Characterization of a Novel KRAB/C2H2 Zinc Finger Transcription Factor Involved in Bone Development*

Andrew H. JheonDagger , Bernhard Ganss§, Sela Cheifetz§, and Jaro SodekDagger

From the § CIHR Group in Periodontal Physiology and the Dagger  Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 3E2, Canada

Received for publication, December 1, 2000, and in revised form, January 30, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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.

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-beta 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-beta 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 Calpha 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.

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha -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 beta -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.

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 beta -actin-specific primers (5'-CACCCTGTGCTGCTCACCGA-3' and 5'- ACCTGGCCGTCAGGCAGCTC-3') were used to PCR-amplify AJ18, Runx2, and beta -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.

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-beta -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 [alpha -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.

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-beta -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).

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).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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.


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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 beta -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.

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.


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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.

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.


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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).

To study DNA binding using the target detection assay, His-tagged proteins were transferred to nitrocellulose and renatured in the absence (-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).

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).


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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 beta -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.

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).


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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

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 TIF1beta /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).

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 TIF1beta /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.

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.

    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.

    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

    ABBREVIATIONS

The abbreviations used are: BMP, bone morphogenetic protein; Runx2, runt domain homeobox gene 2; Cbfa, core binding factor alpha ; Osf, osteogenic-specific factor; TGF-beta , transforming growth factor-beta ; 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-beta -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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