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Molecular and Cellular Biology, December 1999, p. 7951-7960, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
TATA-Binding Protein-Interacting Protein 120, TIP120, Stimulates
Three Classes of Eukaryotic Transcription via a Unique
Mechanism
Yasutaka
Makino,1
Shingo
Yogosawa,1
Kentaro
Kayukawa,1
Frederic
Coin,2
Jean-Marc
Egly,2
Zheng-xin
Wang,3
Robert G.
Roeder,3
Kazuo
Yamamoto,4
Masami
Muramatsu,4 and
Taka-aki
Tamura1,*
Department of Biology, Faculty of Science,
Chiba University, and CREST Japan Science and Technology Corporation,
Inage-ku, Chiba 263-8522,1 and
Department of Biochemistry, Saitama Medical School, Moroyama,
Iruma-gun, Saitama 350-0495,4 Japan;
Institut de Génétique et de Biologie
Moléculaire et Cellulaire, 67404 Illkirch Cédex,
Strasbourg, France2; and Laboratory of
Biochemistry and Molecular Biology, The Rockefeller University, New
York, New York 100213
Received 14 May 1999/Returned for modification 29 June
1999/Accepted 2 September 1999
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ABSTRACT |
We previously identified a novel TATA-binding protein
(TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of
transcription from various kinds of promoters regardless of the
template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the
TBP-binding domain, is required for TIP120 function. Kinetic studies
suggest that TIP120 functions during preinitiation complex (PIC)
formation at the step of RNAP II/TFIIF recruitment to the promoter but
not after the completion of PIC formation. Electrophoretic mobility
shift assays showed that TIP120 enhanced PIC formation, and TIP120 also
stimulated the nonspecific transcription and DNA-binding activity of
RNAP II. These lines of evidence suggest that TIP120 is able to
activate basal transcription by overcoming a kinetic impediment to RNAP
II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven
transcription and binds to RPB5, one of the common subunits of the
eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells,
ectopically expressed TIP120 enhances transcription from all three
classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.
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INTRODUCTION |
The efficiency of transcription is
regulated mainly by two steps, initiation and elongation
(42-44). In the case of RNA polymerase II (RNAP
II)-dependent genes, in vitro studies revealed that the initiation of
transcription from TATA-containing promoters requires multiple general
transcription factors (GTFs) in addition to RNAP II, and early studies
with isolated factors reveal an assembly of these components into a
functional preinitiation complex (PIC) (8, 44, 65). The
initial step involves TFIID binding to the TATA box, which may be
stimulated by TFIIA. TFIIB, TFIIF, and RNAP II then bind to the
TFIID-TATA box complex to form a minimal PIC that, in some case, is
active on supercoiled templates. Further activation and complete PIC
assembly involve recruitment of TFIIE and TFIIH. More recent studies
have described the isolation of RNAP II complexes (RNAP II holoenzyme)
containing associated GTFs and mediators, suggesting a mechanism that
can bypass several steps in PIC assembly (26, 29, 63).
Although the assembly of RNAP II and GTFs on the promoter is
indispensable for transcription, regulation of the assembly through modulation in the levels of these factors is not apparent. Instead, gene-specific DNA-binding regulatory factors have been considered to
play a predominant role in this regulation. These factors may interact
directly or indirectly with RNAP II and GTFs to regulate either the
assembly of the PIC or its subsequent function. Potential mechanisms
include increasing the local concentration of GTFs and RNAP II on a
promoter, activating these factors via stoichiometric interactions
(44), enzymatic modification (52), and modifying chromatin and its associated proteins by the activities of enzymes such
as histone acetyltransferases and deacetylases (51). In some
cases, transcriptional mediators, which do not bind DNA directly, may
serve as bridging factors between gene-specific regulators and GTFs and
RNAP II (22, 44). Apart from functions at the level of
initiation, DNA-binding activators and mediators may also stimulate
transcriptional elongation. RNAP II elongation factors include SII,
SIII, ELL, p-TEFb, DSIF, and TFIIF are thought to act on most class II
genes through binding to RNAP II (42, 56).
The entire gene expression program in a cell is thought to be
systematically and generally controlled in a manner dependent on
various cell activities. For example, if a resting cell enters the
proliferation mode, a number of genes must be simultaneously activated.
Such concerted gene regulation could involve, in part, a
sequence-independent transcriptional regulator that acts on wide
spectrum of regulated genes transcribed by RNAP I, II, and III. In such
a case, the regulatory protein would be expected to interact with a
component common to the RNAP I, II, and III general transcriptional
mechanisms. Such components include common RNA polymerase subunits
(47) and the TATA-binding protein (TBP) (11, 16,
21).
TBP was initially identified as a component of TFIID (TBP plus
TBP-associated factors [TAFs]) in the RNAP II system (9, 14). TBP can bind to the TATA box to nucleate PIC assembly either in isolated form or in the context of TFIID (43). TBP was
also shown to be an essential component of the RNAP I accessory factor SL1/TIF and the RNAP III accessory factor TFIIIB and thus is regarded as a universal transcription factor (7, 53). TBP binds to numerous proteins that include other GTFs, viral transactivators, tumorigenesis-related factors, and other transcriptional activators and
mediators (4, 18, 44). These interactions are presumed to
effect transcriptional regulation through modulations of the efficiency
of PIC formation or function.
TBP-interacting protein 120 (TIP120) was originally identified as one
of several rat liver proteins that bind to a histidine-tagged TBP
(64). Although TIP120 has none of the motifs (leucine
zipper, zinc finger, etc.) usually found in transcription factors, its N-terminal region has a sequence that is related to a region of Drosophila TAF80 (10, 28). Indeed, TIP120
interacts directly with TBP in vitro and is associated with TBP in
nuclear extracts (64), thus suggesting a role in
transcriptional regulation. In this study, we investigated the effect
of TIP120 on basal transcription both in vitro and in vivo, and we
found that TIP120 stimulates the basal level of transcription via
enhancement of RNAP II-containing PIC formation. It is likely that
TIP120 plays an important role in PIC formation at the entry of RNAP II
into a template DNA.
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MATERIALS AND METHODS |
Expression, purification, and antibody for recombinant
TIP120.
The entire open reading frame of TIP120 was linked to an
N-terminal polyhistidine tag (20), subcloned into a
baculovirus vector (His-pBlueBacIII) (Invitrogen), and expressed in
Spodoptera frugiperda Sf9 cells. Sf9 cells expressing TIP120
were harvested, resuspended in a lysis buffer containing 20 mM Tris-HCl
(pH 7.9), 100 mM KCl, 0.1% NP-40, 1 mM 2-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 10% glycerol, and disrupted
by sonication. After centrifugation, the supernatant was applied onto a
Ni-agarose column (Qiagen) equilibrated with lysis buffer, and the
column was washed with BC100 buffer (20 mM Tris-HCl [pH 7.9], 100 mM KCl, 1 mM 2-mercaptoethanol, 1 mM PMSF, 10% glycerol) supplemented with 20 mM imidazole-HCl (pH 7.9). The bound proteins were eluted with
BC100 buffer containing 300 mM imidazole-HCl (pH 7.9), and the peak
fractions were dialyzed against BC50 buffer (BC100 with 50 rather than
100 mM KCl). The Ni column eluate was loaded onto a MonoQ (Pharmacia)
column, and the column was eluted with a linear gradient from 50 to 500 mM KCl in BC buffer. The 0.45 M KCl fraction was collected and dialyzed
against BC100 buffer.
For antibody production, TIP120 was further purified through a
preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel, and the
gel was stained with Coomassie brilliant blue. The band corresponding
to TIP120 was excised, and the protein was electroeluted from the gel
to immunize rabbits. The serum was affinity purified through
TIP120-immobilized HiTrap N-hydroxysuccinimide-activated beads (Pharmacia). The bound antibody was eluted from the column with
0.2 M glycine-HCl (pH 2.5) and neutralized with a 1/10 volume of 1 M
Tris-HCl (pH 8.0).
Reconstituted in vitro transcription. (i) For RNAP II
promoters.
Polyhistidine-tagged mouse TBP (24), human
TFIIB, and human TFIIE (15, 41) were expressed in
Escherichia coli and purified as described previously. The
and 
-
subunits of the human TFIIA coexpressed in E. coli were as reported by Ma et al. (33). Human TFIIF
was purified from Sf9 cells coinfected with recombinant baculoviruses
encoding the RAP74 and RAP30 subunits as described elsewhere
(1). TFIIH was prepared from HeLa cells as previously described (34). For purification of human TFIID, HeLa cell
nuclear extracts were fractionated through phosphocellulose (Whatman) and MonoQ (Pharmacia) columns as previously described (54). RNAP II was purified from calf thymus as described by Hodo and Blatti
(19).
Reconstituted in vitro transcription reactions were performed as
previously described with the promoters of the adenovirus major late
(AdML) and adenovirus E4 (E4) genes followed by a G-free cassette
(40, 46). Twenty microliters of reaction mixture containing
10 mM HEPES-KOH (pH 7.6), 25 mM KCl, 6 mM MgCl2, 3% glycerol, 45 ng of TBP (or 5 µl of TFIID fraction plus 0.1 µg of
TFIIA), 50 ng of TFIIB, 120 ng of TFIIF, 0.2 µg of calf thymus RNAP
II, and 400 ng of supercoiled DNA template (AdML and E4 promoters) was
preincubated for 20 min on ice, and the reaction was initiated by the
addition of ribonucleoside triphosphates (NTPs) to final concentrations
of 620 µM ATP, 620 µM UTP, 25 µM CTP, and 5 µCi of
[-32P]CTP. The standard runoff transcription was
carried out with TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, RNAP II
(34), and AdML and rabbit -globin (61)
promoters as described. Transcription was performed for 45 min at
30°C. Transcripts were resolved on a 5% sequencing gel. When
necessary, transcripts were quantified with a BAS 1500 RI-image
analyzer (Fuji Film).
(ii) For RNAP III promoters.
TFIIIB, TIIIC, and RNAP III
were immunopurified from the nuclear extracts made from FLAG-tagged
cell lines (6, 58-60). The partially purified TFD were
prepared as described previously (59). The adenovirus VA1
gene from 
96 to +212 (AdVA1) and human methionine tRNA gene from
131 to +148 (htRNA[Met]) were used to yield runoff products.
Reconstituted in vitro runoff transcription was performed for 60 min at
30°C in a reaction cocktail similar to that previously described
(58, 59).
(iii) For the mouse rRNA promoter.
The mouse ribosomal RNA
gene (mrDNA) promoter sequence from 330 to +291 in mrDNA
(37) linearized to yield a 320-base transcript was used for
the runoff transcription assay. Reconstituted in vitro transcription
was performed as previously reported (23), using fraction A'
(RNAP I stimulatory factor) (63a), C (RNAP I), and D (SL1
and UBF) prepared from FM3A mouse ascites cells. The reaction mixture
was incubated at 30°C for 60 min in the presence of 100 µg of
-amanitin per ml (23).
EMSA.
Electrophoretic mobility shift assay (EMSA) was
performed with an AdML TATA box probe from 45 to +20 as previously
described (3, 32). The binding mixture (10 µl) consisting
of 50 mM KCl, 12.5 mM HEPES-KOH (pH 7.6), 6.3 mM MgCl2,
0.05 mM EDTA, 0.05% NP-40, 0.5 mM dithiothreitol, 5% glycerol, 80 of
ng poly(dG-dC), 100 ng of bovine serum albumin (BSA), 1 ng of
end-labeled probe, TBP (10 ng), TFIIB (10 ng), TFIIF (50 ng), purified
RNAP II (200 ng), and TIP120 (200 ng) was incubated for 30 min at room
temperature. The reaction products were analyzed by polyacrylamide gel
electrophoresis (PAGE) through a 4% polyacrylamide gel containing 5%
glycerol and running buffer consisting of 25 mM Tris-base (pH 8.3), 190 mM glycine, and 5% glycerol.
Nonspecific transcription of RNAP II.
Nonspecific RNA
synthesis was carried out by the method of Sekimizu et al.
(48). The reaction mixture (50 µl) contained 50 mM
Tris-HCl (pH 7.9), 0.02 mM EDTA, 100 mM
(NH4)2SO4, 3 mM MnSO4,
2 mM MgCl2, 5 mM 2-mercaptoethanol, 1 mM PMSF, 0.5 mM GTP, 0.5 mM UTP, 0.5 mM ATP, 0.1 mM [-32P]CTP (0.5 µCi),
10% glycerol, 20 µg of activated calf thymus DNA, and 0.2 pmol of
purified calf thymus RNAP II. When necessary, 2 to 16 pmol of TIP120 or
16 pmol of GTFs was added to the reaction. Where indicated,
-amanitin (1 µg/ml) was included. The mixture was incubated at
30°C for 10 min, and a 15-µl aliquot was spotted on Whatman DE52
paper. The paper was rinsed five times with 5% Na2HPO4 solution, and radioactivities on the
paper were measured by a liquid scintillation counter.
In vitro binding for TIP120 and RNA polymerase subunits.
TIP120 carrying FLAG tag at its N terminus was purified from Sf9 cells
by passage through M2-agarose and MonoQ columns as described above.
cDNAs of RPB5, RPB6, RPB8, and RPB10, common subunits for RNA
polymerases, were cloned by PCR-mediated techniques. The
histidine-tagged RNA polymerase subunits were expressed in E. coli and purified by using Ni-agarose as described above.
Interaction with TIP120 and each RPB protein was analyzed by affinity
chromatography and pull-down experiments. TIP120 affinity columns (50 µl) were prepared by immobilizing FLAG-TIP120 fusion protein (5 µg)
on M2-agarose beads as described above. The columns were equilibrated with BC100 buffer. Control column contained no TIP120 protein. Purified
RPB proteins (0.5 µg) were loaded onto the columns. After extensive
washing of the columns with BC100 buffer, bound proteins were eluted
with FLAG peptide, analyzed by SDS-PAGE (15% gel), and detected by
silver staining.
Cells and transient luciferase assay.
P19 mouse embryonal
carcinoma cells and HEp-2 cells were cultured in alpha minimal
essential medium and Dulbecco's modified Eagle's medium (Gibco),
respectively, supplemented with 10% fetal bovine serum. For retinoic
acid treatment, all-trans retinoic acid (Sigma) was added
into the medium at a final concentration of 0.5 mM. For the transient
luciferase assay, P19 cells were transfected by the use of
Lipofectamine Plus (Gibco) with each luciferase reporter plasmid (150 ng) together with various amounts of TIP120-expressing effector plasmid
(pRcCMV-HA120). After incubation for 5 h, the cells were
transferred to new dishes at a dilution of 1:2 and further cultured for
40 h. The cells were harvested, and cell extracts (1 ml) were
prepared. The luciferase activity in 20 ml of reaction cocktail was
measured with a luciferase assay system (Promega) and TD20/20
luminometer (Terner Designs). Obtained values were normalized by
protein concentration.
For construction of the effector plasmid, the entire TIP120 cDNA coding
sequence having a hemagglutinin tag at the N terminus
was inserted
downstream from the cytomegalovirus (CMV) enhancer-promoter
regulatory
unit of pRc/CMV DNA (Invitrogen). Reporter plasmids
were constructed
from pGV-B vector DNA (Toyo Ink Co., Ltd.) by
inserting a promoter
sequence into the multicloning site of the
vector. Promoter sequences
included in each plasmid were as follows:
pGV-rDNA, positions
330 to
+291 of the mrDNA promoter; pGV-ML33,
positions
33 to +33 of the AdML
promoter; and pGV-ML677, positions
677 to +33 of the AdML
promoter.
Nucleotide sequence accession number.
Amino acid and
nucleotide sequences of rat TIP120 appears in the GenBank, EMBL, and
DDBJ databases with accession no. D87671.
| |
RESULTS |
Activation of basal transcription of RNAP II genes by TIP120.
To perform biochemical studies on TIP120, we expressed
polyhistidine-tagged TIP120 in Sf9 cells and purified the recombinant protein to near homogeneity by Ni-agarose and MonoQ chromatography (Fig. 1A). To test the effect of TIP120
on transcriptional regulation, we established a minimal system
containing TBP, TFIIB, TFIIF, RNAP II, and supercoiled DNA templates
with AdML and E4 promoters followed by a G-free cassette. The use of
supercoiled templates abrogated the need for TFIIE and TFIIH (39,
40, 55). Both AdML and E4 promoters were transcribed accurately
under our conditions (Fig. 1B, lanes 1, 5, and 9), and the addition of
recombinant TIP120 to the reaction resulted in activation of
transcription from both promoters in a dose-dependent manner (Fig. 1B,
lanes 2 to 4 and 10 to 12). A twofold molar excess of TIP120 per TBP molecule increased the level of transcription from AdML and E4 promoters 8.7- and 14-fold, respectively (Fig. 1B; compare lanes 1 and
4 and lanes 9 and 12), whereas the addition of equivalent amounts of
BSA had little effect (lanes 6 to 8). To further establish that TIP120
itself causes transcriptional activation, we added affinity purified
anti-TIP120 antibody to the reaction. As shown in Fig. 1C, anti-TIP120
antibody repressed TIP120-stimulated transcription (lanes 12 to 14) but
not basal transcription (lanes 9 to 11), whereas an equivalent amount
of control immunoglobulin G had little effect on basal or
TIP120-activated transcription (lanes 3 to 5 and 6 to 8, respectively).
These results further indicate that TIP120 itself was responsible
during the enhanced transcription.
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FIG. 1.
Stimulation of basal transcription in vitro by TIP120.
(A) Expression and purification of recombinant TIP120. Histidine-tagged
TIP120 expressed in Sf9 cells (lane 1) was purified by Ni-agarose (lane
2) and then by MonoQ (lane 3). The proteins were resolved by SDS-PAGE
(7.5% gel) and stained with silver. The arrow indicates the position
of the recombinant TIP120. (B) Effect of TIP120 on in vitro
reconstituted transcription. Transcription reactions were performed as
described in Materials and Methods, using supercoiled AdML (lanes 1 to
8) and E4 (lanes 9 to 12) templates. Reaction mixtures contained 100 ng
(lanes 2 and 10), 200 ng (lanes 3 and 11), or 400 ng (lanes 4 and 12)
of the recombinant TIP120. Equivalent amounts of BSA were added to the
reactions (lanes 5 to 8). TIP120-mediated stimulation is presented as
fold activation relative to controls (lanes 1, 5, and 9) at the bottom.
(C) Anti-TIP120 antibody suppressed the TIP120-mediated transcriptional
stimulation. In vitro transcription was performed with the AdML
promoter as for panel B. Each reaction contained 200 ng of TIP120 or
BSA as indicated. Control immunoglobulin G (IgG) (lanes 3 to 8) and
affinity-purified anti-TIP120 antibody (lanes 9 to 14) were added to
the reaction mixture.
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Since TFIID, rather than the derived TBP, is thought to function in
RNAP II transcription in vivo, we reconstituted a transcription
system
in which TBP was replaced by purified TFIID and TFIIA.
In this case,
TIP120 again stimulated (up to 11.5-fold) transcription
from the AdML
promoter in a dose-dependent manner (Fig.
2A). We
also investigated the effects of
TIP120 on linearized templates
in a complete transcription system that
contained TFIIA, TFIIE,
and TFIIH in addition to the minimal components
(Fig.
2B and C).
As observed in the minimal system with supercoiled
templates (Fig.
1B), transcription from a linear AdML template in the
complete
system was enhanced (up to 4.5-fold) by TIP120 in a
dose-dependent
manner (Fig.
2B). Further studies indicated that TIP120
also significantly
stimulated transcription from
-globin promoter
(Fig.
2C). Some
promoters (e.g., the conalbumin promoter) were
stimulated only
weakly (data not shown), implying that TIP120 may have
a promoter
preference. Thus, the combined results from Fig.
1 and
2
indicate
that TIP120 can stimulate transcription from a variety of
different
promoters, independently of TAFs, TFIIE, and TFIIH or DNA
topology.
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FIG. 2.
TIP120 stimulates the basal transcription in other in
vitro transcription systems. (A) TIP120-stimulated transcription in the
presence of TFIID. The supercoiled AdML G-free template was transcribed
in a transcription system with the minimal set of GTFs including TFIID
and TFIIA instead of TBP. Reaction mixtures contained 200 and 400 ng of
BSA (lanes 2 and 3) and TIP120 (lanes 5 and 6). TIP120-mediated
stimulation is presented as fold activation relative to controls (lanes
1 and 4) at the bottom. (B) TIP120 enhances transcription from
linearized AdML promoter in the complete transcription system with
TFIIB, TBP, TFIIE, TFIIF, TFIIH, and RNAP II. Reaction mixtures
contained 100, 200, and 400 ng of the recombinant TIP120 (lanes 5 to 7, respectively). Equivalent amounts of BSA were added to the reactions
(lanes 2 to 4). TIP120-mediated stimulation is presented as fold
activation relative to the control (lane 1) at the bottom. (C) TIP120
enhances transcription from linearized -globin promoter in the
complete transcription system. The -globin promoter was transcribed
in the complete transcription system as described above. Reaction
mixtures in lanes 2 and 3 contained 200 ng of BSA and TIP120,
respectively. TIP120-mediated stimulation is presented as fold
activation relative to the control (lane 1) at the bottom.
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Domain for transcriptional activation and TBP binding.
To
determine the domain of the TIP120 molecule responsible for
transcriptional activation, we constructed a series of C-terminal deletion mutants as depicted in Fig. 3A.
The full-length TIP120 and the deletion mutants (Fig. 3A) carrying both
FLAG and histidine tags at their N termini were expressed in Sf9 cells
and purified by Ni-agarose, MonoQ, and M2-agarose chromatography to
near homogeneity (Fig. 3B). These highly purified proteins were
analyzed for the ability to activate basal transcription in the
reconstituted system. Mutants DC1-6 (amino acids [aa] 1 to 618) and
DC1-4 (aa 1 to 412), as well as full-length TIP120 (Fig. 3C, lanes 2 to
4), enhanced transcription from AdML promoter, whereas mutant DC1-2 (aa
1 to 271) failed to do so (lane 5). These results suggest that the N-terminal one-third (upstream from aa 412) that includes charged and
acidic regions is sufficient for transcription activation.
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FIG. 3.
The N-terminal one-third of TIP120 is sufficient for
transcriptional stimulation and TBP binding. (A) Structures of deletion
mutants. Several characteristic sequences such as charged, acidic, and
leucine-rich regions are indicated. These four proteins contain both
FLAG and histidine tags at their N termini. Numbers indicate amino acid
positions of TIP120 from the N terminus. Results for transcriptional
stimulation and TBP binding for each construct are summarized. N.D.,
not determined. (B) The proteins depicted in panel A were expressed in
Sf9 cells and purified. One hundred nanograms of the purified proteins
was analyzed by SDS-PAGE and Coomassie brilliant blue staining. (C)
Effects of deletion mutants on basal transcription. Transcription
reactions were performed as described in the legend to Fig. 1B, using
supercoiled AdML template. Equimolar amounts (2.4 pmol) of full-length
TIP120 (lane 2) and the C-terminal deletion mutants (lanes 3 to 5) were
added to the reaction. Lane 1, without TIP120 protein. (D) Mapping of
the TBP-binding region of TIP120. Equimolar amounts (7.5 pmol) of TBP
and the deletion mutants were incubated as indicated. The mixtures were
immunoprecipitated (IP) with anti-TBP antibody (lanes 1 to 4) or
anti-FLAG antibody (lanes 5 to 8). The precipitated proteins were
resolved by SDS-PAGE (10% gel) and analyzed by Western blotting with
anti-FLAG antibody (lanes 1 to 4) or anti-TBP antibody (lanes 5 to
8).
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TIP120 interacts directly with TBP in solution (
64)
but is not stably incorporated into a TBP-DNA complex (data
not shown).
We next examined the ability of truncation
versions to interact
with TBP. Equimolar amounts of full-length TIP120
and truncated
mutants DC1-2 and DC1-4 were incubated with TBP and
immunoprecipitated
with anti-TBP antibody or anti-FLAG antibody.
Anti-TBP antibody
efficiently coimmunoprecipitated full-length TIP120
and DC1-4
(Fig.
3D, lanes 2 and 3) but not DC1-2 (lane 4). Equivalent
results
were obtained in the case of coimmunoprecipitation with
anti-FLAG
antibody (Fig.
3D, lanes 6 to 8). We also observed that
anti-TBP
antibody and anti-FLAG antibody were not able to
immunoprecipitate
DC1-4 (data not shown) and TBP (Fig.
3D, lane 5),
respectively.
These results (summarized in Fig.
3A) clearly demonstrate
the
correlation between the abilities of TBP binding and
transcriptional
stimulation. This correlation supports the idea that
TBP binding
is one of the essential requirements for TIP120-mediated
transcriptional
stimulation.
TIP120 contributes to PIC formation.
The primary event of
transcription entails the assembly of GTFs and RNAP II on a promoter to
form the PIC (17). To investigate whether the
TIP120-mediated stimulation occurs prior to or subsequent to PIC
formation, we used a previously described preincubation-Sarkosyl addition protocol (17). In this assay, transcription factors and DNA are first incubated in the absence of NTPs to allow PIC formation, and transcription is thus allowed to proceed in the presence
of NTPs and a Sarkosyl concentration that inhibits the formation but
not function of the PIC. When 0.01% Sarkosyl was present during PIC
formation, both basal and TIP120-stimulated transcription were
inhibited (Fig. 4A; compare lanes 3 and 4 to lanes 1 and 2). As expected, Sarkosyl did not abolish basal
transcription when added after a time (30 min) sufficient for PIC
formation (Fig. 4A, lanes 5 to 7). TIP120 had no effect when added with Sarkosyl at 30 min, consistent either with TIP120 function during PIC
formation or with a general inhibitory effect of Sarkosyl on TIP120
function (Fig. 4A, lanes 11 to 13). However, TIP120 did enhance
transcription when present during PIC assembly (0-min addition) and
prior to Sarkosyl addition (Fig. 4A, lanes 8 to 10). Since the level of
TIP120-enhanced transcription was comparable to that observed in
standard assay, these results suggest that TIP120 functions mainly
during PIC assembly and not at subsequent initiation or elongation
steps.
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FIG. 4.
TIP120 functions during PIC formation. (A) Effects of
Sarkosyl on TIP120-mediated transcriptional stimulation. Transcription
reactions were performed with the AdML promoter. Template DNA, GTFs,
and RNAP II were mixed at zero time (0'; the time at which
preincubation at 30°C started). After preincubation for 30 min (30'),
transcription was initiated by the addition of nucleotide substrates.
TIP120 was added to the reaction at zero time (lanes 2, 4, and 8 to 10)
or after 30 min (lanes 11 to 13). Sarkosyl (0.01%) was also added at
zero time (lanes 3 and 4) or at 30 min (lanes 5 to 13). The reaction
mixtures contained 100 ng (lanes 8 and 11), 200 ng (lanes 9 and 12), or
400 ng (lanes 2, 10, and 13) of TIP120. Lanes 1 and 2 did not contain
Sarkosyl. Equivalent amounts of BSA were added at zero time (lanes 1, 3, and 5 to 7). (B) Completion of PIC including the three GTFs and RNAP
II impairs the TIP120 effect. The AdML DNA was preincubated with
different subsets of GTFs for 30 min as indicated, and then
transcription was initiated by addition of nucleotide substrates and
remaining factors. Recombinant TIP120 (400 ng) or BSA was added as
indicated.
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To study the mechanism of TIP120-mediated activation in more
detail, we preincubated the DNA template with various combinations
of
GTFs and RNAP II before addition of TIP120, as shown in Fig.
4B.
The absence of any GTFs during the preincubation resulted
in a weak
basal activity (Fig.
4B, lane 1), whereas addition of
TBP alone or with
other factors during the preincubation period
considerably increased
basal transcription (lanes 3, 5, and 7).
This observation is consistent
with previous reports that TBP
binding to the TATA box is a
rate-limiting step for transcription
initiation (
8,
66).
Addition of TIP120 after template preincubation
with no GTFs (Fig.
4B,
lane 2), with TBP (lane 4), or with TBP
plus TFIIB (lane 6)
significantly enhanced the basal level of
transcription. In striking
contrast, TIP120 had no effect on the
basal level of transcription when
added after the template had
been preincubated with all of the
components (TBP, TFIIB, TFIIF,
and RNAP II) necessary for formation of
a minimal stable PIC (Fig.
4B, lane 8 versus lane 7). Inactivation of
GTFs and/or RNAP II
during the preincubation time was negligible (Fig.
4B, lanes 3,
5, and 7). These results, together with those of Fig.
4A,
show
that TIP120 functions during PIC formation at a step that precedes
or is coincident with the entry of RNAP II/TFIIF.
Stimulation of PIC formation by TIP120.
Next, we examined the
effect of TIP120 on complex formation with the AdML TATA box by EMSA.
TIP120 alone showed no stable binding to promoter DNA in EMSA (data not
shown). This finding suggests that TIP120 is not a conventional
DNA-binding protein, which is consistent with the homology search data
(64). An EMSA with TBP, TFIIB, TFIIF, and RNAP II resulted
in three distinct bands (Fig. 5, lanes 2 to 4) which were not observed in the absence of the GTFs and RNAP II
(Fig. 5, lane 1). The formation of those complexes was dependent on TBP
(Fig. 5, lane 2), TFIIB (lane 3), and TFIIF-RNAP II (lane 4), as
previously reported (3, 32). In addition, the binding of
RNAP II to the TBP-TFIIB complex was dependent on TFIIF (data not
shown). The addition of TIP120 significantly reduced the amount of
TBP-DNA complex, whereas the amount of the completed PIC
(TBP-TFIIB-TFIIF-RNAP II) was significantly increased (Fig. 5; compare
lanes 5 and 6 to 7 and 8; the experiment was done in duplicate). The
completed complex found in Fig. 5 (lanes 7 and 8) was not supershifted
by TIP120 and did not contain immunologically detectable TIP120 (data
not shown). Thus, TIP120 evidently is not integrated into the
TBP-DNA-complex even though it can bind to TBP in solution. These
observations suggest that TIP120 acts to increase the amount of the
RNAP II-containing transcription-competent complex and are consistent
with the order of addition experiments of Fig. 4B. However, since it is
unlikely that TIP120 is stably incorporated into the PIC, we anticipate
that TIP120 transiently interacts with the components of the PIC.
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FIG. 5.
TIP120 facilitates the formation of RNAP II-containing
PIC. DNA-protein complexes were formed with various GTFs and RNAP II
and analyzed by EMSA as described in Materials and Methods. A
5'-end-labeled DNA fragment containing the AdML TATA box was used in
the DNA binding assay. Lane 1, no protein. Various combinations of GTFs
and RNAP II, as indicated, were added to the DNA binding assay (lanes 2 to 8). PIC formation was performed in the absence (lanes 5 and 6) or
presence (lanes 7 and 8) of TIP120. An equivalent amount of BSA was
used (lanes 5 and 6). The reactions were done in duplicate. Positions
of the specific complexes are indicated.
|
|
TIP120 stimulates nonspecific transcription by RNAP II.
The
results of Fig. 4 and 5 imply that TIP120 can functionally interact
with RNAP II as well as TBP. To investigate the effect of TIP120 on
RNAP II itself, we assayed TIP120 function in a nonspecific RNAP II
transcription reaction. In experiments shown in Fig.
6A, RNAP II was incubated with template
DNA in the presence or absence of TIP120, and then transcription was
initiated by adding nucleotides. TIP120 dramatically (>7-fold)
increased RNAP II activity in a dose-dependent manner (Fig. 6A, lanes 3 to 5). Neither TBP nor TFIIB affected the RNAP II activity (Fig. 6A,
lanes 8 and 9), whereas TFIIF, which is known to promote the elongation
rate as well as the initiation frequency of RNAP II (2, 12),
facilitated RNAP II activity as expected (lane 7). The TIP120
preparation had no RNAP II activity, and TIP120-stimulated
transcription was sensitive to -amanitin (Fig. 6A, lanes 2 and 6).
These findings clearly demonstrated that TIP120 can stimulate RNAP II
activity. We also found that TIP120 was able to increase the activity
of RNAP II of HeLa and mouse cells prepared by different purification protocols (data not shown). These findings emphasized our supposition that TIP120 targets RNAP II for transcriptional activation as well as
TBP.
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FIG. 6.
Effect of TIP120 on RNAP II activity. (A) Stimulation of
enzyme activity by TIP120. Each RNAP II reaction contains 4 pmol (×1),
8 pmol (×2), or 12 pmol (×3) of TIP120. Lanes 7 to 9 contain 8 pmol
(×2) of TFIIF and 16 pmol (×3) of TFIIB and TBP, respectively. Lane 6 contains -amanitin. The reactions were done in duplicate. (B) TIP120
stimulates the DNA binding of RNAP II. DNA-protein complexes were
formed with the DNA fragment used in the previous experiment (Fig. 5)
and RNAP II and analyzed by EMSA in the absence of a carrier DNA. Lane
1, no protein. Various combinations of RNAP II (200 ng), TIP120, and
BSA, as indicated, were added to the DNA binding assay. Reaction
mixtures contained 100 ng (lanes 3 and 5) and 200 ng (lane 4) of the
recombinant TIP120. Lane 6 contained 100 ng of BSA. Positions of the
specific complexes are indicated.
|
|
The above observations suggest the existence of a mechanism by which
TIP120 stimulates RNAP II recruitment to a template DNA
or the RNAP II
elongation rate. To examine these possibilities,
we tested the effect
of TIP120 on DNA-binding activity of RNAP
II. It is known that RNAP II
is able to bind nonspecifically to
a DNA fragment in vitro (
25,
27). The purified RNAP II was
incubated with the radiolabeled
oligonucleotide used in the previous
experiment (Fig.
5) in the absence
of a carrier DNA. As expected,
an EMSA with only RNAP II resulted in
one distinct band (Fig.
6B, lane 2) which was not detected in the
absence of RNAP II (lane
1). The addition of TIP120 significantly
increased the amount
of RNAP II-DNA complex in a dose-dependent manner
(Fig.
6B, lanes
3 and 4), whereas TIP120 itself did not form a clear
retarded
band (lane 5), and an equivalent amount of BSA had little
effect
on the formation of the RNAP II-DNA complex (lane 6). Moreover,
we found that TIP120 had little effect on the elongation rate
of RNAP
II (data not shown), which agrees with the results of
the Sarkosyl and
kinetic experiments (Fig.
4). These results clearly
demonstrate that
TIP120 facilitates the binding of RNAP II to
DNA. Thus, it is likely
that the stimulation of RNAP II activity
by TIP120 is a consequence of
the ability of TIP120 to enhance
DNA-binding activity of RNAP
II.
Effects of TIP120 on other classes of promoters.
To link the
above observations to our assumption that a target(s) of TIP120 is some
general component or common reaction for transcriptional regulation, we
next investigated the effect of TIP120 on two other transcription
systems, i.e., those utilizing RNAP I and RNAP III. First, mrDNA was
examined in a reconstituted transcription system composed of mouse
factors (Materials and Methods), since in vitro transcription of RNAP I
exhibits a species specificity (14, 45). As shown in Fig.
7A, mrDNA was weakly but significantly
transcribed in this assay system (lane 1), and TIP120 stimulated the
specific transcription (lanes 2 and 3). The activation index obtained
with 500 ng of TIP120 was 15 (compare lanes 1 and 3), nearly the same
as that obtained in the RNAP II system (Fig. 1B).
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FIG. 7.
TIP120 stimulates RNAP I- and III-directed in vitro
transcription. (A) TIP120 enhances transcription from mrDNA. mrDNA was
transcribed in the presence of mouse factors as described in Materials
and Methods). Reaction mixtures contained 250 and 500 ng of the
recombinant TIP120 (lanes 2 and 3, respectively). TIP120-mediated
stimulation is presented as fold activation relative to the control
below the panel. (B) Effect of TIP120 on transcription from class III
promoters. The AdVA1 and htRNA[Met] promoters were transcribed.
Reaction mixtures contained 200 ng (lane 5) or 800 ng (lanes 2 and 6)
of TIP120. Lanes 3 and 7 contained 800 ng of BSA. TIP120-mediated
stimulation is presented as fold activation relative to the controls
(lanes 1 and 4) at the bottom. (C) TIP120-mediated transcriptional
stimulation of the AdVA1 promoter is independent of TDF. The AdVA1
promoter was transcribed as shown in panel B. Transcription reactions
were performed in the absence (lanes 1 to 4) or presence (lanes 3 to 6) of TDF. Reactions contained 200 ng (lane 4) or 800 ng (lanes 2 and 5) of TIP120. Lanes 1 and 6 contained 800 ng of BSA.
TIP120-mediated stimulation is presented as fold activation relative to
the control (lane 1) at the bottom. Parentheses indicate fold
activation relative to lane 3.
|
|
We further examined TIP120 effects on two different RNAP III genes,
AdVA1 and htRNA[Met], in a system reconstituted with human
TFIIIB,
TFIIIC, and RNAP III (
57,
59). Transcription from
the AdVA1
promoter was stimulated by TIP120, with a stimulation
index of 6.1 (Fig.
7B; compare lanes 1 and 2). htRNA[Met] transcription
was also
weakly stimulated by TIP120 (Fig.
7B, lane 6). As TDF
(translation-dependent factor) has been demonstrated to enhance
RNAP
III-driven in vitro transcription (
59), the effect of TIP120
was also examined in the presence of added TDF. As seen in Fig.
7C,
TIP120 stimulated AdVA1 transcription 9.2-fold (compare lanes
1 and 2)
and 4.0-fold (compare lanes 3 and 5) in the absence and
presence of
TDF, respectively. Thus, TIP120-mediated stimulation
is TDF
independent. Significantly, the combined stimulation index
for AdVA1
gene transcription by both TDF and TIP120 was exceptionally
high,
30-fold (Fig.
7C; compare lanes 1 and
5).
To elucidate a mechanism of TIP120-mediated stimulation of three
classes of RNA polymerases, we investigated whether TIP120
interacts
with subunits common to all three RNA polymerases. RPB5,
RPB6, RPB8,
RPB10
, and RPB10
are common subunits of three RNA
polymerases,
whose structures and functions are highly conserved
in the eukaryotes
(
49). Affinity columns were prepared by immobilizing
the
FLAG-TIP120 fusion proteins on M2-agarose beads. Equivalent
amounts of
human RPB5, RPB6, RPB8, and RPB10
were loaded onto
the columns, and
the adsorbed proteins were detected. As shown
in Fig.
8, the TIP120 column significantly
retained RPB5 (lane
3) but not RPB6, RPB8, and RPB10
(lanes 6, 9, and 12). No binding
of those four subunits to M2-agarose was observed
(Fig.
8, lanes
4, 7, 10, and 13), suggesting that binding between
TIP120 and
RPB5 is specific. These results imply that at least RPB5 is
a
possible target for TIP120 in three RNA polymerases.
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FIG. 8.
TIP120 binds to RPB5 in vitro. FLAG-TIP120 fusion
proteins were immobilized on M2-agarose beads (TIP120-ag). As a
control, M2-agarose beads (M2-ag) were used. Recombinant RPB5 (lanes 3 and 4), RPB6 (lanes 6 and 7), RPB8 (lanes 9 and 10), or RPB10 (lanes
12 and 13) was loaded onto the columns. Proteins retained the columns
were analyzed as described in Materials and Methods. Lanes 2, 5, 8, and
11 show 20% of each input subunit. Lane 1, recombinant FLAG-tagged
TIP120. M, molecular weight markers. Note that RPB6 and RPB8 were
scarcely stained with silver (lanes 5 and 8) even though equivalent
amounts of subunits were loaded.
|
|
TIP120 stimulates transcription from three different classes of
promoters in P19 cells.
The above studies have shown that TIP120
stimulates in vitro transcription from three classes of promoters. To
further assess the physiological relevance of these observations, we
studied the contribution of TIP120 to transcriptional regulation in
vivo. P19 mouse cells (35) were used for transient
transfection assays in which various promoter-carrying luciferase
reporter plasmids were introduced into the cells together with the
TIP120 expression plasmid. P19 cells constitutively expressed a low
level of TIP120 protein (see below). As observed in Fig.
9, increased amounts of effector plasmids
enhanced the relative luciferase activity from all of the promoters
used. When 540 ng of effector DNA was used, stimulation indices in
transcription efficiency of pGV-mrDNA for RNAP I, pGV-ML677 (positions
677 to +33 of the AdML promoter), and pGV-ML33 (positions 33 to +33
of the AdML promoter) for RNAP II and of pGV-AdVA1 for RNAP III were
3.1, 3.6, 6.8, and 3.6, respectively. The failure of the control
(empty) effector plasmid to exhibit any stimulation indicates that
ectopically expressed TIP120 is responsible for the observed
stimulation, whereas the failure to see high levels of stimulation (as
observed in vitro) may reflect contributions of endogenous TIP120.
Interestingly, TIP120 exhibited a higher stimulatory effect on the
enhancerless AdML core promoter (pGV-ML33) than on the
enhancer-containing promoter (Fig. 9B). These results are consistent
with the view stated above that TIP120 acts on the basal transcription
apparatus. Taken together, these data indicate that TIP120 enhances
transcription from all three classes of promoters in vivo.
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FIG. 9.
TIP120 stimulates transcription of three classes of
promoters in vivo. Promoter fragments from mrDNA, AdML (677 to +33),
AdML (33 to +33), and AdVA1 were inserted into the promoterless
luciferase vector to construct pGV-mrDNA (A), pGV-ML677 (B), pGV-ML33
(B), and pGV-AdVA1 (C), respectively. Each reporter plasmid (150 ng)
was used to transfect to P19 cells in the presence of the CMV
promoter-driven TIP120-expressing effector plasmid (180, 360, and 540 ng). Enzyme activities are normalized with protein concentration. The
data are presented as activity relative to that without effector
plasmid. The height of each column represents the mean of three
independent transfections performed in duplicate. Standard deviations
are indicated by vertical lines.
|
|
| |
DISCUSSION |
TIP120 commonly stimulates transcription in vitro.
TIP120
stimulated transcription from multiple RNAP II-driven genes in the
reconstituted cell-free systems. Because the recombinant TIP120 was
highly pure (Fig. 1A) and a parallel preparation from mock-infected Sf9
cells did not exhibit an activating property (data not shown), this
stimulatory effect must be due to TIP120 itself and not to
contaminating proteins. In support of this conclusion, an antibody
directed against recombinant TIP120 specifically suppressed the
TIP120-mediated activated transcription (Fig. 1C).
In vitro transcription might be elevated in a nonspecific manner by
proteins that are not true transcription factors but somehow
stabilize
bona fide transcription factors or suppress inhibitory
agents. However,
the following observations indicate that the
effect of TIP120 is
specific. First, TIP120 shows some promoter
specificity, in that
chicken conalbumin (data not shown) and htRNA[Met]
(Fig.
7B)
promoters, in marked contrast to others in the cognate
class, showed
low stimulation by TIP120. Second, TIP120 function
was apparent only
when TIP120 was added at a particular time during
PIC formation, being
essentially ineffective when added after
PIC formation (Fig.
4). Third,
high TIP120-dependent stimulation
levels were seen when TIP120 was
included at nearly equimolar
amounts with GTFs, implying a specific
interaction between TIP120
and a component(s) thereof. Finally, ectopic
TIP120 resulted in
enhanced transcription from all three classes of
promoters in
P19 cells (Fig.
9). In these cases, TIP120-mediated
stimulation
was much more pronounced for the AdML core promoter
(6.8-fold)
than for the enhancer-carrying counterpart (3.6-fold) (Fig.
9B),
in good agreement with the observed effect of TIP120 on AdML core
promoter function in vitro (2.6- to 8.7-fold [Fig.
1B]).
Our analysis showed that a stimulatory effect of TIP120 on
transcription by RNAP II was independent of DNA topology, the type
of
class II promoter, or the specific GTF requirement (Fig.
2).
In the
latter case, the stimulatory effect was independent of
TFIIE, TFIIH,
TFIIA, and TAFs. Moreover, since the degrees of
stimulation were
similar under various RNAP II reaction conditions,
TIP120 appears to
act on a common process in RNAP II-mediated
transcription. Importantly,
TIP120 also stimulated transcription
by RNAP I (Fig.
7A) and by RNAP
III, most notably on the AdVA1
gene in the latter case (Fig.
7B). In
the case of the RNAP III
system, the effect of TIP120 was demonstrated
to be independent
of the stimulatory factor TDF. These observations
indicate that
TIP120 generally stimulates all three transcription
systems in
vitro. To data, no other nonessential transcription factors
have
been clearly demonstrated to activate transcription by all three
RNA polymerases at the basal level. Thus, TIP120 appear to be
unique
among known transcriptional regulators, and the present
study may
provide new insights into mechanisms of gene regulation.
The situation
for TIP120 may be analogous to that of PC4 and topoisomerase
I (Topo
I), earlier-described RNAP II coactivators (
13,
30,
31,
36,
50) that more recently were found in a TFIIIC-containing
complex
and to function in RNAP III transcription (
13,
36,
60). It
was further argued that PC4 and Topo I might function
in transcription
by all three RNA polymerases (
60). In contrast
to TIP120,
PC4 and Topo I affected only the activated or multiple
round
transcription, not basal or single-round
transcription.
Mechanism of TIP120-mediated stimulation.
Since TIP120 could
stimulate transcription by RNAP II only when it was added at a specific
stage in PIC assembly (i.e., before TFIIF-RNAP II is recruited into the
TBP-TFIIB-DNA complex) (Fig. 4), TIP120 may act at a specific time or
affect a particular process in PIC assembly. EMSA clearly indicated
that TIP120 shifted the balance between TBP-DNA complexes (decreased)
and TBP-TFIIB-TFIIF-RNAP II-DNA complexes (increased) (Fig. 5). Hence,
we suggest that TIP120 facilitates PIC assembly and that it must act at
a specific step prior to completion of PIC formation. Consistent with
this idea, TIP120 had little effect on the elongation rate of RNAP II
(data not shown).
What is the target of TIP120, and how does TIP120 facilitate PIC
formation? Since the stimulatory effect of TIP120 was seen
in all RNA
polymerase transcription systems, the logical hypothetical
target would
be a general process or/and a molecule common to
all three reaction
systems. Based on this aspect, TBP is a more
likely target
molecule. First, TBP is included in SL1, TFIID,
and TFIIIB, which are
involved in core promoter recognition events
on genes transcribed by
RNAP I, II, and III, respectively (
7,
9,
53). Second, TIP120
can specifically bind to TBP in solution
(
64), and deletion
analysis demonstrated a correlation between
the ability of TIP120 to
bind TBP and TIP120-mediated stimulation
(Fig.
3). NC2 is a good
contrast to TIP120, because NC2 also binds
to TBP but represses the
basal transcription by RNAP II and III
(
62) and
perhaps RNAP I (
56a). However, even though TIP120
can
bind to TBP in solution, no evidence was obtained for the
stable
binding of TIP120 to TBP-DNA or to the completed PIC. This
suggests an unstable or transient association of TIP120 with
DNA-bound
TBP and higher-order complexes during PIC
assembly.
Additionally, since several polypeptides (RPB5, RPB6, RPB8,
and RPB10
) are shared by all three classes of RNA polymerases
(
47,
49), we expected that such a common RNA polymerase
subunit(s)
would be another target for TIP120. In this study, we
presented
evidence for specific interaction of TIP120 with RPB5 (Fig.
8).
RPB5 was found to interact with human hepatitis B virus X protein
to modulate the function of RNA polymerase (
5). Recent work
also demonstrated RPB5 plays a role in activated transcription
(
38). The protein-protein interaction between TIP120 and
RPB5
can explain the results in Fig.
6. We also found that TIP120 could
stimulate enzymatic activity of RNAP I and III (our unpublished
observations). TIP120 may modify RNAP II function by inducing
a
conformational change in RNAP II through interaction with RPB5
subunit. Eventually, such a mechanism is likely to stimulate the
entry
of RNAP II to PIC. Alternatively, TIP120 could also bind
to DNA
and function as a mediator between RNAP II and
DNA.
We propose a model in which TIP120 transiently interacts with both TBP
(or TBP-TFIIB-DNA) and RNAP II to facilitate specific
integration of
RNAP II into the PIC. TIP120 may dissociate from
the PIC soon after RNA
polymerases are incorporated at the specific
region. Hence, TIP120
might exhibit some chaperone-like activity
toward the RNA polymerases.
Consequently, TIP120 can be categorized
as a novel type of
transcriptional modulator that communicates
with RNA polymerase and
general transcription factor
TBP.
Relevance of TIP120 to in vivo gene regulation.
Since the
transcriptional stimulation function of TIP120 was initially found in
the in vitro reconstituted transcription systems, the question arises
as to whether TIP120 works in gene regulation in the cell. That TIP120
plays a role in vivo is suggested by the following findings. First,
TIP120 was effective in an RNAP II reaction system containing the more
physiological TATA-binding factor TFIID rather than the derived TBP
(Fig. 2A). Second, TIP120 copurified with TFIIIC (data not shown),
which as described above also contains the general coactivators PC4 and
Topo I (60). Third, TIP120 can directly bind to TBP
(64) and a particular subunit of RNA polymerases (Fig. 8).
Both TBP and TIP120 were found in the same nuclear protein complex
(64). Fourth, the TIP120-mediated transcriptional
stimulation by all three RNA polymerases was demonstrated in
cotransfection experiments (Fig. 9).
When cells are exposed to various inducible agents that elicit
proliferation and differentiation, a number of genes must be
regulated
simultaneously at the transcription step. Moreover,
growth stimuli are
thought to influence transcriptional regulation
for multiple sets of
genes that include not only class II genes
but also class I and III
genes. Neither conventional gene-specific
regulators nor GTFs can
account for the occurrence of such total
gene regulation in a cell. We
thus hypothesize the existence of
a global activator which allows
amplification of the expression
of multiple genes, and TIP120 appears
to serve as such a regulator.
If this is the case, analysis of TIP120
will provide new insight
into global gene
expression.
| |
ACKNOWLEDGMENTS |
Y.M. and S.Y. contributed equally to this study.
We thank R. C. Conaway, D. Reinberg, and H. Handa for their
generous gifts of cDNA clones of GTFs. We also thank H. Handa and T. Fukasawa for helpful discussions. This work was supported in part by
grants from the program Grants-in-Aid for Scientific Research on
Priority Areas of The Japanese Ministry of Education, Science, Sports,
and Culture.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho,
Inage-ku, Chiba 263-8522, Japan. Phone: (81) 43-290-2823. Fax: (81)
43-290-2824. E-mail: btamura{at}nature.s.chiba-u.ac.jp.
| |
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Abstract
Introduction
