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
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-
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