Expression of the Transcriptional Repressor Protein Kid-1 Leads
to the Disintegration of the Nucleolus*
Zhiqing
Huang
,
Bärbel
Philippin
,
Eileen
O'Leary§,
Joseph V.
Bonventre§¶,
Wilhelm
Kriz
, and
Ralph
Witzgall
From the
Institute of Anatomy and Cell Biology I,
University of Heidelberg, Germany and the § Renal Unit,
Massachusetts General Hospital and the ¶ Department of
Medicine, Harvard Medical School,
Charlestown, Massachusetts 02129
 |
ABSTRACT |
The rat Kid-1 gene codes for a 66-kDa
protein with KRAB domains at the NH2 terminus and two
Cys2His2-zinc finger clusters of four and nine
zinc fingers at the COOH terminus. It was the first KRAB-zinc finger
protein for which a transcriptional repressor activity was
demonstrated. Subsequently, the KRAB-A domain was identified as a
widespread transcriptional repressor motif. We now present a
biochemical and functional analysis of the Kid-1 protein in transfected
cells. The full-length Kid-1 protein is targeted to the nucleolus and
adheres tightly to as yet undefined nucleolar structures, leading
eventually to the disintegration of the nucleolus. The tight adherence
and nucleolar distribution can be attributed to the larger zinc finger
cluster, whereas the KRAB-A domain is responsible for the nucleolar
fragmentation. Upon disintegration of the nucleolus, the nucleolar
transcription factor upstream binding factor disappears from the
nucleolar fragments. In the absence of Kid-1, the KRIP-1 protein, which
represents the natural interacting partner of zinc finger proteins with
a KRAB-A domain, is homogeneously distributed in the nucleus, whereas coexpression of Kid-1 leads to a shift of KRIP-1 into the nucleolus. Nucleolar run-ons demonstrate that rDNA transcription is shut off in
the nucleolar fragments. Our data demonstrate the functional diversity
of the KRAB and zinc finger domains of Kid-1 and provide new functional
insights into the regulation of the nucleolar structure.
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INTRODUCTION |
Transcriptional repression is increasingly recognized as an
important feature of genetic and epigenetic regulation (for recent reviews, see Refs. 1 and 2). While several proteins have been described
as transcriptional repressors, the number of motifs conferring
transcriptional repressor activity has so far been rather limited. A
widespread transcriptional repressor motif is the KRAB-A domain (3-6),
which is found in approximately one-third of all zinc finger proteins
of the Cys2His2 class (7, 8). Zinc finger
proteins of the Cys2His2 class represent one of
the largest protein families known; the mammalian genome contains several hundred genes coding for such proteins (9), and the KRAB-A
domain therefore represents a very important paradigm of transcriptional repression. Experiments from several laboratories have
shown that the KRAB-A domain represses transcription both upstream and
downstream of a target gene and is able to do so from a distance (5,
10). In addition, the KRAB-A domain has to be tethered to DNA via
fusion to a DNA-binding domain in order to exert its repressor activity
(3). This suggests that the KRAB-A domain interacts with other proteins
and rules out other modes of action such as squelching or steric
hindrance. Interestingly, the KRAB-A domain does not repress
transcription in the context of any promoter, but only works in certain
contexts. Fusion proteins with the KRAB-A domain repress RNA polymerase
II promoters with a TATA box and an initiator element but not promoters
with an initiator element only (11). It has also been reported that in
addition to RNA polymerase II-mediated transcription, the KRAB-A domain
also represses RNA polymerase III-mediated transcription, whereas
transcription by RNA polymerase I and T7 RNA polymerase do not appear
to be inhibited (10). By conventional biochemical techniques and the
two-hybrid cloning protocol, a KRAB-A interacting protein called KAP-1
(12), TIF1
(13), and KRIP-1 (14) was cloned. The
KRIP-1/KAP-1/TIF1
cDNA codes for a protein with a predicted
relative molecular weight of 89 kDa and contains several characteristic
modules that suggest that it is involved in remodeling chromatin, thus
providing a hint to how the KRAB-A domain represses transcription.
The zinc finger protein Kid-1 was cloned as a result of a screen for
transcription factors that are regulated after acute renal failure and
during renal development (15). The Kid-1 protein (kidney,
ischemia, development) contains 13 Cys2His2-zinc fingers at its COOH terminus and
KRAB-A and -B domains at its NH2 terminus; it was the first
KRAB-zinc finger protein for which a transcriptional repressor activity
could be demonstrated (15). By Northern blot and reverse transcription-
polymerase chain reaction analysis, the Kid-1 mRNA is expressed
predominantly in the adult rat kidney (15). In kidneys of newborn rats,
when many nephrons are not fully developed, only low amounts of Kid-1
mRNA can be detected, whereas in the kidneys of adult rats the
Kid-1 mRNA is expressed at its highest levels. During the recovery
phase after ischemic acute tubular necrosis, Kid-1 mRNA levels are
reduced transiently until the injured epithelium is restored (15).
Kid-1 therefore possesses all of the hallmarks of a transcription
factor that plays an important role in the regulation of an advanced
stage of kidney development. We now present a careful analysis of the distribution of the Kid-1 protein demonstrating that expression of
Kid-1 leads to the disruption of the nucleolus.
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MATERIALS AND METHODS |
Construction of Kid-1 Mutant Proteins--
The cDNA and
cDNA fragments coding for the full-length and mutant Kid-1 proteins
were cloned into the mammalian expression vector pMT3. The pMT3 vector
codes for fusion proteins with an epitope tag of the influenza virus
hemagglutinin protein at the NH2 terminus
(HA1 tag). Transcription is
driven by the adenovirus major late promoter; in addition, pMT3
contains the SV40 origin of replication. Sequences of the fusion
proteins are as follows (codons encoding the HA tag are in boldface
type and italicized; the first three codons of the Kid-1 portion are
underlined; numbering of amino acids is according to the rat Kid-1
protein as published in Ref. 15)): pMT3/Kid-1, ATG TAC CCA TAC
GAT GTT CCA GAT TAC GCT GGA ATT CCT CTA GAG GTC GAG GCC ACC
ATG GCT CCT ... (1-576); pMT3/Kid-1, A(
), ATG
TAC CCA TAC GAT GTT CCA GAT TAC GCT GGA AAG CTT GGT ACC GAG
CTC GGA TCC ACT AGT AAC GGC CGC CAG TGT GCT GGA ATT CTG CAG ATG GGA ATT
TCC GGT GGT GGT GGT GGA ATT CTA GAC TCC ATG GCA ...
(52-576); pMT3/Kid-1, AB(
), ATG TAC CCA TAC GAT GTT CCA GAT
TAC GCT GGA ATT GAT CCC TGG ... (72-576);
pMT3/Kid-1C, ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GGA AAG
CTT GGT ACC GAG CTC GGA TCC CCG GGA ATT TCC GGT GGT GGT GGT GGA
TCT TAT TTA ... (174-576); pMT3/Kid-1 (Zf 1-4), ATG
TAC CCA TAC GAT GTT CCA GAT TAC GCT GGA AAG CTT GGT ACC GAG
CTC GGA TCC CCG GGA ATT TCC GGT GGT GGT GGT GGA ATT CAT AAA CGC
TAT ... (186-299); pMT3/Kid-1 (Zf 5-13), ATG TAC CCA
TAC GAT GTT CCA GAT TAC GCT GGA AAG CTT GGT ACC GAG CTC GGA TCC
CCG GGA ATT TCC GGT GGT GGT GGT GGC CGA GAA AAC ...
(296-576).
Two additional plasmids were used: 1) pBXG1/Kid-1N, coding for a fusion
protein between the DNA-binding domain of the yeast GAL4 protein and
the NH2 terminus of the rat Kid-1 protein without the zinc
fingers (amino acids 1-195) (15); 2) pMT3A/KRIP-1, coding for KRIP-1
with an HA epitope tag at the NH2 terminus (14).
Preparation of Polyclonal Anti-Kid-1 and Anti-KRIP-1
Antibodies--
Fragments of the rat Kid-1 protein (amino acids
72-173) and the murine KRIP-1 protein (amino-terminal to the plant
homeodomain (PHD) finger) were used to immunize rabbits according to
standard protocols (16). The specificity of the antibodies was
demonstrated on Western blots and by immunocytochemistry of transiently
transfected COS-7 cells.
Transient Expression Protocols--
COS-7 cells were transfected
with 10-40 µg of expression plasmid according to the DEAE-dextran
protocol (16). CV-1 cells were transfected with 20 µg of expression
plasmid according to the DEAE-dextran (16) and the calcium phosphate
protocols (17). LLC-PK1 cells were transfected with 20 µg
of expression plasmid according to the calcium phosphate protocol
(17).
Preparation of Nuclear Extracts--
Nuclear extracts were
prepared as described by Hoppe-Seyler et al. (18). 2-3 days
after transfection, COS-7 cells were washed twice with PBS and scraped
into a microcentrifuge tube. The cells were centrifuged 5 min at
1250 × g, and the pellet was resuspended in lysis
buffer (150 mM NaCl, 10 mM Tris, pH 7.9, 1 mM EDTA, pH 8.0, 0.6% Nonidet P-40). After an incubation
of 5 min on ice, the cells were centrifuged 5 min at 1250 × g. The supernatant (containing cytoplasmic and
detergent-soluble proteins) was saved, and the nuclear pellet was
resuspended in nuclear extract buffer (1.5 mM
MgCl2, 10 mM Hepes, pH 7.9, 0.1 mM
EGTA, 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 25% glycerol, 420 mM NaCl. Where indicated, 420 mM NaCl was
substituted by 2 M NaCl and 2 M KCl,
respectively). The nuclear suspension was incubated 20 min on ice and
then centrifuged for 5 min at 14,000 × g. The
supernatant (corresponding to the soluble nuclear fraction) was saved,
and the remaining pellet was solubilized by sonication in PBS, 6 M urea.
To control for the role of the conformation of the zinc fingers, the
nuclear extract buffer was modified by the addition of 0.1 mM o-phenanthrolene; 0.1 mM
o-phenanthrolene, 50 mM EDTA; 50 mM
EDTA; or 1 mg/ml N-ethylmaleimide, respectively. Otherwise, the protocol was followed as above.
For a DNase or RNase digest, the nuclei were resuspended in a buffer
containing 10 mM Tris pH 7.0, 50 mM NaCl, 0.5%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml
aprotinin, 2 µg/ml leupeptin, 1 µg/ml pepstatin A. Then either
DNase was added to 1 unit/ml or RNase A was added to 100 µg/ml, and
RNase T was added to 40 units/µl (final concentration). The nuclear suspension was incubated 60 min at 37 °C before adding NaCl to a
final concentration of 420 mM. After a 20-min incubation on ice, the nuclei were pelleted for 5 min at 4 °C, 14,000 × g. The supernatant (corresponding to the nuclear fraction)
was saved, the remaining pellet was solubilized by sonication in PBS, 6 M urea.
Protein concentration in the different fractions was determined
according to the method described by Bradford (19).
Western Blot Analysis--
SDS-polyacrylamide gels and protein
transfers were performed according to standard protocols with
polyvinylidene difluoride membranes from Millipore (Eschborn, Germany)
(16). After the transfer, membranes were blocked overnight at room
temperature in PBS, 5% low fat dry milk, 0.5% Tween 20. The next
morning, the membrane was incubated for 2 h at room temperature
with the primary antibody, followed by a 1-h incubation with the
secondary antibody. Immune complexes were detected with the Renaissance kit from NEN (Bad Homburg, Germany). The following primary antibodies were used: cell culture supernatant from the mouse anti-HA tag hybridoma 12CA5 diluted 1:25, cell culture supernatant from the mouse
anti-Kid-1 hybridoma 5D12 (undiluted), a mouse monoclonal anti-lamin B
antibody diluted 1:30 (kind gift of E. Brigitte Lane, University of
Dundee, Dundee), and a human anti-NuMA antibody (20) diluted 1:1000
(kind gift of Herwig Ponstingl, German Cancer Research Center,
Heidelberg). Horseradish peroxidase-conjugated secondary antibodies
were goat anti-mouse IgG Fab used at a dilution of 1:10,000 and goat
anti-human IgG Fab used at a dilution of 1:100,000 (Sigma, Deisenhofen, Germany).
Single Antibody Immunocytochemistry of Transfected
Cells--
One to two days after transfection, the cells were plated
on coverslips and allowed to grow for an additional 1 or 2 days. Cells
on coverslips were washed with PBS and then fixed 20 min in PBS
containing 2% paraformaldehyde at room temperature. After a blocking
step of 15 min in PBS containing 2% bovine serum albumin, 0.1% Triton
X-100, the cells were incubated 2 h at room temperature with cell
culture supernatant from the hybridoma 12CA5 (diluted 1:30 to 1:100;
the 12CA5 antibody recognizes the HA tag). The primary antibody was
washed off with PBS, after which the cells were incubated for 1 h
at room temperature with the secondary antibody (fluorescein
isothiocyanate-coupled goat anti-mouse IgG from Cappel (Eppelheim,
Germany) diluted 1:100 or Cy3-coupled rat anti-mouse IgG from Dianova
(Hamburg, Germany) diluted 1:300). After incubation with the secondary
antibody, cells were stained for 2 min with the DNA-binding dye Hoechst
33258 (Sigma), washed three times for 5 min each with PBS, and mounted.
Double Antibody Immunocytochemistry of Transfected
Cells--
Cells were prepared as described for single antibody
immunocytochemistry. For double-labeling experiments, cells were
simultaneously incubated for 2 h at room temperature with the two
primary antibodies. After three washes with 1× PBS, the primary
antibodies were detected with fluorescein isothiocyanate-coupled goat
anti-mouse IgG from Cappel (diluted 1:150), Cy3-coupled goat anti-human
IgG from Dianova (diluted 1:300), and Cy3-coupled goat anti-rabbit IgG
from Dianova, respectively. The following combinations of primary
antibodies were used: cell culture supernatant of the mouse anti-HA tag
hybridoma 12CA5 diluted 1:30 and a human anti-upstream binding factor
(UBF) autoantibody diluted 1:300 (kind gift of Ingrid Grummt,
German Cancer Research Center, Heidelberg);
(NH4)SO4-precipitated cell culture supernatant
of the mouse anti-Kid-1 hybridoma 5D12 diluted 1:30; and rabbit
anti-KRIP-1 antiserum diluted 1:30.
Nucleolar Run-on--
Ongoing synthesis of rRNA in the nucleolus
was visualized by incubating transfected COS-7 cells with bromo-UTP
(21, 22). 48 h after the Me2SO shock, cells were
rinsed twice each with 1× PBS and run-on buffer (20 mM
Tris HCl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, pH 8.0, 0.5 mM phenylmethylsulfonyl
fluoride). The cells were permeabilized by incubating for 5 min with
0.05% Triton X-100 in run-on buffer. Following three washes with
run-on buffer, the cells were incubated for 30 min at room temperature
with 0.5 mM each of ATP, CTP, and GTP, 0.2 mM
bromo-UTP, 50 mM
(NH4)2SO4, 50 mM
Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1 mM EGTA, pH 8.0, and 10 µg/ml of amanitin (ATP, CTP, and
GTP were purchased from Boehringer Mannheim, Mannheim, Germany;
bromo-UTP and amanitin were purchased from Sigma). After the run-on
reaction, the cells were rinsed again with 1× PBS, fixed for 20 min at
room temperature with 2% paraformaldehyde, air-dried for 5 min, and
then rinsed again twice with 1× PBS before staining with the primary
antibodies. Newly synthesized rRNA and Kid-1 were detected as described
under double antibody immunocytochemistry using a rabbit anti-Kid-1
antibody at a dilution of 1:1000 and a mouse monoclonal
anti-bromodeoxyuridine antibody at a dilution of 1:50 (Dunn
Labortechnik, Thelenberg, Asbach). Primary antibodies were detected
with fluorescein isothiocyanate-coupled goat anti-mouse IgG from Cappel
(diluted 1:150) and Cy3-coupled goat anti-rabbit IgG from Dianova
(diluted 1:150).
 |
RESULTS |
Distribution of the Full-length Kid-1 and KRIP-1 Proteins in the
Nucleus--
In order to determine the subcellular site of Kid-1
expression, COS-7 cells were transiently transfected with a plasmid
encoding a full-length Kid-1 protein with an HA epitope tag at the
NH2 terminus. 2-3 days after the transfection, cells were
stained with the anti-HA epitope antibody 12CA5 and a fluorescein
isothiocyanate- or Cy3-coupled secondary antibody. By simultaneous
staining of the transiently transfected cells with the DNA-binding dye
Hoechst 33258, it could be clearly seen that Kid-1 was located in the nucleus. The distribution of Kid-1 inside the nucleus, however, varied
between different transfected cells. Whereas some cells showed a
homogeneous nuclear staining (Fig. 1,
a and b), others presented with a patchy (Fig. 1,
c and d) or speckled (Fig. 1, e and
f) nuclear staining. In order to find out how the
distribution of Kid-1 changed depending on when the cells were
harvested after the transfection, we stained transiently transfected
COS-7 cells at various time points after the Me2SO shock
treatment. Soon after the transfection (24 h after shock treatment), we
only detected cells with a homogeneous and patchy
distribution of Kid-1, and it was only at
later time points that the speckles appeared (Fig. 2 and Table
I). In some nuclei, it appeared as if the
patches containing Kid-1 were disintegrating, so that speckles
containing Kid-1 were generated (Fig. 2c). To learn more
about the physiological relevance of the different expression patterns,
COS-7 cells were transfected with a plasmid coding for an HA
epitope-tagged KRIP-1 protein. KRIP-1 (also known as KAP-1 or TIF1
)
has been shown to interact with the KRAB-A domain of
Cys2His2-zinc finger proteins and to mediate
the transcriptional repressor activity of those proteins (12-14).
KRIP-1 was also expressed in the nucleus, but was always distributed
homogeneously in the nucleoplasm, obviously sparing the nucleoli (Fig.
3).

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Fig. 1.
The rat Kid-1 protein is distributed in
different nuclear domains. COS-7 cells were transiently
transfected with a plasmid encoding a full-length Kid-1 protein. The
Kid-1 protein was tagged with an HA epitope of the influenza virus and
could therefore easily be detected by immunofluorescence with the
anti-HA epitope antibody 12CA5 (a, c, and
e), while the nuclei were visualized with the dye Hoechst
33258 (b, d, and f).
Immunofluorescence staining showed a homogeneous (a), patchy
(c), and speckled (e) nuclear distribution of the
Kid-1 protein. Magnification, × 510.
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Fig. 2.
Time course of Kid-1 expression in COS-7
cells. COS-7 cells were transfected with a Kid-1 expression
plasmid and immunohistochemically stained 24 h (a) and
48 h (c) after the Me2SO shock, while
nuclei were detected with the dye Hoechst 33258 (b and
d). 24 h after the Me2SO shock, Kid-1 was
expressed in patches (a), but at 48 h Kid-1 staining
also appeared in nuclear speckles (c). Those speckles seemed
to emanate from disintegrating patches (arrows in
c). Magnification, × 520.
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Table I
COS-7 cells transfected with pMT3/Kid-1
24, 39, and 48 h after the Me2SO shock, cells were stained
with the monoclonal anti-HA epitope antibody 12CA5. 100 positive nuclei
were evaluated according to the nuclear distribution of the Kid-1
protein.
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Fig. 3.
KRIP-1 is a nuclear protein with a
homogeneous distribution. KRIP-1 was transiently expressed in
COS-7 cells. Due to an HA-epitope tag at the NH2 terminus,
the KRIP-1 protein could be visualized by immunofluorescence staining
with the antibody 12CA5 (a), and nuclei were stained with
Hoechst 33258 (b). The KRIP-1 protein was always
homogeneously distributed in nuclei of transfected cells, apparently
sparing the nucleoli. Magnification, × 520.
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The Kid-1 Protein Is Tightly Bound to Nuclear Structures, but
KRIP-1 Is Not--
COS-7 cells, which were transfected with the
expression plasmid coding for a HA-Kid-1 fusion protein, were subjected
to a standard nuclear extraction protocol with 420 mM NaCl.
The Kid-1 protein was not present in the cytosolic nor the nuclear
extracts but was found in the insoluble pellet fraction remaining after the nuclear extraction. When the pellet was solubilized with 6 M urea and the proteins contained in the pellet fraction
were separated on a denaturing polyacrylamide gel, a single band could be observed with the anti-HA-epitope antibody, whereas extracts from
mock-transfected COS-7 cells were unreactive with the anti-HA epitope
antibody (Fig. 4a). The
observed relative molecular weight of the HA-Kid-1 fusion protein of
approximately 59 kDa was smaller than the calculated relative molecular
weight of 68.4 kDa. In order to control our nuclear extraction protocol
for its efficiency, we also transiently expressed the HA-tagged KRIP-1
protein in COS-7 cells. The KRIP-1 protein was easily detected in the
nuclear extract and appeared to a large extent already in the
detergent-soluble fraction (Fig. 4b).

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Fig. 4.
The Kid-1 protein is tightly associated with
nuclear structures, but KRIP-1 is not. Transiently transfected
COS-7 cells were lysed with Nonidet P-40, and the nuclei were separated
from the detergent-soluble proteins by centrifugation. The nuclei were
extracted with a buffer containing 420 mM NaCl, which
yielded a fraction with soluble and "insoluble" nuclear proteins.
Insoluble proteins were homogenized by sonication in PBS, 6 M urea. Proteins were separated on a SDS-polyacrylamide
gel, transferred to a polyvinylidene difluoride membrane, and stained
with the anti-HA epitope antibody 12CA5. Whereas Kid-1 was found in the
fraction comprising the insoluble nuclear proteins (a), a
large portion of the KRIP-1 protein could already be detected in the
fraction containing the detergent-soluble proteins (b).
COS-7 cells transfected with the wild-type expression vector pMT3
showed no staining. C, detergent-soluble proteins;
N, soluble nuclear proteins; P, insoluble nuclear
proteins.
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It has been reported that the treatment of nuclei with high
concentrations of salt (2 M NaCl) results in the release of
most nuclear proteins but leaves behind proteins constituting the
nuclear matrix or proteins tightly associated with it (23). Treatment of transfected COS-7 cells with 2 M NaCl and 2 M KCl, however, did not release the Kid-1 protein from the
nuclei, suggesting that Kid-1 adheres strongly to the nuclear matrix
(Fig. 5b). Incubation of the
nuclei with the SH group modifying agent N-ethylmaleimide, the divalent cation chelator EDTA and the Zn2+ chelator
o-phenanthrolene also did not result in the release of the
Kid-1 protein from the nuclei (Fig. 5a), nor did a digest of
the nuclei with DNase and RNase change the distribution pattern of
Kid-1 (Fig. 5c). In order to control for the integrity of
the protein preparations, the blots were also probed with antibodies against lamin and NuMA, two nuclear matrix-associated proteins (24-26). With either antibody, a distinct band was detected
predominantly in the insoluble fraction after RNase treatment, thus
validating our extracts (Fig. 5d).

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Fig. 5.
Evidence for the binding of Kid-1 to the
nuclear matrix. COS-7 cells were transiently transfected with
pMT3/Kid-1 (encoding an HA epitope-tagged full-length Kid-1 protein).
Transfected cells were subjected to various nuclear extraction
protocols described below, and the fractions were analyzed by Western
blot with the anti-Kid-1 antibody 5D12. a, when the nuclei
of COS-7 cells expressing a full-length Kid-1 protein were treated with
1 mg/ml N-ethylmaleimide (lane 1); a
combination of 0.1 mM o-phenanthrolene, 50 mM EDTA (lane 2); 50 mM
EDTA (lane 3); and 0.1 mM
o-phenanthrolene (lane 4), Kid-1 still
remained in the pellet fraction. Lane 5 represents fractions obtained with the regular nuclear extraction
protocol using 420 mM NaCl but without the addition of any
of the aforementioned chemicals. The protein in lane
1 exhibits a higher molecular weight due to the alkylation
of the cysteine residues of Kid-1 by N-ethylmaleimide.
b, neither extraction with a buffer containing 2 M NaCl nor a buffer containing 2 M KCl resulted
in the release of the full-length Kid-1 protein from the nuclei; the
different fractions obtained with a buffer containing 420 mM NaCl are shown for comparison. c, when nuclei
of COS-7 cells transfected with pMT3/Kid-1 were digested with 1 unit/ml
DNase or a combination of RNase A (100 µg/ml) and RNase T (40 units/µl), the full-length Kid-1 protein still remained in the
insoluble pellet fraction. d and e, in order to
control for the integrity of the extract preparation in the presence of
RNase, the blots were also probed with antibodies against lamin B
(d) and NuMA (e), two nuclear matrix-associated
proteins. Even after a digest with RNase, both proteins remained
predominantly in the insoluble fraction no matter whether
mock-transfected (M) or Kid-1-expressing COS-7 cells
(K) were used, although after a longer exposure they could
also be detected in the other fractions. C,
detergent-soluble proteins; N, soluble nuclear proteins;
P, insoluble nuclear proteins; M,
mock-transfected COS-7 cells; K, COS-7 cells transfected
with the Kid-1 expression plasmid pMT3/Kid-1.
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The Larger Zinc Finger Cluster of Kid-1 Serves as a Nucleolar
Targeting Signal, whereas the KRAB-A Domain Is Responsible for the
Disintegration of the Nucleolus--
The Kid-1 protein contains highly
conserved KRAB-A and -B domains at its NH2 terminus and 13 Cys2His2-zinc fingers clustered in groups of
four and nine zinc fingers at its COOH terminus. Whereas the KRAB-A
domain has been identified as a protein-protein interaction motif
(12-14), the zinc fingers of Kid-1 have been shown to recognize
heteroduplex DNA (27). As a first approach to determine the functional
significance of the various motifs in the Kid-1 protein, we substituted
the zinc finger domain of Kid-1 with the zinc fingers of the yeast
transcription factor GAL4. The DNA-binding domain of GAL4 contains zinc
fingers of the C6 class; furthermore, there are no known
binding sites for GAL4 in the mammalian genome (28). Transient
transfections of COS-7 cells with the plasmid pBXG1/Kid-1N, which codes
for a fusion protein between the DNA-binding domain of yeast GAL4 and
the non-zinc finger portion of rat Kid-1, resulted in a different
staining pattern when compared with COS-7 cells transfected with
pMT3/Kid-1. Transfection of cells with pBXG1/Kid-1N yielded a
homogeneous nuclear staining; a speckled or patchy pattern like that
observed in cells transfected with pMT3/Kid-1 was never detected (data not shown). Treatment of nuclei with a buffer containing 420 mM NaCl resulted in the extraction of a sizable portion of
the GAL4/Kid-1 fusion protein into the soluble fraction (data not shown).
The results obtained with the GAL4/Kid-1 fusion protein suggested that
the zinc fingers of Kid-1 are important for the nonhomogeneous distribution of the full-length Kid-1 protein in the nucleus and its
tight association to nuclear structures. In order to provide direct
evidence for that assumption, we generated mutant Kid-1 proteins with
consecutively larger deletions from the NH2 terminus. Because these mutant proteins were tagged with the HA epitope, they
could easily be detected by immunofluorescence and on a Western blot. A
Kid-1 mutant protein lacking the KRAB-A domain was sorted to the
nucleus (Fig. 6, a and
b), but it was distributed in patches, and we never noticed
those larger speckles seen with the full-length Kid-1 protein
(approximately 1/4 the diameter of an intact nucleolus; see
below) but only much smaller ones (approximately
the
diameter of an intact nucleolus; not shown) in about 40-45% of the
transfected cells. Further deletion of the KRAB-B domain did not change
the overall distribution of the mutant Kid-1 protein (Fig. 6,
d and e). This expression pattern was maintained
when mutant Kid-1 proteins containing all 13 zinc fingers (Fig. 6, g and h) or only the second zinc finger cluster
containing zinc fingers 5-13 were expressed in COS-7 cells (Fig. 6,
m and n). The first zinc finger cluster
comprising zinc fingers 1-4 was sorted to the nucleus but was always
distributed homogeneously in the nucleus (Fig. 6, j and
k).

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Fig. 6.
The nine-zinc finger cluster of Kid-1 is
responsible for the patchy distribution and tight association in the
nucleus. HA epitope-tagged Kid-1 mutant proteins were transiently
transfected into COS-7 cells and detected by immunofluorescence
(a, d, g, j, and
m), while nuclei were stained with Hoechst 33258 (b, e, h, k, and
n). Mutant proteins containing the nine-zinc finger cluster
are distributed in patches (a, d, g,
and m), whereas the four-zinc finger cluster is distributed
homogeneously (j) in nuclei. The patchy distribution
correlates well with the tight association to as yet unidentified
nuclear structures. This is demonstrated by Western blot analysis of
nuclear extracts from COS-7 cells transiently transfected with the same
constructs (c, f, i, l, and
o). The KRAB-A domain, KRAB-B domain, and four- and
nine-zinc finger clusters (from NH2 to COOH terminus) are
shown as boxes, and deleted portions of the Kid-1 protein
are indicated by dashed lines. C,
detergent-soluble proteins; N, soluble nuclear proteins;
P, insoluble nuclear proteins. Magnification, × 400.
|
|
The expression patterns of the various Kid-1 mutants correlated well
with their extractability from the nucleus. All of the mutants
containing zinc fingers 5-13 could only inefficiently be extracted
from the nucleus, whereas the mutant protein comprising the first four
zinc fingers could easily be extracted. The presence of the
KRAB-A and KRAB-B domains had no influence on the extractability of the
proteins from the nucleus (Fig. 6, c, f,
i, l, and o).
In order to identify the nuclear compartment(s) Kid-1 was targeted to,
we used antibodies against various nuclear structures in
double-labeling experiments. Staining with a human autoantibody against
the nucleolar transcription factor UBF resulted in a perfect colocalization with the Kid-1 protein in patches, identifying the
patches as nucleoli (Fig. 7, a
and b). In nuclei with a speckled distribution of Kid-1, UBF
was also located in speckles, although the intensity of UBF staining
clearly was less than in intact nucleoli, and some speckles even lacked
UBF staining altogether (Fig. 7, c and d). The
mutant comprising zinc fingers 5-13 colocalized perfectly with UBF,
thus identifying the larger zinc finger cluster of Kid-1 as a nucleolar
targeting domain (Fig. 7, e and f). The results
obtained in COS-7 cells were corroborated by transiently expressing the
Kid-1 mutant comprising zinc fingers 5-13 in CV-1 cells and
LLC-PK1 cells. The CV-1 cell line represents the parent cell line of COS-7 cells (29); it does not express the large T antigen
of SV40, and therefore the expression levels of exogenous proteins,
encoded by plasmids with an SV40 origin of replication, are much lower
than in COS-7 cells. Both in CV-1 cells and LLC-PK1 cells, a highly differentiated porcine renal epithelial cell line (30),
the larger zinc finger cluster of Kid-1 was sorted to the nucleolus,
indicating that the nucleolar expression pattern of Kid-1 did not
depend on its expression level (Fig.
8).

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|
Fig. 7.
The nine-zinc finger cluster of Kid-1 can
serve as a nucleolar targeting signal. COS-7 cells were
transiently transfected with a plasmid coding for an HA-tagged
full-length Kid-1 protein (a-d) and with a plasmid coding
for an HA-tagged Kid-1 mutant protein comprising zinc fingers 5-13
(e and f). Double staining with a human anti-UBF
antibody (b, d, and f) and the mouse
monoclonal anti-HA epitope antibody 12CA5 (a, c,
and e) clearly demonstrated the colocalization of the Kid-1
proteins and UBI in intact nucleoli (a and b,
e and f). In cells showing a speckled
distribution of the full-length Kid-1 protein, some of the speckles
still contained Kid-1 but had lost UBF (arrows in
c and d). Magnification, × 600.
|
|

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Fig. 8.
The nine-zinc finger cluster of Kid-1 can
serve as a nucleolar targeting signal also in CV-1 cells and
LLC-PK1 cells. CV-1 cells (a, b)
and LLC-PK1 cells (c, d) were
transiently transfected with an expression plasmid coding for the HA
epitope-tagged large zinc finger cluster of Kid-1. It can be easily
seen that also in these cell lines the mutant comprising zinc fingers
5-13 was sorted to the nucleolus as demonstrated by immunocytochemical
staining with the anti-HA-epitope antibody 12CA5 (a and
c). Nuclei were counterstained with Hoechst 33258 (b and d). Magnification, × 600.
|
|
Transcription of the rDNA Genes Is Shut Off in Nucleolar
Fragments--
Expression of the Kid-1 mutant lacking only the KRAB-A
domain did not result in nucleolar disintegration, arguing that
transcriptional repression was necessary for this phenomenon. It was
puzzling, however, that KRIP-1, the adaptor protein for KRAB-zinc
finger proteins, was located in the nucleoplasm and not in the nucleoli in the absence of Kid-1 (Fig. 3). We therefore cotransfected Kid-1 and
KRIP-1 to determine whether Kid-1 could influence the location of
KRIP-1. Interestingly, in cells coexpressing Kid-1 and KRIP-1, KRIP-1
was indeed sorted to the nucleoli (Fig.
9, a and b). The hypothesis, that Kid-1 repressed rDNA transcription through its adaptor
protein KRIP-1, was directly tested by performing nucleolar run-ons
with bromo-UTP either in the absence or presence of Kid-1. Ongoing synthesis of rRNA was detected in intact nucleoli
("patches"), whereas in nucleolar fragments ("speckles") rRNA
synthesis was greatly diminished (Fig. 9, c-f).

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Fig. 9.
Transcription of rDNA is shut off by the
expression of Kid-1. a and b, in order to
determine how the expression of Kid-1 affected the distribution of
KRIP-1, both proteins were simultaneously expressed in COS-7 cells.
Using double antibody immunocytochemistry, the Kid-1 protein was
detected with the mouse monoclonal anti-Kid-1 antibody 5D12
(a) and KRIP-1 with a polyclonal rabbit anti-KRIP-1
antiserum (b). Upon expression of Kid-1, KRIP-1 could also
clearly be detected in the nucleolus, although the nucleoplasmic
staining was still present. c-f, the effect of the
expression of Kid-1 in COS-7 cells on rDNA transcription was
investigated by nucleolar run-ons. Ongoing rRNA synthesis in the
nucleolus was demonstrated by incubating COS-7 cells with bromo-UTP in
the presence of -amanitin to inhibit RNA-polymerase II and
subsequent immunocytochemistry with a mouse monoclonal
anti-bromodeoxyuridine antibody. Expression of Kid-1 was simultaneously
detected with a polyclonal rabbit anti-Kid-1 antiserum. rRNA was still
synthesized (d) in intact nucleoli containing Kid-1
(c), whereas in disintegrating nucleoli containing Kid-1
(e) the synthesis of rRNA was diminished in the remaining
intact nucleoli and almost absent in the nucleolar fragments
(f). Magnification, × 600.
|
|
 |
DISCUSSION |
When the HA epitope-tagged full-length rat Kid-1 protein was
transiently expressed in COS-7 cells, immunocytochemical analysis showed patchy and speckled expression patterns of Kid-1 in the nucleus.
Using a human autoantibody against the nucleolar transcription factor
UBF, a clear colocalization of Kid-1 and UBF in patches was detected,
demonstrating that the full-length Kid-1 protein is sorted to the
nucleolus. The nucleolus consists of at least three ultrastructurally
defined compartments: a fibrillar center, a dense fibrillar component,
and a granular component, which contain distinct proteins and serve
specific functions (for reviews, see Refs. 31-33). Transcription of
the rDNA genes in the nucleolus depends on the presence of UBF, the
TATA-binding protein, and three TATA-binding protein-associated
factors. UBF belongs to the HMG box family of transcription factors and
has been cloned from a variety of different species (e.g.
Refs. 34 and 35). The DNA binding characteristics of UBF are
remarkable, because so far no sequence-specific binding to DNA could be
demonstrated; UBF rather exhibits an affinity for cruciform DNA
(36-38). We have recently shown that both zinc finger clusters of
Kid-1 also can bind in an apparently sequence-independent manner to
heteroduplex or "bubble" DNA (27), but clearly this property is not
sufficient to confer sorting of Kid-1 to the nucleolus, because only
the larger zinc finger cluster comprising zinc fingers 5-13 is
required for nucleolar targeting, whereas the smaller zinc finger
cluster of Kid-1 was distributed homogeneously in the nucleus. The
importance of the zinc finger domains of Kid-1 becomes even more
evident by the fact that the rat, murine, and human homologues of Kid-1 possess a very high homology in their zinc finger regions and also
share the same arrangement of the zinc fingers in clusters of four and
nine zinc fingers (15, 39, 40). Therefore, although both zinc finger
clusters obviously contain nuclear localization signals similar to what
has been described for other zinc finger proteins (e.g.
Refs. 41 and 42) and both of them can bind to heteroduplex DNA (27),
they are functionally different. A similar picture has emerged for UBF
insofar as only the first HMG box is required (together with the acidic
tail of UBF) for nucleolar sorting (43), but the other HMG boxes of UBF
can bind to DNA as well (44, 45). So far we do not know the nature of
this additional signal with respect to Kid-1, but clearly it has to
reside in the large zinc finger cluster. Interestingly, the large zinc
finger cluster of Kid-1 also was responsible for the tight association
inside the nucleus to as yet unknown structures. In the case of UBF, a
tight nuclear association has been described for UBF in mitotic cells
as compared with cells in interphase; on a biochemical level, it is the
phosphorylated form of UBF that can be hardly extracted from the
nucleus (46). For both Kid-1 and UBF, it is unknown how this tight
association is mediated.
While the patchy distribution of the Kid-1 protein could be detected
early after the Me2SO shock, the appearance of the speckles was a late event after transfection, with the speckles emanating from
disintegrating patches. The double staining with the anti-UBF antibody
showed that the patchy distribution of Kid-1 corresponds to intact
nucleoli, while the speckles represent disintegrated nucleoli. The
disintegration of the nucleoli depends on the presence of the KRAB-A
domain, because transfection of cells with a mutant lacking the KRAB-A
domain resulted in a patchy staining pattern. The KRAB-A domain is a
potent transcriptional repressor motif (3-6), and the disintegration
of the nucleolus by Kid-1 therefore probably depends on the
transcriptional repression characteristics of Kid-1. This also explains
the time-dependent increase in the number of cells with
nucleolar fragments, because it requires some time before Kid-1 is
expressed, before it shuts off transcription in the nucleolus and
before the nucleolus disintegrates. Such a model is in some contrast to
the data presented by Moosmann et al. (10), who showed that
the KRAB-A domain did not repress transcription mediated by RNA
polymerase I. In their study, however, they used fusion proteins
between the KRAB-A domain and the DNA-binding domains of LexA and GAL4
on the one hand and a reporter plasmid containing only a short fragment
of the human rDNA gene promoter on the other hand (10). Because we
looked at the effect of the wild-type Kid-1 protein on nucleolar
integrity, our studies were conducted in a more natural context, where
additional cofactors for the repression of nucleolar transcription may
be present. Although there remains the possibility that the nucleolar
disintegration caused by the KRAB-A domain is not based on
transcriptional repression, we consider this unlikely in light of the
well established transcriptional repressor effects by KRAB-A domains
from a variety of different proteins (3-6) and by two additional
pieces of evidence. Whereas in the absence of Kid-1 KRIP-1 was located
in the nucleoplasm and not in the nucleolus, coexpression of Kid-1
resulted in the rerouting of KRIP-1 into the nucleolus. Most
importantly, ongoing rRNA synthesis in nucleolar fragments (speckles)
with Kid-1 labeling was greatly repressed, arguing that transcription
of the rDNA genes was shut off. This was not an immediate effect,
however, because rRNA synthesis could still be demonstrated in intact
nucleoli (patches) with Kid-1 labeling.
Nucleolar fragments can also be observed under other circumstances
(e.g. during apoptosis (47), at the end of mitosis, or experimentally induced). During the cell cycle, the nucleoli
disassemble at prophase and reappear in telophase, when they are
reassembled from the nucleolus organizer region and prenucleolar
bodies. The nucleolus organizer region contains proteins of the rDNA
transcriptional machinery such as RNA polymerase I and UBF
(e.g. Refs. 48-50), which remain bound to the nucleolus
organizer region even during mitosis. Prenucleolar bodies, however,
appear de novo in late mitosis and contain primarily
proteins of the dense fibrillar component such as fibrillarin,
nucleolin, and B23/NO38 (summarized in Ref. 51). The coalescence of
nucleolus organizer regions and prenucleolar bodies can be prevented by
the DNA topoisomerase I inhibitor camptothecin (52) and by
microinjecting antibodies against RNA polymerase I into mitotic cells
(53), which points to the importance of rDNA gene transcription in the
reformation of the nucleolus. Incubation of cells with the adenosine
analogue 5,6-dichloro-1-
-D-ribofuranosylbenzimidazole,
which suppresses mRNA synthesis but does not inhibit or only
moderately inhibits rRNA synthesis, leads to the disintegration of the
nucleolus into a structure called the nucleolar necklace (54). In
contrast to prenucleolar bodies, the nuclear necklace still contains
RNA polymerase I (55). At this point, we do not know whether the speckles generated by the expression of Kid-1 correspond to any of
those structures. The disappearance of UBF from some of the speckles
containing Kid-1 would argue that those speckles correspond to
prenucleolar bodies. Interestingly, one of the mechanisms by which UBF
activates transcription of the rDNA genes probably lies in the
replacement of the Ku antigen, a negative regulator of rDNA gene
transcription, from the rDNA promoter (56). It therefore seems possible
that Kid-1 can replace UBF from the rDNA promoter, but clearly more
markers are needed to characterize the nature of the Kid-1 speckles.
At this point, we do not know in which biological context Kid-1 exerts
its function. Previous studies have shown that Kid-1 is connected to
heterochromatin through its adaptor protein KRIP-1 (12-14, 57). The
connection between the nucleolus and heterochromatin has also been made
for Modulo, a modifier of position-effect variegation in
Drosophila (58), and for Zfp37, a murine
Cys2His2-zinc finger protein with a truncated
KRAB-A domain that is expressed in neuronal cells (59). In the case of
Modulo, it has been hypothesized that the nucleolus balances the amount
of Modulo available for heterochromatin formation and position-effect
variegation (58). Besides Kid-1 and Zfp37, three other zinc finger
proteins have been reported to reside in the nucleolus so far. LYAR, a
protein with a potential RING finger motif (60), has been shown to
enhance tumor formation (61), whereas PAG608, a protein with three
Cys2His2-zinc fingers, can promote apoptosis
(62). So far, no effect on growth has been reported for MOK2, another
Cys2His2-zinc finger protein shown to reside in
the nucleolus (63). Because of the shutdown of nucleolar transcription
by Kid-1 and the vital role of rRNA synthesis for a cell, it is likely
that Kid-1 also affects cell growth. Interestingly, disintegration of
the nucleolus represents one of the earliest morphological changes
during apoptotic cell death (47). In addition to Kid-1, a speckled
distribution has also been reported for the KRAB-zinc finger proteins
ZNF74 (25) and Zfp59 (64), but both ZNF74 and Zfp59 only contain a
truncated KRAB-A domain at their NH2 termini, which very
likely is inactive so that the respective zinc finger domains probably
are important for the targeting of either protein to its specific
nuclear compartment. More information about the protein network, which
these zinc finger proteins are part of, will help us to better
understand their function in molecular and cell biological terms.
 |
ACKNOWLEDGEMENTS |
We acknowledge the kind gifts of materials
from John Kyriakis (pMT3), Ingrid Grummt (anti-UBF antiserum), Herwig
Ponstingl (anti-NuMA antiserum), and E. Brigitte Lane (anti-lamin B
antibody) without which this work would not have been possible. We
thank Rachel Gallagher for critically reading the manuscript. The
expert photographic work of Ingrid Ertel and graphics of Rolf
Nonnenmacher are gratefully acknowledged.
 |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft Grants Wi 1042/2-1 and Wi 1042/2-2 (to R. W.)
and National Institutes of Health Grant DK39773 (to J. V. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: University of
Heidelberg, Institute of Anatomy and Cell Biology I, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany. Tel.: 49-6221-548686; Fax: 49-6221-544951; E-mail: ralph.witzgall{at}urz.uni-heidelberg.de.
 |
ABBREVIATIONS |
The abbreviations used are:
HA, hemagglutinin;
PBS, phosphate-buffered saline;
UBF, upstream binding factor.
 |
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