From the Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
Received for publication, November 27, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Krüppel-associated box-containing
zinc finger proteins (KRAB-ZFPs) repress transcription via functional
interaction with the corepressor KRAB-associated protein-1 (KAP-1).
KAP-1 directly interacts with heterochromatin protein 1 (HP1), a
dose-dependent regulator of heterochromatin-mediated
silencing. Here we show that two KRAB-ZFPs that we previously
identified, KRAZ1 and KRAZ2, are targeted to foci of centromeric
heterochromatin containing HP1 The Krüppel-associated box
(KRAB)1 was originally
identified as an evolutionarily conserved motif that is presumably
contained in about one-third of the several hundred Krüppel-like
zinc finger proteins (ZFPs) (1). It is found almost exclusively in the N terminus of the Krüppel-like ZFPs that contain
C2H2-type zinc finger domains in their C
terminus and is subdivided into KRAB-A and -B domains (1). Several KRAB
domains have been shown to repress transcription when heterologously
tethered to the promoter (2-4). The KRAB-A domain present in all KRAB
motifs is responsible for transcriptional silencing (2-4), and
substitutions of conserved residues in this domain abrogate the
repressor function (2, 3).
KRAB-associated protein-1 (KAP-1) (5), also identified as TIF1 A clue toward elucidation of the mechanisms of KRAB/KAP-1-mediated
silencing was provided by the finding that KAP-1 interacts with
heterochromatin protein 1 (HP1) via its central repressor domain (14,
15). HP1 constitutes a subfamily of the chromatin organization modifier
(chromo) superfamily and is a dose-dependent regulator of
heterochromatin-mediated silencing known as position effect
variegation, suggesting that HP1 is a functional and structural component of heterochromatin (for review see Ref. 16). Three HP1
members, HP1 In this report, we demonstrate that two KRAB-ZFPs, KRAZ1 and KRAZ2,
which we identified previously (9), colocalize with KAP-1 in foci of
centromeric heterochromatin containing HP1 Plasmids--
Recombinant DNA techniques were performed
according to standard protocols (22). The pGL3-G5SV and expression
plasmids for GAL4-, Myc-, and VP16-fused KAP-1 deletions have been
described previously (9). To express full-length KRAZ1 and KRAZ2,
cDNAs encoding amino acids 42-652 of KRAZ1 and 1-606 of KRAZ2
(GenBankTM accession numbers AB024004 for KRAZ1 and
AB024005 for KRAZ2) were inserted into the pEF-BOS-GAL4 or
pEF-BOS-6xMyc-tagged vectors (9). To construct KRAB mutants of KRAZ1/2,
the KRAB-containing regions of full-length KRAZ1/2 were replaced by the
corresponding fragments containing the amino acid substitutions in
KRAB-A domains (aspartic acid-valine to alanine-alanine; DV to
AA) (9). A firefly luciferase reporter plasmid, pGL3-G5T7, was
constructed by subcloning the fragment carrying five GAL4-binding sites
excised from pGL3-G5SV (9) and the fragment carrying seven repeats of
tetracycline operator and the cytomegalovirus promoter excised from
pUHD10-3 (23) into pGL3-Basic vector (Promega). To express tetracycline-controlled transactivator (tTA), the fragment containing tTA excised from pRetro-Off (CLONTECH) was inserted
into pEF-BOS bsr (9).
Transfection and Luciferase Assay--
Transient transfection of
NIH3T3 cells using LipofectAMINETM (Life Technologies,
Inc.) and luciferase assay using the Dual-Luciferase reporter assay
system (Promega) were performed as described (9). Firefly luciferase
activity from pGL3-G5SV or pGL3-G5T7 was normalized for transfection
efficiency with sea-pansy luciferase activity from the internal control
plasmid, pRL-SV40 (Promega). -Fold repression was calculated by
comparing normalized luciferase activity of each GAL4 fusion with that
of GAL4DBD alone, which represents the basal activity of the reporter
used. Each transfection experiment was repeated at least three times in
duplicate, and the results are presented as averages with standard
deviations of duplicates in representative experiments. For TSA
treatment, 16 h after transfection, cells were treated with 100 ng/ml TSA (Wako) or an equal volume of Me2SO as a
vehicle control and incubated for an additional 32 h.
GST Pull-down Assay--
The expression plasmids for GST-HP1 Antibodies--
The primary antibodies (Abs) used were as
follows: (i) rabbit polyclonal Ab against human KAP-1 (nucleotides
1882-2673 that are 96% identical to mouse KAP-1) (25), which was
kindly provided by Dr. S. C. Lee (National Taiwan University,
Taiwan, Republic of China); (ii) mouse anti-mouse HP1 Immunoprecipitation and Western Blotting--
These were
performed as described (9). Briefly, GAL4DBD, GAL4-KRAZ1, or GAL4-KRAZ1
mutant were transiently transfected into NIH3T3 cells, together with
VP16-KAP-1 N835, and immunoprecipitated with rabbit polyclonal
anti-GAL4 Ab (Upstate Biotechnology). Immunoprecipitates were analyzed
by Western blotting using rabbit anti-VP16 Ab.
Immunofluorescent Microscopy--
For conventional and confocal
microscopy analysis, NIH3T3 cells were plated 24 h before
transfection on eight chamber slides (Nunc, Inc.) precoated with 0.1%
gelatin and transfected using LipofectAMINETM as described
(9). Cells were fixed 24-48 h after transfection with 3%
paraformaldehyde in phosphate-buffered saline, followed by
permeabilization with 1% Triton X-100. Cells were then incubated with
primary antibodies for 60 min. After washing three times with 0.2%
Tween 20 in phosphate-buffered saline, cells were incubated with
secondary antibodies for 60 min. DNA was labeled with 0.1 µg/ml
4',6'-diamidino-2-phenylindole (DAPI; Sigma) for 5 min, and the slides
were mounted with PermaFluorTM (Shandon/Lipshaw). All
staining experiments were repeated more than twice, and at least 100 transfected cells were examined in each experiment with conventional
microscopy (DIAPHOT-300; Nikon) or confocal laser microscopy (MRC-1024;
Bio-Rad). The fluorescence signals from the two fluorochromes were
recorded simultaneously in one scan and saved separately on two
channels to be processed independently with the Adobe Photoshop 5.0 software (Adobe Systems Inc.).
KRAZ1 and KRAZ2 Colocalize with KAP-1 and HP1
KRAZ1 functionally interacts with KAP-1 through its KRAB domain (9),
and KAP-1 was shown to directly bind to all three HP1 proteins, HP1 Centromeric Targeting of KRAZ1 Correlates with Silencing--
To
determine whether centromeric localization of KRAZ1 is associated with
KRAZ1-KAP-1 interaction and transcriptional silencing, we first
assessed the silencing activity of full-length KRAZ1 fused with GAL4DBD
(Fig. 2B). KRAZ1 significantly repressed transcription in a
dose-dependent manner, whereas a mutation introduced in
KRAB severely compromised the silencing activity. Immunoprecipitation analysis revealed that full-length KRAZ1 but not its KRAB mutant bound
to KAP-1 (Fig. 2C). These results indicate that the
silencing activity of KRAZ1 is correlated with the ability to interact
with KAP-1. Next, we analyzed localization of the Myc-tagged KRAB
mutant of full-length KRAZ1. Although in multiple experiments we
examined hundreds of transfected cells that expressed the mutant at
various levels, no centromeric localization was ever detected, only a diffuse pattern of staining (Fig. 2A, j-o). This
is not attributed to instability of the KRAZ1 mutant protein, because
comparable expression levels of expected-sized products were detected
for both wild-type and mutant KRAZ1 by Western blotting (data not shown). Thus these data demonstrate that centromeric targeting of KRAZ1
is a specific event that strictly depends on the interaction with KAP-1
via KRAB and that the silencing activity of KRAZ1 is correlated with
the ability to colocalize with KAP-1 to centromeric foci.
Association of KAP-1 Centromeric Localization with Silencing
Activity--
Based on the finding that KAP-1 directly interacts with
HP1 proteins through its core repressor domain (6, 9, 14, 15), we next
addressed the possibility that centromeric localization of KAP-1 in
turn depends on the interaction with HP1 and correlates with its
silencing activity. To this end, we first assessed interaction of KAP-1
deletions (9) with HP1 proteins in vitro. GST pull-down assay revealed that KAP-1 deletions N562, N835, 396C, and 396-711 bound to both GST-HP1
We next investigated subnuclear localization of the KAP-1
deletions tagged with Myc epitope and containing the nuclear
localization sequence of the SV40 large T antigen (9) (Fig. 3,
A and B). KAP-1 N562 and N835 that contain the
RBCC domain and HP1BD colocalized with HP1
To determine whether KAP-1 centromeric localization correlates
with silencing activity, we next re-evaluated the silencing activities
of GAL4-KAP-1 deletions that we previously reported (9) (Fig.
3A). Because GAL4DBD can replace RBCC for centromeric targeting of KAP-1 as shown above, all the KAP-1 constructs containing HP1BD could thus localize to centromeric foci, and all of them except
396-711 showed high silencing activities (N562, N835, and 396C). The
396-711 region contains the core repressor domain that is essential
but not sufficient for full silencing activity; either the N- or
C-terminal portion is also required for full repression (9).
Nonetheless, KAP-1 constructs lacking HP1BD could not localize to
centromeric foci and exhibited weak or almost no silencing activity
(N487 and 697C). This is not attributed to instability of these
deletions, as an expected-sized product was a major fraction detected
for each KAP-1 deletion by Western blotting (data not shown). We thus
conclude that the centromeric targeting ability of KAP-1 is associated
with its silencing activity.
Centromeric Localization of KRAZ1 and KAP-1 Is Functionally
Involved in Silencing--
To determine whether correlation of the
centromeric targeting potential of KRAZ1, as well as KAP-1, with their
silencing activities has functional significance, we used the mammalian
two-hybrid assay by which we previously showed that silencing mediated
by the KRAB-containing region of KRAZ1 fused to GAL4DBD could be converted to strong activation of transcription upon coexpression of
KAP-1 deletions fused to the VP16 transactivation domain (VP16AD) (9).
Like the KRAB-containing region, full-length KRAZ1 fused with GAL4DBD
repressed transcription in comparison with GAL4DBD alone in the
presence of VP16AD (Fig. 4A).
When GAL4-KRAZ1 was coexpressed with a VP16-KAP-1 N487 construct that
binds to KRAB of KRAZ1 but not HP1 (Fig. 3C), however,
transcription was strongly activated (Fig. 4A), indicating
that KAP-1 N487 functionally interacts with full-length KRAZ1. In
contrast, cotransfection with VP16-KAP-1 N562 that binds to both HP1
and KRAB of KRAZ1 (Fig. 3C) inhibited the transcriptional
activation to almost the basal level (Fig. 4A). This result
suggests that although KAP-1 N562 interacts with KRAZ1 in
vivo, the silencing activity of KAP-1, which is probably mediated
via HP1 interaction, dominates over the VP16AD transactivation effect.
We next examined localization of Myc-tagged full-length KRAZ1
coexpressed with VP16-KAP-1 deletions (Fig. 4B). When
coexpressed with KAP-1 N562 that inhibits transactivation by VP16AD,
KRAZ1 could colocalize with KAP-1 N562 and endogenous HP1 Silencing Activity and Centromeric Localization of KRAZ1/KAP-1
Depend on Histone Deacetylation--
The recent observation that TSA,
a specific inhibitor of histone deacetylases (HDACs), can relieve, at
least in part, the silencing activities of KAP-1 and HP1 proteins (15)
prompted us to test whether TSA can also relieve KRAZ1-caused silencing and centromeric localization of KRAZ1, as well as KAP-1. To test the
effect of TSA on silencing, we used a reporter plasmid carrying five
GAL4-binding sites and driven by the tetrocycline repressor and
VP16AD chimera (tTA) (23) (Fig.
5A). Without TSA treatment, GAL4 fusions with KRAZ1, KAP-1 N562, and N835 that can localize to
centromeric foci efficiently repressed transcription, whereas the KRAZ1
mutant that failed to localize in foci did not. TSA treatment, however,
could partially relieve the silencing activities of KRAZ1, as well as
KAP-1 N562 and N835, indicating that KRAZ1/KAP-1-mediated silencing is,
at least in part, dependent on HDAC activity.
We next investigated whether TSA influences localization of KRAZ1 and
KAP-1. Surprisingly, culture with TSA caused redistribution of
endogenous KAP-1 from centromeric foci throughout the nucleus in all
cells (Fig. 5B, a-b). Furthermore, TSA also
caused redistribution of Myc-tagged KRAZ1, KAP-1 N562, and N835 in all
the transfected cells (Fig. 5B, c-n),
accompanied by partial relief of their silencing activities. In
contrast, HP1 In this report, we demonstrated that KRAB-ZFPs are targeted to
foci of centromeric heterochromatin through the interaction with KAP-1
and HP1 TSA-caused redistribution of KRAB/KAP-1 from centromeric foci is
quite rapid, because TSA treatment for only 6 h led to complete redistribution (data not shown), suggesting that inhibition of HDACs
causes primarily dislodgment of KRAB/KAP-1 from foci rather than
prevention of de novo targeting to foci. Therefore, stable anchorage or incorporation of the KRAB/KAP-1-bound target locus into
heterochromatin in the foci may require hypoacetylated nucleosomes in
the target locus. More importantly, TSA treatment simultaneously causes
partial relief of silencing, indicating the involvement of HDAC
activity in the silencing mechanism. This is reminiscent of the finding
that TSA treatment in fission yeast relieves silencing of centromeric
marker genes that require Swi6p, a yeast HP1 homolog (35). Thus,
involvement of the HP1 family proteins and HDACs is evolutionarily
conserved in silencing at centromeric heterochromatin. However, the
finding that HDAC inhibition by TSA could not completely relieve
silencing suggests that, unlike the direct HDAC recruitment to the
localized target locus as previously shown for many repressors (36,
37), HDACs might be indirectly involved in KRAB/KAP-1-mediated silencing, possibly by stabilizing their centromeric localization as
described above or because of fortuitous colocalization within centromeric foci where HDACs are concentrated (29, 30).
Nonetheless, it should be noted that TSA could cause complete
redistribution of KRAB/KAP-1 from centromeric foci despite the incomplete relief of silencing. This suggests that redistribution is
required, but by itself not sufficient, for transcriptional activation
from the formerly silenced locus. This view is consistent with recent
studies using through the interaction with KAP-1.
Centromeric targeting potential of KRAZ1 and KAP-1 is strictly
correlated with their silencing activities; a KRAB mutant of KRAZ1 that
is unable to bind KAP-1 and KAP-1 deletions unable to bind HP1 cannot
localize to centromeric foci nor repress transcription. We provide
evidence that this correlation is likely to be functionally relevant.
First, overexpression of the VP16 transactivation domain fused with the KAP-1 deletion that binds to KRAB but not to HP1 leads to dramatic redistribution of KRAZ1 from centromeric foci and simultaneously converts KRAZ1-mediated silencing into strong transcriptional activation. Second, a specific inhibitor of histone deacetylases, trichostatin A, effectively redistributes KRAZ1 and KAP-1 from centromeric foci and partially relieves their silencing activities. These data strongly suggest that KRAB-ZFPs/KAP-1 silence transcription by dynamic recruitment of the target locus to the specific gene silencing compartment, centromeric heterochromatin, in a histone deacetylase-dependent manner.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(6,
7) or KRIP-1 (8), was isolated as a protein that interacts with
multiple KRAB domains but not with those containing KRAB-A mutations.
KAP-1 is thought to function as a corepressor for the large class of
KRAB-containing ZFPs, because 1) KAP-1 itself represses transcription
when directly targeted to DNA; 2) overexpression of KAP-1 enhances
KRAB-mediated silencing; and 3) KAP-1 functionally interacts with KRAB
in mammalian cells and exerts a repressor function in the complex with
the DNA-bound KRAB domains (5-9). In the N-terminal region, KAP-1
contains the RBCC domain (RING finger, B boxes,
and coiled-coil region) that is essential for
binding to KRAB and participates in multimerization of KAP-1 (5, 6, 9,
10). The central region of KAP-1 is a core domain essential but not
sufficient for the full silencing activity (6, 9). The C-terminal
region that has also been shown to be required for full silencing
activity contains a plant homeodomain finger and a bromodomain
(6, 9). The functions of these domains are still unclear but have been
implicated in chromatin-mediated transcriptional regulation and
interaction with the acetylated histone tail (11-13).
, HP1
(M31/MOD1), and HP1
(M32/MOD2), have been
identified in both human (17-19) and mouse (7, 17). KAP-1 binds
directly with all three HP1 proteins in vitro (14, 15). However, these three subtypes exhibit distinct subnuclear localization patterns depending on the species and cell type (20, 21). For example,
in mouse NIH3T3 cells, HP1
was found almost exclusively in foci of
centromeric heterochromatin (15). HP1
localizes to centromeric foci,
and additional nuclear speckles appear to represent euchromatin (14,
15). HP1
is predominantly distributed in euchromatic regions (14,
15). KAP-1 was shown to colocalize with HP1
in pericentromeric
heterochromatin and with HP1
in euchromatic regions (14), suggesting
that KAP-1 may exert its silencing activity by a
heterochromatin-mediated mechanism. However, there is no evidence
indicating a correlation between subnuclear localization of KAP-1 or
KRAB-ZFPs and their silencing function.
. Centromeric localization
of KRAZ1/2 is dependent on the interaction with KAP-1, and in turn,
binding to HP1 is essential for centromeric recruitment of KAP-1. We
further provide data that strongly suggest that KRAB-ZFPs/KAP-1 repress
transcription by locus targeting to centromeric heterochromatin in a
histone deacetylase-dependent manner.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and GST-HP1
(24) were kindly provided by Dr. H. J. Worman (Centre
National de la Recherche Scientifique (CNRS), Paris, France). Protein
purification and GST pull-down assay were performed as described (9).
Briefly, 1-5 µg of GST or GST fusion proteins immobilized on
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) were incubated
with 35S-labeled VP16-KAP-1 deletions that were
in vitro-translated by using the T7 TNT system (Promega).
After washing, bound proteins were separated by 10-12% SDS
polyacrylamide gel electrophoresis and analyzed by a Bio-image analyzer
(Fujix BAS 5000).
monoclonal
antibody (mAb) 2HP-1H5 (15), which was kindly provided by
Dr. P. Chambon (CNRS, Strasbourg, France); (iii) rabbit polyclonal Ab
against human HP1
(amino acids 181-191 that are 91%
identical to mouse HP1
) (21), which was kindly provided by
Dr. J. C. Courvalin (CNRS, Paris, France); (iv) mouse anti-GAL4 mAb
(RK5C1; Santa Cruz Biotechnology); (v) rabbit anti-VP16 polyclonal Ab
(CLONTECH); and (vi) unlabeled and Cy3-labeled
mouse anti-Myc mAb (9E10; Santa Cruz Biotechnology). Labeling was
performed by a Cy3 labeling kit (Amersham Pharmacia Biotech). All of
the following secondary Abs were purchased from Jackson ImmunoResearch
Laboratories, Inc.: donkey Texas Red-labeled anti-mouse or rabbit IgG
and donkey FITC-labeled anti-mouse or rabbit IgG. Species specificities
of the secondary antibodies were confirmed by control staining.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
to Centromeric
Foci--
We previously reported that N-terminal KRAB-containing
regions of KRAZ1 and KRAZ2 repress transcription when heterologously targeted to the nucleus as fusions with GAL4DBD (9). We
observed, however, that these regions alone did not localize to the
nucleus (data not shown), whereas full-length KRAZ1 and KRAZ2
containing zinc fingers in the C terminus (15 and 9 repeats,
respectively; see Fig. 1A)
were nuclearly localized (Fig. 1B). We thus further investigated the subnuclear localization of KRAZ1/2 within this more
physiological context of full-length proteins. Each Myc-tagged full-length protein was transiently expressed in mouse NIH3T3 cells and
examined by immunofluorescent staining with an anti-Myc antibody (Fig.
1B). In ~20% of transfected cells, KRAZ1 exhibited a
unique dot-like structure in interphase nuclei, whereas the remaining
80% presented a diffuse distribution (Fig. 1B, a
and c). KRAZ2 also displayed similar nuclear dots though to
a lesser extent than KRAZ1 (Fig. 1B, e and
g), suggesting that these dot-like structures might be a
common characteristic in many KRAB-containing ZFPs. Although the levels
of KRAZ1/2 expression varied widely among individual transfected cells,
there was no correlation between the presence of the dot-like
structures and the expression level (data not shown), suggesting that
dot formation is not likely because of artifacts caused by
overexpression. Moreover, KRAZ1/2 nuclear dots coincide with
DAPI-stained dots that correspond to foci containing centromeric
heterochromatin (Fig. 1B, d and h) (21, 26), suggesting that KRAZ1/2 specifically localize to centromeric
foci. We focused on KRAZ1 for further investigation, because the cell
population showing nuclear dots was consistently larger for KRAZ1 than
KRAZ2. However, basically similar data were obtained for KRAZ2.
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Fig. 1.
A fraction of KRAZ1 and KRAZ2 localize to the
heterochromatic dot-like structures. A, a
schematic diagram of the KRAZ1 and KRAZ2 proteins and amino acid
sequences of their KRAB domains. The KRAB-A, KRAB-B, and the multiple
zinc finger domains are shown. The consensus sequence of the KRAB
domains and DV residues substituted to AA in the KRAB-A mutants are
indicated. B, localization of full-length KRAZ1 and KRAZ2 in
interphase nuclei. NIH3T3 cells were transiently transfected with
Myc-tagged full-length KRAZ1 and KRAZ2 and stained with Cy3-labeled
anti-Myc mAb. Conventional microscope images of representative cells
exhibiting a diffuse or dot-like pattern are
shown in left panels. Right panels represent
counterstaining of the same nuclei with DAPI.
,
HP1
, and HP1
in vitro (14, 15). Thus, we examined
whether KRAZ1 colocalizes with KAP-1 and HP1s within the nucleus by
using immunofluorescent confocal microscopy. Among the HP1 members, we
focused on HP1
, because endogenous HP1
exclusively localized to
centromeric foci as reported previously (see Refs. 15 and 21 and Fig.
2A, b). Consistent
with the previous observation (14), endogenous KAP-1 was concentrated
in dot-like structures in nearly 50% of the nuclei (Fig.
2A, a). KAP-1 dots colocalized exclusively with
HP1
(Fig. 2A, c), indicating that KAP-1
localized to foci of centromeric heterochromatin. Overlaying of the
corresponding images revealed that the nuclear dots of KRAZ1 coincided
entirely with endogenous KAP-1 and HP1
in centromeric foci (Fig.
2A, d-i). Furthermore, like other proteins that
are specifically targeted to centromeric foci such as HP1
/MOD1, CAF1
p150, and Suv39 h1 (26, 27), dot-like structures by KRAZ1 were
resistant to Triton X-100 extraction (data not shown), suggesting that
KRAZ1 is specifically targeted to centromeric foci and tightly
associated with heterochromatin. The colocalization of KRAZ1 dots with
KAP-1 and HP1
were further confirmed by using a different
combination of antibodies, anti-mouse KAP-1 (1Tb-1A9, which was kindly
provided by Dr. P. Chambon; see Ref. 15), and anti-human HP1
antibodies (21) (data not shown).
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Fig. 2.
Centromeric localization of KRAZ1 is
associated with silencing activity. A, localization of
KRAZ1 but not its KRAB mutant, together with endogenous KAP-1 and
HP1 . Confocal microscope images of single optical sections of
representative cells are shown. NIH3T3 cells were transiently
transfected with Myc-tagged full-length KRAZ1 or its KRAB mutant
(KRAZ1 mut) and stained with Cy3-labeled anti-Myc Ab
(red; d, g, j, and
m). Endogenous KAP-1 was stained with anti-hKAP-1 Ab
followed by Texas Red-labeled anti-rabbit IgG (red;
a) or FITC-labeled anti-rabbit IgG Ab (green;
e and k). Endogenous HP1
were stained with
anti-mHP1
and FITC-labeled anti-mouse IgG Abs (green;
b, h, and n). Right panels
show the merged images of left and middle panels
(yellow). B, transcriptional silencing by
full-length KRAZ1 but not its KRAB mutant. NIH3T3 cells were
transiently transfected with the reporter plasmid pGL3-G5SV (100 ng)
carrying five GAL4-binding sites upstream of the SV40 promoter,
together with increasing amounts (2, 10, and 50 ng) of the expression
plasmids for GAL4DBD, GAL4-KRAZ1, or GAL4-KRAZ1 mut. Transcriptional
silencing activity is shown as -fold repression relative to GAL4DBD
alone. Luc, luciferase. C, in vivo
interaction of KAP-1 with full-length KRAZ1 but not its KRAB mutant.
NIH3T3 cells were transiently transfected with VP16-KAP-1 N835 and each
GAL4 fusion as indicated. GAL4 fusions were immunoprecipitated with
anti-GAL4 Ab. Immunoprecipitates and 10% of inputs were run by 10%
SDS polyacrylamide gel electrophoresis and analyzed for the presence of
KAP-1 by Western blotting with anti-VP16 Ab.
and -
but not to GST alone (Fig.
3, A and C). In
contrast, no binding was detected for KAP-1 N487 and 697C in this
assay, indicating that amino acids 487-562 of KAP-1 are necessary for
interaction with HP1 in vitro. These results are consistent
with the reports showing that amino acids 396-562 constitute the core
repressor domain (9) and that amino acids 483-510 constitute the HP1
binding domain (BD) (14, 15).
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Fig. 3.
Centromeric localization of KAP-1 is
associated with its silencing activity. A, schematic
diagram of KAP-1 deletion constructs and their silencing activities
(taken from Ref. 9). Silencing activities are shown as -fold repression
relative to GAL4DBD alone. Note that those of the top three
constructs fused with GAL4DBD are shown in parentheses and
that the corresponding images in B are of Myc-tagged
constructs. Bromo, bromodomain; PHD, plant
homeodomain. B, localization of KAP-1 deletions transiently
expressed in NIH3T3 cells and endogenous HP1 in interphase nuclei.
Confocal microscope images of representative cells are shown. In the
upper six rows, Myc-KAP-1 deletions
(red; left panels) were stained with Cy3-labeled
anti-Myc Ab, and endogenous HP1
(green; middle
panels) were stained with anti-mHP1
and FITC-labeled anti-mouse
IgG Abs. In the lower three rows, GAL4-fused
KAP-1 deletions that lack KRAB BD (red; left
panels) were stained with anti-GAL4 and Texas Red-labeled
anti-mouse IgG Abs, and endogenous HP1
(green;
middle panels) were stained with anti-hHP1
and
FITC-labeled anti-rabbit IgG Abs. Right panels show the
merged images of left and middle panels
(yellow). C, in vitro interaction
between KAP-1 deletions and HP1 proteins. VP16-KAP-1 deletions shown in
A were 35S-labeled in vitro and
incubated with GST alone, GST-HP1
, or GST-HP1
. Bound proteins and
10% of the input were subjected to 10-12% SDS polyacrylamide gel
electrophoresis and analyzed by a Bio-image analyzer (Fujix BAS
5000).
to centromeric foci,
whereas KAP-1 N487 and 694C lacking HP1BD could not, indicating that
HP1BD is essential for centromeric targeting. Surprisingly, however,
KAP-1 396C and 396-711 that retain HP1BD but not RBCC could not
localize to centromeric foci, indicating that HP1BD alone is not
sufficient for centromeric targeting in vivo but that RBCC
might also be required. Considering the fact that RBCC serves as the
interface for KAP-1 homo-oligomerization upon binding to KRAB (10), the
above result suggests that KAP-1 oligomerization is required for
centromeric targeting. To address this possibility, we examined the
localization of RBCC deletions of KAP-1 fused with GAL4DBD that can
form a homodimer (28). Although GAL4-KAP-1 697C that lacks HP1BD still
could not localize to centromeric foci, GAL4-KAP-1 396C and 396-711
that contain HP1BD became targeted to centromeric foci (Fig.
3B), indicating that the requirement for RBCC can be
replaced with GAL4DBD. These results demonstrate that HP1BD and
oligomerization through RBCC are required and sufficient for
centromeric targeting of KAP-1.
View larger version (30K):
[in a new window]
Fig. 4.
A VP16AD-fused KAP1 deletion causes
redistribution of KRAZ1 from centromeric foci and transcriptional
activation. A, transcriptional activity by GAL4-KRAZ1
and VP16-KAP-1 in mammalian-two hybrid assay. NIH3T3 cells were
transiently transfected with the reporter plasmid pGL3-G5SV (100 ng)
and expression plasmids for GAL4DBD or GAL4 full-length KRAZ1 (50 ng)
and for VP16 AD, VP16-KAP-1 deletions N487 or N562 (200 ng) were
used. The schematic diagram of VP16-KAP-1 deletions and relative
luciferase activities are shown. B, redistribution of
full-length KRAZ1 from centromeric foci by VP16-KAP-1 N487 but not
N562. Confocal microscope images of representative cells are shown.
NIH3T3 cells were transiently transfected with Myc-tagged full-length
KRAZ1, and VP16-KAP-1 deletions are indicated. In left
panels, Myc-KRAZ1 expression was visualized with anti-Myc and
FITC-labeled anti-mouse IgG Abs (green; a and
g) or with Cy3-labeled anti-Myc Ab (red;
d and j). In middle panels, VP16-KAP-1
deletions (red; b and h) were detected
with anti-VP16 and Texas Red-labeled anti-rabbit IgG Abs, and
endogenous HP1 (green; e and k) was
detected with anti-mHP1
and FITC-labeled anti-mouse IgG Abs.
Right panels show the merged images of left and
middle panels (yellow).
to
centromeric foci (Fig. 4B, g-l). This result
suggests that KRAZ1 interacts with KAP-1 N562 within centromeric foci
wherein strong transcriptional activation by VP16AD is inhibited by
KAP-1 silencing activity via HP1 interaction. In contrast, upon
coexpression of KAP-1 N487 that leads to strong transcriptional
activation, KRAZ1 was dramatically redistributed from centromeric foci,
together with KAP-1 N487, to exhibit a diffuse nuclear staining,
whereas HP1
remained in centromeric foci (Fig. 4B,
a-f). This observation suggests that overexpressed KAP-1
N487 interacts with KRAZ1 and displaces KRAZ1 from centromeric foci in
a dominant manner over endogenous KAP-1, resulting in activation of
reporter gene transcription elsewhere in the nucleus away from foci.
View larger version (37K):
[in a new window]
Fig. 5.
TSA causes redistribution of KRAZ1/KAP-1 from
centromeric foci and partial relief of their silencing activity.
A, effect of TSA on KRAZ1- and KAP-1-mediated
transcriptional silencing. NIH3T3 cells were transiently transfected
with the reporter plasmid pGL3-G5T7 (100 ng) carrying five GAL4-binding
sites upstream of seven repeats of the tetrocycline operator (Tet
O) and the cytomegalovirus promoter, together with the expression
plasmids for tTA (10 ng) and for GAL4DBD or GAL4-KRAZ1, KRAZ1 mutant,
and KAP-1 deletions (125 ng). The relative luciferase (Luc)
activities of GAL4 fusions are shown as percentages of GAL4DBD alone in
the absence or presence of TSA (50 ng/ml). B, redistribution
of KRAZ1 and KAP-1 from centromeric foci by TSA. Confocal microscope
images of representative cells are shown. In the top panels,
endogenous KAP-1 in NIH3T3 cells treated with (b) or without
(a) TSA for 24 h were stained with anti-hKAP-1 and
Texas Red-labeled anti-Rabbit IgG Abs. In the lower panels,
NIH3T3 cells were transiently transfected with Myc-tagged, full-length
KRAZ1 (c and f), KAP-1 N562 (i), or
KAP-1 N835 (l) and treated with TSA from 24 to 36 h
after transfection. Cells were stained with anti-Myc and FITC-labeled
anti-mouse IgG Abs (green; c) or with Cy3-labeled
anti-Myc Ab (red; f, i, and
l). Endogenous KAP-1 (red; d) was
detected with anti-hKAP-1 and Texas Red-labeled anti-rabbit IgG.
Endogenous HP1 (green; g, j, and
m) were stained with anti-mHP1
and FITC-labeled
anti-mouse IgG Abs. Right panels show the merged images of
left and middle panels (yellow).
remained in the foci with TSA, indicating that
centromeric localization of HP1
does not depend on HDACs. Taken
together, these data demonstrate that centromeric localization of KRAZ1
and KAP-1 requires TSA-sensitive HDAC activity and further suggest that
their centromeric targeting ability is functionally relevant to the
silencing activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and that the ability of KRAB/KAP-1 to localize to foci is
strictly correlated with their silencing activities. This correlation
is likely to be functionally relevant, because overexpression of a
VP16AD-KAP-1 N487 that binds to KRAB but not to HP1 led to dramatic
redistribution of KRAB-ZFPs from centromeric foci and simultaneously to
reversion of KRAB-mediated silencing to strong transcriptional
activation. In addition, HDAC inhibition by TSA also resulted in
redistribution of KRAB-ZFPs, as well as KAP-1, from centromeric foci
and in partial relief of their silencing activities. Furthermore,
VP16AD-KAP-1 N562 that binds to both KRAB and HP1 could indeed
colocalize with KRAB-ZFPs to centromeric foci; however, their
interaction did not lead to transcriptional activation. This indicates
that centromeric foci are indeed a transcriptionally inert compartment
where even strong transactivation by VP16AD is severely suppressed.
Therefore, these data strongly suggest that one of the mechanisms for
KRAB/KAP-1-mediated silencing is sequestration of target gene loci to
the transcriptionally inert compartment, centromeric foci, where HP1
proteins and HDACs are concentrated (29, 30), but the RPB1 subunit of
RNA polymerase II is excluded (15). A similar mechanism has been
implicated for the gene regulation by the lymphocyte-specific Ikaros
protein (29, 31-34). However, transcriptional silencing when assessed even in a bulk cell population is quite efficient despite the fact that
only a small fraction of cells exhibit centromeric localization of
KRAB/KAP-1. This indicates that another mechanism must operate, at
least in the cells that do not exhibit KRAB/KAP-1 dots. For example,
KRAB-ZFPs may recruit KAP-1 and HP1 to the target gene loci, leading to
local conversion of euchromatic regions into transcriptionally
inactive, higher order heterochromatin-like structures (14, 15). Based
on the following findings, we propose that both mechanisms might be
functional within the individual cells and that active transition may
occur between the two silencing states. First, KAP-1 localizes to both
centromeric heterochromatin and euchromatic regions (14, 15). Although
only HP1
exclusively localizes to centromeric foci, KAP-1 can bind
to not only HP1
but also HP1
and -
(14, 15), which are not
exclusively associated with centromeric heterochromatin (14, 15, 20).
HP1
and -
might thus be the components of local
heterochromatin-like structures that are formed on the
KRAB/KAP-1-targeted loci and may be involved in the transition between
centromeric and local euchromatic regions. Alternatively, it is
conceivable that such local heterochromatic domains defined by HP1
and HP1
might constitute fine local compartments for gene silencing.
Second, in both the two-hybrid assay and the TSA experiment,
localization of KRAB/KAP-1 and hence the presumable target locus itself
is not static, but dynamically mobile in the nucleus, whereas the
position of HP1
was relatively fixed. It would be intriguing to test
whether the dynamic localization of KRAB-ZFPs might be also regulated
by the cell cycle as previously postulated for KAP-1 (14).
-globin transgenes, which have shown that localization
away from centromeric heterochromatin is required for general
hyperacetylation and an open chromatin structure of the locus but not
sufficient for fully active transcription (38) and that a functional
enhancer suppresses silencing of a transgene and prevents its
localization close to centromeric heterochromatin (39). Consistent with
this idea, the presence of VP16AD is absolutely required for
transcriptional activation in both our mammalian two-hybrid assay and
the partial relief of silencing by TSA. The same KAP-1 deletion N487
tagged with only Myc epitope without VP16AD caused similar
redistribution of KRAZ1 but did not activate transcription or relieve
silencing even to the basal level (data not shown). We thus speculate
that, in the absence of functional enhancers and activators, the
silencing status might be persistent and dominant even after
redistribution of KRAB/KAP-1 from centromeric foci. The molecular
nature of the persistent silencing status might be the stable
heterochromatin complex that is formed within centromeric foci and
moves together with the target locus after redistribution. This idea
would provide another explanation for the fact that silencing is quite
efficient despite the small cell fraction showing KRAB/KAP-1 dots.
Finally, identification of the target genes that are regulated by
KRAB/KAP-1 will provide the opportunity, by using in situ
hybridization, to directly test whether the target locus can be
localized to centromeric foci in the repressed state and excluded from
foci when activated.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. P. Chambon, J. C. Courvalin, S. C. Lee, and H. J. Worman for materials, to Dr. H. Kurooka for valuable suggestions and discussion, and to Drs. K. Shibahara and R. Yu for critical reading of the manuscript. We thank S. Hirano for technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan.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.
These authors equally contributed to this work.
§ To whom correspondence should be addressed. Tel.: 81-75-751-4189; Fax: 81-75-751-4190; E-mail: shimizu@virus.kyoto-u.ac.jp.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010663200
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ABBREVIATIONS |
---|
The abbreviations used are: KRAB, Krüppel-associated box; ZFP(s), zinc finger protein(s); DBD, DNA-binding domain; AD, activation domain; GST, glutathione S-transferase; BD, binding domain; HDAC(s), histone deacetylase(s); KAP-1, KRAB-associated protein-1; HP1, heterochromatin protein 1; tTA, tetracycline-controlled transactivator; TSA, trichostatin A; Ab(s), antibody(ies); mAB, monoclonal antibody; FITC, fluorescein isothiocyanate; DAPI, 4',6'-diamidino-2-phenylindole.
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