From the Center for Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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ABSTRACT |
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We have isolated two novel Krüppel-like
zinc finger proteins containing the evolutionarily conserved
Krüppel-associated box (KRAB), KRAZ1 and KRAZ2, and demonstrated
that they repress transcription when heterologously targeted to DNA.
Their repression activity appeared to be mediated by the putative
corepressor KAP-1 (KRAB-associated protein-1), because KRAZ1/2 bind to
KAP-1, but KRAB mutants of KRAZ1/2 that are unable to interact with
KAP-1 lack repression activity, and KAP-1 has intrinsic repressor
activity and potentiates KRAZ1/2-mediated repression. We dissected the KAP-1 protein into a KRAB-interacting domain and a region necessary for
repression. Using a mammalian two-hybrid assay, we further demonstrated
that KAP-1 deletions lacking repression activity fused to the VP16
transactivation domain strongly activated transcription when
coexpressed with KRAZ1. In contrast, VP16-KAP-1 fusions retaining repression activity resulted in repression. These results provide the
first evidence that KAP-1 functionally interacts with KRAB in mammalian
cells and seems to exert repressor activity in the DNA-bound KRAB-KAP-1
complex, and they further support the hypothesis that KAP-1 functions
as a corepressor for the large class of KRAB-containing zinc finger proteins.
A large number of studies on transcriptional factors has revealed
that functional domains of many transcriptional factors are modular.
They can be structurally and functionally separated into DNA-binding
domains and effector (activation or repression) domains. DNA-binding
domains can be classified according to common structural motifs, which
are well conserved throughout evolution (1). One of such motifs is the
C2H2-type zinc finger repeat that has been
estimated to be present in several hundreds of genes, thus being
identified as a major family of DNA-binding proteins (2-4). The vast
majority of the C2H2-type zinc finger proteins (ZFPs)1 are classified as
Krüppel-like on the basis of the fact that they share a highly
conserved stretch of seven amino acids (the H/C link) connecting
multiple tandem repeats of the zinc finger domain (5). In contrast to
the DNA-binding domains, analysis of the structure or targets of
effector domains has been hampered by the lack of amino acid sequence
homology or structural motifs common among them. Nevertheless, some
effector domains can be loosely categorized according to the primary
amino acid content. It has been reported that activation domains are
often rich in acidic amino acids and/or proline and glutamine (6, 7). Less is known about repression domains; however, some of them are rich
in alanine, proline, or charged amino acids (8).
The Krüppel-associated box (KRAB) was first identified as an
evolutionarily conserved motif consisting of 75 amino acids that has
been assumed to be present in about one-third of the Krüppel-like
ZFPs (9). It is found almost exclusively in the N terminus of the
Krüppel-like ZFPs that contain zinc finger domains in their C
terminus, and is subdivided into KRAB-A and B domain (9). Recently
several KRAB domains have been shown to act as potent repressors when
heterologously tethered to the promoter (10-13). The KRAB-A domain
present in every KRAB domain, but not the B domain, is responsible for
such transcriptional repression (10, 11). The KRAB domain is rich in
charged amino acids and predicted to fold into two amphipathic This prediction was recently substantiated by the isolation and
characterization of a novel protein that interacts with KRAB, KRAB-associated protein-1 (KAP-1) (14). KAP-1 was also identified as
TIF1 A highly related protein to KAP-1 in the overall structure including
the conserved domains is TIF1 In this report, we describe the isolation and characterization of two
novel KRAB-ZFPs, KRAZ1 and KRAZ2, which repress transcription and
interact with KAP-1. We further investigated the functional interaction
of KRAZ1/2 with KAP-1 using a mammalian two-hybrid assay based on the
detailed molecular characterization of the functional domains in KAP-1.
Our results present the first evidence that KAP-1 functionally
interacts with KRAB in mammalian cells and that KAP-1 seems to function
as a corepressor of KRAB-ZFPs in the complex with the DNA-bound KRAB domains.
Isolation of Full-length cDNAs of KRAZ1 and
KRAZ2--
Isolation of the cDNAs encoding zinc finger domains
using degenerative polymerase chain reaction (PCR) was described
previously (25). The EcoRI-BamHI fragments of
Mszf 42 and Mszf 49 (25) were used as
hybridization probes to isolate cognate full-length cDNAs from a
Screening with the Mszf 42 probe resulted in the
identification of one cDNA clone carrying a sequence (nt 949-1207)
that was highly similar but not identical to Mszf 42 (85%
identity at nucleotide level) and a region encoding the KRAB-A domain
(nt 276-395). This Mszf 42-like zinc finger gene was thus
referred to as KRAZ1. Two cDNA clones isolated using the
Mszf 49 probe contained an identical sequence to
Mszf 49 (nt 1569-1839, numbered in the composite cDNA), but did not extend to the 5'-end of the mRNA. To generate the full-length cDNA, the 5'-end of the cDNA was amplified by the 5'-rapid amplification of cDNA ends technique from cDNAs
derived from a murine B-lymphoma CH12 F3-2 (27) with specific primers (49AS5; 5'-GGTGTGTGACAGATTCTGCAGTTCC-3' complementary to nt 815-839 or
49AS6; 5'-TCTGCGGATTTTCTGATGAAGACTC-3' complementary to nt 1219-1243)
using the Marathon cDNA amplification kit
(CLONTECH) according to the manufacturer's
instructions, and subcloned into pGEM-T vector (Promega). Sequence
analysis of seven clones resulted in the identification of an
additional 690-nt sequence that precedes the 5'-end of the original
cDNA fragments (1571 nt) and contains a region encoding the KRAB-A
and B domains (nt 335-514). Mszf 49 was therefore renamed
KRAZ2 in order to simplify the nomenclature used in this report.
Plasmids--
Recombinant DNA works were performed according to
standard protocols (28). A firefly luciferase reporter plasmid,
pGL3-G5SV, was constructed by subcloning the
PvuII-XbaI fragment carrying five GAL4-binding
sites excised from pG5BCAT (29) into the MluI (blunted) and
NheI sites upstream of the SV40 promoter of pGL3-Promoter vector (Promega, designated pGL3-SV in this report). Another reporter plasmid, pGL3-G5B, was constructed in two steps. The
XhoI-SmaI fragment containing five GAL4-binding
sites and the E1b TATA promoter excised from pG5BCAT was inserted into
the XhoI and EcoRV sites of pBluescript KS (+),
and the insert was excised with KpnI and SpeI and
subcloned into the KpnI and NheI sites of the
pGL3-Basic vector (Promega).
All of the constructs described below except for VP16-KAP-1 were cloned
into the mammalian expression plasmid pEF-BOS bsr, which was produced
by subcloning the 2.9-kilobase PvuII fragment excised from
pEF-BOS (30) into the PvuII sites of pSV2bsr (Kakenseiyaku). To construct GAL4 fusions, the KRAB-containing regions of KRAZ1 (amino
acids 43-216) and KRAZ2 (7-312) were amplified by PCR from the
cDNA clones, and the KRAB-containing region of KOX1 (1-90) (10)
and various regions of the KAP-1 protein (14) indicated in Fig. 5
(except for KAP-1 Cells, Transfection, and Luciferase Assay--
NIH 3T3 and 293T
cells were grown in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum. For the
luciferase assay, NIH 3T3 cells plated 24 h prior to transfection
at 2 × 104 cells/well in 24-well plate were
transfected with 100 ng of the firefly luciferase reporter plasmid, 4 ng of the sea-pansy luciferase reporter plasmid pRL-SV40 (Promega), and
the amounts of expression plasmids indicated in each figure using
LipofectAMINE according to the manufacturer's instructions (Life
Technologies, Inc.). Total amounts of expression plasmids were adjusted
by adding the empty expression plasmid pEF-BOS bsr if necessary. After
48 h of incubation, preparation of cell lysates and dual
luciferase assays were carried out using the dual-luciferase reporter
assay system according to the manufacturer's instructions (Promega). Firefly and sea-pansy luciferase activities were quantified using a
Lumat LB9507 luminometer (Berthold), and firefly luciferase activity
was normalized for transfection efficiency as determined by sea-pansy
luciferase activity. Each transfection experiment was performed at
least three times in duplicate. The results are presented as the
average with standard deviations of duplicates in a representative experiment.
Immunoprecipitation--
293T cells plated 24 h before
transfection at 5 × 105 cells/dish in 60-mm dishes
were transfected with 0.4 µg of the expression plasmid for
6MT-NLS-KAP-1 N835 together with 0.4 µg of the plasmids encoding
GAL4DBD alone, GAL4-KRAZ1, GAL4-KRAZ2, GAL4-KOX1, or their mutants
using the CellPhect transfection kit according to the manufacturer's
instructions (Amersham Pharmacia Biotech). After 48 h of
incubation, cells were washed in phosphate-buffered saline and lysed in
lysis buffer (50 mM HEPES, pH 7.5, 250 mM NaCl,
0.1% Nonidet P-40, 1 mM EDTA, and 1 mM
dithiothreitol) containing 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml leupeptin and 1 µg/ml aprotinin. The whole-cell
lysates were precleared with protein G-Sepharose (Amersham Pharmacia
Biotech) for 2 h at 4 °C. To the precleared lysates, 1 µl of
rabbit anti-GAL4DBD antibody (Upstate Biotechnology) was added and
incubated for 15 h at 4 °C, and then 10 µl of a 50% slurry
of protein G-Sepharose was added and incubated for 4 h at 4 °C.
After washing five times in lysis buffer and boiling in
SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer, the
precipitated proteins were separated by 10% SDS-PAGE and transferred
to polyvinylidene difluoride filters (Millipore). After blocking in
phosphate-buffered saline and 0.05% Tween 20 containing 5% skim milk,
the filters were cut into upper and lower strips and incubated with
anti-Myc monoclonal antibody (9E10; Roche Molecular Biochemicals) and
mouse anti-GAL4DBD monoclonal antibody (RK5C1; Santa Cruz Biotech),
respectively, followed by peroxidase-conjugated goat anti-mouse IgG
antibody (Southern BioTech) and developed using the ECL Plus Western
blotting detection system (Amersham Pharmacia Biotech).
GST Pull-down Assay--
Glutathione S-transferase
(GST)-KRAB fusion proteins were produced by subcloning the
KRAB-containing regions of KRAZ1, KRAZ2, and KOX1 identical to those
used for GAL4 fusions into pGEX-4T-1 (Amersham Pharmacia Biotech),
expressed in Escherichia coli BL21 (DE3) and purified using
glutathione-Sepharose 4B (Amersham Pharmacia Biotech) according to the
manufacturer's instructions. Whole-cell lysates were prepared from
293T cells transfected with the expression plasmids for 6MT-NLS-KAP-1
deletions as described for immunoprecipitation. The cell lysates were
incubated with 1-5 µg of GST fusion proteins immobilized to
glutathione Sepharose 4B for 2-4 h at 4 °C. After washing five
times in lysis buffer and boiling in SDS-PAGE loading buffer, the bound
proteins were subjected to 10% SDS-PAGE and analyzed by Western
blotting using anti-Myc monoclonal antibody as described for immunoprecipitation.
Isolation of Two Novel KRAB-containing ZFPs, KRAZ1 and
KRAZ2--
We previously cloned 88 mouse cDNAs encoding 60 different Krüppel-like zinc finger domains using the degenerative
PCR method (25) based on the structural characteristics of these
factors such as the highly conserved H/C link connecting consecutive
finger repeats (5). To further investigate the molecular features and
biological functions of the novel zinc finger genes identified, several
full-length cDNAs were isolated by screening a cDNA library of
mouse spleen cell using cognate zinc finger genes as probes (for
details, see under "Experimental Procedures"). Sequence analysis revealed that two of the isolated cDNAs encoded novel
Krüppel-like ZFPs containing the KRAB-A domain (9). We thus refer
to these two genes as KRAZ1 and KRAZ2
(KRAB-containing zinc finger proteins) (Fig.
1). The KRAZ1 cDNA (2191 nt) contains an open reading frame (nt 117-2075) encoding a 652-amino
acid protein with a calculated molecular mass of 76.0 kDa. The
KRAZ2 cDNA (2261 nt) contains an open reading frame (nt
308-2128) for a polypeptide of 606 amino acids with a molecular mass
of 71.2 kDa. Both KRAZ1 and KRAZ2 contain the KRAB-A domain at the N
terminus and repeated zinc finger domains located in the C terminus (15 and 9 repeats) (Fig. 1A). However, the KRAB-A domain of
KRAZ2 is followed by KRAB-B (9), whereas that of KRAZ1 is not (Fig. 1).
By Northern blot analysis, KRAZ1 and KRAZ2 were found to be
ubiquitously expressed in all mouse tissues tested; brain, heart, lung,
thymus, spleen, lymph node, bone marrow, liver, kidney, muscle, testis,
ovary, and uterus (data not shown).
KRAZ1 and KRAZ2 Repress Transcription When Heterologously Targeted
to the Promoter--
To address first the possibility that KRAZ1 and
KRAZ2 possess transcriptional repressor activity when recruited to the
promoter, the KRAB-containing region of KRAZ1 (amino acids 43-216) and
KRAZ2 (7-312) were fused to GAL4DBD (Fig.
2A). Expression plasmids for these fusions were transfected into NIH 3T3 cells with a luciferase reporter plasmid carrying five GAL4-binding sites upstream of the SV40
promoter (pGL3-G5SV). This reporter exhibited a high basal level of
transcriptional activity when transfected with the parent expression
plasmid or the plasmid encoding GAL4DBD alone (Fig. 2C).
However, transcriptional activity was significantly repressed by
GAL4-KRAZ1 and GAL4-KRAZ2 (Fig. 2B, lanes 4 and
5) in a dose-dependent manner (Fig.
2C), as well as by GAL4-KOX1 (Fig. 2B, lane 3), a
GAL4 fusion with the KRAB-containing region of KOX1 (1-90) that was
described previously to repress transcription (10, 13-15). These
GAL4-KRAB fusions did not affect transcription from a reporter plasmid
lacking the GAL4-binding site (pGL3-SV) (Fig. 2B, lanes
12-16), and a non-DNA-bound form of KRAB did not significantly
repress transcription (lanes 9-11), indicating that repression by KRAB is dependent on site-specific DNA binding and did
not result from a nonspecific toxic effect on the transfected cells or
titration of the basal transcription machinery. Such specific
repression was also observed using another reporter plasmid driven by
the thymidine kinase promoter of herpes simplex virus (data not shown),
suggesting that KRAB-mediated repression is a universal phenomenon
rather than a specific effect on some classes of transcription factors.
Furthermore, substitution mutations at the highly conserved amino acids
in the KRAB-A domains (DV to AA) disrupted repression activity (Fig.
2B, lanes 6-8), indicating that KRAB-A is responsible for
repression.
KRAZ1 and KRAZ2 Share a Common Cellular Cofactor with KOX1 to
Repress Transcription--
As reported previously (14), repression by
KOX1 was dose-dependently abrogated by overexpression of
free wild-type KOX1, whereas free KOX1 harboring the DV/AA mutation did
not significantly interfere with repression (Fig.
3A), suggesting that a
titratable cellular cofactor is required for repression and that the
repression ability of KRAB correlates to the interaction with such a
cofactor. To determine whether the same cofactor is required for
KRAZ1/2-mediated repression, we performed a similar squelching
experiment. As shown in Fig. 3B, repression by GAL4-KRAZ1/2
was efficiently squelched by free KOX1 in a dose-dependent
manner (lanes 5-8 and 14-17), whereas the KOX1
DV/AA mutant did not significantly abrogate repression (lanes
9-12 and 18-21). These results strongly suggest that
KRAZ1 and KRAZ2 share a common specific cellular cofactor with KOX1 to
exert their repressor activity.
KRAZ1 and KRAZ2 Interact with a Putative Corepressor,
KAP-1--
KAP-1 was identified as such a cofactor that binds to
several KRAB domains including KOX1 but not to KRAB mutants lacking repression activity (14-16). To explore the possibility that KAP-1 represents the cofactor shared by KRAZ1/2 with KOX1, we examined the
interaction of KRAZ1/2 with KAP-1 by immunoprecipitation using a
Myc-tagged KAP-1 (30-835, designated N835) construct that was expressed in 293T cells together with the GAL4-KRAB fusions. Each GAL4
fusion protein was immunoprecipitated with anti-GAL4DBD antibody, and
the precipitated proteins were examined by Western blotting. As shown
in Fig. 4, comparable amounts of all GAL4
fusions were precipitated, and KAP-1 was coprecipitated with
GAL4-KRAZ1/2 and GAL4-KOX1 (lanes 3, 5, and
7), but not with GAL4DBD alone (lane 2).
Furthermore, coprecipitation of KAP-1 with GAL4-KRAB mutants (lanes 4, 6, and 8) was strongly abrogated,
indicating that KRAB-A is responsible for interaction and the ability
of KRAB to interact with KAP-1 correlates to the repression activity.
Taken together, these results demonstrate that KAP-1 specifically
interacts with KRAB-A of KRAZ1/2 and are consistent with that KAP-1
represents the cellular cofactor required for repression by KRAZ1/2.
The interaction between KRAB and KAP-1 seems to be direct, because in vitro-translated KAP-1 also bound to bacterially
expressed GST-KRAB fusion proteins (data not shown), although it
remains to be excluded that proteins in the extract for in
vitro translation might be bridging the interaction.
Mapping of the KRAB-binding Domain in KAP-1--
To further
characterize the interaction between KRAZ1/2 and KAP-1, we next
attempted to delineate the KRAB-binding domain in KAP-1 by GST
pull-down assays using a number of Myc-tagged KAP-1 deletions (Fig.
5) expressed in 293T cells and GST-KRAB fusion proteins. As shown in Fig. 6,
KAP-1 N835 was able to bind with GST-KRAZ1/2 or GST-KOX1 (lanes
18-20), but not with GST alone (lane 17). In a series
of N- or C-terminal deletions of KAP-1, further deletions extending
from amino acid 239 to 421 failed to bind to GST-KRAZ1 (Fig. 6).
Although expression of KAP-1 697C, 145-421 and 239-421 are
consistently low for unknown reason (lanes 33, 36, and
39), 145-421 appeared to bind quite efficiently, and 239-421 alone also could bind (lanes 38 and 41),
indicating that KAP-1 239-421, which almost corresponds to the
coiled-coil region, is necessary and sufficient for the interaction
with KRAB in vitro. Completely identical results were
obtained with GST-KRAZ2 and GST-KOX1 and also by immunoprecipitation
using GAL4-KRAZ1 (data not shown).
Intrinsic Repressor Activity of KAP-1 and Mapping of Its Repression
Domain--
One of the definitive criteria for corepressors is their
intrinsic ability to repress transcription when directly targeted to
the promoter. We thus constructed KAP-1 N835 directly fused to GAL4DBD
and assessed its ability to repress transcription. As shown in Fig.
7A, GAL4-KAP-1 N835
efficiently repressed transcription (lane 7). Similar to
KRAB, repression by GAL4-KAP-1 was dependent on site-specific DNA
binding, because it did not affect transcription from the reporter
lacking the GAL4-binding site, and free KAP-1 did not repress
transcription (data not shown).
We next attempted to determine the repressor domain of KAP-1 using
GAL4-KAP-1 deletions (Fig. 7A). An internal deletion
construct, GAL4-KAP-1 Overexpression of KAP-1 Potentiates KRAZ1/2-mediated
Repression--
Although KAP-1 interacts with KRAZ1/2 and has
intrinsic repressor activity, it remains to be proved that KAP-1 would
function in vivo as a mediator of repression by KRAZ1/2 when
targeted to DNA through the interaction with KRAB. To address this
issue, we first examined whether overexpression of KAP-1 influenced the efficiency of KRAZ1/2-mediated repression. As shown in Fig.
7B, expression of increasing amounts of KAP-1 N835 resulted
in an enhancement of GAL4-KRAZ2-mediated repression (lanes
7-11) that was more apparent when repression was partially
squelched by free KOX1 (lanes 12-16), whereas
overexpression of KAP-1 N835 did not affect transcription in the
presence of GAL4DBD alone (lanes 1-6). Moreover, even in
the presence of free KOX1, repression was not potentiated by KAP-1
396C, which failed to bind to KRAB (lanes 17-20),
suggesting that the functional interaction of KAP-1 with KRAB is
required to potentiate KRAB-mediated repression. Repression by
GAL4-KRAZ1 was also potentiated by KAP-1 N835 though less efficiently (data not shown). These data suggest that KAP-1 positively regulates KRAZ1/2-mediated repression.
KRAZ1 and KRAZ2 Functionally Interact with KAP-1 in Mammalian
Cells--
The results described above, however, do not provide direct
evidence that KAP-1 interacts with KRAB in mammalian cells and KAP-1
mediates repression in the resultant DNA-bound KRAB-KAP-1 complex. In
addition, although KAP-1 was coprecipitated with KRAB expressed in
mammalian cells (Fig. 4), even if KAP-1 and KRAB were separately
expressed and then mixed with each other in vitro, they were
coprecipitated (data not shown). Therefore, interaction proved by such
immunoprecipitation does not necessarily indicate actual in
vivo interaction.
Thus, we next examined the in vivo functional interaction of
KRAB with KAP-1 using a mammalian two-hybrid assay in which GAL4-KRAB was coexpressed with the KAP-1 deletions fused to the VP16
transactivation domain (VP16AD) in NIH 3T3 cells. If GAL4-KRAB can
functionally interact with KAP-1 in cells, VP16AD fused to KAP-1 would
be recruited to the vicinity of the promoter and expected to activate
transcription. As shown in Fig. 5, GAL4-KRAZ1 and GAL4-KOX1 repressed
transcription in the presence of VP16AD alone, whereas GAL4DBD alone
did not significantly affect transcription in the presence of VP16AD
alone or any VP16-KAP1 deletions. Repression by GAL4-KRAZ1 and
GAL4-KOX1 was unaffected by VP16-KAP1 N244, 396C, 554C, or 697C, all of which lack the KRAB-binding domain (Figs. 5 and
8A, lanes 3 and 11-13), suggesting that these KAP-1 deletions fail to
interact with KRAB also in vivo. In contrast, when
GAL4-KRAZ1 or GAL4-KOX1 was coexpressed with VP16-KAP1 N421, N487 or
145-421, which contains the KRAB-binding domain but not repression
activity, transcription was strongly activated above the basal level
(Figs. 5 and 8A, lanes 4, 5, and 14) in a
dose-dependent manner (for N487, see Fig. 8B).
This result clearly indicates that these KAP-1 deletions functionally
interact with KRAZ1 and KOX1 in mammalian cells. Although coexpression
of GAL4-KRAZ2 with these VP16-KAP-1 deletions did not result in strong
activation, repression by GAL4-KRAZ2 was dose-dependently
counteracted by VP16-KAP1 N487 (Fig. 8B, lanes 16-20),
suggesting that KRAZ2 can also interact with KAP-1 in vivo.
KAP-1 145-421 containing the B1/B2 finger and coiled-coil region
appeared to interact with KRAB in vivo (Fig. 8A, lane
14) as well as in vitro. Although KAP-1 239-421
further lacking the B1/B2 finger could bind to KRAB in vitro
(Fig. 6), VP16-KAP1 239-421 failed to activate transcription (Fig.
8A, lane 15), suggesting that the B1/B2 finger is required
for the interaction with KRAB only in vivo but not in
vitro.
KAP-1 Exerts Repressor Activity in the DNA-bound KRAB-KAP-1
Complex--
More surprisingly, coexpression of VP16-KAP-1 N562, N711,
N835, 145C, or 239C harboring both KRAB-interacting and repression activity did not result in significant activation of transcription but
remained repression especially for N835, 145C, or 239C (Figs. 5 and
8A, lanes 6-10). This result suggests two possibilities. First, they cannot functionally interact with KRAB in vivo,
although they can interact in vitro. Second, they indeed
interact in vivo, but their repressor activity dominate to
transactivation by VP16AD, thereby transcription would not be activated
but rather repressed. To discriminate between these possibilities, we
carried out a competition experiment in which in vivo
binding of the Myc-tagged KAP-1 deletions to KRAB was assessed by
assaying their capability to suppress the strong transcriptional
activation caused by the interaction of GAL4-KRAZ1 with VP16-KAP-1
N487. Coexpression of GAL4-KRAZ1 with VP16-KAP-1 N487 again resulted in
strong activation (Fig. 8C, lane 4), but this activation was
dose-dependently suppressed by coexpression of Myc-tagged
KAP-1 N487 (Fig. 8C, lanes 5-8). This suppression of
activation appeared to be dependent on the interaction of KAP-1 with
KRAB, because KAP-1 396C lacking the KRAB-binding domain failed to
suppress activation (Fig. 8C, lanes 13-16). These results
indicate that Myc-tagged KAP-1 N487 but not 396C can compete with
VP16-KAP-1 N487 for binding to KRAZ1. Furthermore, KAP-1 N835 also
counteracted activation as efficiently as N487 (Fig. 8C, lanes
9-12), and a similar counteraction was observed for KAP-1 N421,
N562, and N711 (data not shown), indicating that they can also interact
with KRAB in vivo. Although KAP-1 145C, 239C, and 239-421
could bind to KRAB in vitro, and 145-421 could bind
in vivo as well as in vitro, KAP-1 145C only
partially suppressed activation, and KAP-1 239C, 239-421, and 145-421
did not (data not shown), suggesting that the RING-B1/B2 finger is required to efficiently displace VP16-KAP-1 N487 from the KRAB domain
in vivo.
In conclusion, it is most likely that KAP-1 N562, N711 and N835 can
interact with KRAB in vivo and that their repression
activity dominates transactivation of VP16AD; therefore, the
corresponding VP16-KAP-1 fusions would not enhance transcription but
rather cause repression. This prediction is further supported by the careful observation that residual transcriptional enhancement by
VP16-KAP-1 N562, N711, or N835 correlates reciprocally to the repressor
activity of these KAP-1 deletions reproducibly when coexpressed with
either GAL4-KRAZ1 or GAL4-KOX1 (Figs. 5 and 8A, lanes
6-8).
To further directly prove that KAP-1 dominantly suppresses
transactivation ability of VP16AD, the KAP-1 deletions were fused to
the GAL4DBD-VP16AD chimeric protein (GAL4-VP16). To exclude the
influence of other cellular transcription factors, a reporter plasmid,
pGL3-G5B, containing the minimal E1b TATA promoter, which shows very
low basal transcription (Fig. 8D, lane 1), was used. GAL4-VP16 strongly activated transcription in a
dose-dependent manner (Fig. 8D, lanes 5-7), but
GAL4DBD alone did not (Fig. 8D, lanes 2-4). Activation by
GAL4-VP16 was not affected when fused to KAP-1 N244 completely lacking
repressor activity (Fig. 8D, lanes 8-10) but was severely
suppressed by fusion with KAP-1 N835 containing full repression
activity (Fig. 8D, lanes 11-13). Similar suppression was
observed for GAL4-VP16-KAP-1 N562 and N711 (data not shown). These data
clearly indicate that the repressor activity of KAP-1 is dominant to
transactivation by VP16AD and further strengthen the notion that
VP16-KAP-1 N562, N711, and N835 interact with promoter-bound GAL4-KRAB
and dominantly repress transcription in the mammalian two-hybrid assay.
Taken together, these results further support the hypothesis that KAP-1
functions as a corepressor for KRAB-ZFPs to exert repressor activity in
a physiological context where complexed with DNA-bound KRAB in
mammalian cells.
It has recently been thought to be a universal molecular mechanism
that DNA-binding transcriptional repressors exert their repression
activity by recruitment of corepressors in many systems from yeast and
Drosophila to mammals (14). Our study, together with the
previous reports (14-16), revealed that KAP-1 functions as a universal
corepressor for the large class of KRAB-ZFPs, because it fulfills the
criteria for corepressors: first, KAP-1 interacts in mammalian cells as
well as in vitro with several different KRAB domains
including novel ones we isolated, but not with KRAB mutants defective
in repression. Second, KAP-1 has intrinsic repressor activity, because
KAP-1 efficiently represses transcription when targeted to the
promoter. Third, overexpression of KAP-1 potentiates KRAB-mediated
repression in a manner dependent on the interaction with KRAB. Fourth,
KAP-1 seems to exert the repressor activity in a physiological context
of the DNA-bound KRAB-KAP-1 complex. These findings further support the
model that KRAB-ZFPs bind to cis-regulatory sequences by
their putative DNA-binding domains, zinc fingers, recruit the
corepressors including KAP-1 to the promoter by their KRAB domains, and
thereby repress transcription (14-16).
We demonstrated that amino acids 239-421 of KAP-1, which almost
correspond to the coiled-coil region, are essential and sufficient for
the interaction with KRAB in vitro, but the RING-B1/B2
region is dispensable (Fig. 6). However, involvement of the RING-B1/B2 region in the in vivo interaction with KRAB seems to be
complicated but important. The RING finger is essentially dispensable
in vivo and also in vitro, but the B1/B2 finger
is required only in vivo but not in vitro. These
results are in agreement with the report by Moosmann et al.
(15) suggesting that the coiled-coil region of KAP-1/TIF1 Amino acids 396-562 of KAP-1, which reside between the coiled-coil and
the PHD finger, are considered to be a core domain essential for
repression but not sufficient for full repression. Several explanations
can be made why the N-terminal portion (the RBCC domain) or the most
C-terminal portion (the bromo-like domain) should be required for full
repression in addition to the core domain. First, the N- or C-terminal
region may contribute to the functional conformation or stability of
the KAP-1 protein. Second, KAP-1 might possess two or more independent
repression modules that are composed of the N- or C-terminal region
together with the core domain. The core domain may thus be shared with
both repression modules and would be essential, whereas the N- or
C-terminal region would be dispensable as long as the other module
remains functional. Third, KAP-1 can function only within a
multiprotein complex, and the N or C terminus is required for efficient
incorporation of KAP-1 into such a complex through specific
protein-protein interaction that is the possible function of these
domains, or more simply, there might be a definite threshold of
molecular mass necessary for proper formation of such a
multiprotein complex.
Several hypotheses have been proposed for the mechanism(s) underlying
KRAB/KAP-1-mediated repression (15, 17, 36). One is the reorganization
of chromatin into a repressive state by heterochromatin formation,
because in the yeast two-hybrid assays, both KAP-1 and TIF1 At present, the most favorable model for KRAB/KAP-1-mediated repression
is active repression (8), in which KRAB or KAP-1 interacts in an
inhibitory manner with specific proteins of the basal transcription
machinery, thereby interfering with the assembly and/or function of
them (15, 36). Several lines of evidence support the active repression
model rather than the formation of repressive chromatin (15, 36).
First, KRAB/KAP-1-mediated repression is quite efficient even in short
term transient transfection assays as in this study, as well as in an
in vitro transcription experiment (36), in which the bulk of
the reporter DNA templates would not be fully assembled into chromatin.
Second, it was recently demonstrated that KRAB is able to repress
transcription by RNA polymerase II and III, but neither by RNA
polymerase I nor T7 RNA polymerase in mammalian cells, suggesting that
KRAB exerts its repression activity by interfering with some
component(s) for RNA polymerase II and III transcription rather than by
altering the chromatin structure into a repressive state (37). There are two potential molecular mechanisms proposed for active repression, direct and quenching repression (8). For KRAB/KAP-1-mediated repression, direct repression seems to be more likely than quenching repression, because KRAB has been demonstrated to repress transcription from a number of different polymerase II-dependent
promoters (12, 15) and suppress the activating function of various
transcriptional activators (36), and moreover, KRAB is able to repress
basal promoter activity in addition to activated transcription in an in vitro transcription experiment (36).
The active repression mechanism has been considered to involve specific
inhibitory protein-protein interactions; however, it is also possible
that active repression is accomplished without direct interaction with
any components of the transcription machinery, but rather by
modification of them. With regard to this possibility, the recent
report by Fraser et al. (38) seems to be instructive. They
showed that TIF1 It was recently proposed that TIF1 It is clearly necessary to elucidate the mechanism(s) and identify the
molecular target(s) in KRAB/KAP-1-mediated repression. In addition, it
is also important to investigate the biological significance of
KRAB/KAP-1-mediated repression. In the mammalian two-hybrid assay, we
clearly demonstrated that repression of transcription by GAL4-KRAB can
be converted to strong activation when coexpressed with the VP16-fused
KAP1 deletions that contain the KRAB-interacting domain but no
repression activity, suggesting that these VP16-KAP1 deletions would
function as dominant negative mutants with regard to the physiological
function of KRAB-ZFPs and/or KAP-1. Therefore, functional investigation
using these deletions as a molecular probe in mammalian cells as well
as in transgenic animals will facilitate the elucidation of the
physiological importance of transcriptional repression by the large
class of KRAB-ZFPs and their corepressor KAP-1.
INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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helices, which might thus serve as a protein-protein interaction
surface (9). Unlike other alanine- or proline-rich repression domains,
the KRAB domain is highly conserved in a subfamily of the
Krüppel-like ZFPs sharing a common amino acid sequence and a
predicted secondary structure, suggesting that the KRAB domains in many
different ZFPs may share a common cellular target(s) (10).
or KRIP-1 (15, 16) (hereafter referred to as KAP-1) and found
to interact with several different KRAB domains but not with KRAB-A
mutants deficient in repression, to enhance KRAB-mediated repression
and to repress transcription when directly targeted to DNA (14-16).
KAP-1 is a member of the RBCC subfamily of the RING finger proteins
that contain the RING finger motif defined as
C3HC4 finger and followed by one or two B
box-type fingers and a putative coiled-coil domain (14-17). Although
the functions of these domains are so far unknown, they have been
thought to be involved in nucleic acid-protein and/or protein-protein
interactions (18, 19). In the C-terminal portion, KAP-1 also contains a C4HC3 zinc finger called PHD finger followed by
a domain similar to the bromodomain (14-17). The PHD finger is often
found in concert with the bromodomain and is also present in a number
of proteins implicated in chromatin-mediated transcriptional
regulation, thereby suggesting that it might be involved in
interactions with chromatin components (20). The bromodomain has also
been identified in several proteins that regulate transcription in the
context of large multiprotein complexes and/or through interaction
with, or modification of, chromatin (21-23). Thus, it has been
proposed to mediate the protein-protein interactions influencing the
assembly and/or activity of such complexes, or to be involved in
interactions with chromatin (21).
, which was originally identified as a
putative coactivator of the nuclear receptor (24). However, it was
subsequently reported to interact with KRAB and repress transcription
when directly recruited to the promoter (15, 17). Therefore, KAP-1 and
TIF1
have been postulated to constitute a new family of
transcriptional intermediary factors and function as mediators of
transcriptional repression for the large class of KRAB-containing ZFPs
(17). However, there is no compelling evidence directly indicating that
KAP-1 or TIF1
indeed interacts with KRAB in mammalian cells and
exerts the repression function in a physiological context of the
resultant DNA-bound KRAB-KAP-1/TIF1
complex.
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gt10 cDNA library that was constructed from C57BL/6 mouse spleen
cells stimulated with lipopolysaccharide and interleukin-4 (25). The
cDNAs were subcloned into pBluescript KS (+) (Stratagene) and
sequenced on both strands using the Dye Terminator cycle sequencing kit
with AmpliTaq DNA polymerase, FS (Applied Biosystems), and analyzed on
a model 373A or 310 automatic DNA sequencer (Applied Biosystems).
Sequence homology search was performed using the BLAST program
(26).
404-696) were obtained by PCR from Jurkat cell
cDNA using corresponding primers tagged with appropriate restriction sites. For all the KAP-1 fragments, 5'- and 3'-primers were
tagged with EcoRI and SalI sites, respectively.
The PCR-amplified fragments were digested with relevant restriction
enzymes and fused in-frame to the C terminus of the yeast GAL4
DNA-binding domain (GAL4DBD, 1-147) excised from KS(+)-GAL4 (31).
Amino acid substitutios in the KRAB domains (DV to AA) were introduced by PCR-mediated site-directed mutagenesis (32). KAP-1
404-696 was
constructed by ligating the EcoRI-StuI (30-403)
and EcoRI (blunted)-SalI (697-835) fragments
prepared from the KAP-1 N421 and 697C constructs, respectively. To
express non-DNA-bound forms of the KRAB domains properly in the
nucleus, the KRAB regions were tagged N-terminally with the
hemagglutinin (HA) epitope preceded by methionine in a Kozak consensus
sequence (33) and followed by the nuclear localization signal (NLS) of
the SV40 large T antigen (34) as follows. The
ClaI-EcoRI fragment in KS(+)-GAL4 containing GAL4DBD was replaced by a linker (Met-HA CE;
5'-CGATATCGCCACCATGGTGTACCCATACGACGTCCCAGACTACGCG-3') encoding the initiator methionine (italics) and the HA epitope (underlined), and subsequently the
EcoRI-SalI sequence was replaced by a linker
(SV40LT-NLS;
5'-AATTTGCCGAAAAAGAAGAGAAAGGTGGAATTCTCGAG-3') carrying the NLS of the SV40 large T antigen (underlined) and a
new EcoRI site (italics), resulting in pKS+HA-NLS. The
wild-type or mutated KRAB regions of KRAZ1, KRAZ2, and KOX1 were then
fused in-frame to the C terminus of the HA-NLS tag excised from
pKS+HA-NLS. To construct the KAP-1 deletions tagged N-terminally with
six repeats of the Myc epitope (6MT) and NLS, the
ClaI-EcoRI fragment in KS(+)-GAL4 was replaced by
the ClaI-EcoRI fragment carrying 6MT excised from
pCS2+MT mNotchIC (35), and subsequently the EcoRI-SalI sequence was replaced by the
SV40LT-NLS linker, resulting in pKS+6MT-NLS. The KAP-1 deletions were
then fused in-frame to the C terminus of the 6MT-NLS tag excised from
pKS+6MT-NLS. Fusions of the KAP-1 deletions with the activation domain
(AD) (413-490) of herpes simplex virus VP16 were constructed by
replacing the EcoRI-SalI sequence in pCMX-VP16-N
with the SV40LT-NLS linker and subcloning the KAP-1 deletions into the
new EcoRI and SalI sites in-frame to the C
terminus of VP16AD-NLS. For generation of GAL4-VP16-fused KAP-1
deletions, GAL4DBD was amplified by PCR from KS(+)-GAL4 with 5'-M13
reverse primer and 3'-BglII-tagged primer, and VP16AD was
amplified by PCR from pCMX-VP16-N with 5'-BglII- and
3'-EcoRI-tagged primers. The amplified GAL4DBD and VP16AD
regions were digested with ClaI/BglII and
BglII/EcoRI, respectively, and subcloned between
the ClaI and EcoRI sites of KS(+)-GAL4, resulting
in pKS+GAL4-VP16. The KAP-1 deletions were then fused in-frame to the C
terminus of GAL4-VP16 excised from pKS+GAL4-VP16. All the PCR-derived
sequences were confirmed by sequencing on both strands. Equivalent
expression of the proteins with the expected molecular mass and nuclear
localization were confirmed by Western blot analysis and immunohistochemistry.
RESULTS
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Fig. 1.
Structures and amino acid sequences of KRAZ1
and KRAZ2. A, schematic representation of the
structures of the KRAZ1 and KRAZ2 proteins (each upper
portion). The KRAB-A and KRAB-B domains and the multiple zinc
finger domains are indicated by black, gray, and
shaded boxes, respectively. Positions of the sequences
similar to Mszf 42 (nt 949-1207) and identical to
Mszf 49 (nt 1569-1839) are indicated by an
arrow. The predicted amino acid sequences of the KRAZ1 and
KRAZ2 proteins are indicated in each lower portion. The
KRAB-A and KRAB-B domains are double and single
underlined, respectively. The multiple zinc finger domains are
boxed, and cysteine and histidine residues are highlighted
with black. B, alignment of the amino acid sequences of the
multiple KRAB domains. The KRAB domains of 15 zinc finger proteins
including KOX1 are aligned with the KRAZ1 and KRAZ2
sequences. The consensus sequence and DV residues that
were substituted to AA in the KRAB-A mutants are shown at the
bottom, and conserved residues corresponding to
the consensus are shown on a black background. Data base
accession numbers are as follows: KOX-1, X52332; ZFP90/NK10, X79828;
ZNF91/HTF10, L11672; ZNF140, U09368; ZNF133, U09366; ZNF85,
U35376; mKID-1, L77247; rKID-1, M96548; ZNF141, L15309; ZNF90, M61870;
ZNF157, U28687; ZNF43, X59244; ZNF7, M29580; ZNF177, U37263; and ZNF45,
L75847.
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Fig. 2.
KRAZ1/2 repress transcription when
heterologously targeted to the promoter. A, schematic
representation of the reporter and expression plasmids used in
luciferase assays. pGL3-G5SV and pGL3-SV reporter plasmids contain the
firefly luciferase gene (Luc+) driven by the
SV40 promoter with or without five GAL4-binding sites (5XGAL4),
respectively. The indicated regions (amino acids) of KOX1, KRAZ1, and
KRAZ2 were fused to GAL4DBD. The identical regions tagged with HA-NLS
were expressed as non-DNA-bound forms of the KRAB domains
(e.g. HA-KRAZ1). The corresponding regions carrying the
substitution mutation (DV to AA) in the KRAB-A domains were also fused
to GAL4DBD or HA-NLS (e.g. GAL4-KRAZ1 mut). B,
DNA binding-dependent transcriptional repression by
GAL4-KRAB. The expression plasmids (250 ng) were transfected into NIH
3T3 cells with the reporter plasmid (100 ng) indicated at the
bottom. The result is shown as fold repression relative to
GAL4DBD alone. C, dose-dependent transcriptional
repression by GAL4-KRAB. The indicated amounts of the expression
plasmids (open circle, GAL4DBD; filled circle,
GAL4-KOX1; filled square, GAL4-KRAZ1; filled
triangle, GAL4-KRAZ2) were transfected with the reporter plasmid
pGL3-G5SV (100 ng) into NIH 3T3 cells. The result is shown as relative
luciferase activity.
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Fig. 3.
KRAZ1/2 share a common cellular cofactor with
KOX1. All the expression plasmids were transfected into NIH 3T3
cells with the reporter plasmid pGL3-G5SV (100 ng). The result is shown
as relative luciferase activity. A, squelching of
GAL4-KOX1-mediated repression by free KOX1. The GAL4DBD or GAL4-KOX1
expression plasmid (2 ng) was transfected with 250 ng (+) or the
indicated amounts of the expression plasmid for HA-KOX1 or HA-KOX1
DV/AA mutant (mut). The diagram indicated at the
left illustrates how overexpression of free KOX1 may result
in squelching of GAL4-KOX1-mediated repression. PIC,
preinitiation complex. B, squelching of
GAL4-KRAZ1/2-mediated repression by free KOX1. The GAL4DBD or
GAL4-KRAZ1/2 expression plasmid (10 ng) was transfected as in
A.
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Fig. 4.
KAP-1 was coimmunoprecipitated with KRAZ1/2
as well as KOX1, but not with KRAB mutants. The plasmid encoding
KAP-1 (30-835) tagged with 6MT-NLS (6MT-KAP-1) was transfected into
293T cells with the empty plasmid pEF-BOS bsr or the expression plasmid
for GAL4DBD alone, GAL4-KRAB, or GAL4-KRAB mutant (mut)
described in Fig. 2. Whole-cell lysates were prepared and
immunoprecipitated with anti-GAL4DBD antibody, and the precipitated
proteins (IP) and 3% of the lysates (Input) were
subjected to 10% SDS-PAGE and analyzed by Western blotting using
anti-Myc antibody (upper two panels) or anti-GAL4DBD
antibody (lower two panels). Positions of the molecular mass
markers and the corresponding proteins are indicated on the
left and right sides, respectively.
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Fig. 5.
KAP-1 deletion constructs and summary of the
characterization of the KRAB-binding and repression domain in
KAP-1. Schematic representation of the KAP-1 deletion constructs
used in this study is shown at the left, with amino acid
numbers at both ends. KRAB binding ability of the various KAP-1
deletions was assessed by the GST pull-down assay using GST-KRAZ1 shown
in Fig. 6 and is presented as + (bound) and (not bound).
ND, not determined. Intrinsic repressor activity of the
KAP-1 deletions was measured by the luciferase assay using GAL4-KAP-1
deletions shown in Fig. 7A and is presented as fold
repression relative to GAL4DBD alone. In vivo KRAB binding
ability of the KAP-1 deletions was determined by the mammalian
two-hybrid assay shown in Fig. 8A (for GAL4-KRAZ1), in which
VP16AD alone (Control) or VP16-KAP-1 deletions were
coexpressed with GAL4DBD, GAL4-KOX1, or GAL4-KRAZ1 and assayed for
their effects on the transcriptional activity of the pGL3-G5SV reporter
plasmid. The results are presented as relative luciferase activity
(percentage of the activity in the presence of GAL4DBD and VP16AD). The
positive results in each assay are boxed. The essential
repression domain (396-562) and the KRAB-binding domain (239-421)
depicted by these assays are indicated at the bottom.
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Fig. 6.
Mapping of the KRAB-binding domain in
KAP-1. 293T cells were transfected with the expression plasmids
for 6MT-KAP-1 deletions indicated in Fig. 5. Whole-cell lysates were
prepared and incubated with GST alone (G), GST-KOX1
(KO), GST-KRAZ1 (K1), or GST-KRAZ2
(K2) immobilized to glutathione-Sepharose 4B, and bound
proteins and 3% of the input (I) were subjected to 10%
SDS-PAGE and analyzed by Western blotting using anti-Myc antibody. The
results shown were obtained with GST-KRAZ1, but identical results were
obtained with GST-KOX1 and GST-KRAZ2 (lanes 18 and
20 for KAP-1 N835, data not shown). Positions of the
molecular mass markers are indicated on the left side.
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Fig. 7.
KAP-1 repressed transcription when
heterologously targeted to the promoter and enhanced KRAB-mediated
repression. All of the expression plasmids were transfected into
NIH 3T3 cells with the reporter plasmid pGL3-G5SV (100 ng).
A, mapping of the repression domain in KAP-1. The expression
plasmids (250 ng) for the GAL4-KAP-1 deletions used are indicated at
the left. The result is shown as fold repression relative to
GAL4DBD alone. B, enhancement of KRAB-mediated repression by
overexpression of KAP-1. The GAL4DBD or GAL4-KRAZ2 expression plasmid
(10 ng) was transfected with the indicated amounts of the expression
plasmid for 6MT-KAP-1 N835 or 396C. The HA-KOX1 expression plasmid (25 ng) was added to partially squelch GAL4-KRAZ2-mediated repression. The
result is shown as relative luciferase activity.
404-696, failed to repress transcription
significantly (lane 16), indicating that the deleted region
is necessary for repression. Furthermore, the region between the
coiled-coil and PHD finger (396-562) seems to be a core domain
essential for repression, because further deletions in a series of N-
or C-terminal deletions considerably abolished repression activity.
However, this region alone was not sufficient for full repression,
because GAL4-KAP-1 396-711 repressed transcription less efficiently
(7.7-fold repression) than 396C (52.5-fold) or N711 (21.3-fold), which
included an additional portion, the C- or N-terminal region,
respectively (Figs. 5 and 7A).
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Fig. 8.
KRAZ1/2 functionally interact with KAP-1 in
mammalian cells and KAP-1 dominantly suppresses transactivation by
VP16AD. The expression plasmids were transfected into NIH 3T3
cells with the reporter plasmid (100 ng) pGL3-G5SV (A-C) or
pGL3-G5B (D). The result is shown as relative luciferase
activity. A, a mammalian two-hybrid assay using GAL4-KRAZ1
and VP16-KAP-1 deletions. The GAL4DBD or GAL4-KRAZ1 expression plasmid (10 ng) was transfected
with the expression plasmids (250 ng) for VP16AD or VP16-KAP-1
deletions indicated in Fig. 5. The diagram shown at the left
illustrates how interaction of GAL4-KRAB with VP16-KAP-1 deletions may
result in repression or activation of transcription. B,
dose-dependent transcriptional activation by GAL4-KRAB and
VP16-KAP-1 N487. The expression plasmid (10 ng) for GAL4DBD, GAL4-KOX1,
or GAL4-KRAZ1/2 was transfected with the indicated amounts of the
VP16-KAP-1 N487 expression plasmid. C, KAP-1 N835 suppressed
the transcriptional activation by GAL4-KRAZ1 and VP16-KAP-1 N487. The
expression plasmid (10 ng) for GAL4DBD or GAL4-KRAZ1 was transfected
with 50 ng of the plasmid encoding VP16AD or VP16-KAP-1 N487 in the
absence or the presence of the indicated amounts of the expression
plasmid for 6MT-KAP-1 N487, N835, or 396C. The diagram shown at the
left illustrates how overexpression of the KAP-1 deletions
may suppress the transcriptional activation by GAL4-KRAZ1 and
VP16-KAP-1 N487. D, the transcriptional activation by
GAL4-VP16 was suppressed by fusion with KAP-1 N835. The reporter
plasmid pGL3-G5B (100 ng) was transfected with the indicated amounts of
the expression plasmid for GAL4DBD alone, GAL4-VP16, GAL4-VP16-KAP-1
N244, or GAL4-VP16-KAP-1 N835. The diagram shown at the left
illustrates how GAL4-VP16-KAP-1 N835 may suppress transactivation by
VP16AD.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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or TIF1
is essential but not sufficient for the interaction with KRAB in a
yeast two-hybrid assay. They clearly indicated that the B1/B2 finger of
KAP-1/TIF1
is required for the functional interaction, whereas the
RING finger is dispensable (15). Nevertheless, the RING finger is
suggested to contribute to the efficient interaction with KRAB in
vivo by the observation in our mammalian two-hybrid assay that
activation by VP16-KAP1 145-421 is consistently lower than that of
VP16-KAP1 N421 (Fig. 8A, compare lanes 4 and
14). Moreover, the competition experiment revealed that
KAP-1 145C lacking the RING finger could partially compete with
VP16-KAP-1 N487 for binding to KRAZ1, and KAP-1 239C, 145-421, or
239-421 could not (data not shown). It might thus also be possible
that a third molecule(s) is required for the stable interaction between
KAP-1 and KRAB in vivo and that the RING-B1/B2 finger region
of KAP-1 is necessary for the interaction with such a third molecule(s).
were
found to be associated with mHP1
and mMOD1, which are mouse homologs
of Drosophila heterochromatin protein 1 that repress
transcription by the formation of heterochromatin or a similar
structure (17). However, we observed that KAP-1 N562 lacking the
putative mHP1
/mMOD1-interacting domain (amino acids 571-579) (17)
could repress transcription more effectively (17.8-fold) than KAP-1
554C containing this site (7.7-fold) (Figs. 5 and 7A), in
agreement with the finding that a mutation of TIF1
within the
HP1-interacting domain that disrupts the interaction in yeast did not
affect repression activity in transfected mammalian cells (17). Thus,
the formation of repressed chromatin by direct interaction with mHP1
and/or mMOD1 does not seem to be a principle mechanism of
KAP-1/TIF1
- and TIF1
-mediated repression, although lack of
interaction with mHP1
/mMOD1 in KAP-1 N562 remains to be confirmed.
possesses intrinsic protein kinase activity responsible for autophosphorylation as well as for phosphorylation of
TFIIE
, TAFII28, and TAFII55. Although functional involvement of such
phosphorylation in transcriptional repression and whether KAP-1/TIF1
also has similar activity should be further studied, it is intriguing
that KRAB/KAP-1-mediated repression can be regulated through
phosphorylation of specific components in the basal transcription complex. It should also be noted that active direct repression and
repression by chromatin reorganization are not mutually exclusive (15).
The possible existence of multiple repression modules in KAP-1 (this
study) and TIF1
(17) might imply repression through both mechanisms.
and KAP-1/TIF1
constitute a
new family of transcriptional intermediary factors (TIFs) that might
play a dual role in the control of transcription at the chromatin level
(17), because TIF1
was originally identified as a protein enhancing
transcription by the nuclear receptor in yeast (24), and KAP-1/TIF1
was recently reported to activate transcription by glucocorticoid
receptor and C/EBP
(39), also because they share common properties
not only to repress transcription when targeted to the promoter but
also to interact with mHP1
/mMOD1 and with the KRAB repressor
domains. Therefore, it seems possible that TIFs might function as
corepressors or coactivators in a way dependent on the chromatin
status, the promoter context, or the combination of certain types of
transcription factors that bring them to the promoter.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. M. Fujii, S. Minoguchi, H. Kurooka, K. Umesono, and M. Maeda for providing pG5BCAT, pEF-BOS bsr/pCS2+MTmNotchIC, KS(+)-GAL4, pCMX-VP16-N, and Jurkat cell cDNA, respectively, and helpful discussions. We thank Dr. M. Nazarea for critical reading of the manuscript. We also thank Dr. S. Hirano for technical assistance.
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FOOTNOTES |
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* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB024004 and AB024005 for KRAZ1 and KRAZ2, respectively.
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{at}virus.kyoto-u.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: ZFP, zinc finger protein; KRAB, Krüppel-associated box; PCR, polymerase chain reaction; nt, nucleotide(s); DBD, DNA-binding domain; HA, hemagglutinin; NLS, nuclear localization signal; 6MT, six repeats of the Myc epitope; AD, activation domain; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; TIF, transcriptional intermediary factor.
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