC4 on the promoter
(Fig. 5). Thus, the role for ERCC3 helicase activity may be to overcome
the topological constraint conferred by PC4 on negatively supercoiled
templates, a process that could potentially prompt the release of PC4
from the promoter region (21). Our results, however, rule out the possibility that the cdk7 kinase of TFIIH phosphorylates PC4 (15, 21)
and facilitates its release from the promoter (21).
In light of our study as well as a previous study (14), we propose two
possible mechanisms by which PC4 represses promoter-independent transcription: i.e. "direct" and "indirect"
mechanisms. In the direct mechanism, PC4 binds to ssDNA regions
via its ssDNA binding ability, competing directly with RNAPII, and thus
physically displaces RNAPII from ssDNA regions (Fig.
6A). By contrast, in the
indirect mechanism PC4 binds dsDNA regions via its dsDNA binding
ability and renders DNA more "rigid" so that the free energy stored
in negative superhelicity (24-26) will not generate transiently melted ssDNA regions that permit RNAPII to initiate random transcription (Fig.
6B). It is conceivable that the indirect mechanism provides the primary protection against spurious transcription and the direct
mechanism provides a backup. In this scenario, PC4 bound to dsDNA
regions may also serve as a reservoir that can be recruited quickly to
ssDNA regions where the possibility of spurious transcription is
greater. In agreement with the recruitment of PC4 from dsDNA to ssDNA,
PC4 binds to ssDNA more strongly than to dsDNA (14).
-
bond hydrolysis of ATP. In addition, the alleviation does not
require either ERCC2 helicase or cyclin-dependent kinase 7 kinase activity. We also show that, as complexed within TFIIH, the cyclin-dependent kinase 7 kinase does not possess the
activity to phosphorylate PC4. Thus, TFIIH appears to protect
promoters from PC4-mediated repression by relieving the topological
constraint imposed by PC4 through the ERCC3 helicase activity rather
than by reducing the repressive activity of PC4 via its phosphorylation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets rich in positively charged and
aromatic residues involved directly in binding to ssDNA (16, 17).
Interestingly, in contrast to its coactivator activity, the ssDNA
binding activity of PC4 is augmented by phosphorylation of its
N-terminal region (8). Further studies indicate that PC4-mediated
repression of specific transcription from promoters is alleviated by
TFIIH, possibly through its enzymatic activities that require
-
hydrolysis of ATP (14, 15). However, the identity of the enzymatic
activity responsible for the alleviation as well as the mechanism by
which TFIIH alleviates PC4-mediated repression remains unknown.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP, 20 ng TFIIA, 10 ng TFIIB, 4 ng f:TBP, 10 ng TFIIE, 20 ng TFIIF, 20 ng recombinant TFIIH, 100 ng RNAPII, and the
indicated amount of PC4. All the transcription reactions
contained negatively supercoiled pML
53 (100 ng) as a template. The
reactions were performed at 30 °C for 1 h, stopped by the
addition of 20 mM EDTA, 0.2% SDS, and 5 µg of proteinase
K, and further incubated at 37 °C for 1 h. After
phenol/chloroform extraction and ethanol precipitation, the transcripts
were analyzed by electrophoresis on a 5% denaturing polyacrylamide
gel, followed by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
Bond Hydrolysis of ATP for the Alleviation
of PC4-mediated Repression by TFIIH--
To investigate the functional
relationship between TFIIH and PC4, we prepared recombinant TFIIH
reconstituted in the insect cells infected with three baculoviruses
that expressed TFIIH subunits (18, 19). In vitro
transcription assays were performed with recombinant TBP, TFIIB, TFIIE,
TFIIF, and TFIIH together with RNAPII purified from HeLa cells (Fig.
1A), using a linearized pML
53C2AT template that contained the adenovirus major late promoter fused with a 380-bp G-less cassette (19). The specific 390-nt transcript was observed only in the presence of all factors. No transcription was observed when one of the factors was omitted from the
reaction, indicating that there was no cross-contamination among the
factors (Fig. 1B).
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Fig. 1.
Transcription factors used for in
vitro transcription. A, purified proteins
were separated by 10% SDS-PAGE and silver stained. B,
reconstituted transcription analysis in vitro. Transcription
reactions were performed with a linearized pML C2AT that requires
TFIIH for the production of the 390-nt-specific transcript.
Transcription assays were performed in the presence of all factors
(lane 1) or in the absence of TBP (lane 2), TFIIB
(lane 3), TFIIE (lane 4), TFIIF (lane
5), TFIIH (lane 6), or RNAPII (lane 7). The
arrow indicates the position of the 390-nt transcript.
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Fig. 2.
Requirement of
-
bond hydrolysis of ATP
for alleviating PC4-mediated repression by TFIIH. A,
recombinant TFIIH was used for alleviating PC4-mediated transcriptional
repression. Transcription reactions consisting of TBP, TFIIB, TFIIE,
TFIIF, and RNAPII contained 200 ng of PC4 (lanes 2-6)
together with 5 (lane 3), 10 (lane 4), 20 (lane 5), or 40 ng (lane 6) of TFIIH. The
arrow indicates the position of the 390-nt transcript from
pML
C2AT. B, transcription reactions contained TBP, TFIIB,
TFIIE, TFIIF, and RNAPII, with (lanes 1-7) or without TFIIH
and PC4 (lanes 8-14). The reactions also contained 100 µM ATP (lanes 1 and 8), no ATP
(lanes 2 and 9), 100 µM dATP
(lanes 3 and 10), 100 µM AMP-PNP
(lanes 4 and 11), 100 µM AMP-PNP
and 100 µM dATP (lanes 5 and 12),
100 µM ATP-
S (lanes 6 and 13),
or 100 µM ATP-
S and 100 µM dATP
(lanes 7 and 14).
-
bond hydrolysis by substituting ATP with
adenylyl-imidodiphosphate (AMP-PNP) and
adenosine-5'-O-(thiotriphosphate) (ATP-
S), both of which
can be incorporated into growing RNA chains during transcription but
cannot be hydrolyzed at the
-
bond. When ATP was replaced by
non-hydrolyzable AMP-PNP or ATP-
S in the transcription reactions
containing both PC4 and TFIIH, virtually no transcription was observed
(Fig. 2B, lanes 4 and 6), indicating that
-
bond hydrolysis of ATP was absolutely required for
counteracting PC4-mediated repression. Transcription was restored,
however, when AMP-PNP and ATP-
S were further supplemented with dATP
(Fig. 2B, lanes 5 and 7), which could
provide
-
bond hydrolysis. These results show the requirement for
-
bond hydrolysis of ATP (or dATP) for alleviating PC4-mediated
repression even in the highly pure transcription system. Because TFIIH
is the only known factor that utilizes
-
bond hydrolysis of ATP
in this well defined transcription system, the results clearly
demonstrate the involvement of the enzymatic activities of TFIIH in the alleviation.
-
bond
hydrolysis suggested that one of the enzymatic activities of TFIIH was
required for alleviating the repression by PC4. To determine which
enzymatic activity of TFIIH was responsible for the alleviation, we
utilized three recombinant TFIIH mutants, each of which is defective in
either cdk7 kinase, ERCC3 helicase, or ERCC2 helicase activities (Fig.
3, A and B). These
mutants have alanine instead of the conserved lysine within the ATP
binding site of Walker type A motifs, at the 41st residue of cdk7,
346th residue of ERCC3, and 48th residue of ERCC2, respectively (Fig.
3A) (18, 20). Substitution of the lysine with either arginine or alanine in these motifs is known to eliminate the ability
to hydrolyze ATP, resulting in the inactivation of each enzymatic
activity. As shown in Fig. 3C, TFIIH with the mutated cdk7
kinase (K41A) and with the mutated ERCC2 helicase (K48A) alleviated
PC4-mediated repression as well as wild-type TFIIH, whereas TFIIH with
the mutated ERCC3 helicase (K346A) could not alleviate the repression
at all. These results demonstrate that ERCC3 helicase activity is the
sole enzymatic activity required for alleviating PC4-mediated
repression, and neither the cdk7 kinase nor the ERCC2 helicase plays
any role in alleviating PC4-mediated repression through ATP
hydrolysis.
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Fig. 3.
Requirement of ERCC3 helicase activity for
alleviating PC4-mediated repression. A, diagram of
cdk7, ERCC3, and ERCC2, indicating portions of amino acid sequences
including alanine residues (boldfaced) that were introduced
in place of lysine. The lysine residues (residues 41, 346, and 48 of
cdk7, ERCC3, and ERCC2, respectively) in the conserved ATP binding
domains, as shown in filled boxes, were mutated to alanine
using oligonucleotide-directed mutagenesis. B, purified
recombinant wild-type TFIIH (WT) and TFIIH mutants that have
a mutation in cdk7 (K41A), ERCC3 (K346A), or in
ERCC2 (K48A). C, alleviation of PC4-mediated
repression requires ERCC3 helicase activity. The transcription
reactions contained TBP, TFIIB, TFIIE, TFIIF, PC4, and RNAPII, together
with 5, 10, 20, and 40 ng of wild-type TFIIH (lanes 3-6),
K41A (lanes 7-10), K346A (lanes 11-14), or K48A
(lanes 15-18) as indicated.
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Fig. 4.
Phosphorylation of PC4. A,
phosphorylation of GST-CTD by casein kinase II (CKII) and
TFIIH were tested. Phosphorylation reactions contained GST-CTD as a
substrate together with 2 (lane 1) and 10 (lane
2) units of casein kinase II or with 100 ng of wild-type TFIIH
(lane 4), K41A (lane 5), K346A (lane
6), and K48A (lane 7). The arrows indicate
the position of hyperphosphorylated (IIO) and
hypophosphorylated (IIA) forms of GST-CTD. B,
phosphorylation reactions contained casein kinase II and TFIIH mutants
as shown in panel A, with 200 ng of PC4 as a
substrate in place of GST-CTD. The top and middle
panels show the short (1.5 h) and long exposures (20 h) of the
autoradiogram; the bottom panel shows Coomassie Blue
staining of the same gel. The arrows indicate the positions
of the phosphorylated (p-PC4) and non-phosphorylated
(PC4) forms of PC4, which migrated as ~20 and ~15 kDa,
respectively.
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Fig. 5.
Repression of transcription by phosphorylated
and non-phosphorylated PC4 in the absence of the ERCC3 helicase
activity. The relative levels of transcription are shown. The
level of transcription in the absence of PC4 was arbitrarily defined as
1.0. Transcription reactions contained TBP, TFIIB, TFIIE, TFIIF, and
RNAPII in the presence of either wild-type TFIIH (WT) or the
ERCC3-deficient TFIIH mutant (K346A), together with
indicated amounts of either non-phosphorylated or phosphorylated PC4.
The values indicate the level of transcription in the presence of
wild-type TFIIH and non-phosphorylated PC4 (filled squares,
solid line), wild-type TFIIH and phosphorylated PC4
(open squares, dotted line), K346A and non-phosphorylated
PC4 (filled triangles, solid line), and K346A and
phosphorylated PC4 (open triangles, dotted line).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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Fig. 6.
Model for PC4 in the regulation of
transcription from non-promoter and promoter regions.
A, PC4 binds to dsDNA and ssDNA regions and prevents
the binding of RNAPII to ssDNA regions by a direct competition. PC4
bound to dsDNA regions may also serve as a reservoir for PC4 recruited
to ssDNA regions. B, PC4 binds dsDNA regions and prevents
the binding of RNAPII to DNA by restricting the formation of transient
ssDNA regions. C, the PIC (dotted line)
containing TFIIH can initiation transcription from the promoter DNA
that is bound by PC4. D, the PIC whose TFIIH activity is
repressed (such as by FBP interacting repressor) fails to
initiate transcription from the promoter DNA that is bound by
PC4.
PC4-mediated repression of transcription from non-promoter regions as described above may facilitate the efficient allocation of the limiting amount of RNAPII in vivo (27, 28), which could be otherwise sequestered onto transiently melted ssDNA regions. In the living cells, DNA is predominantly negatively supercoiled and is also undergoing dynamic topological changes during DNA replication, transcription, and repair, possibly exposing melted ssDNA regions frequently. Spurious transcription from these melted ssDNA regions is likely to be suppressed mainly by phosphorylated PC4, which constitutes ~95% of PC4 in vivo (13), because phosphorylated PC4 can strongly suppress promoter-independent (and thus, general transcription factor-independent) transcription from the melted DNA region in vitro (14).
PC4 may also play a role in preventing spurious transcription from promoters, which in vivo is likely to be negatively supercoiled and from which transcription could be potentially initiated in the absence of TFIIH. When the ERCC3 helicase of TFIIH is active within the general transcriptional machinery, transcription is probably not repressed by PC4 in vivo (Fig. 6C) because the TFIIH ERCC3 helicase activity counteracts the repressive activity of phosphorylated PC4 at the physiological concentration (~1 µM) (Fig. 5). Indeed, when PC4 is overexpressed in cells in the absence of the HIV transactivator, transcription from the HIV promoter is only marginally reduced or not reduced at all, depending upon the assay conditions (10). However, if the ERCC3 helicase activity of TFIIH is inhibited (Fig. 5), such as by negative regulator of activated transcription and by FBP interacting repressor (29, 30), phosphorylated PC4 may further reduce the low background transcription from promoters even at the physiological PC4 concentration (Fig. 6D). Because TFIIH appears to be sub-stoichiometric (20-30%) to other general transcription factors in vivo (27), a fraction of PIC might even lack TFIIH and could be repressed by PC4, though this possibility must be rigorously examined in vivo. In any event, regulation of promoter-dependent transcription with a high level of dynamic range in vivo is likely to be contingent upon the presence of PC4, because negatively supercoiled DNA in vivo may permit inadvertent transcription from promoters and could potentially reduce the dynamic range of transcriptional regulation.
Several lines of evidence suggest the importance of PC4 in regulating transcription in vivo. First, a yeast homolog of human PC4, SUB4, enhances transcriptional activation by the activators GCN5 and HAP4 (12), and though PC4 is not essential for viability, its deletion results in inositol auxotrophy, a phenotype observed in the mutations of transcriptional regulators such as SNF/SWI, SRB, and the CTD of RNA polymerase II (31-34). Second, PC4 enhances TAT-dependent transcription from the HIV promoter (10) and restores the reduced AP-2 activity in the ras-transformed cell lines by relieving AP-2 self-interference (9). Finally, PC4 may play a role as a tumor suppressor in lung and bladder cancers, because the loss of heterogeneity of the PC4 gene is often observed in these cancer cells (35, 36). These results demonstrate the importance of PC4 as a regulator of transcription and possibly as a tumor suppressor in vivo. Though the importance of PC4 in vivo has been mainly interpreted in the context of its coactivator activity, the predominance of the repressive form of PC4 in vivo (13) suggests that some of the observed effects may well be attributed to the reduced precision of transcriptional regulation caused by the loss of the repressive activity of PC4.
In conclusion, the repressive activity of PC4 may be
essential for the intricate regulation of transcription in conjunction with the ERCC3 helicase of TFIIH. The repressive activity of PC4, and
possibly of other ssDNA-binding proteins, may play an important but yet
under-appreciated role for more elaborate and fine-tuned regulation of
reactions involving DNA molecules.
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ACKNOWLEDGEMENT |
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We thank M. Suganuma for technical assistance.
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FOOTNOTES |
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* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and by the Maruki Memorial Prize of Saitama Medical School.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.
A Research Fellow of the Japan Society for the Promotion of Science.
§ To whom correspondence should be addressed. Tel.: 81-49-276-1490; Fax: 81-49-294-9751; E-mail: kojihisa@saitama-med.ac.jp.
Published, JBC Papers in Press, February 17, 2003, DOI 10.1074/jbc.M213172200
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ABBREVIATIONS |
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The abbreviations used are: PC4, positive cofactor 4; ERCC, excision repair cross-complementing; cdk, cyclin-dependent kinase; PIC, preinitiation complex; dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; RNAPII, RNA polymerase II; TBP, TATA box-binding protein; CTD, carboxyl-terminal domain; Ni-NTA, nickel-nitrilotriacetic acid; TF, transcription factor; nt, nucleotide; HIV, human immunodeficiency virus.
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