From the Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea
Received for publication, November 22, 2002
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ABSTRACT |
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Latency-associated nuclear antigen (LANA) of
Kaposi's sarcoma-associated herpesvirus plays an important role in
maintenance of the viral genome during latent infection. LANA
additionally participates in the transcriptional regulation of several
viral and cellular promoters. When tethered to constitutively active promoters, the protein exhibits transcriptional repressor activity. In
this report, we further characterized cell type-, promoter-, and
domain-specific transcriptional repression by LANA. We additionally speculated on the mechanism underlying transcriptional repression by
the C terminus of the protein. Subnuclear localization patterns and
association with heterochromatin suggested a possible link between LANA
and heterochromatin protein 1, a representative
heterochromatin-associated protein. In vivo and in
vitro binding and immunofluorescence assays revealed that LANA
associates with heterochromatin protein 1 in an isotype-specific
manner. Furthermore, biochemical fractionation and transient
replication assays supported the possibility that this interaction
contributes to transcriptional repression, targeting to subnuclear
structures, and latent DNA replication activity of LANA.
The nonhistone chromosomal protein, heterochromatin protein 1 (HP1),1 is tightly associated
with heterochromatin. It was originally identified by expression
library screening with a monoclonal antibody against a fraction of
DNA-binding nuclear proteins of Drosophila melanogaster (1).
The chromosomal rearrangement of euchromatic genes adjacent to the
heterochromatic regions results in gene silencing, designated
"position effect variegation." Mutations in a Drosophila
suppressor of the variegation 2-5 gene encoding HP1 result in
dosage-dependent dominant suppression of position effect
variegation (2, 3). In addition, HP1 is an essential gene in
Drosophila (3). Its mutation affects the segregation of
chromosomes (4) and causes multiple telomeric fusions (5). HP1 homologs
have been identified in diverse organisms, from fission yeast to
mammals (for reviews see Refs. 6 and 7). Three isotypes of mammalian
HP1 protein, denoted HP1 Kaposi's sarcoma is the most common neoplasm in persons with acquired
immunodeficiency syndrome. Kaposi's sarcoma-associated herpesvirus
(KSHV) was originally identified from Kaposi's sarcoma tissues of
acquired immunodeficiency syndrome patients, using representational
difference analyses (30), and later shown to associate with several
lymphoproliferative diseases, including body cavity-based
lymphoma/primary effusion lymphoma and some cases of multicentric
Castleman's disease (31, 32). KSHV switches its infection cycle
between latent and lytic states. During latent infection, viral gene
expression is restricted to a small subset of the entire open reading
frame of the KSHV genome (33), and the circularized viral genome is
maintained as a multicopy episome (34, 35) by the latency-associated
nuclear antigen (LANA) encoded by open reading frame 73 (36, 37). LANA associates with the mitotic chromosome (38-40) and binds to
sites within the 801-bp terminal repeat (TR) sequences of the viral
genome (41-45), thereby allowing the maintenance of plasmids
containing this region in the long term selection of drug resistance of
stably transfected cells (38, 41). Recently, we and others (44, 45)
reported that LANA is required for the DNA replication of KSHV
TR-containing plasmids, based on data from a transient replication
assay using a methylation-sensitive restriction enzyme. Moreover, the
protein interacts with components of origin recognition complexes
(ORCs) (45), similar to Epstein-Barr virus nuclear antigen-1, a
functional analog of KSHV LANA in the latent replication of the viral
genome (46-48). These data suggest that LANA maintains the KSHV
genome, not only by tethering the viral episome to host chromosomes
during host cell mitosis but also by actively participating in the DNA replication of the viral genome, possibly through interactions with
cellular replication machinery, such as ORCs. In addition to
maintenance of the viral genome, LANA interacts with several cellular
transcription factors (49-53) and regulates their activities in
cellular and viral promoters (43, 50, 52, 54-58). LANA represses
transcription from KSHV TR by direct binding (43-45), and exerts
transcriptional repression activity when tethered to a heterologous
promoter via the GAL4 DNA-binding domain (DBD) (50, 59). We are
interested in determining the molecular mechanism underlying the
transcriptional repression activity of LANA. In this report, we
speculate on the functional association between LANA and HP1, and the
consequent effects on transcriptional activity, subnuclear structure
targeting, and latent replication activity.
Plasmids--
Plasmids pcDNA3 LANA, pFLAG-CMV2 LANA, pEBG
LANA, and their derivatives encoding deletion mutants of LANA were
described previously (45, 51, 53). Samples of cDNA corresponding to
specific regions of LANA were amplified from pcDNA3 LANA by PCR,
with appropriate sets of primers, and inserted into CMV G4 (45) to
express GAL4 DBD-fused proteins in mammalian cells. The pGEX2T hHP1 Cell Culture, Transfection, and Reporter Assay--
293T cells
were maintained and transfected as described previously (51). The human
cervical cancer cell line, C33A, was maintained in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum and
transfected using the calcium phosphate method. The quantity of total
DNA used in transfection was kept constant by including an appropriate
blank vector. The transient reporter assay was performed as described
in a previous report (51).
In Vivo and in Vitro Binding Assays--
293T cells in 100-mm
dishes were cotransfected with specific combinations of expression
vectors. After ~36 h of transfection, cells were harvested and
pellets stored at Immunofluorescence Microscopy--
293T cells grown on
coverslips were cotransfected with the specified combinations of
expression vectors. At ~24 h post-transfection, cells were either
fixed in methanol at Fractionation of Cellular Proteins--
Subcellular
fractionation was performed as described previously (63) with minor
modifications. 293T cells in 60-mm dishes were transfected with
expression vectors containing GAL4 DBD fusion proteins and harvested
~36 h after transfection. Cells were lysed in cytoskeleton buffer (10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 0.1 mM ATP, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, and 10 mM
NaF) containing 0.1% Triton X-100 at 4 °C for 15 min. After centrifugation at 4000 rpm for 3 min, soluble fractions extracted by
Triton X-100 were further clarified by centrifugation at 12000 rpm for
15 min. Triton-extracted nuclei were washed once in cytoskeleton buffer
containing 0.1% Triton X-100 and subjected to chromatin digestion in
the above buffer containing 1000 units/ml DNase I (Sigma) at room
temperature for 30 min. DNase I-extractable and -resistant proteins
were separated by low speed centrifugation. A schematic representation
of subcellular fractionation is depicted in Fig. 5A. GAL4
DBD fusion proteins were detected with anti-GAL4 DBD monoclonal
antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Transient Replication Assay--
The transient replication assay
was performed as described previously (45). C33A cells on 100-mm dishes
were cotransfected with 2 µg of p4TR-luc, 2 µg of pGL2-basic as an
internal control, and 8 µg of FLAG-CMV2 derivatives expressing
FLAG-tagged LANA mutants. At ~36 h post-transfection, cells were
trypsinized, collected by centrifugation, and diluted into fresh media.
Cells were harvested 96 h after transfection, and low molecular
weight DNA was extracted by the Hirt lysis method (64). Supernatants
were successively extracted with phenol/chloroform/isoamyl alcohol and
chloroform. Ethanol-precipitated DNA was dissolved in 50 µl of
distilled water containing RNase. DpnI and Alw44I
were used to digest unreplicated DNA and linearize pGL2-basic
derivatives, respectively. DpnI digestion was confirmed by
monitoring the complete digestion of the unreplicated internal control,
pGL2-basic. Southern blot hybridization and detection of plasmids
containing the luciferase gene were performed as described previously
(45).
Promoter- and Cell Type-specific Transcriptional Repression Domain
of KSHV LANA--
Earlier reports (50, 59) suggest that LANA functions
as a transcriptional repressor when tethered to constitutively active promoters via a heterologous DNA-binding domain. However, the domain of
LANA responsible for transcriptional repression remains to be
conclusively identified. Schwam et al. (59) demonstrated that both the N and C termini of LANA contain a transcriptional repression domain, in contrast to reports by Krithivas et
al. (50) that only the N terminus of LANA mediates transcriptional repression, possibly via interactions with the mSin3 corepressor complex. Because different cell lines and reporters were employed in
the two sets of experiments, it is possible that the cellular or
promoter context affected the transcriptional activity of LANA. Accordingly, we examined the transcriptional activity of full-length LANA and derivatives fused to GAL4 DBD in a transient reporter assay,
using two cell lines and four different reporters containing tandem
repeats of GAL4 DBD-binding sites. The reporters include pFR-luc,
pG4M2-luc, pGx5SV-luc, and pGal4TK-luc containing the basic promoter
element (TATA box), adenovirus major late promoter, SV40 promoter, and
thymidine kinase promoter downstream of the tandem GAL4 DBD-binding
sites, respectively. As shown in Fig. 1A, both full-length and
N-terminal LANA corresponding to amino acids 1-340 fused to GAL4 DBD
displayed comparable transcriptional repressor activity on all
reporters tested in transiently transfected 293T cells. However,
transcriptional repression by C-terminal LANA corresponding to amino
acids 950-1162 fused to GAL4 DBD was promoter-specific; pG4M2-luc was
slightly repressed, and pGX5SV-luc was not affected. LANA and deletion
mutants not fused to GAL4 DBD included in the same reporter assay did
not affect the transcriptional activity of reporters (data not shown).
Therefore, it seems unlikely that the observed promoter specificity is
because of the effect of C-terminal LANA itself on transcription from
each promoter independently of GAL4 DBD sites. Similar transient
reporter assays were performed in C33A cells (Fig. 1B).
Unexpectedly, the N terminus of LANA fused to GAL4 DBD did not display
transcriptional repression activity on any of the reporters tested. In
contrast, the C terminus of LANA fused to GAL4 DBD repressed
transcriptional activity with similar promoter specificity as that
observed in 293T cells. To determine the transcriptional repression
domain within the C terminus of LANA, we constructed a series of
deletion mutants fused to GAL4 DBD (Fig. 1C). As shown in
Fig. 1D, the central region of the C terminus is responsible
for transcriptional repression in both cell lines. The inability of
LANA C In Vivo and in Vitro Binding of KSHV LANA to HP1--
KSHV LANA
associates preferentially with the border of heterochromatin in
interphase nuclei (65). Because HP1 is a representative nonhistone
chromosomal protein tightly associated with heterochromatin, we
speculate that the association of LANA with heterochromatin is mediated
by interactions with HP1. Additionally, the transcriptional repression
activity of GAL4 DBD-fused LANA may be partly explained by a gene
silencing mechanism involving the recruitment of HP1 and subsequent
heterochromatinization of the promoter. We initially examined physical
interactions between KSHV LANA and HP1 by using in vivo and
in vitro binding assays. 293T cells were cotransfected with
FLAG-tagged LANA and either a green fluorescent protein (GFP)-tagged human HP1
To date, three isoforms of HP1 have been identified in mammals (8-11).
Despite similar modular structures, the proteins display different
subnuclear localization and phosphorylation patterns (12). Recent
reports (66) indicate that HP1
To determine the HP1
The results collectively confirm that KSHV LANA interacts with HP1
proteins in an isotype-specific manner. Moreover, the region required
for transcriptional repression of the LANA C terminus is necessary for
binding to HP1 Subnuclear Localization of KSHV LANA and HP1--
KSHV LANA
displays characteristic speckled nuclear localization in latently
infected cells, and this punctate distribution is mediated by the C
terminus (37, 59, 65, 67). HP1 also displays a distinct cell type- and
isotype-specific subnuclear localization pattern (12, 61, 68). We
speculate that the subnuclear distribution of LANA, and possibly
association with heterochromatin, is mediated by interactions with HP1.
Immunofluorescence assays were employed to determine whether the two
proteins colocalize in cotransfected cells. LANA and FLAG-tagged
expression vectors encoding the indicated HP1 isotypes were
cotransfected into 293T cells, and subnuclear localization was detected
using rabbit polyclonal anti-LANA serum (green) and mouse
anti-FLAG M2 monoclonal antibody (red), respectively. As
shown in Fig. 4, LANA and hHP1 Subcellular Fractionation of C-terminal Deletion Mutants of LANA
Fused to GAL4 DBD--
Because confocal microscopy data revealed that
LANA and HP1 colocalize into specific subnuclear structures with
characteristic speckled spots, we speculated that the transcriptional
repression domain of the LANA C terminus correlates not only with the
HP1 Transient Replication Assay Using KSHV oriP-containing Plasmid and
hHP1
Previously, we reported that p4TR-luc containing KSHV TR, but not
pGL2-basic, replicates only in the presence of LANA in transiently transfected 293T and BJAB cells (45). Similar results were obtained with the human cervical cancer cell line, C33A (data not shown). To
determine whether chromo domain or chromo shadow domain of hHP1 The transcriptional repression activity of LANA, including LANA
derivatives fused to GAL4 DBD and reporters with tandem repeats of GAL4
DBD-binding sites, was previously identified from transient reporter
assays (50, 59). LANA represses the transcriptional activity of KSHV TR
by direct binding (43-45). Transcriptional repression activity of
C-terminal LANA fused to GAL4 DBD was controversially observed,
although the C terminus alone was necessary and sufficient for
transcriptional repression of KSHV TR (43-45). We further
characterized the transcriptional activity of LANA derivatives in the
GAL4 system. A transient reporter assay using two cell lines and four
different reporters revealed cell type- and promoter-specific repressor activity of LANA derivatives fused to GAL4 DBD (Fig. 1). In view of
earlier reports that the N terminus of LANA interacts with and
represses the mSin3 corepressor complex (50), we were interested in
determining the mechanism of transcriptional repression by C-terminal
LANA.
Association of LANA with heterochromatin (65) and localization of the
C-terminal region to subnuclear structures (59) suggested a possible
link between LANA and HP1, a representative heterochromatin-associated
protein. In vivo and in vitro binding assays
revealed isotype-specific interactions between the two proteins (Figs.
2 and 3). Immunofluorescence data using cotransfected 293T cells
further supported the binding assay results (Fig. 4). A targeting
signal to a DNase I-resistant structure was also biochemically identified within C-terminal LANA (Fig. 5). Data from a series of LANA
deletion mutants, in addition to our recent finding that C-terminal
LANA interacts with ORCs (45), are summarized in Table
I. The HP1- and ORC-binding region of
LANA was determined from experiments with LANA-(
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, HP1
, and HP1
, have been reported
(8-11). All isotypes contain common structural motifs, specifically an
N-terminal chromo domain and C-terminal chromo shadow domain connected
by a hinge region. However, their subnuclear localization and
phosphorylation states during the cell cycle are distinct (12, 13),
suggesting participation in different chromosomal events. Analysis of a
conserved sequence motif in Drosophila Polycomb protein and
HP1 led to the identification of a chromo domain termed for chromosome
organization modifier (14), and a second chromo domain-like motif in
HP1, known as chromo shadow domain (15) that mediates most
protein-protein interactions, including self-association (6, 7, 10,
16-18). The chromo domain is present in diverse proteins that are
potentially involved in chromosomal organization (18-20). Recent
reports (21, 22) suggest that the chromo domain of HP1 is responsible
for the recognition of methylated lysine 9 on histone H3. These
results, in addition to the finding that HP1 physically interacts with suv39h1, a histone methyltransferase responsible for the methylation of
lysine 9 on histone H3 (23, 24), suggests a self-propagating mechanism
of heterochromatin. Interestingly, suv39h1-HP1 complexes are involved
not only in heterochromatic gene silencing but also in the
transcriptional regulation of the euchromatic cyclin E promoter (25,
26), which was shown previously to be regulated partly by the
recruitment of histone deacetylase via a retinoblastoma protein (27).
These data collectively implicate an additional molecular mechanism
supporting the histone code hypothesis that differential and
combinatorial post-translational modifications of the N-terminal tail
of histone, including acetylation, methylation, and phosphorylation,
epigenetically mark the chromosomal state as open or closed for
transcriptional activity (28). However, the enzymes responsible for
histone demethylation or removal of methylated histone tail remain to
be identified (29).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
plasmid (60) was a gift from Dr. A. Dejean (Institut Pasteur, France). pGEX2T mHP1
, mHP1
, and mHP1
(61) were generously supplied by
Dr. R. Losson (Institut de Genetique et de Biologie Moleculaire et
Cellulaire, France). A BamHI/EcoRI fragment of
pGEX2T hHP1
was inserted into the corresponding restriction sites of
pcDNA3 (Invitrogen) and the BglII/EcoRI sites
of pEGFP C1 (Clontech, Palo Alto, CA). A
BamHI/NotI fragment of pcDNA3 hHP1
was
inserted in the corresponding sites of pEBG to express glutathione
S-transferase (GST)-fused hHP1
in mammalian cells.
cDNAs corresponding to hHP1
, mHP1
, and mHP1
were amplified
by PCR with appropriate sets of primers and inserted into the
EcoRI/XhoI site of pME18S (62) to express
FLAG-tagged proteins in mammalian cells. Deletion mutants of hHP1
were constructed using similar procedures. pFR-luc was purchased from
Stratagene (San Diego, CA). A BamHI fragment of pFR-luc
containing five tandem GAL4-DBD-binding sites was inserted into the
BglII site of the pGL3 promoter (Promega, Madison, WI), and
the resulting construct was designated pGx5SV-luc. pG4M2-luc was a gift
from Dr. H. G. Stunnenberg (University of Nijmegen, The
Netherlands). The p4TR-luc construct was described earlier (45).
70 °C before use. Cells were lysed in 500 µl
of ice-cold phosphate-buffered saline containing 0.5% Nonidet P-40 and
1 mM phenylmethylsulfonyl fluoride, with brief sonication.
Cell debris was removed by centrifugation. For coimmunoprecipitation,
the supernatant was incubated with anti-FLAG M2 monoclonal antibody
(Sigma) at 4 °C for 1 h. Following the addition of protein
G-Sepharose (Amersham Biosciences), the supernatant was further
incubated at 4 °C for 3 h. For the GST pull-down assay, the
supernatant was incubated with glutathione-Sepharose 4B (Amersham
Biosciences) at 4 °C for 3 h. Beads were washed three times in
1 ml of lysis buffer, and bound proteins were eluted with SDS gel
loading buffer. Eluted proteins were separated by SDS-PAGE, transferred
to a nitrocellulose membrane, immunoblotted with the indicated
antibodies, and detected using ECLTM (Amersham
Biosciences). The in vitro binding assay was performed as
described previously (45).
20 °C for 10 min or 3.7% formaldehyde at
room temperature for 30 min and permeabilized in phosphate-buffered
saline containing 0.2% Triton X-100 at 4 °C for 25 min. LANA was
detected with rabbit polyclonal anti-LANA serum (a gift from Dr. J. Jung, Harvard Medical School) and fluorescein isothiocyanate-conjugated
goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West
Grove, PA). FLAG-tagged proteins were observed using an anti-FLAG M2
monoclonal antibody (Sigma) and rhodamine-conjugated goat anti-mouse
secondary antibody (Jackson ImmunoResearch Laboratories). Coverslips
were mounted with Vectashield® (Vector Laboratories, Inc.,
Burlingame, CA) and examined by confocal laser scanning microscopy
(Pascal, Carl Zeiss, Jena, Germany).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
161 and LANA C1107 fused to GAL4 DBD to repress transcription
was not due to lower expression levels in transfected cells, as
verified by Western blotting with anti-GAL4 DBD antibody (data not
shown). The results collectively suggest that both the N and C termini
of LANA display cell type- and promoter-specific transcriptional
repression activity.
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Fig. 1.
Transcriptional repression of LANA
derivatives fused to GAL4 DBD in a transient reporter assay.
A, 293T cells in 60-mm dishes were cotransfected with 1 µg
of reporter plasmid, 0.5 µg of CMV G4 derivatives, and 3 µg of
pcDNA3. B, C33A cells in each well of the 6-well plates
were cotransfected with 1 µg of reporter plasmid and 2 µg of CMV G4
derivatives. C, schematic diagram of C-terminal LANA
deletion mutants fused to GAL4 DBD used in the transient reporter assay
for mapping the transcriptional repression domain. P-rich,
proline-rich region; DED, aspartate/glutamate-rich repeat
region; Q-rich, glutamine-rich region; ZIP,
putative leucine-zipper domain. D, 293T cells in 60-mm
dishes were cotransfected with 1 µg of indicated reporter plasmid and
0.1 µg of CMV G4 derivatives. C33A cells were cotransfected as in
B. Transfection efficiency was routinely monitored by
cotransfected -galactosidase activity, and activation folds were
calculated relative to luciferase activity in the presence of blank
vector, which was set as 100%. Data presented are an average of three
independent experiments, and standard deviations are indicated with
error bars.
(hHP1
) expression vector or an appropriate blank
vector. After 36 h of transfection, cell extracts were prepared
and immunoprecipitated with an anti-FLAG antibody. GFP-tagged hHP1
,
but not GFP alone, precipitated only in the presence of FLAG-tagged
LANA, confirming physical interactions between the two proteins in
mammalian cells (Fig. 2A). To
map the LANA-binding region within hHP1
, mammalian expression
vectors encoding wild-type hHP1
or deletion mutants fused to GST
were constructed (Fig. 2B, top) and cotransfected with the FLAG-tagged LANA expression vector into 293T cells. Proteins precipitated from cell extracts with glutathione-Sepharose 4B were
immunoblotted with anti-FLAG or anti-GST antibodies (Fig. 2B, bottom). As depicted in Fig. 2B,
FLAG-tagged LANA was pulled down by GST-hHP1
and GST-hHP1
CD, but
not GST alone or GST-hHP1
CSD, suggesting that LANA binds to the N
terminus of hHP1
containing a chromo domain.
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Fig. 2.
In vivo and in vitro
association of KSHV LANA with HP1. A, in
vivo coimmunoprecipitation of FLAG-tagged LANA and GFP-tagged
hHP1 . FLAG-tagged LANA and GFP or GFP-tagged hHP1
expression
vectors were cotransfected into 293T cells. Following cell lysis,
proteins immunoprecipitated (IP) with anti-FLAG antibody
were immunoblotted with rabbit polyclonal anti-GFP serum
(top). Total lysates from transfected cells were also
immunoblotted with rabbit polyclonal anti-GFP serum (middle)
and anti-FLAG antibody (bottom). B, in
vivo GST pull-down assay between FLAG-tagged LANA and GST-hHP1
derivatives. FLAG-tagged LANA expression vector was transfected into
293T cells, along with expression vectors of GST or GST- hHP1
derivatives (top). After cell lysis, proteins pulled down by
glutathione-Sepharose beads were immunoblotted with anti-FLAG antibody
(top) and anti-GST antibody (middle). Total
lysates from transfected cells were also immunoblotted with anti-FLAG
antibody (bottom). CD, chromo domain;
CSD, chromo shadow domain. C, in vivo
GST pull-down assay between GST-LANA and FLAG-tagged HP1 isotypes.
Expression vectors of GST or GST-LANA and indicated HP1 isotypes were
cotransfected into 293T cells. Proteins precipitated with
glutathione-Sepharose beads were immunoblotted with anti-FLAG
(top) and anti-GST antibodies (bottom). Total
lysates from transfected cells were additionally immunoblotted with
anti-FLAG antibody (middle). D, Coomassie Blue
staining of indicated GST fusion proteins at the top, which were
bacterially expressed, purified, and used for in vitro
binding. Protein size markers are shown on the left of the
Coomassie stain panel, and GST fusion proteins with appropriate sizes
are indicated by asterisks. E, binding assay
between in vitro translated LANA and GST-HP1 isotypes.
In vitro translated LANA in the presence of
[35S]methionine was incubated with glutathione-Sepharose
4B precoated with indicated GST fusion proteins. Where specified, 1 mM ATP was included in the binding buffer. Bound proteins
were subjected to autoradiography. Input, 10% in
vitro translated protein.
and HP1
(but not HP1
)
specifically interact with human TAFII130, suggesting that
each HP1 isotype participates in distinct chromosomal and transcriptional events. We determined the isotype specificity of
interactions between KSHV LANA and HP1. The GST or GST-fused LANA
expression vector was cotransfected with the indicated FLAG-tagged HP1
expression vector in 293T cells, and a GST pull-down assay was
similarly performed using extracts of transfected cells. FLAG-tagged HP1
, but not mouse HP1
, was efficiently precipitated with
GST-LANA (Fig. 2C). FLAG-tagged mouse HP1
also weakly
bound GST-LANA. To further confirm this isotype-specific interaction
between KSHV LANA and HP1, isoforms of GST-fused HP1 were bacterially
purified, and equivalent amounts were used in in vitro GST
pull-down assays (Fig. 2D). In vitro translated
LANA in the presence of [35S]methionine was included in
the binding reaction with indicated GST fusion proteins. After
precipitation using glutathione-Sepharose 4B, bound proteins were
subjected to SDS-PAGE and autoradiography. Our data reveal that only
HP1
interacted with LANA. Furthermore, this interaction was enhanced
in the presence of 1 mM ATP in the binding buffer (Fig.
2E).
-interacting domain of LANA, a series of
deletion mutants was constructed, translated in vitro, and
employed in similar in vitro binding assays. The
HP1
-interacting domain of LANA was mapped to the C-terminal region
(Fig. 3). By using small truncation and
internal deletion mutants of the C terminus of LANA, we demonstrated
that the region encompassing amino acids 1047-1062 of LANA is
responsible for interactions with HP1
. Interestingly, the region
required for HP1
binding coincided with the transcriptional repression domain of the LANA C terminus, with the exception that amino
acids 1063-1162 did not interact with HP1
but displayed moderate
transcriptional repressor activity.
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Fig. 3.
In vitro binding assay between
GST-hHP1 and LANA deletion mutants. A
schematic diagram of LANA deletion mutants used in the in
vitro binding assay is depicted in the left panel. Each
LANA derivative was translated in vitro in the presence of
[35S]methionine, and a pull-down assay was performed as
in Fig. 2E. Input, 10% in
vitro-translated protein. P-rich, proline-rich region;
DED, aspartate/glutamate-rich repeat region;
Q-rich, glutamine-rich region; ZIP, putative
leucine-zipper domain.
, supporting the hypothesis that transcriptional
repression activity of the C terminus of LANA may be at least partly
mediated by recruiting HP1 to the promoter.
clearly colocalized into relatively large subnuclear dots, although their punctate patterns did not accurately coincide. In contrast, we observed
marginal or no significant colocalization of LANA with mHP1
and
mHP1
. These results are consistent with the in vivo and
in vitro binding data and further support the hypothesis
that the punctate subnuclear distribution of LANA may be partly
mediated by associations with HP1.
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Fig. 4.
Colocalization of LANA and HP1
isotypes in 293T cells. Cells grown on coverslips were
cotransfected with 1 µg of pcDNA3 LANA and 1 µg of ME18S-F HP1
derivatives expressing the indicated FLAG-tagged HP1 isotypes
(left column). At ~24 h post-transfection, cells were
fixed and immunostained. LANA was detected using a fluorescein
isothiocyanate-conjugated secondary antibody against rabbit polyclonal
anti-LANA serum (green), and FLAG-tagged HP1 isotypes were
identified using a rhodamine-conjugated secondary antibody against
anti-FLAG monoclonal antibody (red). Two representative
confocal microscopic images per HP1 isotype are shown.
-binding region but also with the region responsible for
targeting to subnuclear structures. To verify this theory, we
biochemically fractionated total proteins from transfected 293T cells
expressing GAL4 DBD fusion proteins, which were used in the transient
reporter assay to identify the transcriptional repression domain within
the C-terminal LANA (Fig. 1D). A schematic procedure for
subcellular fractionation is depicted in Fig.
5A. As shown in Fig.
5B, GAL4 DBD alone displayed little 0.1% Triton
X-100-extractable fraction, but the majority was equivalently observed
in both DNase I-extractable and DNase I-resistant fractions.
Fractionation patterns of a series of LANA C-terminal deletion mutants
fused to GAL4 DBD revealed that the targeting signal for association
with DNase I-resistant structures resides in amino acids 1026-1062 of
LANA, which includes the HP1
-binding region. Similar results were
obtained using 0.5% Triton X-100 instead of 0.1% (data not shown).
This finding supports the possibility that targeting the LANA C
terminus to subnuclear structures is mediated by associations
with HP1 protein.
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Fig. 5.
Subcellular fractionation of C-terminal LANA
deletion mutants fused to GAL4 DBD. A, scheme of
biochemical procedures for fractionating cellular proteins.
B, schematic diagram of C-terminal LANA deletion mutants
fused to GAL4 DBD is depicted in the left panel. 293T cells
in 60-mm dishes were transfected with 4 µg of indicated CMV G4
derivatives and harvested 36 h after transfection. Fractionation
of cellular proteins from transfected cells was performed as shown in
A, and Western blots of each fraction with anti-GAL4 DBD
monoclonal antibody are shown on the right. Fraction
S, Triton X-100-extractable; Fraction C, DNase
I-extractable; Fraction M, DNase I-resistant.
-LANA Hybrid Proteins--
Previously, we characterized the
requirement of cis- and trans-elements of KSHV latent replication using
a transient replication assay with a methylation-sensitive restriction
enzyme (45). To investigate the functional role of interactions between
LANA and HP1 in KSHV latent replication, we performed similar transient replication assays. Transient overexpression of hHP1
had no
significant effect on the replication of p4TR-luc containing 4 tandem
KSHV terminal repeats (TRs) by LANA in conditions where the
overexpression of other trans-elements affected transient replication
by LANA (data not shown). Because all internal deletion mutants of the C terminus of LANA failed to bind KSHV TR in an electrophoretic mobility shift assay, we did not include HP1-binding defective mutants
of LANA in the transient replication assay, as in the case of origin
recognition complexes binding defective mutants to check their ability
in KSHV latent replication (45). Therefore, we constructed two-hybrid
proteins in which the N or C terminus of hHP1
containing a chromo or
chromo shadow domain, respectively, was fused to the C terminus of LANA
that binds KSHV TRs and ORCs/HP1 to mediate the replication of a
TR-containing plasmid (Fig.
6A), and we examined their
contribution to KSHV latent replication. Appropriate sizes and
comparable levels of FLAG-tagged CD-LANA C and CSD-LANA C were verified
by Western blotting of total cell extracts from transfected 293T cells
with an anti-FLAG M2 antibody (data not shown). Subcellular
localization was determined by immunofluorescence assays using the same
antibody. LANA-(
91-949), which mediates KSHV TR replication
comparable with full-length LANA in transfected 293T cells (45),
localized to the nucleus with a speckled pattern (Fig. 6B).
Similar to full-length HP1, CD-LANA C also displayed more punctate
subnuclear localization than LANA C alone. However, CSD-LANA C was more
heterogeneous in the population of transfected cells and localized to
both the nucleus and cytoplasm.
View larger version (38K):
[in a new window]
Fig. 6.
Transient replication assay of KSHV
TR-containing plasmids, p4TR-luc, using
hHP1 -LANA C hybrid constructs in C33A
cells. A, schematic diagram of LANA deletion mutants
and hHP1
-LANA C hybrids. CD, chromo domain;
CSD, chromo shadow domain. B, subcellular
localization of LANA deletion mutants and hHP1
-LANA C hybrids in
293T cells. Cells grown on coverslips were transfected with 1 µg of
FLAG-CMV2 derivatives expressing FLAG-tagged LANA derivatives and
hHP1
-LANA C hybrids. At ~24 h post-transfection, cells were fixed
and immunostained. FLAG-tagged proteins were detected using an
anti-FLAG monoclonal antibody and a rhodamine-conjugated secondary
antibody. C, transient replication assay in C33A cells.
Cells in 100-mm dishes were cotransfected with 2 µg of p4TR-luc, 2 µg of pGL2-basic, and 8 µg of the indicated FLAG-CMV2 derivatives.
Cells were split at ~36 h post-transfection and harvested at ~96 h.
Hirt-extracted DNA from transfected C33A cells was digested with
Alw44I/DpnI (right), and 20% samples
with Alw44I alone as input (left). Digested DNA
was separated by 0.8% agarose gel electrophoresis and analyzed by
Southern blot hybridization with a probe specific for the luciferase
gene.
fused to the C terminus of LANA supports the replication of
TR-containing plasmids, expression vectors of these hybrid proteins
along with p4TR-luc and pGL2-basic were cotransfected into C33A cells,
and a transient replication assay was performed (Fig. 6C).
Unexpectedly, TR-containing plasmids weakly replicated in the presence
of LANA-(
91-949), compared with full-length LANA, in
contrast to data obtained from 293T cells. However, LANA C alone did
not support p4TR-luc replication, similar to the behavior observed in
293T cells. Interestingly, CD-LANA C showed the replication activity of
TR-containing plasmids (albeit very weakly), compared with CSD-LANA C
and LANA C alone. The internal control, pGL2-basic, was employed to
confirm complete digestion of unreplicated DNA by DpnI. The
expression level of the trans-acting element was largely excessive,
which excluded the possibility that distinct replication activities of
each mutant were due to different expression levels of them. The
results imply that the N terminus of hHP1
containing a chromo domain
functionally replaces the N terminus of LANA containing a
chromosome-binding domain, at least in part, and support the
possibility that associations between LANA and HP1 contribute to the
replication activity of LANA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1002-1062).
The mutant did not bind HP1 or associate with DNase I-resistant
structures but interacted with ORCs and possessed moderate
transcriptional repression activity, supporting the hypothesis that
transcriptional repression and targeting to subnuclear structures is a
result of associations with HP1 protein. Because ORCs showed the
transcriptional repression activity of reporter when tethered to a
heterologous promoter via GAL4 DBD (data not shown), the residual
transcriptional repression activity of LANA-(
1002-1062) may
be explained by interactions with ORCs.
Summary of transcriptional repression, targeting to DNase I-resistant
structures, and HPI/ORC binding activities of LANA deletion mutants
In view of the finding that LANA binds KSHV TR and exerts
transcriptional repression and replication activity, the association between LANA and HP1 presents an attractive model. LANA may repress transcription from KSHV TR, at least in part, by recruiting HP1 proteins so that the gene-silencing mechanism works in heterochromatic chromosomes. To investigate this possibility, we examined the effect of
overexpressed HP1 on the transcriptional activity of p4TR-luc in the
presence or absence of LANA. In our transient reporter assay,
overexpressed HP1
did not have a significant effect on
transcriptional repression of p4TR-luc by LANA, compared with basal
transcriptional activity of KSHV TR in the absence of LANA (data not
shown). A transient reporter assay using the GAL4 system revealed
similar data. It is possible that interactions with other factors, such
as ORCs, play a dominant role in the transcriptional repression
activity of LANA. Alternatively, endogenous HP1 may be abundant in
transfected cells relative to exogenously expressed protein, or only a
small fraction of HP1 may participate in transcriptional repression by
LANA. A transient reporter assay involving this type of overexpression
system did not give conclusive results on the effects of HP1 on
transcriptional repression by LANA at the physiological level, as
discussed by Vassallo and Tanese (66).
Although we could not experimentally prove the heterochromatinization
of KSHV TR by LANA via possible interactions with HP1, this model
benefits viral infection in several ways. KSHV contains 30-40 copies
of the TR sequence within its genome. This tandem repeat sequence is
more prone to gradual loss during DNA replication or recombination
events. Therefore, KSHV must have a protection mechanism to prevent the
loss of TR, which acts as a latent origin of replication required for
maintaining the genome. Heterochromatinization of TR may be one
solution. In addition, targeting of the viral genome to subnuclear
structures, coupling of viral replication to that of heterochromatin by
associations between LANA and HP1, or the centromere function of HP1
(69) may partly contribute to viral genome replication, which was
preliminarily verified by the transient replication assay using
HP1-LANA C hybrids in this report (Fig. 6). With respect to
transcriptional regulation, heterochromatinzation of TR may affect the
activity of other regions within the viral genome by intramolecular
contacts. This type of trans effect (70) may globally restrict
transcriptionally active viral promoters during the latent infection
cycle or in the absence of appropriate signals. More detailed studies
are required for validating these models and possibilities.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Research Laboratory Program of the Korea Institute of Science and Technology Evaluation and Planning, the Korea Science and Engineering Foundation through the Protein Network Research Center at Yonsei University, and the BK21 Program of the Ministry of Education, Korea.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 82-42-869-2630;
Fax: 82-42-869-5630; E-mail: jchoe@mail.kaist.ac.kr.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M211912200
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ABBREVIATIONS |
---|
The abbreviations used are: HP1, heterochromatin protein 1; KSHV, Kaposi's sarcoma-associated herpesvirus; LANA, latency-associated nuclear antigen; TR, terminal repeat; ORC, origin recognition complex; DBD, DNA-binding domain; GST, glutathione S-transferase; GFP, green fluorescent protein; PIPES, 1,4-piperazinediethanesulfonic acid; CMV, cytomegalovirus.
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