From the Transcription and Disease Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore-560 064, India
Received for publication, January 31, 2001
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ABSTRACT |
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The human positive coactivator 4 (PC4) acts as a
general coactivator for activator-dependent transcription,
the activity of which is regulated negatively by phosphorylation. We
report here that PC4 can be acetylated specifically by another
coactivator, p300. Interestingly, phosphorylation of PC4 by casein
kinase II inhibits the p300-mediated acetylation. Mass spectral
analysis revealed that there are at least two lysine residues
acetylated in PC4, as a result of which its DNA binding activity is stimulated.
Human positive coactivator 4 (PC4)1 was isolated from a
mammalian cofactor activity, upstream stimulatory activity,
independently by two different groups. It mediates
activator-dependent transcription by RNA polymerase II,
apparently through interactions with the transcriptional activator and
basal transcription machinery (1, 2). PC4 binds double-stranded DNA in
a sequence-independent manner. The regions required for the binding and
for coactivation overlap in PC4. However, the coactivator function also
depends on its ability to interact with the preinitiation complex (1, 3). Presumably both DNA binding and interaction with general transcription factors (predominantly TATA box-binding protein and TFIIA) are involved in coactivation by PC4. Recently, it was reported that PC4 binds tightly to melted double-stranded and single-stranded DNA through its novel C-terminal fold. It has been
demonstrated that interactions of this unique C-terminal domain with
the melted DNA repress transcription that can be attenuated by TFIIH
and phosphorylation of PC4 (4-6).
PC4 is subjected to in vivo phosphorylation events that
negatively regulate its coactivator function and its interactions with
the activator and TATA box-binding protein/TFIIA (2, 7). Phosphorylation also inhibits double-stranded DNA binding but not the
single-stranded DNA binding (3). Mutation and mass spectrometric
analyses suggest that phosphorylation of PC4 in vivo is
mediated by casein kinase II (CKII), and it is restricted to seven
serine residues between 2 and 28 at the N terminus (7). Interestingly
95% of the total cellular PC4 is phosphorylated and thus inactive
in vivo, whereas only 5% is in the nonphosphorylated active form.
Among the different human PCs, PC1/Poly (ADP-ribose) polymerase,
PC3/topoisomerase I, PC4, PC52, high mobility group protein 1 (HMG-1),
and HMG-2 are relatively abundant nuclear proteins that display
somewhat generalized, sequence-independent DNA binding properties and
are involved in diverse nuclear processes besides RNA polymerase
II-mediated transcription (reviewed in Refs. 8 and 9). Thus it raises
the possibility that PC4 may be a chromatin protein having
transcriptional coactivator function.
The reversible acetylation of nucleosomal histones and presumably
non-histone chromatin proteins plays a significant role in
chromatin-mediated transcription regulation. The idea that histone
acetylation is related causally to transcriptional activation has
received solid support from the discovery that a number of transcriptional coactivators have histone acetyltransferase (HAT) activity. These include GCN5 and PCAF, p300 and CBP, nuclear
receptor coactivators SRC1 and ACTR, and TATA box-binding
protein-associated factor TAFII250 and HIV-1-tat
interactive protein, TIP60 (10, 11). Among these HATs, the HAT activity
of yeast GCN5 and human p300 has been shown to be involved directly in
chromatin-mediated transcriptional activation (12, 13). However,
histones are not the only substrates of HAT proteins. For example,
CBP/p300 can acetylate p53, resulting in an enhancement of its DNA
binding activity. CBP/p300 also can acetylate basal transcription
factors such as TFIIE In support of this possibility we demonstrate that PC4 can be
acetylated specifically by p300 among the different HATs tested. Mass
spectral analysis and acetylation of recombinant PC4 revealed that
there are two lysine residues that are acetylated by p300. Most
interestingly, we found that phosphorylation of PC4 by CKII inhibits
the p300-mediated acetylation of PC4. However, acetylation does not
influence phosphorylation of PC4. Furthermore, acetylation of PC4
stimulates its double-stranded DNA binding activity. These findings may
open a new chapter in the understanding of PC4 function in
vivo.
Purification of Recombinant Proteins--
Recombinant PC4 was
expressed in Escherichia coli and purified as described
elsewhere (19). Briefly, the clear bacterial lysate was passed through
a heparin-Sepharose column, and the bound protein was eluted with BC
buffer (20 mM Tris-HCl, pH 7.9, at 4 °C, 20% (v/v)
glycerol, 10 mM Purification of Human Core Histones--
Human core histones
were purified from HeLa nuclear pellet as described previously
(23).
HAT Assay--
HAT assays were performed as described elsewhere
(23). Indicated amounts of proteins (see figure legends) were incubated at 30 °C for 30 min in a 30-µl final reaction volume consisting of
50 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, 0.1 mM EDTA, pH 8.0, 10 mM sodium
butyrate, and 0.5 µl of 3.3 Ci/mmol [3H]acetyl
Coenzyme A (acetyl-CoA). The reaction mixture then was blotted onto
P-81 filter paper. Radioactive counts were recorded on a Wallac 1409 liquid scintillation counter. To visualize the radiolabeled acetylated
protein, the reaction products were precipitated with 25%
trichloroacetic acid, washed with ice-cold acetone, and resolved
electrophoretically on 15% SDS-polyacrylamide gel and subjected to
fluorography using a solution containing 22.5% 2,5-diphenyloxazole in
Me2SO (24). Gels were dried, and autoradiography
was performed at Mass Spectral Analysis of PC4--
For the mass spectral
analysis of PC4, the protein was acetylated by FLAG epitope-tagged
recombinant p300 HAT domain (amino acids 1195-1810). In a typical
reaction mixture, 30 µg of PC4 and p300 HAT domain was incubated in
the presence of 50 µM acetyl-CoA at 37 °C for 1.5 h. To have the efficient acetylation, p300 and acetyl-CoA were added at
every 30-min interval. The reaction volume was scaled up to 1000 µl
and then concentrated to 100 µl using a 10K cutoff Centricon
concentrator (Amicon). Acetylation of protein was confirmed by
analyzing the protein on 15% acid-urea-polyacrylamide gel. The
acetylated protein was dialyzed against water before being subjected to
mass spectral analysis. Electrospray ionization mass spectral analysis
was done in an HP:1100 machine using acidified water (pH 3.0). The
solvent flow rate used was 0.03 ml/min. Nitrogen gas was used for
drying and nebulization.
In Vitro Phosphorylation of PC4--
In the phosphorylation
reaction, 0.25 µg of the recombinant PC4 was incubated for 30 min at
30 °C in the phosphorylation buffer (50 mM
HEPES-K+, pH 7.6, 125 mM NaCl, 10 mM MgCl2, 6% (v/v) glycerol, 5 mM
dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride)
containing 5 mM ATP with 10 milliunits of rat liver CKII in
a total volume of 20 µl. For large scale purification of
phosphorylated PC4, the reaction volume was scaled up to 1000 µl and
concentrated to a final volume of 100 µl using a 10K cutoff Centricon
concentrator (Amicon). To determine the effect of acetylation on
phosphorylation, the acetylated PC4 (0.25 µg) was subjected to
in vitro phosphorylation using 1 µCi of
[ Electrophoretic Mobility Shift Assay--
The electrophoretic
mobility shift assay was carried out essentially as described
previously (3) with some modifications. The sequence of the
oligonucleotide comprising of HIV-1 core promoter sequences upstream of
the initiator region of the adenovirus major late promoter
(5'-CCCTCAGATGCTGCATATAAGCAGCTGCTTTTTGCCCGTCCTCACTCTCTTCCGCATCGC-3') was used for the assays. The indicated amounts of different proteins were incubated with 40 fmol of either double-stranded labeled oligonucleotide for 30 min at 30 °C in a buffer containing 10 mM Tris-HCl, pH 7.4, 5% (v/v) glycerol, 1 mM
dithiothreitol, 1 mM EDTA, pH 8.0, 0.4 M KCl,
and 0.1% Nonidet P-40. The reaction mixtures then were resolved on a
6% native polyacrylamide gel at 130 V in TGE buffer (25 mM
Tris, 100 mM glycine, and 10 mM EDTA, pH 8.0)
for 3 h. The gels were dried and autoradiogrammed. Quantitation of
the DNA-protein complex was carried out using a Fuji phosphorimaging analyzer.
To test whether PC4 could be acetylated by known HATs, full-length
His6-tagged p300 (21) and FLAG epitope-tagged PCAF (22) from respective baculovirus-infected Sf21 cells (Fig.
1B) were purified. The native
recombinant human PC4 (19), Gal4 DNA-binding domain, and the nucleosome
assembly protein 1 (NAP1) (23) also were purified from E. coli for this purpose. The highly purified human core histones
were isolated from HeLa nuclear pellet (Fig. 1A) (23). The
Gal4 DNA-binding domain and NAP1 were used in the protein
acetyltransferase assay as negative controls. The human core histones
were included as positive controls in all the assays. The authenticity
of the purified human PC4 was checked by Western blotting using
polyclonal antibodies (Fig. 1C). The reaction mixtures
containing equivalent amounts of either p300 or PCAF as normalized by
filter binding assay using highly purified human core histones (Fig.
2A) and the indicated amount
of proteins (see figure legends) were incubated with
[3H]acetyl-CoA for 30 min at 30 °C. The products were
analyzed by autoradiography of acetylated reaction products resolved on
SDS-PAGE gels. As shown in Fig. 2B it was observed that PC4
can only be acetylated by p300 but not with equivalent activity of PCAF
(compare lanes 5 and 6). As expected, p300 could
not acetylate NAP1 and Gal4 (Fig. 2B, lanes 7 and
8), showing the specificity of p300-mediated acetylation.
The acetylation of PC4 was completely dependent on the presence of both
[3H]acetyl-CoA and p300, confirming the p300-mediated
acetylation of PC4 (Fig. 2C).
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and TFIIF, the roles of which are not known
(14, 15). It is known that HMG proteins also are reversibly acetylated in vivo. In duck erythrocytes, two acetylation sites in
HMG-1 and HMG-14 and three sites in HMG-17 were identified (16).
Recently it has been reported that PCAF is the enzyme responsible for
the acetylation of HMG-17 at position 2. Apparently binding of HMG-14 and HMG-17 to nucleosome cores inhibits the PCAF-mediated acetylation of histone H3. Thus the presence of HMG-17 affects the ability of PCAF to acetylate chromatin, whereas the acetylation of HMG-17 reduces its binding affinity to chromatin (17). PCAF and CBP can also
acetylate HMG-I(Y) either in solution or in the context of the
enhanceosome (18). Acetylation of HMG-I(Y) by CBP but not by PCAF
decreases its DNA binding activity and results in enhanceosome
destabilization and disassembly. However, both CBP and PCAF HAT
activities are required for activation, whereas only CBP HAT activity
is required for postinduction turnoff of interferon-
expression.
Thus it seems acetylation of HMG protein is an important means of
regulating transcription or biological function of them. Because PC4
very closely resembles HMG proteins, it was not too hard to imagine
that PC4 also would be substrate for at least one of the several
histone acetyltransferases.
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-mercaptoethanol, 0.2 mM
EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% Nonidet
P-40) containing 500 mM KCl. The peak protein fractions
containing PC4 were pooled and chromatographed onto a 1-ml
phosphocellulose P11 column. FLAG epitope-tagged Gal4 and p300
HAT domain (amino acids 1195-1810) also were expressed in E. coli and immunopurified on M2-agarose as described previously
(20). His6-tagged nucleosome assembly protein 1 (NAP1) was
purified with Ni2+-nitrilotriacetic acid (Qiagen). FLAG
epitope-tagged full-length PCAF and His6-tagged full-length
p300 were expressed and purified by infection of Sf21 cells with
recombinant baculoviruses followed by affinity chromatography of
whole-cell extracts on M2-agarose and Ni2+-nitrilotriacetic
acid column as described elsewhere (21, 22).
70 °C for 1-2 days.
-32P]ATP and 1 mM cold ATP. To visualize
the phosphorylated proteins, the reaction products were precipitated
with 25% trichloroacetic acid, washed with ice-cold acetone, and
resolved electrophoretically on 15% polyacrylamide gel. Gels were
stained and/or autoradiographed. The extent of phosphorylation was
quantitated using a Fuji phosphorimaging analyzer.
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View larger version (46K):
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Fig. 1.
Purified proteins used in different
experiments. A, lane 1, broad range marker
(Bio-Rad); lane 2, 2.3 µg of
His6-tagged mouse NAP1; lane 3, 2 µg of
human core histones; lane 4, 1 µg of FLAG epitope-tagged
Gal4 DNA-binding domain; lane 5, 0.8 µg of purified
recombinant human PC4. B, baculovirus expressed full-length
His6-tagged p300 (lane 1) and FLAG
epitope-tagged PCAF (lane 2). 0.5 µg of each protein was
analyzed on 8% polyacrylamide gel and visualized by Coomassie Blue
staining. C, authenticity of PC4 verified by Western
blotting. The protein was resolved by 15% SDS-PAGE and subjected to
either staining (lane 1) or Western blotting (lane
2).
View larger version (37K):
[in a new window]
Fig. 2.
In vitro acetylation of PC4.
A, normalization of the amounts of histone
acetyltransferases p300 (lane 2) and PCAF (lane
3) to be used for PC4 acetylation using 1 µg of highly purified
HeLa core histones. Lane 1 represents the control without
enzyme. The [3H]acetate incorporation by each HAT
was measured by the filter binding assay. B, p300 acetylates
PC4. Purified proteins were incubated with [3H]acetyl-CoA
for 30 min at 30 °C and then separated by SDS-PAGE (15%) and
visualized by fluorography. 1 µg of purified core histones without
any HAT (lane 1), with p300 (lane 2), and with
PCAF (lane 3) and bacterially expressed PC4 without any HAT
(lane 4), with p300 (lane 5), with PCAF
(lane 6), Gal4 with p300 (lane 7), and NAP1 with
p300 (lane 8) were incubated. C, substrate
and enzyme requirements. Solution HAT assay products processed as above
after incubation of various combinations of PC4, full-length p300, and
[3H] acetyl-CoA are shown.
The present results demonstrate clearly that PC4 is acetylated
specifically by p300 among the different HATs we have tested. In
addition to PCAF, the equivalent HAT activity of GCN5 (short form) (25)
and TIP60 (11) also could not acetylate PC4 under the identical
conditions (data not shown). There are several non-histone proteins
that are substrates of p300 and can be acetylated also by PCAF. These
include p53, TFIIB, TFIIF, and HMG-17 (15, 17). However, HMG-14,
which is related closely to HMG-17, can be acetylated only by p300 but
not PCAF at the nucleosomal binding domain similar to what we report
for PC4. It would be interesting to find out the mechanisms behind this
p300 specificity of PC4 acetylation.
To find out the number of lysine residues that are getting acetylated
in PC4, an acetylation reaction was carried out using purified FLAG
epitope-tagged, recombinant p300 HAT domain (20). To obtain the
complete acetylation of PC4, the reaction was performed at 37 °C for
a longer time, and the HAT domain and acetyl-CoA were added every 30 min (see "Experimental Procedures"). The complete acetylation was
confirmed by analyzing the acetylated protein on 15%
acid-urea-polyacrylamide gel (data not shown). The acetylated protein
then was dialyzed thoroughly against water and subjected to
electrospray ionization mass spectral analysis. As shown in Fig.
3A, unmodified mock control
protein (treated with p300 HAT domain without acetyl-CoA) gave a major
peak with a molecular weight of 14,265, confirming the identity of
native PC4 as described previously (7). The acetylated protein showed a
peak of 14,346 (Fig. 3B). The mass difference between the
acetylated and the unmodified proteins suggests that there are at least
two lysine residues getting acetylated by p300.
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Isolation of native PC4 from HeLa nuclear extract showed 95% of the
protein present as the heavily phosphorylated form in vivo
(7). The phosphorylation negatively regulates PC4-mediated transcriptional coactivator activity. Presumably, phosphorylation imposes a structural change in PC4 which in turn may effect
p300-mediated acetylation of PC4. To investigate this possibility, PC4
was phosphorylated to saturation levels in vitro by casein
kinase II as depicted in Fig.
4A. The optimum conditions for
this complete phosphorylation was standardized for 0.25 µg of PC4, 10 milliunits of casein kinase II, and 5 mM ATP (Fig.
4A, compare lanes 1 and 4). Consistent with our prediction, we found that indeed phosphorylation of PC4 modulates the acetylation negatively as of any other functional consequence of the phosphorylation. In an in vitro
acetylation reaction, an equivalent amount of unmodified PC4 subjected
to a mock-phosphorylation reaction (without ATP) was acetylated
efficiently in a p300- and
[3H]acetyl-CoA-dependent manner, whereas
phosphorylated PC4 was not acetylated at all (Fig. 4B,
compare lanes 2 and 4). This interesting result
tempted us to find out what happens in the reverse consequences, i.e. how acetylation of PC4 influences its phosphorylation.
To address this question, PC4 was acetylated in vitro with
p300 and cold acetyl-CoA as described under "Experimental
Procedures." The acetylation of PC4 was confirmed by mass spectral
analysis of the acetylated protein as described in Fig. 3. The
acetylated protein then was subjected to an in vitro
phosphorylation reaction by casein kinase II (Fig. 4C) and
[-32P]ATP. We found that both acetylated and
unacetylated PC4 was phosphorylated efficiently by CKII (Fig.
4C, lanes 1 and 5 versus lane
6). The quantitation of phosphorylation by phosphorimaging analysis showed that acetylation of PC4 has no effect on
phosphorylation under the present reaction condition.
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Phosphorylation of substrates or the enzyme (HAT) itself may be one of the key regulators of acetylation of histones and non-histone proteins. Phosphorylation of CBP by cyclin E-Cdk2 in the C-terminal region enhances its activity almost 5-fold (26). Probably phosphorylation induces a structural change in CBP that leads to the activation of acetylation function. Similarly phosphorylation of ATF-2 (27), a transcription factor that stimulates CRE-dependent transcription, enhances its intrinsic HAT activity, whereas GCN5-HAT activity is inhibited by Ku-DNA-dependent protein kinase complex-mediated phosphorylation (28). On the other hand phosphorylation of histone H3 stimulates acetylation (10-fold) by GCN5 (29). Recently it was reported that phosphorylation of p53 enhances its acetylation, which consequently enhances its DNA binding ability as well as the repair activity (14, 30). The negative regulation of acetylation upon the phosphorylation of the substrate is somewhat unique for PC4. As per our knowledge there is no report yet in which phosphorylation of a substrate inhibits acetylation. PC4 has a serine-rich acidic domain at the N terminus followed by a lysine-rich domain that is linked to a unique single-stranded DNA-binding domain (4-6). It may be possible that the phosphorylation of serine residues in the serine-rich acidic domain may induce a conformational change in PC4 that masks the acetylation sites in or around the lysine-rich domain, resulting in a complete abolition of acetylation. This possibility needs to be addressed further by detailed structural studies using site-directed mutagenesis followed by NMR spectroscopy or x-ray crystallography. However, because acetylation does not affect the phosphorylation of PC4, it may be assumed that acetylation of PC4 does not lock it in an active form. At this juncture it is difficult to predict how these two posttranslational modifications (acetylation and phosphorylation) are regulated in vivo to manifest PC4 function. Most likely there are few other proteins having phosphatase as well as deacetylase activity that also are involved in the regulation of PC4 function in the cell.
The negative effect of PC4 phosphorylation on acetylation suggests its
functional consequence for acetylation. Because phosphorylation also
prevents double-stranded DNA binding, which is correlated directly to
its positive coactivator function, it is important to examine how
acetylation affects the DNA binding ability of PC4. To address this
possibility gel-mobility shift assays were done using a 60-base
pair-long oligonucleotide comprising the HIV-1 core promoter sequences
upstream of the initiator region of the adenovirus major late promoter
with phosphorylated PC4 and acetylated PC4. The DNA binding of
unmodified PC4 showed cooperativity with increasing concentration of
PC4, yielding an uncompact complex (Fig.
5, lanes 2-6).
Although 0.25 µg of unmodified PC4 produce a fairly good amount of
complex, the equivalent amount of phosphorylated PC4 could not bind
to the DNA (compare lanes 3 and 9).
Interestingly, the similar amount of acetylated PC4 (by p300) enhances
the double-stranded DNA binding significantly as compared with the
equivalent amount (0.25 µg) of unmodified protein (compare
lanes 3 and 7), whereas the addition of p300 only
did not stimulate the DNA binding (compare lanes 3 and
8). It is to be noted that to achieve a similar amount of complex, nearly 3-fold more unmodified protein was needed as
compared with the acetylated proteins (compare lanes 6 and 7). This experiment was repeated independently five times,
and the average enhancement of DNA binding upon acetylation was found to be 40% (±5%) based on the phosphorimaging analysis
quantitation.
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In the present study we observe that a relatively very high amount of
PC4 (0.3 µg) is required to get the proper PC4-DNA complex, which is
consistent with the previous reports. The uncompact nature of the
complex indicates the nonspecific binding of PC4. Although the
stimulation of DNA binding by PC4 upon acetylation is not dramatic, it
was consistent and very significant for the positive cofactor activity
of PC4. Because the double-stranded DNA binding activity was shown to
be correlated directly to the coactivation, presumably acetylation also
will enhance the transcription. Our results show that phosphorylation
that prevents the double-stranded DNA binding also inhibits the
acetylation. Thus it is quite possible that phosphorylation-mediated
negative regulation of PC4 function operates through two different
pathways: first, it prevents the double-stranded DNA binding, direct
interaction with TFIIA and TATA box-binding protein complex, and
interaction with activators, and second, it inhibits the acetylation.
It is tempting to speculate that this phosphorylation and acetylation
balance ultimately regulates the PC4 function in vivo, where
acetylation would be more relevant under the nucleosomal context.
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ACKNOWLEDGEMENTS |
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We thank R. G. Roeder for the PC4 expression clone and antibody, Y. Nakatani for the PCAF baculovirus expression vector and p300 HAT domain expression vector, L. Kraus and J. Kadonaga for the p300 baculovirus expression vector, P. Balaram for providing Department of Biotechnology, New Delhi, mass spectral facilities, and M. R. S. Rao and K. S. Ullas for helpful discussion.
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FOOTNOTES |
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* This work was supported in part by Jawaharlal Nehru Center for Advanced Scientific Research and the Council of Scientific and Industrial Research.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.
Council of Scientific and Industrial Research Junior Research Fellow.
§ To whom correspondence should be addressed. Tel.: 91-80-8462750 (Ext. 257); Fax: 91-80-8462766; E-mail: tapas@jncasr.ac.in.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M100934200
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ABBREVIATIONS |
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The abbreviations used are: PC, positive coactivator; TFII, transcription factor II; CKII, casein kinase II; HAT, histone acetyltransferase; HMG, high mobility group; CBP, CREB-binding protein; HIV, human immunodeficiency virus; PAGE, polyacrylamide gel electrophoresis; PCAF, p300/CBP-associated factor; ACTR, activator of retinoid receptor; SRC, steroid receptor coactivator.
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