From the § CIHR Group in Periodontal Physiology and the
Department of Biochemistry, University of Toronto,
Toronto, Ontario M5S 3E2, Canada
Received for publication, December 1, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Osteogenic differentiation involves a cascade of
coordinated gene expression that regulates cell proliferation and
matrix protein formation in a defined temporo-spatial manner. Here we have used differential display to identify a novel zinc finger transcription factor (AJ18) that is induced during differentiation of
bone cells in vitro and in vivo. The 64-kDa
protein, encoded by a 7- kilobase mRNA, contains a
Krüppel-associated box (KRAB) domain followed by 11 successive
C2H2 zinc finger motifs. AJ18 mRNA, which
is also expressed in kidney and brain, is developmentally regulated in
embryonic tibiae and calvariae, with little expression in neonate and
adult animals. During osteogenic differentiation in vitro
AJ18 mRNA is expressed as cells approach confluence and declines as
bone formation occurs. Using bacterially expressed, His-tagged AJ18 in
a target detection assay, we identified a consensus binding sequence of
5'-CCACA-3', which forms part of the consensus element for
Runx2, a master gene for osteogenic differentiation. Overexpression of AJ18 suppressed Runx2-mediated transactivation of an
osteocalcin promoter construct in transient transfection assays and reduced alkaline phosphatase activity in bone morphogenetic protein-induced C3H10T1/2 cells. These studies, therefore, have identified a novel zinc finger transcription factor in bone that can
modulate Runx2 activity and osteogenic differentiation.
The characterization of bone morphogenetic proteins
(BMPs1), their
serine/threonine kinase membrane receptors, and downstream Smad
effectors (reviewed in Refs. 1-3) along with the identification of
Runx2/Cbfa-1/Osf2 (Runt domain
factor 2/core binding factor BMPs were originally identified by their ability to induce ectopic bone
formation (6, 7). This unique bone-inductive activity indicates that
BMPs provide the primordial signals for osteodifferentiation, as
supported by the BMP-induced expression of Runx2. As a sub-group of the
TGF- To characterize genes involved in osteoblastic differentiation, we used
differential display to identify genes expressed by proliferating fetal
rat calvarial cells (FRCCs) that were up-regulated by BMP-7 (osteogenic
protein-1; OP-1). From an initial screen we identified a novel gene,
provisionally named AJ18 (19). Here we report the
characterization of AJ18 as a zinc finger transcription factor that is
transiently up-regulated during osteoblastic differentiation and that
appears to modulate osteogenic differentiation through effects on
Runx2 activity.
Cell Culture--
Primary FRCCs and rat bone marrow cells
(RBMCs) were prepared and cultured as described previously (20).
FRCCs, RBMCs, and the rat osteosarcoma cell line, ROS 17/2.8, were
grown in 10% fetal bovine serum, RNA Extraction--
Total RNA was extracted from cell culture
and tissues as described by Chomczynski and Sacchi (21).
Differential Display--
Differential display was performed
using the RNAimage kit (GenHunter, Nashville, TN) following the
manufacturer's instructions and is described in Jheon et
al. (19).
5'-Rapid Amplification of cDNA Ends (5'-RACE)--
The
Marathon cDNA amplification kit (Clontech, Palo Alto, CA) was used
according to the manufacturer's instructions. Briefly, using 1 µg of
poly(A) RNA from rat brain, a library of adapter-ligated double-stranded cDNA was constructed. An antisense oligonucleotide (5'-ACTGATTGGCTGACCCAGAGTAT-3') specific for the 3'-untranslated region
of AJ18 was used with the kit primer AP-2 to PCR-amplify the upstream
sequence. The 5'-RACE product was subcloned into pBluescript II SK
(Stratagene, La Jolla, CA) and sequenced.
Northern Blot Hybridization--
Preparation of cDNA probes
for bone sialoprotein, osteopontin, osteocalcin, osteonectin/SPARC,
collagen-1, alkaline phosphatase, and glyceraldehyde-3-phosphate
dehydrogenase are described in Li et al. (22); mouse
Runx2/Osf2 (pLA-Oa4) cDNA was provided by Dr. G. Karsenty
(Baylor College of Medicine, Houston, TX). Various tissues from
developing embryonic and adult rat tissues were prepared as described
previously by Chen et al. (23). A mouse embryonic total RNA
blot was purchased from CLONTECH. Northern blot
hybridization was performed as described in Jheon et al. (19).
Semi-quantitative PCR and Southern Blot--
Total RNA (1 µg)
from various rat tissues and RBMCs was reverse-transcribed using
Moloney Reverse Transcriptase (Life Technologies). AJ18-specific
primers (5'- GGAGAACTAAGAAGGGAAATGGCTG-3' and
5'-CAGGCTTCTCCCCCTTCAGACACCT-3'), rat Runx2 primers
(5'-AACCGCACCATGGTGGAGATCAT-3' and 5'-TGAGGCGGGACACCTACTCTCATA-3'), and
Bacterial Expression and Target Detection Assay--
Full-length
AJ18 was PCR-amplified using primers
(5'-CCGCATGCCTGTGGATTTGCTGGC-3' and
5'-CCTCTCTGCTTGTGTCCTGGATCA-3') and inserted in-frame into
SphI and SmaI sites downstream of the N-terminal 6xHis tag of pQE32 (Qiagen, Ontario, Canada) to produce His-AJ18 (His-AJ). Truncated AJ18 (His-ZF) was prepared by excising the zinc
finger region with SacI and SalI, and re-ligating
this fragment in-frame into pQE32. The Escherichia coli
strain, M15[pREP4] (Qiagen), was transformed with full-length or
truncated AJ18-pQE32 constructs and plated on Luria-Bertani (LB)
agarose (Difco Laboratories, Detroit, MI) plates containing 25 µg/ml
kanamycin and 100 µg/ml ampicillin. Single colonies were grown in
culture, and the expression of full-length and truncated His-AJ18 was
induced with 1 mM
isopropyl- Anti-AJ18 Polyclonal Antibodies--
Peptides spanning amino
acid residues 2-13 (AVDLLAARGTEP; anti-AJ18-1) and 158-169
(EDGIPTDPEELK; anti-AJ18-2) of the AJ18 sequence were synthesized and
conjugated to keyhole limpet hemocyanin protein and to bovine serum
albumin (BSA) by Alberta Peptide Institute (Alberta, Canada). The
keyhole limpet hemocyanin peptides were used by Cedarlane Laboratories
(Ontario, Canada) to generate antiserum in rabbits from which
affinity-purified antibodies were isolated using BSA immobilized to
CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech). The IgG was
eluted from the column using glycine-HCl buffer (0.05 M
glycine, 0.15 M NaCl, pH 2.3), and was immediately adjusted
to pH 7 with 0.1 N NaOH. Anti-HisG monoclonal antibody was
purchased from Invitrogen (Carlsbad, CA).
Immunohistochemical Analysis--
Immunoperoxidase staining for
AJ18 protein in formalin-fixed paraffin-embedded sections of tibia from
4-week-old rats was performed using the Vectastain ABC kit (Vector
Laboratories, Burlingame, CA) following the manufacturer's
instructions. Briefly, 6-µm sections were mounted on SuperFrost/Plus
glass slides (Fisher, Ontario, Canada), dewaxed, and rehydrated through
graded alcohols to water. Sections were incubated in blocking solution
(5% BSA, 2% normal goat serum) for 1 h. Affinity-purified
anti-AJ18-1, or anti-AJ18-2, antibodies were applied, and tissue
sections were incubated for 1 h. The sections were washed and
treated with biotinylated anti-rabbit IgG for 30 min, followed by
incubation with peroxidase-labeled streptavidin for 30 min, and
subsequently incubated with diaminobenzidine tetrahydrochloride and
H2O2 for 15 min. All incubations were performed at room temperature (21 °C). Sections were counterstained with hematoxylin. The stained sections were visualized under a light microscope and photographed.
Transient Transfections and Fluorescence
Microscopy--
Full-length AJ18 and truncated AJ18 were PCR-amplified
with KlenTaq (CLONTECH) using antisense primer
5'-GCGGTACCAGGGATGAATTAAGGTCCTCAGGCTTCT-3' and sense
primers 5'-AAGGTACCCGCCACCATGGCTGTGGATTTGCTGGCTGCTCGA-3' and 5'-AAGGTACCCGCCACCATGTATCACACTTCAGAGAAAGATTTA-3',
respectively. The fragments were digested with KpnI
and inserted in-frame into a KpnI site of the pEGFP-N1
plasmid (clontech) to produce AJ18-GFP and ZF-GFP. ROS 17/2.8 cells
were plated at 20,000 cells/well on 8-well chamber slides and grown
overnight. Cells were transfected with 2 µg of plasmid using
LipofectAMINE 2000 (Life Technologies), grown for 24 h, and fixed
in 4% paraformaldehyde-phosphate-buffered saline. The nuclei were
stained with 4',6'-diamidino-2-phenylindole (DAPI) for 5 min and
visualized under a fluorescence microscope and photographed.
Transcription Assay--
AJ18 was PCR-amplified using antisense
primer (5'-GCGGTACCTGGATCAGAGGGATGAATTAAGGTC-3') and
primers described above and inserted into a KpnI site of the
pcDNA plasmid (Invitrogen) to produce AJ18-pcDNA and
ZF-pcDNA. Sense and antisense plasmids of AJ18-pcDNA and
ZF-pcDNA were purified using a midi-prep procedure (Qiagen). C3H10T1/2 cells were plated at 50,000 cells/well in 24-well dishes and
grown for 24 h. Total DNA (2 µg), including 0.5 µg of
p6OSE2-luc (6xOSE2) and 0.3 µg of Runx2/Osf2 (both plasmids
generously provided by Dr. G. Karsenty), 0.01-1 µg of AJ18-pcDNA
(sense, AJ18-S; antisense, AJ18-AS) or 0.01 µg and 1 µg of
ZF-pcDNA (ZF-S), and 0.2 µg of pSV- Alkaline Phosphatase Activity--
Plasmids were transfected
into C3H10T1/2 cells as described in the transcription assay above.
BMP-7 was added 5 h after transfection, and the cells were grown
for 48 h. The cells were fixed in 4% paraformaldehyde and
alkaline phosphatase activity was observed by staining the cells using
Naphthol AS-MX Phosphate and Fast Red TR (Sigma) in 100 µl of 1 M Tris-HCl and 0.1 M MgCl2. Total alkaline phosphatase activity was measured as described by Li et
al. (22).
Sequence Analysis--
A search of the non-redundant and
expressed sequence tag data bases (GenBankTM/EBI Data Bank) for
the 5'-RACE product was performed using the BLAST program (27). The
open reading frame and its translated product were determined using the
Analyze program (MacMolly Tetra version 2.5). Amino acid sequences of
KRAB domains were retrieved from GenBankTM/EBI and aligned using the
ClustalW algorithm (28).
Identification and Sequence Analysis of AJ18--
We first
performed differential display using proliferating (80% confluence)
and differentiating (confluent) FRCCs to show that AJ18 is induced
during osteoblastic differentiation (Fig. 1A). The 313-bp gene fragment
was subcloned and subsequently used as a probe to perform Northern blot
hybridization to confirm the differential display results (Fig.
1B). An mRNA of ~7 kb was visualized that was
expressed at 5-fold levels higher in differentiating FRCCs. In
comparison, the levels of another transcription factor, Ets1, were not
altered significantly. A 2514-nucleotide fragment was generated by
5'-RACE amplification of the 313-bp gene fragment. Sequence analysis
revealed an open reading frame of 560 amino acid residues extending
from the first ATG codon, located at nucleotide 175, to a termination
TGA codon present at nucleotide 1855 (Fig. 2). The N-terminal 85 amino acids
constitute a KRAB domain and, beginning at amino acid 220, a series of
11 C2H2 zinc finger motifs was identified that
extend almost to the end of the protein sequence (Fig. 2). Alignment of
N-terminal sequence of AJ18 with the KRAB domains of several
C2H2 zinc finger proteins revealed at least two
distinct subfamilies of KRAB/C2H2 proteins
(Fig. 3). Those sequences shown
above the AJ18 sequence represent a subfamily of proteins
containing KRAB A box alone, whereas sequences below AJ18
represent a subfamily of proteins with both KRAB A and B box
domains.
AJ18 Is Expressed during Osteoblast Differentiation and Bone
Development--
Northern hybridization analysis of RNA isolated from
FRCCs at various stages of osteodifferentiation showed that AJ18 is
first detected as cells approached confluence, reaching maximal levels as the cells multilayer and is subsequently down-regulated as the
mineralization of the bone-like nodules begins (Fig.
4). These stages of osteodifferentiation
are characterized by the expression of bone matrix proteins (reviewed
in Ref. 29) with alkaline phosphatase and osteopontin mRNAs being
expressed as early differentiation markers, the increase in collagen
mRNA reflecting early bone nodule formation, and the expression of
bone sialoprotein and osteocalcin mRNAs as early and late
indicators of mineralization (20), respectively. Notably, osteopontin
expression is markedly elevated in response to mineralization, whereas
the mRNAs for the other matrix proteins, with the exception of
osteonectin/SPARC, are down-regulated. In vivo, AJ18
mRNA expression could be observed by Northern hybridization during
embryonic bone formation in rat calvariae and tibiae, but little
expression was evident in neonate and adult bone tissues (Fig.
5). The expression of AJ18 mRNA was
not limited to bone but was also expressed during the embryonic
development of kidney and brain with strong expression still evident in
the adult rat brain (Fig. 5). The expression of AJ18 protein was shown
by immunohistochemical staining sections of tibia from 4-week-old rats
using anti-AJ18 polyclonal antibodies (Fig.
6). AJ18 protein was detected in the nuclei of hypertrophic chondrocytes and osteoblastic cells in the
transition zone of the growth plate where osteogenesis is still
evident. Both antisera gave identical results.
AJ18 Is Localized to the Nucleus through Its Zinc Finger
Region--
To determine whether AJ18, as a putative zinc finger
transcription factor, is localized in the nucleus of osteoblastic
cells, we transfected ROS 17/2.8 cells at 50% confluence with an empty pEGFP-N1 vector, or the same vector in which either a full-length AJ18
cDNA (AJ-GFP) or a truncated AJ18 cDNA (ZF-GFP) were
incorporated. ZF-GFP lacked the coding region for the KRAB domain and
120 of the 150 amino acids comprising the linker region but retained the sequence for all the zinc fingers. After 24 h, the cells were fixed and visualized by fluorescence microscopy (Fig.
7). Although cells transfected with the
empty vector showed a generalized fluorescence in the cytoplasm and
nucleus, cells transfected with either AJ-GFP or ZF-GFP showed distinct
nuclear localization. Thus, the KRAB domain was not required for
nuclear localization, whereas the presence of the zinc finger region
and/or remaining 30 amino acids of the linker region appeared to be
sufficient for directing nuclear localization of AJ18.
AJ18 Shows Selective Binding to dsDNA--
The presence of zinc
finger motifs in AJ18 is indicative of an ability of this protein to
bind to dsDNA. Therefore, to determine the DNA-binding ability of AJ18,
a His-tagged version of AJ18 (HIS-AJ) was prepared by expressing the
protein in bacteria. SDS-PAGE analyses revealed a major IPTG-induced
protein band of 64 kDa, corresponding to the hypothetical molecular
mass of AJ18 (Fig. 8A,
I versus NI). In addition, a second
band was also evident at ~35 kDa. To produce a protein in which the
zinc finger domain was isolated from the KRAB domain, AJ18 was
truncated at the 5'-end creating HIS-ZF, producing a construct similar
to ZF-GFP described above. A protein band consistent with the predicted
size of the truncated protein (44 kDa) was induced with IPTG (Fig.
8B). Western blot analyses showed that both the 64- and
35-kDa proteins generated by HIS-AJ18 and the 44-kDa protein generated
by HIS-ZF were recognized by a monoclonal anti-HisG antibody (HIS) and
a polyclonal antibody raised to a peptide corresponding to amino acids
158-169 in the AJ18 protein sequence. Thus, the 35-kDa protein appears
to be a spontaneously truncated form of AJ18 that represents the
N-terminal half of the protein and retains the His-tag.
To study DNA binding using the target detection assay, His-tagged
proteins were transferred to nitrocellulose and renatured in the
absence ( AJ18 is Co-expressed with Runx2 and Modulates Its Transcriptional
Activity--
Because the DNA binding studies indicated that AJ18
could modulate the activity of Runx2, we first examined the temporal
expression of AJ18 and Runx2 in primary rat bone marrow cells (20),
grown in the presence of 10 nM dexamethasone to stimulate
osteogenic differentiation (Fig.
9A). Similar expression
profiles for the two proteins were evident, with both AJ18 and Runx2
mRNA being expressed at maximal levels as the cells reached
confluence and began to differentiate. Although AJ18 and Runx2
expression was down-regulated as mineralized bone nodules were being
formed, both mRNAs increased again at 21 days, after bone nodule
formation had been completed. The maximal expression of these
transcription factors at the onset of bone formation corresponded to
their expression profile in vivo (Fig. 9B). To
determine whether AJ18 might disrupt Runx2 transcriptional activity by
competing for the OSE2, transient transfection assays were performed
using an osteocalcin-luciferase reporter construct in which six OSE2
sequences had been incorporated (30). When a full-length AJ18
expression vector (AJ18-S) was co-transfected with the
Runx2/Osf2 expression vector, the induction of transcription
observed with Runx2/Osf2 was markedly suppressed in a
dose-dependent manner, whereas the expression vector with antisense AJ18 (AJ18-AS) was without effect (Fig.
10). Similarly, a
dose-dependent reduction of Runx2/Osf2-induced
transcription was observed with the truncated AJ18 vector (ZF-S).
Overexpression of AJ18 Suppresses Alkaline Phosphatase
Expression--
Because alkaline phosphatase is an early marker of
osteogenesis that is induced downstream of Runx2, following stimulation of C3H10T1/2 cells with BMP (31), we examined the effects of AJ18 on
the BMP-7-induced osteogenesis in these cells. When C3H10T1/2 cells
were transiently transfected with the AJ18 expression vector, the
induction of alkaline phosphatase by BMP-7 (400 ng/ml) observed in
non-transfected cells and cells transfected with the empty vector, was
suppressed. This was evident in cultures stained for alkaline
phosphatase activity, and from quantitative assessment, a
reduction in alkaline phosphatase activity of almost 40% was calculated (Fig. 11).
Although more than one hundred members of the
KRAB/C2H2 zinc finger protein family have been
described (32, 33), little is known of their biological function.
Moreover, few target DNA sequences or target genes have been identified
for KRAB/C2H2 proteins (34). In this study, we
have isolated and characterized a novel zinc finger transcription
factor, provisionally named AJ18, that is developmentally
expressed in bone and that appears to regulate osteoblastic
differentiation. AJ18 contains 11 C2H2 zinc
finger motifs and an N-terminal Krüppel-associated box domain
(KRAB), which is believed to function as a repressor of transcription. We show that the zinc finger region of the molecule is involved in
nuclear targeting and for selective binding of AJ18 to dsDNA containing
a 5'-CCACA-3' sequence, including the OSE2 for Runx2. Our studies
further show that AJ18 can suppress the osteogenic effects of Runx2, a
transcription factor that is crucial for bone development (5), and
alkaline phosphatase activity in BMP-7-stimulated C3H10T1/2 cells. To
our knowledge, this study is the first to identify a
KRAB/C2H2 protein that is involved in
regulating osteoblastic differentiation.
The C2H2 or TFIIIA/Krüppel-type zinc
finger proteins comprise a large family of genes that is divided into
two classes according to the number of zinc finger motifs contained
within the protein sequence (35). In one class are zinc finger genes
that code for proteins such as Egr-1, Sp1, and
WT1, with fewer than five zinc finger motifs. The proteins
in the group have generally been identified as transcriptional
activators or repressors involved in cell proliferation and
differentiation. The proteins expressed by the second class of zinc
finger genes have more than five zinc finger motifs and include AJ18.
Although these genes are more abundant, apart from TFIIIA, which binds
to both the 5S RNA gene and to 5 S RNA (36) and MZF1, which
regulates the CD34 gene (37), the biological function of the
proteins expressed by these genes is largely unknown.
Approximately, one-third of all C2H2 zinc
finger proteins contain a KRAB domain, which is not present in yeast
proteins and appears to have evolved with multicellular organisms as a
transcriptional repressor (38). KRAB domains can be separated into
three subfamilies based on nucleic acid sequence alignment (34):
subfamilies containing a KRAB A box alone, both A and B boxes, or an A
box with a divergent B box. Based on amino acid (Fig. 3) and nucleic
acid (data not shown) sequence alignments, AJ18 appears to be a member
of the subfamily of genes possessing a classical KRAB A and divergent B
box. Notably, the A domain alone is sufficient for repressor activity,
whereas the B domain has a lesser contribution (38-40). The KRAB
domain associates with TIF1 Consistent with zinc finger proteins acting as transcription factors,
we have shown, using an AJ18-GFP fusion protein and immunohistochemical
analysis, that AJ18 is localized to the nucleus (Fig. 6, 7) and that it
binds a consensus DNA binding site that includes a 5'-CCACA-3' sequence
(Fig. 8C). Moreover, analyses of the expression of a
truncated form of AJ18 lacking the N-terminal KRAB domain revealed that
the KRAB motif is not required for nuclear targeting or for DNA
binding. We recognized that the DNA binding sequence for AJ18 is
present within the consensus binding sequence for Runx2 (OSE2), which
is present in the promoters of several genes, including
osteopontin and osteocalcin that are involved in
bone formation (31). Our studies show that the OSE2 binds, under high
stringency conditions of the target detection assay, to AJ18 in the
presence of Zn2+ (Fig. 8D). Notably, we were
unable to establish conditions for electrophoretic mobility shift
assays, suggesting that stabilization of the protein structure,
afforded by the target detection assay, may be required for DNA
binding. In this regard, ZNF74, a KRAB/C2H2 protein whose primary sequence is similar to AJ18, has been shown to
interact strongly with the nuclear matrix (46). Thus in
vivo, full-length AJ18 could strongly suppress transcriptional
activity induced by Runx2 in a 6xOSE2-luciferase reporter gene in a
dose-dependent manner (Fig. 10). This suppression could be
mediated by the KRAB domain recruiting co-repressors such as
TIF1 That AJ18 may modulate Runx2-mediated osteogenic differentiation is
indicated by the suppression of alkaline phosphatase expression in
BMP-7-stimulated CH310T1/2 cells transfected with an AJ18 expression vector (Fig. 11). Although not an immediate target of Runx2, alkaline phosphatase is an early marker of osteogenic differentiation
that is required for mineralization (47). Analysis of the
temporal expression of AJ18 and Runx2 during bone formation in
vivo and in vitro is also consistent with an
interactive role of these transcription factors (Fig. 9), although the
relative level of expression of the two proteins within the same cell
is currently unknown. Both AJ18 and Runx2 are up-regulated early and
are maximally expressed as osteoblastic differentiation occurs; the
mRNA expression of both proteins being down-regulated as bone
tissue formation is underway. However, the expression of AJ18 in other
embryonic tissues, including kidney and brain, indicates a more general role for AJ18 in organogenesis.
In summary, we have characterized a novel zinc finger transcription
factor, expressed early in bone formation, which has the
potential to modulate the osteo-inductive activities of Runx2.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1/osteoblast specific factor 2;
reviewed in Ref. 4) as a master gene for osteogenesis (5) has
established a template for osteogenic differentiation. However, the
molecular mechanisms linking the osteogenic effects of BMPs and Runx2
and the expression of a bone matrix by osteoblastic cells are still
largely unknown.
superfamily, BMPs signal through type I and type II
serine/threonine receptors on the cell surface (8). Upon ligand
stimulation, the type I receptor phosphorylates a family of Smad
proteins. Smad1, Smad5, and Smad8 mediate BMP signaling (9-11) whereas
Smad2 and Smad3 mediate TGF-
signaling (12, 13). These
receptor-regulated Smads form a complex with the common partner Smad
(Smad4) and translocate to the nucleus, where they interact with other
transcription factors, including Xenopus FAST1 and its
mammalian homologues and also the c-Jun·c-Fos complex, to
regulate the transcription of target genes. A murine isoform of Runx2
was first identified as a binding protein of an osteoblast-specific
"AACCACA" enhancer element (OSE2) of the osteocalcin
gene (14). Runx2-dependent gene expression increases in
parallel with osteogenic differentiation (14, 15), and loss of
Runx2 by homozygous gene deletion in mice arrests skeletal tissue development (16, 17). Moreover, Runx and Smads have been shown
to functionally interact and stimulate transcription of the germline Ig
C
promoter for which binding of both factors to their specific
binding sites is essential (18). Although the Runx·Smad complex
likely regulates genes involved in the differentiation of bone, target
genes for this complex have yet to be identified.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium (Sigma
Chemical Co., St. Louis, MO), and antibiotics (100 µg/ml penicillin
G, 50 µg/ml gentamicin, and 0.3 µg/ml fungizone). Dexamethasone (10 nM) was added to the RBMC cultures. For time-course
experiments, FRCCs and RBMCs were plated at 90,000 cells/60-mm dish,
50 µg/ml ascorbate was added at confluence, and 10 mM
-glycerophosphate was added at the onset of nodule formation. The
mouse fibroblast-like cell line, C3H10T1/2, was obtained at passage 8 from the American Type Culture Collection (Rockville, MD). All
experiments on these cells, which were maintained in 10% fetal bovine
serum and basal medium essential (Life Technologies, Ontario,
Canada), were performed between passage 10 and 15. All cells were grown
in a humidified air/CO2 (19:1) mixture at 37 °C.
-actin-specific primers (5'-CACCCTGTGCTGCTCACCGA-3' and
5'- ACCTGGCCGTCAGGCAGCTC-3') were used to PCR-amplify AJ18, Runx2, and
-actin, respectively. The PCR products were amplified for 22 cycles with Taq polymerase (Life Technologies) and
separated on a 1.5% agarose gel. The gel was placed in denaturation
buffer (0.5 M NaOH, 1.5 M NaCl) for 30 min and
washed in neutralization buffer (0.5 M Tris, 0.2× SSC) for
30 min. The DNA was transferred and probed for AJ18 or Runx2
essentially as described in "Northern Blot hybridization" above.
-D-thiogalactoside (IPTG) 5 h, and the
whole lysate collected. Whole cell lysates were separated by SDS-PAGE
and transferred to a nitrocellulose membrane (Schleicher and Schuell,
Keene, NH). The target detection assay was adapted from the methods
described by Thiesen and Bach (24), and Sukegawa and Blobel (25). The
membrane was renatured overnight at 4 °C in 10 ml of 50 mM Tris-Cl, 100 mM KCl, 1% Triton X-100, 10%
glycerol (pH 7.5) in the absence (+50 mM EDTA + 10 mM dithiothreitol) or presence (+1 mM
ZnCl2) of zinc. Double-stranded (ds) DNA was prepared from
oligonucleotide TDA
(5'-CGCTCTAGAACTAGTGGATC-N12-ATCGATACCGTCGACCTCGA-3') using
KS primer (5'-TCGAGGTCGACGGTATCGAT-3') in a 50-µl reaction containing
1 µM TDA oligonucleotide, 1 µM KS primer,
1.5 µM MgCl2, 1 mM dNTPs, 10 µCi of [
-32P]dCTP (Amersham Pharmacia Biotech,
Quebec, Canada), 1× PCR buffer, and 2.5 units of Taq
polymerase that was heated to 94 °C for 30 s, annealed at
52 °C for 2 min, and extended at 72 °C for 10 min. DNA was
purified though a ProbeQuant column (Amersham Pharmacia Biotech), and
1 × 105 cpm/ml was hybridized to the membrane in
renaturation buffer (see above) in the absence (+10 mM EDTA + 2 mM dithiothreitol) and presence (+0.1 mM
ZnCl2) of zinc at 4 °C overnight. The membrane was
washed for 6 h at 4 °C in 100 mM KCl, and the
amount of DNA bound was visualized using a PhosphorImager and
ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA). The positive
band was aligned to the blot and excised. The excised membrane was
washed in 400 mM KCl and then eluted with 500 µl of 1 M KCl of which 5 µl was amplified by PCR using SK
(5'-CGCTCTAGAACTAGTGGATC-3') and KS primers using conditions described
above but amplified for 20 cycles. The amplified product was used for
the next round of selection and was repeated for a total of five
rounds. After five rounds, the product was PCR-amplified for 35 cycles,
5'-end-phosphorylated using T4 polynucleotide kinase (Life
Technologies), ligated into pBluescript, and the clones sequenced.
-Gal (Amersham Pharmacia
Biotech) was transfected using LipofectAMINE 2000, and the cells were
grown for 48 h. Luciferase assays were performed as we have
described previously (26).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of a 313-bp gene fragment
up-regulated in differentiating bone cells. A,
differential display was performed using primers polyT-A
(5'-AAGCTTTTTTTTTTTTA-3') and arbitrary primer 18 (5'-AAGCTTTTCGGAC-3'). Experiments were performed in duplicate using
0.2 µg (0.2) or 1 µg (1) of starting total RNA, from proliferating
(P) and confluent (C) FRCCs, to minimize
artifactual amplification. A differentially amplified 313-bp fragment
(marked by an arrow) separated on a 6% sequencing gel was
identified by radioautography and excised from the gel, re-amplified,
cloned, and sequenced. B, Northern blot hybridization was
performed on the original RNA isolated from the FRCCs using the 313-bp
gene fragment to probe for the expression of the corresponding
mRNA. A ~7-kb mRNA, identified by the 313-bp probe, was
expressed at ~5-fold higher levels in confluent cells (C),
whereas the expression of another transcription factor, Ets1, was not
altered significantly. A cDNA probe to 18 S rRNA was used as a
control for RNA loading.
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Fig. 2.
Primary structure of rat AJ18.
Nucleotide and predicted amino acid sequence of rat AJ18.
Krüppel-associated box A (KRAB A) domain is dot
underlined; KRAB B domain is dash underlined; 11 conserved C2H2 zinc finger motifs are
underlined. A polyadenylation signal (AATAAA) in the
3'-untranslated region is underlined. The 313-bp gene
fragment isolated from the differential display is in
italics.
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Fig. 3.
Amino acid sequence alignment of multiple
KRAB domains. The KRAB domains of 16 zinc finger proteins are
aligned with AJ18, using the ClustalW program. All sequences show a
conserved KRAB A box domain. Amino acid sequences shown
above the AJ18 sequence lack the KRAB B box, while sequences
beneath the AJ18 sequence show conservation of the KRAB B
box domain. The consensus sequence is shown at the bottom,
and conserved sequences are on a gray background. Data base
accession numbers for the zinc finger proteins are as
follows: MZF13, AAF79949; ZNF136, P52737; KRAZ1, AB024224;
MZF31, AAF79951; ZFP93, Q61116; rKr2, AAB60512; ZFP97, NP_035895; HZF4,
Q14588; ZNF45, NP_003416; HZF6, AAD12728; ZNF85, Q03923; ZNF91, Q05481;
KOX1, g549835; MZF22, AAF79950; KOX31, Q06730; and ZFP228,
Q9UJU3.
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Fig. 4.
Temporal expression of AJ18 and
bone-associated genes during FRCC differentiation. Total RNA (15 µg) extracted at various days during FRCC differentiation was
hybridized with AJ18 cDNA probe. The blot was stripped and
rehybridized with cDNA from bone sialoprotein (BSP),
osteopontin (OPN), osteocalcin (OC),
osteonectin/SPARC (OCN), collagen-1 (COL), and
alkaline phosphatase (ALP). Glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as loading controls.
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Fig. 5.
AJ18 is differentially expressed during rat
development. Various tissues were isolated from rats at different
embryonic (E), neonate (N), and adult stages.
A, the expression levels of AJ18 mRNA from calvaria
(C), tibia (T), liver (L), kidney
(K), and brain (B) were analyzed using
semi-quantitative PCR and Southern blot hybridization. Levels of
-actin were used as loading controls for PCR. B, to
compare expression of AJ18 in a range of tissues from an adult rat,
various tissues were isolated, and the expression of AJ18 mRNA
analyzed by Northern blot hybridization. Lanes 1-9 in the
top panel contain total RNA from muscle (M),
kidney, liver, lung (Lu), brain, ovary (O),
spleen (S), intestines (I), and heart
(H), respectively. The lower panel shows ethidium
bromide-stained 28 S and 18 S rRNA, which were used as loading
controls.
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Fig. 6.
Expression of AJ18 protein in the growth
plate of rat tibiae. The left panel shows a
section of the tibial growth plate from a 4-week-old rat that has been
immunostained with an affinity-purified anti-AJ18 polyclonal antibody.
AJ18 is localized to the nucleus in hypertrophic cartilage cells and
osteogenic cells, as shown by a red/brown stain.
The right panel shows immunostaining without primary
anti-AJ18 antibody. The sections were counterstained with hematoxylin.
Black arrows indicate hypertrophic chondrocytes; white
arrows indicate osteoblastic cells on the surface of newly forming
bone.
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Fig. 7.
KRAB domain is not required for nuclear
localization. The left panels show full-length AJ18-GFP
fusion protein, truncated AJ18 (ZF-GFP) or empty pEGFP
vector expressed in ROS 17/2.8 cells and visualized under blue
fluorescence. The right panels show the corresponding
nuclear staining as detected with DAPI. Note the nuclear location of
the GFP for both the full-length and truncated AJ18-GFP fusion proteins
whereas the GFP protein expressed alone is found throughout the
cell.
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Fig. 8.
Bacterially expressed AJ18 binds dsDNA in the
presence of zinc. A, cell lysates from
non-induced (NI) and IPTG-induced (I) M15
bacterial cells, containing expression plasmid for 6xHis-AJ18, were
separated on 10% SDS-PAGE and stained with Coomassie Blue. A second
gel loaded with the same samples was transferred to a nitrocellulose
membrane, and Western blot hybridization was performed using an
affinity-purified polyclonal AJ18 antibody (AJ) and an
anti-His monoclonal antibody (His). Both antibodies revealed
a 64-kDa protein corresponding to full-length 6xHis-AJ18 fusion protein
(His-AJ; indicated by an arrow), and a 35-kDa
fragment. A target detection assay was also performed in the presence
(+Zn) and absence of zinc ( Zn) after the
proteins were transferred to a nitrocellulose membrane. Radiolabeled
ds-oligonucleotide probes selectively hybridized to the 64- and 35-kDa
proteins in the presence of zinc. B, to determine whether
the KRAB domain was required for DNA binding, the cDNA coding for
the N-terminal half of AJ18 was removed using restriction enzymes
SacI and SalI, and the truncated AJ18 retaining
the 11 zinc finger motifs re-inserted into pQE32. The target detection
assay was repeated revealing zinc-dependent binding to the
truncated 6xHis-AJ18 fusion protein (His-ZF; indicated by an
arrow). C, a consensus DNA binding site (in
boldface) was identified by aligning the sequences of 17 ds-oligonucleotides that bound to AJ18 in the target detection assay.
D, a radiolabeled ds-oligonucleotide encompassing the Runx2
regulatory element (OSE2) was incubated with immobilized HIS-AJ, and
shown to bind under the target detection assay conditions (indicated by
an arrow).
Zn) and presence of zinc (+Zn), prior to hybridization with
radiolabeled dsDNA. Randomized 12-mer ds-oligonucleotides with flanking
primer sequences were incubated with the nitrocellulose-immobilized His-AJ18 and His-ZF in the absence and presence of zinc (Fig. 8,
A and B). Zinc-dependent binding of
ds-oligonucleotides was observed in the three bands identified by
immunoblotting. After five cycles of hybridization, as described under
"Experimental Procedures," the bound DNA was eluted, amplified,
subcloned, and sequenced. Alignment of the sequences generated was used
to identify a consensus binding site of 5'-CCACA-3' (Fig.
8C). Because a "CCACA" sequence is typically found
within the consensus element (OSE2) utilized by Runx2, the ability of
an OSE2 ds-oligonucleotide to bind to the immobilized HIS-AJ18 was
investigated and shown to bind to AJ18 under stringent binding
conditions of the target detection assay (Fig. 8D).
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Fig. 9.
AJ18 and Runx2 are co-expressed during RBMC
differentiation and mouse embryonic development. A,
levels of mRNA for AJ18 and Runx2 were determined using
semi-quantitative PCR and Southern blot hybridization analyses of total
RNA isolated from RBMCs over the course of osteogenic
differentiation. Levels of -actin were used for RNA template
controls. B, levels of mRNA expression of AJ18 and Runx2
were determined by Northern blot hybridization on total RNA isolated
from mouse embryos at days 7, 11, 15, and 17.
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Fig. 10.
AJ18 represses Runx2/Osf2
transactivation of 6xOSE2 in a dose-dependent manner.
p6OSE2-luc (containing six copies of OSE2; 6xOSE2) was transfected with
Runx2/Osf2 into C3H10T1/2 fibroblast-like cells with increasing
amounts (0.01-1 µg) of AJ18-S, 0.01 µg or 1 µg of truncated AJ18
(ZF-S), or without ( ) AJ18-S, ZF-S, or AJ18-AS. Resultant
luciferase activities are expressed relative to the level of luciferase
activity observed with cells transfected with 6xOSE2 alone.
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Fig. 11.
Alkaline phosphatase activity is repressed
by AJ18 in C3H10T1/2 cells treated with BMP-7. A,
C3H10T1/2 cells were transfected with AJ18-pcDNA or empty vector
and grown with (+) or without ( ) BMP-7. The cells were fixed and
stained for alkaline phosphatase (ALP) activity.
B, C3H10T1/2 cells were transfected as in A, and
the level of alkaline phosphatase activity was measured using a soluble
assay as described under "Experimental Procedures." This experiment
was done in triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/KAP-1 (41-43), which serves as a
universal co-repressor for KRAB-containing transcription factors
involved in silencing RNA pol II- and III-, but not pol I-, dependent
transcription (44). However, until recently neither target genes nor
DNA target sequences have been identified for these transcription
factors. Characterization of ZBRK1, has identified a novel 60-kDa zinc
finger protein, with a KRAB domain and eight zinc fingers, that
recognizes a GGGXXXCAGXXXTTT DNA sequence found in the growth regulatory gene GADD45. ZBRK interacts with
BRCA1, which is required as a co-repressor in the regulation of genes involved in cell growth and differentiation (45).
/KAP-1, or involve competition of the AJ18 and Runx2/Osf2
for OSE2 binding sequence. Because truncated AJ18, which lacks the KRAB
domain, also suppressed Runx2-induced transcription of a
6xOSE2-luciferase reporter gene, the modulation of Runx2/Osf2
appears to involve competition between AJ18 and Runx2 for the OSE2
(Fig. 10). Because of the low transfection efficiency of the ROS17/2.8
cells used in transient transfection assays, we have not been able to
determine whether AJ18 suppresses the endogenous expression of either
osteocalcin or osteopontin. To answer this question, we are currently
preparing stable transfectants of a rat bone marrow clonal cell line
that undergoes osteogenic differentiation in vitro.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Kuber Sampath (Creative Biomolecules, Hopkington, MA) for providing the BMP-7 (OP-1) and to Dr. Gerard Karsenty (Baylor College of Medicine, Houston, TX) for providing the mouse Runx2/Osf2 cDNA (pLA-Oa4) and the p6OSE2-luc reporter. We also thank Kam-Ling Yao and Dr. Jun Chen for their expert technical assistance.
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FOOTNOTES |
---|
* These studies were supported by a grant from the Canadian Institute of Health Research (MOP 37786).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) AF321874.
¶ To whom correspondence should be addressed: CIHR Group in Periodontal Physiology, Rm. 239, Fitzgerald Bldg., 150 College St., University of Toronto, Toronto, Ontario M5S 32E, Canada. Tel.: 416-978-6624; Fax: 416-978-5956; E-mail: a.jheon@utoronto.ca.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M010885200
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ABBREVIATIONS |
---|
The abbreviations used are:
BMP, bone
morphogenetic protein;
Runx2, runt domain homeobox gene 2;
Cbfa, core
binding factor ;
Osf, osteogenic-specific factor;
TGF-
, transforming growth factor-
;
OSE2, osteoblast-specific
cis-acting element 2;
FRCC, fetal rat calvarial cell;
OP-1, osteogenic protein-1;
RBMC, rat bone marrow cell;
PCR, polymerase chain
reaction;
IPTG, isopropyl-
-D-thiogalactoside;
PAGE, polyacrylamide gel electrophoresis;
GFP, green fluorescent protein;
DAPI, 4',6'-diamidino-2-phenylindole;
KRAB, Krüppel-associated box;
kb, kilobase(s);
bp, base pair(s);
ds, double-stranded;
SPARC, secreted protein acidic and rich in
cysteine.
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