MOLECULAR AND CELLULAR BIOLOGY, Mar. 2004, p. 2153–2168
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 5
Direct Interaction between Nucleosome Assembly Protein 1 and the
Papillomavirus E2 Proteins Involved in Activation of Transcription
Manuela Rehtanz, Hanns-Martin Schmidt,† Ursula Warthorst, and Gertrud Steger*
Institute of Virology, University of Cologne, 50935 Cologne, Germany
Received 5 November 2003/Accepted 9 December 2003
Using a yeast two-hybrid screen, we identified human nucleosome assembly protein 1 (hNAP-1) as a protein
interacting with the activation domain of the transcriptional activator encoded by papillomaviruses (PVs), the
E2 protein. We show that the interaction between E2 and hNAP-1 is direct and not merely mediated by the
transcriptional coactivator p300, which is bound by both proteins. Coexpression of hNAP-1 strongly enhances
activation by E2, indicating a functional interaction as well. E2 binds to at least two separate domains within
hNAP-1, one within the C terminus and an internal domain. The binding of E2 to hNAP-1 is necessary for
cooperativity between the factors. Moreover, the N-terminal 91 amino acids are crucial for the transcriptional
activity of hNAP-1, since deletion mutants lacking this N-terminal portion fail to cooperate with E2. We provide
evidence that hNAP-1, E2, and p300 can form a ternary complex efficient in the activation of transcription. We
also show that p53 directly interacts with hNAP-1, indicating that transcriptional activators in addition to PV
E2 interact with hNAP-1. These results suggest that the binding of sequence-specific DNA binding proteins to
hNAP-1 may be an important step contributing to the activation of transcription.
The transcriptional activation of genes repressed by nucleo-
somes requires the presence of activators that bind sequence
specifically. These increase the efficiency of assembly of the
transcriptionally competent preinitiation complex (PIC) and
counteract the repressive effects of chromatin through the re-
cruitment of chromatin remodeling complexes and histone
acetyltransferases (HATs) (2, 15, 30, 31, 40, 52, 53, 64). Chro-
matin remodeling is accomplished by large, ATP-dependent
chromatin remodeling complexes that alter chromatin struc-
ture by transiently disrupting histone-DNA interactions (re-
viewed in references 6, 30, and 63). Posttranslational modifi-
cations of chromatin, such as acetylation by transcriptional
coactivators, also contribute to gene regulation (22). HATs are
thought to catalyze the addition of acetyl groups to the N-
terminal tails of core histones, a process which usually corre-
lates with the activation of transcription (55). The transcrip-
tional coactivator p300 and its homologue CREB binding
protein (CBP) possess HAT activity, and both are implicated
in the regulation of transcription by a large number of se-
quence-specific activator proteins (reviewed in references 11,
19, and 21). p300 and CBP are associated with other HATs,
such as p/CAF, ACTR, and SRF, in a multiprotein complex.
Functional studies have shown that the coactivator function of
p300 and CBP requires their acetyltransferase activity (32, 33,
41). Moreover, additional functions of these cofactors, such as
the interaction with components of the PIC, are necessary for
their stimulatory activity (3, 45, 51).
The assembly of nucleosomes is tightly linked to DNA rep-
lication. The naked daughter strands of newly replicated DNA
are rapidly assembled into chromatin by a multistep process.
Chromatin assembly factor 1 and replication-coupling assem-
bly factor/anti-silencing function 1 protein (ASF1) act as his-
tone chaperones to deposit histones H3 and H4. Nucleosome
assembly protein (NAP) is a histone chaperone responsible for
the incorporation of two histone H2A-H2B dimers to complete
the nucleosome (reviewed in reference 62). NAP-1 may act as
a nucleocytoplasmic shuttling protein that delivers H2A-H2B
dimers from the cytoplasm to the chromatin assembly machin-
ery in the nucleus (47). In addition to its function in chromatin
assembly, NAP-1 also may play a role in cell cycle progression.
Yeast genetic experiments have shown that NAP-1 has a role
in cell cycle progression during G1phase and mitosis. NAP-1
binds to cyclin B (29) and a kinase, Gin4p (1).
Furthermore, histone chaperones seem to facilitate tran-
scriptional activation through their chromatin-modifying activ-
ity. Recent data suggest that HAT complexes as well as ATP-
dependent chromatin remodeling complexes cooperate with
histone chaperones in altering chromatin structure during the
activation of transcription. ASF1 was found to functionally
interact with the Brahma (SWI/SNF) ATP-dependent chroma-
tin remodeling complex, involved in the activation of transcrip-
tion (48). A functional interaction between p300/CBP and
NAPs also has been reported (4, 27, 58). It has been demon-
strated that the acetylation of histones by p300 facilitates the
transfer of histones H2A and H2B to NAP-1 in vitro. Thus, the
structure of the histones may be altered by histone acetylation
facilitating the loss of H2A-H2B dimers that have been remod-
eled by the action of ATP-dependent chromatin remodeling
complexes (27). This model is supported by the observation
that NAPs may augment activation by factors which use p300
as a coactivator (58). Furthermore, NAP-1 has been shown to
stimulate the binding of transcription factors to their binding
sites, a process which is accompanied by disruption of the
histone octamer (65). Although hints for a role of histone
chaperones such as NAP-1 in the activation of transcription
* Corresponding author. Mailing address: Institute of Virology, Uni-
versity of Cologne, Fu ¨rst-Pu ¨ckler-Str. 56, 50935 Cologne, Germany.
Phone: 49-221-478-3926. Fax: 49-221-478-3902. E-mail: Gertrud.
† Present address: Amaxa GmbH, 50829 Cologne, Germany.
are accumulating, it is not known how histone chaperones may
be targeted to actively transcribing promoter regions.
The papillomavirus (PV) E2 protein is a transcription factor
which regulates PV transcription and is also required for the
replication of viral DNA (14, 18, 46). For bovine PV type 1
(BPV1), E2 strongly activates several BPV1 promoters by
binding to its high-affinity palindromic recognition sequence,
ACCGN4CGGT, present in multiple copies within the PV
genome (60). The activation of transcription by viral E2 pro-
teins involves the binding of the activation domain (AD) to
transcription factors TFIIB and TBP and cofactors AMF1/
Gps2 and p/CAF. All of these interactions seem to be impor-
tant for activation by E2 (7, 9, 36, 44, 70). Furthermore, E2
interacts with p300/CBP (37, 49, 56). It was shown that the
coexpression of p300 can potentiate activation by E2, indicat-
ing that binding to p300 may be a rate-limiting step for acti-
vation by E2 (49).
Here, we describe a yeast two-hybrid system that uses tran-
scriptionally competent BPV1 E2 protein. This system allowed
the identification of human NAP-1 (hNAP-1) as an interaction
partner for E2. We present data demonstrating that a direct
interaction between E2 and hNAP-1 is important for the acti-
vation of transcription by BPV1 E2. However, additional func-
tions located within the N terminus of hNAP-1 are required.
We show that hNAP-1 and E2 can bind simultaneously to p300
and that this stable ternary complex efficiently contributes to
the activation of transcription. Furthermore, p53 also directly
binds to hNAP-1. Our data suggest that the binding to NAP-1
may be an essential step for sequence-specific transcriptional
activators that use p300 as a coactivator to activate transcrip-
MATERIALS AND METHODS
Plasmid constructions. All yeast plasmids were shuttle vectors, and cloning
was performed with Escherichia coli strain XL1Blue. The yeast reporter con-
struct expressing the lacZ gene under the control of the synthetic promoter
containing four E2 binding sites in front of the TATA box of human PV (HPV)
type 18 (HPV18) P105was obtained in several steps. The SalI-BglII promoter
fragment was isolated from construct p4E223T105(24), ligated to an oligonucle-
otide encoding the junction between the promoter and the lacZ gene and fol-
lowed by a BspEI restriction site, and cloned into the SalI-BspEI restriction site
of pBR322. Subsequently, this promoter fragment was inserted into the XhoI-
BspEI restriction sites of p?UAS (34), resulting in the displacement of the CYC
promoter by the 4E223P105promoter. Finally, the HindIII-BamHI promoter
fragment isolated from this construct was inserted into the HindIII-BamHI
vector fragment of placZi (Clontech) to give rise to 4E223P105lacZi. 5E2-pHISmin
was constructed by inserting oligonucleotides encoding E2 binding sites into the
XbaI binding site of pHIS-i1 (Clontech). The yeast E2 expression vector
pGBT9-E2 was obtained by removing the GAL4 DNA binding domain (DBD)
from pGBT9 (Clontech) by digestion with BamHI and partial digestion with
HindIII. The E2-encoding HindIII-BamHI fragment was isolated from
pTZE2mHIII (34) and cloned into pGBT9. The HindIII-BamHI fragment de-
rived from pTZE2?192– 282 (42) was inserted in a similar way to obtain pGBT9-
To express the AD of GAL4 fused to the DBD of E2, the StuI-BamHI
fragment of E2 derived from pTZE2mHIII (42) was cloned into vector
pGAD424 (Clontech). The full-length hNAP-1 clone was isolated from the
keratinocyte cell line HaCaT cDNA library (expressing cDNA-encoded proteins;
Clontech) by amplification with specific primers and cloned in frame with the
hemagglutinin (HA) tag into vectors pXJ41 (68) and pcDNA3.1 (Invitrogen).
Plasmids expressing a glutathione S-transferase (GST)– hNAP-1 fusion protein
were obtained by cloning PCR products encoding full-length hNAP-1 into
pGEX-5X-2 and pGEX-2T (Pharmacia Biotech) or deletion mutants into
pGEX-2T. Some of the hNAP-1 deletion constructs were subsequently trans-
ferred into eukaryotic vector pXJ41 to express them fused to the HA tag. To
express the E2 proteins of BPV1, HPV8, and HPV18 fused to a FLAG tag, the
corresponding open reading frames were amplified by PCR, followed by cloning
into vector pCMV2-FLAG (Kodak), as were BPV1 E2 lacking the AD and E2
expressing the AD. Expression vector pC59, expressing BPV1 E2 under the
control of the simian virus 40 (SV40) early promoter, was described previously
(69). Expression vectors for HPV8 E2 and HPV18 E2 and the HPV8 noncoding
region-luciferase reporter construct also were described previously (49), as was
the expression vector for p300 (13). The regulatory region of BPV1 from nucle-
otides 6958 to 475 (long control region [LCR]) was amplified by PCR and cloned
into pALuc (12) to obtain BPV1 LCR-Luc. p53 expression vector pCp53wt was
described by Nigro et al. (54), and the synthetic p53-responsive reporter con-
struct was described by Funk et al. (17). The pG5-Luc reporter construct and the
vector expressing the DBD of GAL4 (pM) were derived from a mammalian
two-hybrid system (Clontech). PCR products encoding the AD of TEF-1 or
HPV8 E2 were cloned into vector pM.
Yeast two-hybrid system. Yeast maintenance, transformation, and storage
were performed according to the instructions of the manufacturer of the yeast
two-hybrid system (Clontech). Yeast strain YM954 (67) (genotype: MATa
ura3-52 his3-200 ade2-101 lys2-801 leu2-3,112 trp1-901 gal4-542 gal80-538) was
kindly provided by P. Bartel, Stony Brook, N.Y. ?-Galactosidase (?-Gal) activity
was determined by a filter lift assay and liquid ?-Gal assays according to proto-
cols from Clontech.
Cell culture and transfection. RTS3b, a keratinocyte cell line established from
an HPV-negative skin lesion from a renal transplant recipient (57), was culti-
vated in E medium (43). For transient transfection, 105RTS3b cells were used to
seed one six-well plate on the day before transfection. Transfection was per-
formed with FuGene reagent according to the manufacturer’s recommendations
(Roche Diagnostics), and the cells were harvested 48 h later. Luciferase activities
were determined as described previously (49). To generate a stable cell line
expressing the luciferase reporter gene under the control of the BPV1 LCR
integrated into the cellular genome, RTS3b cells were transfected with the BPV1
LCR-Luc construct. After 48 h, the cells were placed in medium containing 800
?g of G418/ml. After another 48 h, the medium was replaced with medium
containing 400 ?g of G418/ml; this medium was changed every second day for 2
to 3 weeks until resistant colonies had grown. The resistant colonies then were
pooled. The presence of the intact BPV1 LCR-Luc cassette within the cells was
confirmed by PCR (data not shown). At 24 h after transfection of this stable cell
line with the E2 or hNAP-1 expression plasmids, the medium was replaced with
medium supplemented with 400 ?g of trichostatin A (TSA)/ml; incubation was
continued for another 24 h before luciferase activities were determined as de-
scribed above. To detect the effect of hNAP-1 on the activation of p21 expression
by p53, RTS3b cells were transiently transfected with the corresponding expres-
sion vectors (see Fig. 6). A total of 90 ?g of total cell extract was loaded onto a
sodium dodecyl sulfate–15% polyacrylamide gel and analyzed for p21 by West-
ern blotting with an antibody directed against p21 (Pharmingen).
Coimmunoprecipitation and pull-down assays. GST pull-down assays were
performed as previously described (49). To detect a direct protein-protein in-
teraction, His-tagged proteins were expressed in bacteria, purified as described
previously (25), incubated with immobilized GST– hNAP-1 fusion protein or
GST, and further treated as described elsewhere (49). To detect an interaction
between various E2 proteins and hNAP-1 in vivo, 293T cells were transfected
with an expression vector for FLAG-tagged BPV1 E2 protein (see Fig. 2E) or
with vectors for various FLAG-tagged E2 proteins (see Fig. 2D) and for HA-
tagged hNAP-1. After 48 h, the cells were washed in ice-cold phosphate-buffered
saline, followed by four freeze-thaw cycles in LSDB buffer (50 mM Tris-HCl [pH
7.9], 10% glycerol, 0.5 mM EDTA, 1 mM dithiothreitol, 0.2% NP-40) containing
100 mM KCl. Cell debris was removed by centrifugation for 10 min at 4°C. The
supernatants were incubated with 20 ?l of anti-FLAG M2 affinity resin (Kodak).
The samples were incubated at 4°C for 2 h with gentle mixing, followed by four
washes in LSDB buffer with 200 mM KCl or 250 mM KCl and one wash in LSDB
buffer with 100 mM KCl. The presence of hNAP-1 was analyzed by Western
blotting with a monoclonal antibody directed against the HA epitope (Roche
Diagnostics) or with a monoclonal antibody directed against hNAP-1 (kindly
provided by Y. Ishimi) to detect an interaction between E2 and endogenous
hNAP-1. E2 proteins were detected with anti-FLAG M5 antibody. To detect an
interaction between endogenous p53 and endogenous hNAP-1 in vivo, 85 ?g of
nuclear extracts from normal neonatal human epidermal keratinocytes (pur-
chased from Clonetics) and 85 ?g of nuclear extracts from RTS3b cells were
incubated with 5 ?l of anti-p53–agarose conjugate (DO-1; Santa Cruz) at 4°C for
2 h with gentle mixing, followed by four washes in LSDB buffer with 200 mM KCl
and one wash in LSDB buffer with 100 mM KCl. The presence of the endogenous
proteins was analyzed by Western blotting with antibodies directed against
hNAP-1 and p53 (DO-1).
2154REHTANZ ET AL.MOL. CELL. BIOL.
Density gradient analysis of the ternary complex. hNAP-1, E2 with a deletion
of amino acids 326 to 420 (E2?326–420), and a fragment of p300 from amino
acids 1195 to 1761 (p300 1195 to 1761) were expressed with a tag of six histidines
in bacteria and purified. Totals of 800 ng of p300 1195 to 1761, 600 ng of hNAP-1,
and 200 ng of E2?326–420 were incubated for 2 h in the presence of 25 mM
HEPES-KOH (pH 7.9)–0.1 mM EDTA–12.5 mM KCl–10% glycerol–100 mM
KCl in various combinations (see Fig. 5C). Next, the proteins were loaded onto
a 2-ml 7.5 to 30% glycerol gradient prepared as described by Tanese et al. (61)
and then centrifuged for 8 h at 50,000 rpm with a TL100 tabletop ultracentrifuge
(Beckman) and a TLS-55 swinging-bucket rotor. Finally, 36 fractions of 60 ?l
each were carefully removed beginning from the top of the gradients. The
presence of various proteins was analyzed by Western blotting.
Identification of cellular factors binding to PV E2 proteins
by the yeast two-hybrid system. To identify cellular factors
which may play a role in E2-mediated activation of transcrip-
tion, we set up a yeast two-hybrid system. Since we wished to
screen with wild-type E2 protein, which is a strong activator of
transcription in yeast cells (34), we modified the classical yeast
two-hybrid system. Previously, Ham et al. showed that in mam-
malian cells, E2 could not efficiently activate the transcription
of a minimal promoter composed of E2 binding sites and a
TATA box only but was an efficient activator of more complex
promoters (24). In contrast, most cellular sequence-specific
activator proteins, among them GAL4, are able to stimulate
such a minimal promoter (66). To test whether E2 would be
unable to activate transcription from this minimal promoter in
yeast cells, we inserted a fragment expressing such a minimal
promoter into vector placZi (Fig. 1). E2 yielded a three- to
FIG. 1. Identification of hNAP-1 as an interaction partner for the PV E2 protein. (A) Schematic representation of two reporter constructs.
5E2-pHISminexpresses the yeast HIS3 gene under the control of its minimal promoter, pHISmin, and five E2 binding sites. 4E223P105placZi
expresses the lacZ gene under the control of a synthetic promoter composed of the TATA box of the early promoter of HPV18 (P105) and four
E2 binding sites located 23 bp further upstream. For the yeast two-hybrid system, both reporter constructs were integrated into the cellular genome
of YM954. (B) Yeast expression vector pGBT9-E2, expressing the E2 protein of BPV1 (E2), or pGBT9-E2DBD-GAL4AD (E2DBD-GAL4-AD),
expressing the AD of GAL4 fused to the DBD of BPV1 E2, or the vector alone (?) was transformed into the yeast strain harboring the two
reporter constructs shown in panel A. ?-Gal activities of the respective strains were determined. The activity in the presence of the vector alone
(?) was assigned an arbitrary value of 1, and the fold activation was calculated. The columns represent the averages of three independent
experiments performed with two different clones in each case; error bars are shown. (C) ?-Gal activities of parental yeast strain YM954
transformed with a yeast expression vector for E2 or E2?195–282 (an E2 protein lacking the internal hinge region) and with plasmid pACT-c-
?NAP-1, expressing hNAP-1 fused to the AD of GAL4, were determined. The latter was isolated by the yeast two-hybrid screen. The fold
activation in relation to that of the strain transformed with the expression vector was calculated as described for panel B. The columns represent
the averages for three independent clones; error bars are shown.
VOL. 24, 2004FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-12155
FIG. 2. E2 proteins and hNAP-1 interact in vitro. (A) GST pull-down assays were carried out with purified GST, full-length hNAP-1 fused to
GST (GST–hNAP-1), and35S-labeled E2 proteins of BPV1, HPV8, and HPV18, obtained by in vitro translation with a rabbit reticulocyte lysate.
Ten percent of the input is shown in lanes 1, 4, and 7. The positions of the marker proteins are indicated. (B) (Top panel)35S-labeled full-length
E2 of BPV1 (E2), two activation-deficient N-terminal deletion mutants (E2?1–161 and E2?1–203), and a C-terminal deletion mutant lacking the
DBD (E2?326–420) were incubated with GST, GST–hNAP-1 or, as a negative control, GST–p300-2, expressing a fragment of p300 which was
shown previously not to interact with E2 (49). Ten percent of the E2 protein and its derivatives used in one interaction assay is shown in the lanes
labeled 10% input. (Middle panel) E2 proteins with point mutations in the AD, E2 E39A, which is replication deficient, and E2 I73A, which is
impaired in transcription (14), were used in a GST pull-down assay. (Bottom panel) Structure of the E2 protein showing the positions of the amino
acids (aa) in the various domains. (C) His-tagged, bacterially expressed, purified E2 protein of BPV1 (His-E2) or His-tagged E2?326–420 was
incubated with purified GST or GST–hNAP-1. Bound E2 proteins were detected by Western blotting with an antibody directed against the His
tag (Qiagen). The position of the 31-kDa marker protein is indicated. (D) (Upper panel) 293T cells were transfected with an expression vector
expressing a FLAG-tagged full-length E2 protein of BPV1 (lanes 1, 3, 5, 7, 10, and 14), a FLAG-tagged BPV1 E2 mutant lacking the N-terminal
AD (FLAG-E2?1–203; lanes 12 and 16), or FLAG-tagged AD (FLAG-E2?204–420; lanes 11 and 15) and an expression vector for HA-tagged
hNAP-1 (lanes 2, 3, 6, 7, and 9 to 16). Cell extracts were incubated with the FLAG affinity gel, and bound hNAP-1 was detected with an antibody
directed against the HA epitope (lanes 1 to 4 and lanes 9 to 12). In lanes 5 to 8 and lanes 13 to 16, 1/40 the input cellular extract was included,
and the expression of HA-tagged hNAP-1 was analyzed with the HA antibody. The presence of FLAG-tagged E2 proteins was determined by
reprobing of the blot shown in lanes 1 to 4 and lanes 10 to 12 with the FLAG M5 antibody. IP, immunoprecipitation; WB, Western blotting. (Lower
panel) Western blot developed with the antibody directed against the HA epitope in a coimmunoprecipitation similar to that shown above but with
cell extracts from 293T cells that had been transfected with an expression vector for FLAG-tagged HPV18 E2 (lanes 17, 19, 22, and 24) or HPV8
E2 (lanes 18, 20, 23, and 25) or an expression vector for HA-tagged hNAP-1 (lanes 17, 18, 21, 22, 23, and 26). (E) (Upper panel) Cell extracts of
293T cells that had been transfected with an expression vector for FLAG-tagged BPV1 E2 (lanes 3 and 6) or FLAG-tagged bacterial alkaline
2156 REHTANZ ET AL.MOL. CELL. BIOL.
fourfold activation of the expression of the lacZ gene from
reporter construct 4E223P105placZi integrated into the cellular
genome. A fusion protein composed of the DBD of E2 and the
AD of GAL4 led to a 14-fold activation (Fig. 1B), indicating
that the mode of activation mediated by the two ADs is distinct
in yeast cells also. Furthermore, E2 only weakly activated the
HISminpromoter located behind five E2 binding sites and
controlling the expression of the HIS gene in plasmid 5E2-
pHISmin(Fig. 1A). Therefore, plasmid 5E2-pHISminwas used
to select for yeast growth in the presence of 100 to 300 mM
phosphatase (BAP) as a negative control (lanes 2 and 5) or with an empty vector (lanes 1 and 4) were incubated with the FLAG antibody coupled
to Sepharose (lanes 4 to 6). Bound, endogenous hNAP-1 was detected by Western blotting with an antibody directed against hNAP-1. In lanes 1
to 3, 1/160 the cellular extract used for immunoprecipitation was loaded as an input control. (Lower panel) Part of the blot in the upper panel was
reprobed with the FLAG antibody to detect the presence of FLAG-tagged proteins. A cellular protein cross-reacting with both antibodies is
indicated by an asterisk.
VOL. 24, 2004 FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-12157
3-amino-1,2,4-triazole, an inhibitor of the HIS gene product. It
was necessary to include this additional selection to suppress
yeast cell growth mediated by the weak activation of pHISmin
A cDNA library derived from keratinocyte cell line HaCaT
and allowing the expression of cDNA-encoded proteins fused
to the GAL4 AD was transformed into the yeast strain har-
boring the two reporter genes in addition to expression vector
pGBT9-E2. Out of 107yeast clones, the two reporter genes
were activated significantly in 41 clones. The cDNA-encoded
proteins of two independent clones were identical to the 300
C-terminal amino acids of hNAP-1, which has 391 amino acids
in total. The interaction between hNAP-1 and E2 could be
confirmed with yeast strains which were retransformed with
the corresponding plasmids (Fig. 1C). The interaction involved
the N-terminal AD of BPV1 E2, since the C-terminal 94 amino
acids of BPV1 E2, containing the DBD, did not reveal in-
creased ?-Gal activity in the presence of hNAP-1 (data not
shown). However, E2?195–282, which lacks the internal hinge
region, retained similar cooperativity with hNAP-1 (Fig. 1C).
The reduced absolute levels of activation of this deletion mu-
tant correlated with reduced protein levels (data not shown).
E2 proteins bind to hNAP-1 in vitro and in vivo. To confirm
an interaction between E2 and h-NAP-1 in vitro, the hNAP-1
fragment isolated by the yeast two-hybrid system and encoding
hNAP-1 lacking the N-terminal 91 amino acids was inserted
into vector pGEX-5X-2. A GST–hNAP-1 fusion protein re-
tained the full-length BPV1 E2 protein obtained by in vitro
translation with a rabbit reticulocyte lysate (Fig. 2A). The E2
proteins of different PV types are rather conserved within the
N-terminal AD and the C-terminal DBD, in contrast to the
variable hinge region, which was suggested to function as a
linker between the AD and the DBD of E2 (20, 42). In vitro-
translated E2 proteins of HPV8 and HPV18 bound to GST–
hNAP-1 (Fig. 2A), indicating that the interaction with hNAP-1
may be conserved among E2 proteins of different PV types.
In correlation with the data obtained with yeast cells, the AD
of BPV1 E2 is required for binding to hNAP-1 in vitro. E2?1–
FIG. 3. E2 and hNAP-1 cooperate in the activation of gene expres-
sion. (A) (Top panel) RTS3b cells, immortalized skin keratinocyte
cells (57), were transfected with a luciferase reporter construct con-
taining the regulatory region of BPV1 called the LCR, the structure of
which is shown. The different promoters are indicated, and the 12 E2
binding sites are depicted as black boxes. (Middle panel) Either 5 or 20
ng of expression vector for BPV1 E2, under the control of the SV40
promoter, was cotransfected together with 400 ng of expression vector
for HA-tagged hNAP-1. The graph shows the results of one represen-
tative experiment. (Bottom panel) Transient transfection experiments
with the BPV1 LCR-Luc reporter plasmid, an expression vector for
HA-tagged hNAP-1, and expression vectors for full-length E2 (E2), E2
E39A (the replication-deficient mutant), E2 I73A (the mutant im-
paired in transcription), E2?1–203 (lacking the AD), and E2?195– 282
(lacking the internal hinge region). The activity of each of the E2
proteins in the absence of coexpressed HA-tagged hNAP-1 was arbi-
trary defined as 1. The fold enhancement of E2-mediated activation by
hNAP-1 was calculated. The graph represents the averages of at least
three independent experiments; error bars are shown. The activation
of each E2 mutant protein in the absence of coexpressed hNAP-1,
compared to that of the wild-type protein, which was set at 100%, is
given below the graph. (B) RTS3b cells, containing the BPV1 LCR-
Luc reporter construct integrated into the cellular genome, were co-
transfected with expression vectors for HA-tagged hNAP-1 and for E2.
At 24 h after transfection, TSA was added to the medium for another
2158 REHTANZ ET AL.MOL. CELL. BIOL.
161, which lacks the N-terminal 161 amino acids and which is
a naturally occurring N-terminally truncated mutant that re-
presses activation by the full-length E2 protein (35), still inter-
acted with hNAP-1; in contrast, E2?1–203, which lacks the
entire AD, did not bind at all. An E2 protein which lacks the
C-terminal DBD, E2?386–420, retained binding, as shown in
Fig. 2B. The various E2 proteins average 30% amino acid
identity within the AD. Many of the conserved residues
throughout the AD are important for both replication and
transcription. However, two amino acid substitutions clearly
separated these two capacities. Changing Ile at position 73 to
Ala (I73A) destroyed transcriptional activation while leaving
replication function intact, whereas replacing Glu at position
39 with Ala (E39A) had the inverse phenotype (14). Neither
amino acid change affected the binding of BPV1 E2 to GST–
hNAP-1, as shown in Fig. 2B.
In order to exclude the possibility that the interaction be-
tween BPV1 E2 and hNAP-1 is mediated by a factor present
within the reticulocyte lysate or in yeast cells, we incubated
GST–hNAP-1 with His-tagged, bacterially expressed, purified
full-length BPV1 E2 or E2?386–420. As shown in Fig. 2C, both
E2 and E2?386–420 were specifically retained by GST–
hNAP-1, indicating that the interaction is direct. In this exper-
iment, the binding of full-length E2 was weaker than that of
E2?386–420. This result may have been due to the lower
concentration of full-length E2. We were not able to purify
full-length E2 in the same amounts as E2?386–420. Further-
more, a faster-migrating product, which appears to be an N-
terminal proteolytic form, since it retains the His tag, showed
a stronger interaction with hNAP-1, supporting the notion that
the interaction is mediated by the AD (Fig. 2C).
E2 and hNAP-1 also interact within the cell, as shown in Fig.
2D. 293T cells were transiently transfected with expression
vectors for FLAG-tagged full-length BPV1 E2, an E2 protein
that lacks the N-terminal AD (FLAG-E2?1–203), or the AD
of BPV1 E2 (FLAG-E2?204–420) and an expression vector
for HA-tagged full-length hNAP-1. Whereas in the presence of
full-length BPV1 E2 or its AD, HA-tagged hNAP-1 could be
coprecipitated by the FLAG antibody, E2?1–203 did not me-
diate this effect, confirming that the interaction within the cell
is mediated by the AD of BPV1 E2 as well. The E2 proteins of
HPV8 and HPV18 also were able to coprecipitate HA-tagged
hNAP-1 (Fig. 2D).
Furthermore, E2 binds to endogenous hNAP-1. In all cer-
vical carcinoma cell lines infected with PV, the HPV DNA is
integrated into the cellular genome, resulting in the disruption
of the open reading frame for E2. Therefore, cell lines express-
ing E2 at physiological levels during HPV infection do not
exist, and to analyze the binding of E2 to endogenous hNAP-1,
we overexpressed FLAG-tagged E2 in 293T cells. As shown in
Fig. 2E, the FLAG antibody was able to precipitate endoge-
nous hNAP-1 from extracts of cells transfected with the vector
for FLAG-tagged BPV1 E2 and not from extracts of cells
transfected with the empty expression vector or with a vector
expressing a control protein, bacterial alkaline phosphatase
fused to the FLAG epitope.
Taken together, the data shown in Fig. 1 and 2 demonstrate
that E2 proteins from different PV types interact with hNAP-1.
For BPV1 E2, we can demonstrate that this interaction is
direct and involves the AD.
E2 and hNAP-1 cooperate in the activation of BPV1 gene
expression. As already mentioned, NAP-1 and NAP-2 act as
histone chaperones to which functions in both transcription
and DNA replication have been ascribed (4, 26–28, 39, 50, 58).
Here, we focused on an involvement of the interaction of E2
with hNAP-1, observed in vitro, in the activation of transcrip-
tion. In BPV1, E2 strongly activates transcription from several
BPV1 promoters by binding to the 12 E2 binding sites located
within the regulatory region called the LCR (60). A reporter
construct containing the entire LCR of BPV1 in front of the
luciferase gene was cotransfected into human cell line RTS3b
(57) derived from human keratinocytes, the natural target cells
of PV, together with vectors for HA-tagged hNAP-1 and BPV1
E2. As shown in Fig. 3A, E2 could activate 100- to 130-fold,
depending on the amount of expression vector transfected.
The expression of hNAP-1 on its own stimulated promoter
activity 2-fold; however, the coexpression of hNAP-1 and small
amounts of E2 increased luciferase activity 500-fold. This co-
operativity between hNAP-1 and BPV1 E2 was reduced in the
presence of larger amounts of E2. However, at these high E2
concentrations, cooperativity between E2 and hNAP-1 was still
As shown in Fig. 2B, point mutations abolishing the repli-
cation or transcription function of E2 did not affect binding to
hNAP-1 in vitro. In our experiments, the activation-deficient
E2 I73A mutant retained 33% the activation capacity of wild-
type E2 (data not shown), a finding which is in good agreement
with previous findings (14). However, even this residual acti-
vation was stimulated about fivefold after cotransfection with
hNAP-1, indicating that the step involving binding to hNAP-1
is not affected by this mutant. The same is true for the repli-
cation-deficient E2 E39A mutant (Fig. 3A). As expected, the
activation of transcription by E2 is necessary for the effect of
hNAP-1, since a mutant lacking the AD, E2?1–203, was not
stimulated by hNAP-1 coexpression, in contrast to the mutant
lacking the internal hinge region, E2?195–282 (Fig. 3A). The
activity of the SV40 early promoter, which had driven the
expression of BPV1 E2 in the previous transient transfection
experiments, was not stimulated by coexpression of the
amounts of E2 and hNAP-1 used here (data not shown). This
result excludes the possibility that the enhancing effect is due
to elevation of the expression of E2 by hNAP-1. These results
demonstrate that E2 and hNAP-1 cooperate in the activation
of transcription, which correlates with a direct interaction of
the two proteins.
Because the role of NAP-1 as a histone chaperone is known,
it is possible that hNAP-1, in cooperation with E2, also affects
the state of chromatin during activation by E2. Until now, we
have used an E2-responsive reporter construct, which has been
transiently transfected. In order to test the effect of hNAP-1 on
activation by E2 on reporter constructs organized into a high-
er-order chromatin structure, we established a cell line harbor-
ing reporter construct BPV1 LCR-Luc integrated into the cel-
lular genome. After transiently transfecting this stable cell line
with the expression vector for E2, we were not able to observe
any activation, independent of whether the hNAP-1 vector was
cotransfected or not (data not shown). Since we suspected that
the tight packing of chromatin may inhibit the access of E2 to
the DNA, we treated the cells at 24 h after transfection with
TSA, an inhibitor of histone deacetylases. This treatment en-
VOL. 24, 2004FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-1 2159
abled E2 to stimulate luciferase activity weakly; this effect was
slightly enhanced by the coexpression of hNAP-1 (Fig. 3B).
These results demonstrate that with promoters organized in
cellular chromatin, hNAP-1 also stimulates activation by E2,
although to a much lesser extent than transiently transfected
Full-length hNAP-1 is required for cooperation with E2. In
order to gain insights into the mechanism of the cooperativity
between hNAP-1 and E2 proteins, we determined the domains
within hNAP-1 which are bound by E2 and analyzed their roles
in the enhancement of activation by E2. A series of N- and
C-terminal deletion mutants of hNAP-1 were expressed as
GST fusion proteins. Although the N-terminal region from
residues 1 to 162 possessed marginal binding activity (GST–
hNAP-1?4, GST–hNAP-1?7, and GST–hNAP-1?10), an in-
ternal domain from residues 162 to 290 (GST–hNAP-1?6) and
the C-terminal region from residues 291 to 392 (GST– NAP-
1?5) bound with greater efficiency to
(Fig. 4A). These results suggest that E2 binds to at least two
separable domains within hNAP-1, one internal from amino
acids 162 to 290 and one C terminal from residues 291 to 392.
To analyze the contributions of the domains of hNAP-1 that
interact with E2 to the stimulation of activation by E2, hNAP-1
deletion mutants (Fig. 4B) were fused at the N terminus to an
HA tag and expressed. The results obtained in the cotransfec-
tion experiments shown in Fig. 4B demonstrate that the N-
35S-labeled BPV1 E2
FIG. 4. The binding of E2 to hNAP-1 is necessary but not sufficient for the stimulation of E2-mediated activation. (A) Different regions of
hNAP-1 were fused to GST. Bacterially expressed, purified GST– hNAP-1 fusion proteins (shown in the sodium dodecyl sulfate [SDS] gel at the
bottom, stained by Coomassie blue) were incubated with35S-labeled BPV1 E2. Bound proteins were detected by autoradiography. The binding
of E2 to hNAP-1 is summarized (?, no binding; ?/?, weak binding; ?, binding; ??, strong binding). In some cases, the presence of GST– hNAP-1
fusion proteins in the gel might have affected the migration of the E2 protein slightly. aa, amino acids; fl, full length. (B) (Upper left panel) RTS3b
cells were cotransfected with the BPV1 LCR reporter construct and an expression vector for HA-tagged hNAP-1 or HA-tagged deletion mutants
of hNAP-1, either alone or together with 5 ng of the E2 expression vector. (Upper right panel) Effects of the various hNAP-1 mutants on BPV1
promoter activity in the absence of E2. The activity of the BPV1 LCR in the absence of hNAP-1 was arbitrarily defined as 1. (Lower right panel)
Effects of the hNAP-1 mutants on activation by E2. Here, the activity in the presence of E2 without hNAP-1 (lane ?) was set at 1, and the effects
of the various hNAP-1 mutants were calculated. Both graphs represent the averages of at least three independent experiments with two different
plasmid DNA preparations; error bars are shown. (Lower left panel) Levels of expression of the various HA-tagged hNAP-1 deletion mutants
analyzed by Western blotting (WB) with extracts of cells that had been transiently transfected with the corresponding expression vectors and a
high-affinity antibody against the HA epitope. Asterisks indicate the positions of the respective hNAP-1 mutants. Lanes 1 to 9, 10% polyacrylamide
gel; lanes 10 to 12, 15% polyacrylamide gel.
2160 REHTANZ ET AL.MOL. CELL. BIOL.
terminal 91 residues are indispensable for hNAP-1 to enhance
activation by E2. All hNAP-1 mutants lacking the N-terminal
91 amino acids failed to cooperate with E2 in activation. Most
of the mutants repressed activation by E2 from 2-fold (hNAP-
1?8) to 10-fold (hNAP-1?1, hNAP-1?2, hNAP-1?5, and
hNAP-1?7). This repression is not related to squelching due to
unphysiologically high concentrations of these mutants com-
pared to that of full-length hNAP-1 in transfected cells, since
all hNAP-1 derivatives were present in similar amounts. The
sole exception is mutant hNAP-1?8, which was produced at
reduced levels, as shown in the Western blot in Fig. 4B. Fur-
thermore, titration of these proteins revealed constant, dose-
dependent repression (data not shown). Mutant hNAP-1?4,
retaining the N-terminal domain but lacking an interaction
with E2, had no effect on activation by E2. Mutants hNAP-1?3
and hNAP-1?9, containing the N-terminal domain and one E2
binding motif, respectively, were able to enhance activation by
E2, although to a reduced extent compared to that obtained
with wild-type hNAP-1. These results indicate that in addition
to the N-terminal 91 amino acids, at least one E2 binding motif
is required for hNAP-1 to stimulate activation by E2, although
it seems that amino acids 162 to 290 of hNAP-1 are more
The various hNAP-1 mutants also modulated the activity of
the BPV1 promoters in the absence of E2. However, there was
no clear correlation between the effects of each mutant on
activation by E2 and on basal promoter activity. For example,
hNAP-1?5 and hNAP-1?9 both slightly reduced basal pro-
moter activity, but hNAP-1?5 strongly repressed activation by
E2 and hNAP-1?9 still weakly stimulated activation by E2.
These results suggest that the effects of hNAP-1 may be spe-
cific for E2. Moreover, these data imply that hNAP-1 has
another function in transcription, since some of the mutants,
e.g., hNAP-1?7 and hNAP-1?10, which do not interact with
E2 in vitro, strongly inhibited activation by E2; the latter result
may be related to their inhibitory effects on promoter activity
E2, hNAP-1, and p300 can form a ternary complex in vitro.
As already mentioned, NAP-1 and NAP-2 have been shown to
interact with p300. Like that of E2, the binding of p300 to
hNAP-1 may involve at least two domains, one from amino
acids 123 to 230 and a second one C terminal to amino acid 290
(4, 58). Thus, the regions in hNAP-1 required for activation by
E2 also include the p300 interaction domains, and we cannot
rule out the possibility that the binding of hNAP-1 to p300 may
be necessary as well. Since it has been demonstrated that E2
proteins of HPV8, HPV18, and BPV1 directly and functionally
interact with p300 (37, 49, 56), as does hNAP-1, we were
interested in the effects of hNAP-1 and p300 on E2-mediated
activation. Unfortunately, coexpression of p300 with reporter
VOL. 24, 2004 FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-12161
the control of the regulatory region of HPV8 was cotransfected with
expression vectors for HPV8 E2, p300, and HA-tagged hNAP-1, either
alone or in combination. The luciferase activity in the presence of the
empty expression vector was set at 1, and the fold activation of any of the
proteins was calculated. The graph represents the averages of five inde-
pendent experiments; error bars are shown. (Bottom panel) Structure of
the regulatory region of HPV8, including the two promoters and the E2
binding sites (black boxes). (B) GST or GST–p300-4 was incubated with
increasing amounts of His-tagged, bacterially expressed, purified hNAP-1
for 2 h at 4°C. After several washes to remove unbound His-tagged
hNAP-1, incubation with35S-labeled BPV1 E2 or hNAP-1 for another 2 h
followed. The binding of radiolabeled E2 or hNAP-1 was analyzed by
autoradiography. The radioactive signals were quantified with a Phospho-
rImager; the percentages bound to GST–p300-4 were calculated. Ten
percent of the input of35S-labeled E2 or 20% of the input of35S-labeled
22 and 23) or presence (lanes 26 and 27) of
(C) Glycerol density sedimentation analysis performed as described pre-
viously (61) with 600 ng of His-tagged hNAP-1, 200 ng of His-tagged
E2?326–420 (E2), and 800 ng of p300 1195 to 1761. Samples were sub-
jected to 7.5 to 30% glycerol gradient centrifugation, and a total of 36
fractions were collected, starting from the top of the gradient. Since an
initial analysis revealed that all proteins fractionated in fractions 10 to 31,
only these fractions are shown here after analysis by Western blotting with
an antibody against the His tag. The presence of E2, hNAP-1, and p300
in the various fractions is indicated on the right.
hNAP-1 and E2 can form a ternary complex with p300.
35S-labeled BPV1 E2.
construct BPV1 LCR-Luc, used in the experiments described
so far, led to the inhibition of promoter activity (data not
shown). However, Mu ¨ller et al. showed previously that HPV8
E2 and coexpressed p300 cooperated in the activation of
HPV8 gene expression (49). Since we demonstrated here that
in addition to BPV1 E2, HPV8 E2 also bound to hNAP-1 (Fig.
2A and D) and coexpressed hNAP-1 enhanced activation by
HPV8 E2 (data not shown; see Fig. 7), we used the HPV8
system to study the effects of hNAP-1 and p300 on E2-medi-
ated activation. In agreement with previous observations, E2
and p300 cooperated, since they stimulated promoter activity
15.5-fold, compared to 4-fold by each of the proteins alone
(Fig. 5A). As shown previously, the overexpression of p300
does not affect the expression of the E2 protein (49). hNAP-1
increased activation by either E2 or p300 by up to 23- or
17-fold, respectively. Thus, hNAP-1 can functionally interact
with both proteins, in correlation with the direct binding of
hNAP-1 to E2 (Fig. 2) and p300 (4, 58). The overexpression of
all three proteins together resulted in 134-fold activation. This
activation in the presence of E2, hNAP-1, and p300 was far
beyond the sum of the effects of the single components, sug-
gesting that a ternary complex of E2, p300, and hNAP-1 likely
contributes very efficiently to the activation of transcription.
To analyze possible ternary complex formation among the
three proteins in vitro, we used a fragment of p300 from amino
acids 1453 to 1882 fused to GST (GST–p300-4). Mu ¨ller et al.
observed previously that E2 binds to this segment of p300,
which colocalizes with the C/H3 domain (49). In addition to
the KIX domain (4), NAP-2 and NAP-1 were shown to require
amino acids 1572 to 1818 for binding to p300 (58), like E2. To
confirm the formation of a ternary complex in vitro, we per-
formed a competition experiment. As shown in Fig. 5B, GST
or the GST–p300-4 fusion protein was incubated with increas-
ing amounts of bacterially expressed, purified His-tagged
hNAP-1 for 2 h. After several washing steps to remove un-
bound His-tagged hNAP-1, either35S-labeled E2 or hNAP-1,
each obtained by in vitro translation, was added; after incuba-
tion for another 2 h, binding was analyzed. The signals were
quantified with a PhosphorImager. While in the absence of
unlabeled His-tagged hNAP-1 59% of radiolabeled hNAP-1
was precipitated by GST–p300-4, preincubation of increasing
amounts of unlabeled hNAP-1 reduced the binding of labeled
hNAP-1 to 19% (Fig. 5B, lanes 6 to 10). This competition is
consistent with the notion that hNAP-1 binding sites within
p300-4 have been occupied by the nonlabeled protein. In con-
trast, preincubation of GST–p300-4 with the same increasing
VOL. 24, 2004 FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-12163
amounts of His-tagged hNAP-1 did not affect the binding of
radiolabeled E2. A total of 17 to 20% of the input E2 proteins
were precipitated by GST–p300-4, regardless of preincubation
with increasing amounts of His-tagged hNAP-1 (Fig. 5B, lanes
1 to 5). As expected, purified His-tagged hNAP-1 also was
bound by GST–p300-4 in the presence of E2, as revealed by
Western blotting with a monoclonal antibody against hNAP-1
(Fig. 5B, lanes 21 to 27). These results suggest that E2 can still
interact with p300 when hNAP-1 is bound and that a ternary
complex among E2, p300, and hNAP-1 is likely.
To further address the formation of a ternary complex
among E2, p300, and hNAP-1 in vitro, we investigated the
fractionation of the E2 protein in the presence of hNAP-1 and
p300 in a density gradient. After incubation of His-tagged,
bacterially expressed, purified E2?326–420 with His-tagged
hNAP-1 and p300 1195 to 1761, the binding reaction was
subjected to glycerol gradient centrifugation as described by
Tanese (61). A total of 36 fractions were collected, and the
presence of E2?326–420, hNAP-1, and p300 1195 to 1761 was
monitored by Western blotting. Since all three proteins easily
were distinguishable due to their characteristic sizes, we used
an antibody directed against the His tag. To ensure that most
of E2 was complexed by p300 and hNAP-1, we added E2 in
limiting amounts to the binding reaction. An initial analysis
revealed that all three proteins sedimented near the middle of
the gradient within fractions 10 to 31. Therefore, only these
fractions were used for the detailed analysis shown in Fig. 5C.
Most of E2 was present in fractions 22 to 24. After incubation
with hNAP-1, E2 sedimented more toward the top of the
gradient, with the highest concentrations in fractions 19 to 22
(Fig. 5C, panel I, E2 vs. E2?hNAP-1). Thus, the interaction
between E2 and hNAP-1 can be demonstrated in this glycerol
gradient analysis, as indicated by the observation that hNAP-1
cofractionated (Fig. 5C, panel II, hNAP-1 vs. hNAP-1?E2). In
FIG. 6. p53 interacts directly with hNAP-1. (A) The GST– hNAP-1 fusion protein or GST was incubated with His-tagged, bacterially expressed,
purified p53. After the beads were washed with a buffer containing 200 mM KCl, bound p53 was revealed by an antibody directed against the His
tag. (B) The p53-negative cell line RTS3b was cotransfected with a synthetic p53-responsive luciferase reporter construct, 5 ng of an expression
vector for p53, and 400 ng of the vector for HA-tagged hNAP-1. The luciferase activity in the presence of the vector only was set at 1. Error bars
are shown. (C) Extracts (90 ?g) from RTS3b cells transiently transfected with an expression vector for p53 (lanes 5, 7, and 8) or for HA-tagged
hNAP-1 in two different amounts (lanes 6 to 8) were used for Western blotting (WB) to detect endogenous p21. The blot was reprobed with an
antibody against p53 to detect p53 protein levels. (D) Nuclear extracts from p53-negative RTS3b cells (lanes 2 and 4) and from neonatal human
epidermal keratinocytes (NHEK) (lanes 1 and 3), which express wild-type p53, were subjected to immunoprecipitation (IP) with a p53 antibody.
Bound endogenous hNAP-1 was analyzed by Western blotting with a monoclonal antibody against hNAP-1 (lanes 3 and 4). In lanes 1 and 2,
one-sixth of the input of nuclear extracts was loaded to detect the level of expression of hNAP-1 in both types of cells. Lanes 3 and 4 were reprobed
with the p53 antibody to confirm the pattern of expression of p53. A cross-reacting cellular protein is indicated by an asterisk.
2164 REHTANZ ET AL.MOL. CELL. BIOL.
contrast to the addition of hNAP-1, the addition of p300 to E2
had the consequence that E2 sedimented more toward the
bottom of the gradient (Fig. 5C, panel I, E2?p300). In addi-
tion, E2 cofractionated with p300, which was detectable in
higher fractions after incubation with E2, compared to free
p300, confirming the interaction between E2 and p300 (Fig.
5C, compare panel I, E2?p300, with panel III, p300?E2). In
the presence all three proteins, E2 again was shifted toward the
top of the gradient (Fig. 5C, panel I, E2?hNAP-1?p300).
However, the E2 pattern differed from that obtained after
incubation with hNAP-1 alone, since now a larger portion of
E2 was detectable in fractions 13 to 16, accompanied by a
reduction in fractions 24 and 25. Significant amounts of p300
and hNAP-1 also were found in fractions 13 to 22 when all
three proteins were incubated (Fig. 5C, panels II and III, all
three proteins). Thus, the cofractionation of E2, hNAP-1, and
p300 and the change in the sedimentation of E2 after simul-
taneous incubation with both hNAP-1 and p300 correlate with
ternary complex formation among E2, hNAP-1, and p300.
Together, transient transfection, GST pull-down, and den-
sity gradient sedimentation experiments suggested that multi-
ple protein-protein interactions (E2-p300, E2–hNAP-1, and
hNAP-1–p300) may contribute to the formation of a stable
ternary complex efficient in the activation of transcription.
A direct interaction between p53 and hNAP-1 also is in-
volved in activation mediated by p53. Previously, it was sug-
gested that NAP-2, which is closely related to NAP-1, aug-
ments transcription by activators such as p53 and E2F, which
use p300 as a coactivator, through a direct interaction with
p300 (58). In order to test whether, in addition to p300–
hNAP-1 binding, a direct interaction between p53 and hNAP-1
(like that for PV E2) may be involved, we incubated His-
tagged, bacterially expressed, purified p53 with a GST–
hNAP-1 fusion protein. As shown in Fig. 6A, p53 was precip-
itated by the GST–hNAP-1 fusion protein and not by GST.
The interaction could be confirmed with GST-p53 and His-
tagged, purified hNAP-1 (data not shown). These results dem-
onstrate that the interaction between hNAP-1 and p53 is direct
and not merely mediated by other factors, such as p300, as has
been suggested elsewhere (58). When a synthetic reporter con-
struct containing one p53 binding site upstream of the Hsp70
promoter was transiently transfected into the p53-negative cell
line RTS3b, hNAP-1 also potentiated activation by p53 (Fig.
6B), confirming that the interaction is functional, as has been
shown for NAP-2 (58). On the basis of these results, we sus-
pected that hNAP-1 is also able to stimulate the activation by
p53 of an endogenous p53-responsive promoter in the absence
of the coexpression of p300. We analyzed the effect of the
coexpression of p53 and hNAP-1 on the p21 level in cell line
RTS3b. Under the Western blotting conditions shown in Fig.
6C, endogenous p21 was not visible. Cotransfection of the p53
expression vector or of small amounts of the hNAP-1 expres-
sion vector did not induce p21 expression to detectable levels.
However, the coexpression of p53 and large amounts of
hNAP-1 led to the appearance of the p21 protein. In addition,
endogenous p53 and hNAP-1 can form a stable complex, as
shown in Fig. 6D. Extracts from RTS3b cells, which do not
express a detectable level of the p53 protein, and from primary
neonatal human epidermal keratinocytes, which express high
levels of p53 (Fig. 6D, lower panel), were subjected to immuno-
precipitation with a monoclonal antibody against p53. Only in
neonatal human epidermal keratinocytes could endogenous
hNAP-1 be coprecipitated. The results shown in Fig. 6 imply
that p53 and hNAP-1 interact directly with each other.
In order to test whether a functional interaction with
hNAP-1 is a general strategy for activators or is restricted to
activators interacting with p300, we used the AD of TEF-1.
Various investigators have shown that TEF-1 neither binds to
p300 in vitro nor cooperates with coexpressed p300 (49, 59). As
shown in Fig. 7, the AD of TEF-1 failed to interact with
GST–hNAP-1 in vitro. The TEF-1 AD fused to the DBD of
GAL4 stimulated transcription from a reporter containing
GAL4 binding sites about fourfold; this effect was not en-
hanced after the coexpression of hNAP-1. However, as a con-
trol, activation by a hybrid protein composed of the AD of
HPV8 E2 and the DBD of GAL4 was further stimulated by
There is growing evidence demonstrating that hNAP-1 plays
important roles during the activation of transcription. NAP-1
and other histone chaperones, such as ASF1 and chromatin
assembly factor 1, have been shown to cooperate with ATP-
dependent chromatin remodeling complexes (4, 28, 48, 50).
Furthermore, NAP-1 and NAP-2 are part of the p300/CBP
coactivator complex (4, 27, 58). Thus, it has been suggested
that NAP-1 may serve as a point of interaction between tran-
scriptional coactivators and chromatin. We identified hNAP-1
as a target of the AD of PV transcription factor E2 by a yeast
two-hybrid screen. In contrast to a previous screen in which a
transactivation-defective point mutant of E2 was applied (9),
we were able to use wild-type E2 protein as bait, since our
reporter construct was only weakly activated by E2 in compar-
ison to the GAL4 AD. We were able to confirm the interaction
between E2 and hNAP-1 by GST pull-down assays and coim-
munoprecipitation experiments. Thus, using three different
methods and proteins synthesized in different ways, i.e., in
yeast, bacterial, and eukaryotic cells, we were able to demon-
strate an interaction between E2 and hNAP-1. Moreover, E2
also precipitates endogenous hNAP-1. Therefore, it is unlikely
that the interaction is artifactual as a result of protein misfold-
ing, but is indeed specific.
In addition to the E2 protein of BPV1, the E2 proteins of
two HPV types, HPV18 (infecting the genital mucosa and
associated with cervical cancer) and HPV8 (infecting the skin),
also contact hNAP-1 in vitro and in vivo, indicating that the
binding to hNAP-1 may be common to E2 proteins. The in-
teraction between BPV1 E2 and hNAP-1 is direct, since bac-
terially expressed purified proteins also coprecipitate. Further-
more, we show that another transcriptional activator, p53, also
directly binds to hNAP-1. This finding demonstrates that the
interaction does not have to be mediated by a bridging factor.
p300 may be such a factor, since hNAP-1 interacts with p300,
as do PV E2 and p53 (4, 5, 23, 49, 56, 58).
The binding of BPV1 E2 to hNAP-1 is mediated by its AD,
as revealed by the yeast two-hybrid system, GST pull-down
experiments, and coimmunoprecipitation. The concept of an
interaction between E2 and hNAP-1 is reinforced by the ability
of hNAP-1 overexpression to stimulate activation by E2. Even
VOL. 24, 2004 FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-12165
in the presence of saturating E2 concentrations, hNAP-1 and
E2 cooperated, indicating that binding to hNAP-1 is a rate-
limiting step for E2. The observation that the activation-com-
promised mutant E2 I73A (14) was stimulated by hNAP-1 to
the same extent as wild-type E2 suggests that binding to
hNAP-1 is not affected in this mutant, in correlation with the in
vitro binding data shown in Fig. 2B. Thus, binding to hNAP-1
may be one of several steps, performed by E2, leading to the
activation of transcription. These include contacts with com-
ponents of the PIC, such as TBP and TFIIB (8, 44). Further-
more, E2 binds to coactivators such as AMF-1/Gps, CBP/p300,
and p/CAF (36, 37, 49, 56). Interestingly, Lefebvre et al.
showed previously that the activation of transcription by E2
correlates with a change in the chromatin structure in yeast
cells (38), where the interaction with NAP-1 may play a role.
While E2 strongly activated transcription from reporter con-
structs which had been transiently transfected, it was not able
to activate transcription when the enhancer-promoter region
was integrated into the cellular genome. Transiently trans-
fected plasmids are thought to assemble with histones into a
nucleosome-like structure (10, 36). However, it is possible that
the overall structure is not organized as cellular chromatin
(10). The observation that treatment with TSA partially re-
stores the ability of E2 to activate and to act in synergy with
hNAP-1 strengthens the notions that a compact chromatin
structure may inhibit the access of E2 to chromosomal DNA
and that hNAP-1 can enhance E2-mediated activation only
after E2 has bound. Thus, hNAP-1 may not be able to stimu-
late the binding of E2 to sites organized in chromatin, as has
been shown for GAL4 in vitro (65).
How does hNAP-1 enhance activation by E2? With the use
of deletion mutants, we demonstrated that all of hNAP-1 is
required to stimulate E2 activity. The interaction of E2 with
hNAP-1 may be necessary, since mutants hNAP-1?3, hNAP-
1?4, and hNAP-1?9, which lack either one or both E2-inter-
acting motifs, failed or showed a reduced capacity to stimulate
activation by E2. In addition to the C-terminal 301 amino
acids, which are bound by both E2 and p300, the N-terminal 91
amino acids of hNAP-1 are crucial. All N-terminally truncated
mutants tested here not only lacked stimulating activity but
also had a dominant-negative effect on activation by E2. Some
of these N-terminally truncated overexpressed hNAP-1 mu-
tants may compete with endogenous, wild-type hNAP-1 for
binding to E2. Thus, the recruitment of N-terminally deleted
hNAP-1 by E2 would lead to nonfunctional complexes, unable
to support transcription. Moreover, the hNAP-1 domains ex-
pressed from these mutants may bind to and sequester other
cellular factors necessary for the activation of transcription.
This may be the case with hNAP-1 mutants still repressing
activation by E2 but lacking binding to E2 in vitro (such as
hNAP-1?7 or hNAP-1?10). The notion that targets other than
E2 or p300 that are involved in transcription are bound by
hNAP-1 is supported by the observation that some of these
mutants not only repressed activation by E2 but also inhibited
the basal activity of the promoters.
What is the role of the N-terminal part of hNAP-1? Accord-
ing to published data, it does not participate in binding to p300
(58). It has been suggested that the import of histones H2A
and H2B into the nucleus is essential for NAP-1 function in the
activation of transcription, since yeast NAP-1 mutations inac-
tivating a leucine-rich nuclear export signal (NES) within the N
terminus led to reduced transcription of some genes (47). In
FIG. 7. TEF-1 does not interact with hNAP-1. (Left panel) An expression vector for the DBD of GAL4 fused to either the AD of TEF-1
(pG4-TEF AD) (68) or the AD of HPV8 E2 (pG4-8E2 AD) was cotransfected either alone or together with the expression vector for HA-tagged
hNAP-1 and the GAL4-responsive reporter construct, which is shown schematically below the graph. The data represent the averages of four
experiments, and error bars indicate standard deviations. (Right panel)35S-labeled TEF-1 AD was incubated with GST or GST–hNAP-1. After
the samples were washed with a buffer containing 100 mM KCl, the reaction was analyzed by autoradiography. Ten percent of the input is shown
in lane 1.
2166 REHTANZ ET AL.MOL. CELL. BIOL.
our study, mutations of corresponding conserved residues in
hNAP-1 did not lead to such inhibition. In addition, the mu-
tations also did not affect the localization of hNAP-1, indicat-
ing that the human counterpart may have an NES at a different
position (M. Rehtanz and G. Steger, unpublished results).
However, localization studies with our hNAP-1 mutants re-
vealed stronger nuclear staining of all N-terminal deletion mu-
tants than of full-length hNAP-1 or of C-terminal truncation
mutants. A precise mutational analysis of the N-terminal 91
amino acids of hNAP-1 must show whether the nuclear import
of H2A and H2B, which requires a functional NES motif, is
essential for hNAP-1 to activate transcription. Furthermore,
other functions which have been attributed to this domain,
such as dimerization or multimerization of NAPs (58) or nu-
cleosome assembly activity (16), may be important.
The recruitment of hNAP-1 to the promoter through fusion
with the DBD of GAL4 did not lead to the activation of
transcription (M. Rehtanz and G. Steger, unpublished results),
indicating that hNAP-1 must act in concert with an AD. The
observation that the enhancing effect of hNAP-1 on E2-medi-
ated activation was much stronger in the presence of subsatu-
rating amounts of E2 may be explained by the fact that
hNAP-1 affects the interaction of E2 with another factor. p300
may be such a factor. p300 has been shown to bind to two
separable domains within the C-terminal 300 amino acids of
hNAP-1 (58), as does E2. Our data demonstrate that in vitro
the formation of a ternary complex among hNAP-1, p300, and
E2 is possible. First, a competition experiment revealed that
hNAP-1 cannot displace E2 from p300 (Fig. 5B). Second, re-
sults from glycerol gradient density sedimentation correlated
with ternary complex formation. Purified hNAP-1 and p300
1195 to 1761 affect the sedimentation of E2?386–420, a result
which is consistent with direct interactions of both proteins
with E2. In the presence of both hNAP-1 and p300, the sedi-
mentation of E2 is changed again, indicating that E2 is com-
plexed not only with either p300 or hNAP-1 but also with both
proteins simultaneously. Moreover, E2, hNAP-1, and p300 co-
fractionate when incubated together (Fig. 5C). This ternary
complex may be very efficient in the activation of transcription,
since overexpressed E2, hNAP-1, and p300 strongly cooperate
in the activation of HPV8 gene expression. Ito et al. demon-
strated that the acetylation of histones by p300 helps transfer
histones H2A and H2B from nucleosomes to NAP-1 (27). In
vitro, the absence of H2A and H2B correlates with increased
gene activity, probably by decreasing the level of chromatin
folding (reviewed in reference 11). Thus, E2 may induce the
recruitment of hNAP-1 and p300, which then participate in the
creation of such an H2A- and H2B-free environment.
The importance of hNAP-1 seems not to be restricted to a
single factor, since we provide further evidence that bacterially
expressed, purified p53 can bind directly to hNAP-1 in vitro
and that endogenous p53 and hNAP-1 interact as well. This
interaction seems to be functional, a conclusion which is sup-
ported by the observation that overexpressed hNAP-1 alone
can stimulate p53-mediated activation from a synthetic re-
porter construct and from a p53-responsive endogenous pro-
moter, that of p21, which does not necessarily require the
coexpression of p300, as has been suggested elsewhere (58).
Since we were able to demonstrate that TEF-1, devoid of
binding to p300, also lacks a functional interaction with
hNAP-1, hNAP-1 may function specifically in concert with
p300. However, future experiments will need to confirm
whether binding to hNAP-1 is a phenomenon only for activa-
tors binding to p300. Our data demonstrate for the first time
that in addition to p300, hNAP-1 is an essential target for
activator proteins to activate transcription. The result may be
sufficiently high local concentrations of hNAP-1 and p300 on
actively transcribed promoters, which are essential for efficient
We thank Y. Ishimi for the gift of the monoclonal antibody against
hNAP-1, D. Livingston and M. Scheffner for providing plasmid DNAs,
and P. Bartel for providing yeast strain YM954. We also thank Herbert
Pfister and Andrew Barker for critical reading of the manuscript and
Karin Schnet z for helpful support.
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB274-A8, STE604/3-1, and STE604/3-2) and by the BMBF (Ak-
tenzeichen 0312708F). G.S. was the recipient of a Lise-Meitner Ha-
1. Altman, R., and D. R. Kellogg. 1997. Control of mitotic events by NAP1 and
the Gin4 kinase. J. Cell Biol. 138:119–130.
2. Armstrong, J. A., J. J. Bieker, and B. M. Emerson. 1998. A SWI/SNF-related
chromatin remodeling complex, E-RC1 is required for tissue specific tran-
scriptional regulation by EKLF in vitro. Cell 95:93–104.
3. Asahara, H., B. Santoso, E. Guzman, K. Du, P. A. Cole, I. Davidson, and M.
Montminy. 2001. Chromatin-dependent cooperativity between constitutive
and inducible activation domains in CREB. Mol. Cell. Biol. 21:7892–7900.
4. Asahara, H., S. Tartare-Deckert, T. Nakagawa, T. Ikehara, F. Hirose, T.
Hunter, T. Ito, and M. Montminy. 2002. Dual roles of p300 in chromatin
assembly and transcriptional activation in cooperation with nucleosome as-
sembly protein 1 in vitro. Mol. Cell. Biol. 22:2974–2982.
5. Avantaggiati, M. L., V. V. Ogryzko, K. Gardner, A. Giordano, S. A. Levine,
and K. Kelly. 1997. Recruitment of p300/CBP in p53 dependent signal
pathways. Cell 89:1175–1184.
6. Becker, P. B. 2002. Nucleosome sliding: facts and fiction. EMBO J. 21:4749–
7. Benson, J. D., and P. M. Howley. 1995. Amino-terminal domains of the
bovine papillomavirus type 1 E1 and E2 proteins participate in complex
formation. J. Virol. 69:4364–4372.
8. Benson, J. D., R. Lawande, and P. M. Howley. 1997. Conserved interaction
of the papillomavirus E2 transcriptional activator proteins with human and
yeast TFIIB proteins. J. Virol. 71:8041–8047.
9. Breiding, D. E., F. Sverdrup, M. J. Grossel, N. Moscufo, W. Boonchai, and
E. J. Androphy. 1997. Functional interaction of a novel cellular protein with
the papillomavirus E2 transactivation domain. Mol. Cell. Biol. 17:7208–7219.
10. Cervoni, N., and M. Szyf. 2001. Demethylase activity is directed by histone
acetylation. J. Biol. Chem. 276:40778–40787.
11. Chan, H. M., and N. B. La Thangue. 2001. p300/CBP proteins: HATs for
transcriptional bridges and scaffolds. J. Cell Sci. 114:2363–2373.
12. Dong, X.-P., F. Stubenrauch, E. Beyer-Finkler, and H. Pfister. 1994. Preva-
lence of deletions of YY1-binding sites in episomal HPV 16 DNA from
cervical cancers. Int. J. Cancer 58:803–808.
13. Eckner, R., M. E. Ewen, D. Newsome, M. Gerdes, J. A. DeCaprio, J. B.
Lawrence, and D. M. Livingston. 1994. Molecular cloning and functional
analysis of the adenovirus E1A-associated 300-kD protein (p300) reveals a
protein with properties of a transcriptional adaptor. Genes Dev. 8:869–884.
14. Ferguson, M. K., and M. R. Botchan. 1996. Genetic analysis of the activation
domain of bovine papillomavirus protein E2: its role in transcription and
replication. J. Virol. 70:4193–4199.
15. Fryer, C. J., and T. K. Archer. 1998. Chromatin-remodelling by the glucocor-
ticoid receptor requires the BRG1 complex. Nature 393:89–91.
16. Fujii-Nakata, T., Y. Ishimi, A. Okuda, and A. Kikuchi. 1992. Functional
analysis of nucleosome assembly protein, NAP-1. J. Biol. Chem. 267:20980–
17. Funk, W. D., D. T. Pak, R. H. Karas, W. E. Wright, and J. W. Shay. 1992. A
transcriptionally active DNA binding site for human p53 protein complexes.
Mol. Cell. Biol. 12:2866–2871.
18. Gillitzer, E., G. Chen, and A. Stenlund. 2000. Separate domains in E1 and E2
proteins serve architectural and productive roles for cooperative DNA bind-
ing. EMBO J. 19:3069–3079.
19. Giordano, A., and M. L. Avantaggiati. 1999. p300 and CBP: partners for life
and death. J. Cell. Physiol. 181:218–230.
VOL. 24, 2004FUNCTIONAL INTERACTION BETWEEN E2 AND NAP-12167
20. Giri, I., and M. Yaniv. 1988. Structural and mutational analysis of E2 trans-
activating proteins of papillomaviruses reveals three distinct functional do-
mains. EMBO J. 7:2823–2829.
21. Goodman, R. H., and S. Smolik. 2000. CBP/p300 in cell growth, transfor-
mation and development. Genes Dev. 14:1553–1577.
22. Grunstein, M. 1997. Histone acetylation in chromatin structure and tran-
scription. Nature 389:349–352.
23. Gu, W., X.-L. Shi, and R. G. Roeder. 1997. Synergistic activation of tran-
scription by CBP and p53. Nature 387:819–823.
24. Ham, J., G. Steger, and M. Yaniv. 1994. Cooperativity in vivo between the E2
transactivator and the TATA box binding protein depends on core promoter
structure. EMBO J. 13:147–157.
25. Hoffmann, A., and R. G. Roeder. 1991. Purification of his-tagged proteins in
non-denaturing conditions suggests a convenient method for protein inter-
action studies. Nucleic Acids Res. 19:6337–6338.
26. Ito, T., M. Bulger, R. Kobayashi, and J. T. Kadonaga. 1996. Drosophila
NAP-1 is a core histone chaperone that functions in ATP-facilitated assem-
bly of regularly spaced nucleosomal arrays. Mol. Cell. Biol. 16:3112–3124.
27. Ito, T., T. Ikehara, T. Nakagawa, W. L. Kraus, and M. Muramatsu. 2000.
p300-mediated acetylation facilitates the transfer of histone H2A-H2B
dimers from nucleosomes to a histone chaperone. Genes Dev. 14:1899–1907.
28. Jiang, W., S. K. Nordeen, and J. T. Kadonaga. 2000. Transcriptional analysis
of chromatin assembled with purified ACF and dNAP-1 reveals that acetyl-
CoA is required for preinitiation complex assembly. J. Biol. Chem. 275:
29. Kellogg, D. R., A. Kikuchi, T. Fujii-Nakata, C. W. Turck, and A. W. Murray.
1995. Members of the NAP/SET family of proteins interact specifically with
B-type cyclins. J. Cell Biol. 130:661–673.
30. Kingston, R. E., and G. J. Narlikar. 1999. ATP-dependent remodeling and
acetylation as regulators of chromatin fluidity. Genes Dev. 13:2339–2352.
31. Kowenz-Leutz, E., and A. Leutz. 1999. A C/EBP ? isoform recruits the
SWI/SNF complex to activate myeloid genes. Mol. Cell 4:735–743.
32. Kraus, W. L., and J. T. Kadonaga. 1998. p300 and estrogen receptor coop-
eratively activate transcription via differential enhancement of initiation and
reinitiation. Genes Dev. 12:331–342.
33. Kraus, W. L., E. T. Manning, and J. T. Kadonaga. 1999. Biochemical anal-
ysis of distinct activation functions in p300 that enhance transcription initi-
ation with chromatin templates. Mol. Cell. Biol. 19:8123–8135.
34. Lambert, P. F., N. Dostatni, A. A. McBride, M. Yaniv, P. M. Howley, and B.
Arcangioli. 1989. Functional analysis of the papilloma virus E2 trans-activa-
tor in Saccharomyces cerevisiae. Genes Dev. 3:38–48.
35. Lambert, P. F., B. A. Spalholz, and P. M. Howley. 1987. A transcriptional
repressor encoded by BPV1 shares a common carboxy-terminal domain with
the E2 transactivator. Cell 50:69–78.
36. Lee, D., S. G. Hwang, J. Kim, and J. Choe. 2002. Functional interaction
between p/CAF and human papillomavirus E2 protein. J. Biol. Chem. 277:
37. Lee, D., B. Lee, J. Kim, D. W. Kim, and J. Choe. 2000. cAMP response
element-binding protein-binding protein binds to human papillomavirus E2
protein and activates E2 dependent transcription. J. Biol. Chem. 275:7045–
38. Lefebvre, O., G. Steger, and M. Yaniv. 1997. Synergistic transcriptional
activation by papillomavirus E2 protein occurs after DNA binding and cor-
relates with a change in chromatin structure. J. Mol. Biol. 266:465–478.
39. LeRoy, G., A. Loyola, W. S. Lane, and D. Reinberg. 2000. Purification and
characterization of a human factor that assembles and remodels chromatin.
J. Biol. Chem. 275:14787–14790.
40. Li, Q., A. Imhof, T. N. Collingwood, F. D. Urnov, and A. P. Wolffe. 1999. p300
stimulates transcription instigated by ligand-bound thyroid hormone recep-
tor at a step subsequent to chromatin disruption. EMBO J. 18:5634–5652.
41. Martinez-Balbas, M. A., A. J. Bannister, K. Martin, P. Haus-Seuffert, M.
Meisterernst, and T. Kouzarides. 1998. The acetyltransferase activity of CBP
stimulates transcription. EMBO J. 17:2886–2893.
42. McBride, A. A., J. C. Byrne, and P. M. Howley. 1989. E2 polypeptides
encoded by the bovine papillomavirus type 1 form dimers through the com-
mon carboxyl-terminal domain: transactivation is mediated by the conserved
amino-terminal domain. Proc. Natl. Acad. Sci. USA 86:510–514.
43. Meyers, C., M. G. Frattini, and L. A. Laimins. 1994. Tissue culture tech-
niques for the study of human papillomaviruses in stratified epithelia. Aca-
demic Press, Inc., Orlando, Fla.
44. Miller Rank, N., and P. F. Lambert. 1995. Bovine papillomavirus type 1 E2
transcriptional regulators directly bind two cellular transcription factors,
TFIID and TFIIB. J. Virol. 69:6323–6334.
45. Mink, S., B. Haenig, and K.-H. Klempnauer. 1997. Interaction and func-
tional collaboration of p300 and C/EBP?. Mol. Cell. Biol. 17:6609–6617.
46. Mohr, I. J., R. Clark, S. Sun, E. J. Androphy, P. MacPherson, and M. R.
Botchan. 1990. Targeting the E1 replication protein to the papillomavirus
origin of replication by complex formation with the E2 transactivator. Sci-
47. Mosammaparast, N., C. S. Ewart, and L. F. Pemberton. 2002. A role for
nucleosome assembly protein 1 in the nuclear transport of histones H2A and
H2B. EMBO J. 21:6527–6538.
48. Moshkin, Y. M., J. A. Armstrong, R. K. Maeda, J. W. Tamkun, C. P.
Verrijzer, J. A. Kennison, and F. Karch. 2002. Histone chaperone ASF1
cooperates with the Brahma chromatin-remodelling machinery. Genes Dev.
49. Mu ¨ller, A., A. Ritzkowsky, and G. Steger. 2002. Cooperative activation of
human papillomavirus type 8 gene expression by the E2 protein and the
cellular coactivator p300. J. Virol. 76:11042–11053.
50. Nakagawa, T., M. Bulger, M. Muramatsu, and T. Ito. 2001. Multistep chro-
matin assembly on supercoiled plasmid DNA by nucleosome assembly pro-
tein-1 and ATP-utilizing chromatin and remodeling factor. J. Biol. Chem.
51. Nakajiama, T., C. Uchida, S. F. Anderson, J. D. Parvin, and M. Montminy.
1997. Analysis of a c-AMP-responsive activator reveals a two component
mechanism for transcriptional induction via signal dependent factors. Genes
52. Neely, K. E., A. H. Hassan, C. E. Brown, H. LeAnn, and J. L. Workman.
2002. Transcription activator interactions with multiple SWI/SNF subunits.
Mol. Cell. Biol. 22:1615–1625.
53. Nie, Z., Y. Xue, D. Yang, S. Zhou, B. J. Deroo, T. K. Archer, and W. Wang.
2000. A specificity and targeting subunit of a human SWI/SNF family-related
chromatin-remodeling complex. Mol. Cell. Biol. 20:8879–8888.
54. Nigro, J. M., S. J. Baker, A. C. Preisinger, J. M. Jessup, R. Hostetter, K.
Cleary, S. H. Bigner, N. Davisdson, S. Baylin, P. Devilee, T. Glover, F. S.
Collins, A. Weston, R. Modali, C. C. Harris, and B. Vogelstein. 1989. Mu-
tations in the p53 gene occur in diverse human tumor types. Nature 342:
55. Ogryzko, V. V., R. L. Schiltz, V. Russanova, B. H. Howard, and Y. Nakatani.
1996. The transcriptional coactivators p300 and CBP are histone acetyltrans-
ferases. Cell 87:953–959.
56. Peng, Y.-C., D. E. Breiding, F. Sverdrup, J. Richard, and E. J. Androphy.
2000. AMF-1/Gps2 binds p300 and enhances its interaction with papilloma-
virus E2 proteins. J. Virol. 74:5872–5879.
57. Purdie, K. J., C. J. Sexton, C. M. Proby, M. T. Glover, A. T. Williams, J. N.
Stables, and I. M. Leigh. 1993. Malignant transformation of cutaneous
lesions in renal allograft patients: a role for human papillomavirus. Cancer
58. Shikama, N., H. M. Chan, M. Krstic-Demonacos, L. M. Smith, C.-W. Lee, W.
Cairns, and N. B. La Thangue. 2000. Functional interaction between nu-
cleosome assembly proteins and p300/CREB-binding protein family coacti-
vators. Mol. Cell. Biol. 20:8933–8943.
59. Slepak, T. I., K. A. Webster, J. Zang, H. Prentice, A. O?Dowd, M. N. Hicks,
and N. H. Bishopric. 2001. Control of cardiac-specific transcription by p300
through myocyte enhancer factor-2D. J. Biol. Chem. 276:7575–7585.
60. Spalholz, B. A., P. F. Lambert, C. L. Lee, and P. M. Howley. 1987. Bovine
papillomavirus transcriptional regulation: localization of the E2-responsive
elements of the long control region. J. Virol. 61:2128–2137.
61. Tanese, N. 1997. Small-scale density gradient sedimentation to separate and
analyze multiprotein complexes. Methods 12:224–234.
62. Tyler, J. 2002. Cooperation between histone chaperones and ATP-depen-
dent nucleosome remodeling machines. Eur. J. Biochem. 269:2268–2274.
63. Vignali, M., A. H. Hassan, K. E. Neely, and J. L. Workman. 2000. ATP-
dependent chromatin-remodeling complexes. Mol. Cell. Biol. 20:1899–1910.
64. Wallberg, A. E., K. E. Neely, A. H. Hassan, J.-A. Gustafsson, J. L. Workman,
and A. P. Wright. 2000. Recruitment of the SWI-SNF chromatin remodeling
complex as a mechanism of gene activation by the glucocorticoid receptor ?1
activation domain. Mol. Cell. Biol. 20:2004–2013.
65. Walter, P. P., T. A. Owen-Hughes, J. Cote, and J. L. Workman. 1995.
Stimulation of transcription factor binding and histone displacement by
nucleosome assembly protein 1 and nucleoplasmin requires disruption of the
histone octamer. Mol. Cell. Biol. 15:6178–6187.
66. Webster, N., J. R. Jin, S. Green, M. Hollis, and P. Chambon. 1988. The yeast
UASG is a transcriptional enhancer in human HeLa cells in the presence of
the GAL 4 trans-activator. Cell 52:169–178.
67. Wilson, R. B., A. A. Brenner, T. B. White, M. J. Engler, J. P. Gaughran, and
K. Tatchell. 1991. The Saccharomyces cerevisiae SRK1 gene, a suppressor of
bcy1 and ins1, may be involved in protein phosphatase function. Mol. Cell.
68. Xiao, J. H., I. Davidson, H. Matthes, J.-M. Garnier, and P. Chambon. 1991.
Cloning, expression, and transcriptional properties of the human enhancer
factor TEF-1. Cell 65:551–568.
69. Yang, Y.-C., H. Okayama, and P. M. Howley. 1985. Bovine papillomavirus
contains multiple transforming genes. Proc. Natl. Acad. Sci. USA 82:1030–
70. Yao, J.-M., D. E. Breiding, and E. J. Androphy. 1998. Functional interaction
of the bovine papillomavirus E2 transactivation domain with TFIIB. J. Virol.
2168 REHTANZ ET AL.MOL. CELL. BIOL.