INTRODUCTION

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Cartilage serves as the template for the growth and development of most bones. It contains an extensive extracellular matrix and provides mechanical strength to resist compression in joints. Cartilage development is initiated by mesenchymal cell condensation, followed by a series of chondrocyte maturation processes, including resting, proliferative, and hypertrophic chondrocytes. Type II collagen, a homotrimer of the 1(II) chain (Col2a1), is a major extracellular matrix protein in cartilage. It forms collagen fibrils and provides a structural framework for cartilage matrix. Type II collagen is synthesized primarily by proliferating chondrocytes but not by hypertrophic chondrocytes (9). Disruption of Col2a1 expression leads to degenerative joint disorders and a variety of chondrodysplasias (24), suggesting that fidelity of type II collagen expression is essential for maintaining normal cartilage structure and function. Transcriptional regulation of Col2a1 is mediated by tissue-specific regulatory elements located within the promoter and first intron. We have shown that a 100-bp segment within the first intron is the minimal sequence sufficient for high-level, cell type-specific expression of Col2a1 (23). Recent reports suggest that Sox9, a member of the transcription factor family with a high-mobility-group-type DNA binding domain homologous to that of SRY (16, 45, 46), plays an important role in chondrocyte differentiation and cartilage formation (6). Mutations in the gene for Sox9 cause camptomelic dysplasia, a severe dwarfism syndrome, which affects all cartilage-derived structures (11, 42, 44). Sox9 binds to a high-mobility group box-like sequence in the Col2a1 enhancer and upregulates the enhancer activity (25). Expression of recombinant Sox9 under the control of the COL2A1 enhancer trans-activates the reporter gene harboring Sox9 binding sites in the cartilage of transgenic mice (5). Coexpression of Sox9 and Col2a1 during chondrogenesis in vivo was also demonstrated (34, 47).

A-crystallin binding protein 1 (CRYBP1) is a family of zinc finger DNA binding proteins originally cloned as a murine nuclear factor bound to the A-crystallin gene promoter (33). It is one of the largest zinc finger proteins (molecular mass, 300 kDa) with two sets of C₂H₂-type zinc finger domains widely separated between the amino and carboxyl termini, and is expressed in many tissues (7). Homologues of CRYBP1 are found in Drosophila melanogaster (Schnurri) (2, 15, 41), Caenorhabditis elegans (SEM-4) (4), rat (AT-BP2) (30), and humans (PRDII-BF1/MBP1/HIV-EP1) (3, 10, 28) genes. The human homologue was shown to interact with the beta interferon promoter and the enhancer in human immunodeficiency virus type 1 long terminal repeat (3, 10, 28). Alternative splicing of the PRDII-BF1 gene gives rise to two proteins of 200 and 68 kDa, containing either of the two zinc finger binding domains (31). Comparable alternative splicing or processing of CRYBP1 also forms 50- and 90-kDa proteins detected by antibodies against the carboxyl terminus of CRYBP1, indicating the presence of protein-containing zinc fingers at the carboxyl terminus alone (22). Recent studies have suggested the possible involvement of this protein family in growth factor-mediated signaling pathways and in mesoderm development (2, 4, 15, 41).

In the present study, we have searched for protein factors that bind to the Col2a1 enhancer using the yeast one-hybrid screening system (27, 43) and identified CRYBP1 as a binding protein for the enhancer. We found that CRYBP1 expression was inversely correlated with the expression of Col2a1 mRNA during chondrocyte differentiation in vitro and mesenchymal condensation for cartilage development in mouse embryos. In developing cartilage, CRYBP1 mRNA was highly expressed in the hypertrophic zone but weakly expressed in the resting and proliferating zones. We showed that CRYBP1 inhibited Col2a1 enhancer activity by competing with binding to the enhancer with Sox9. These results suggest a negative regulatory mechanism for Col2a1 expression.

	MATERIALS AND METHODS

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Yeast strains and gene constructs. S. cerevisiae YM4271 (MATa ura3-52 his3-200 leu2-3,112 trp1-903) and reporter vectors pHISi and pLacZi were obtained from CLONTECH (Palo Alto, Calif.). The reporter construct was generated by inserting six head-to-tail copies of a double-stranded oligonucleotide (5'-TGCGCTTGAGAAAAGCCCCATTCATGAGAGGCAAGGCCCA-3'), which corresponds to the mouse Col2a1 enhancer sequence (+2206 to +2245) (29), into the EcoRI and XbaI sites of pHISi or the EcoRI and SalI sites of pLacZi. These plasmids were linearized and integrated into yeast YM4271 genomes. The yeast host strain was maintained by selection on synthetic dextrose medium lacking histidine and uracil. For a GAL4 activation domain-tagged cDNA library, poly(A)⁺ RNA was extracted from the limb buds of 13.5-day-old mouse embryos with the Micro-FastTrack kit (Invitrogen, Carlsbad, Calif.). An oligo(dT)-primed cDNA library was constructed in the HybriZap phage vector (Stratagene, La Jolla, Calif.). The plasmid (pAD-GAL4) library was obtained by in vivo excision according to the manufacturer's instructions (Stratagene). The library had a complexity of 2.2 × 10⁶ PFU and an average insert size of about 1.7 kb.

Screening of the GAL4 activation domain-tagged cDNA library. Screening of the cDNA library was performed in a yeast strain carrying HIS3 and lacZ reporter genes containing six copies of the Col2a1 enhancer sequence with a lithium acetate method as described by Schiestl and Gietz (37). The transformed yeast cells were plated under selective conditions in synthetic dextrose medium lacking histidine and leucine. The cells grown on the selective plates were transferred onto nitrocellulose filters. The membranes were frozen in liquid nitrogen and assayed for -galactosidase activity. An estimated 2.4 × 10⁶ transformants were selected, and 12 positive clones were obtained from the first screening. Four positive clones were recovered in the secondary screening.

Cell lines. ATDC5 chondrocytic cells (39) and RCS rat chondrosarcoma cells (32) were obtained from Yuji Hiraki and James H. Kimura, respectively. NIH 3T3, BALB/c 3T3, and 10T1/2 mouse cells, L6 rat myoblast cells, C2C12 mouse myoblast cells, MC3T3 mouse osteoblastic cells, and ROS17/2.7 rat osteosarcoma cells were obtained from the American Type Culture Collection (Manassas, Va.).

Northern hybridization. Total RNA was extracted from various cell lines and newborn mouse tissues using the RNeasy Mini kit (Qiagen). Northern blot analysis was performed by electrophoresing 20 µg of total RNA or 2 µg of poly(A)⁺ RNA and transferring the RNA onto a Nytran membrane (Schleicher & Schuell) as previously described (36). cDNAs were labeled with [-³²P]dCTP with the Prime-it II kit (Stratagene). The membranes were hybridized with the labeled probes at 42°C in 50% formamide, washed first at room temperature in 1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate (SDS) and then at 60°C in 0.1× SSC and 0.1% SDS, and exposed to autoradiography film.

Western blotting. Cell lysates and nuclear extracts were prepared as described previously (25). Two micrograms of protein samples were fractionated by SDS-polyacrylamide gel electrophoresis and transferred onto a nylon membrane. The blots were incubated with anti-CRYBP1 antibodies, and signals were detected with an ECL Kit (Amersham). The anti-CRYBP1 polyclonal antibodies were raised against the C-terminal portion of CRYBP1 (amino acid residues 2160 to 2187) (7) by immunizing rabbits. The antibodies were purified using an ImmunoPure IgG Purification kit (Pierce, Rockford, Ill.).

In situ hybridization. Digoxigenin-11-UTP-labeled single-strand RNA probes for Col2a1, the 1(X) collagen chain (Col10a1), and CRYBP1 were prepared using the DIG RNA labeling kit (Boehringer Mannheim, Indianapolis, Ind.) according to the manufacturer's instructions. In situ hybridization was performed as described previously (18). After deparaffinization, the sections were treated with 10 µg of proteinase K per ml for 15 min at room temperature and subjected to 0.2 N HCl to inactivate endogenous alkaline phosphatase. Hybridization was performed at 50°C in 50% formamide, and washes were carried with 2× SSC containing 50% formamide at 55°C. Then, the slides were subjected to 10 g of RNase A per ml in TNE (10 mM Tris-HCl [pH 8.0], 500 mM NaCl, 1 mM EDTA) at 37°C for 30 min to digest nonhybridized transcripts and were then washed. A Genius Detection System (Boehringer Mannheim) was used to detect signals according to the manufacturer's instructions.

Electrophoretic mobility shift assays (EMSAs). The expression vector pCA1F (Yamada et al., unpublished data) was used to express Flag-tagged CRYBP1 fusion proteins in vitro. A PCR product for the amino-terminal zinc finger domain (amino acid residues 1 to 1118) (7) of CRYBP1 was obtained from clones MBC-1 and MBC-2 (gifts from James P. Brady) and cloned into the pCA1F vector (pCA1F-NZF). pCA1F-CZF, containing the carboxy-terminal zinc finger domain (amino acid residues 2023 to 2688) (7) was also generated by PCR with cDNA from clone B25, a positive clone from the yeast screening. S-tagged rat Sox9 (GenBank accession no. R47011) was prepared by the TNT Coupled Reticulocyte Lysate System (Promega, Madison, Wis.) with the pCITE-4a vector (Novagen, Madison, Wis.). Nuclear extracts from various cell lines were prepared as described previously (25).

DNA transfection assay. DNA was transfected into various cells using Fugene 6 (Boehringer Mannheim) according to the manufacturer's instructions. The expression vectors pCA1F-NZF and pCA1F-CZF were used to express zinc finger domains of CRYBP1 in RCS cells. The expression construct for full-length PRDII-BF1, a human homologue of CRYBP1, was a gift from Richard B. Gaynor (38). The expression construct pCA1-Sox9 was used to express Sox9 protein. The reporter constructs pKN185luc, pKN159luc, and pKN159Bx6luc contained 640 bp, 100 bp, and six tandem copies of the Col2a1 enhancer sequences used for one-hybrid screening, respectively, linked to the luciferase reporter gene (23, 29). The reporter construct pKN159Mluc was the same as pKN159luc, except there was a substitution mutation at the Sox9 binding site (CATTCAT to CAGGCAT). pGL3-Control and pRL-SV40 (Promega) were respectively used as a positive control and an internal control for normalization of transfection efficiency. The transfected cells were harvested 48 h after transfection and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega).

	RESULTS

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Isolation of cDNA clones for proteins interacting with the Col2a1 enhancer. A 13.5-day-old mouse embryo limb bud cDNA library was screened by the yeast one-hybrid screening method with a yeast strain harboring six tandem copies of the Col2a1 enhancer as a target sequence. After secondary screening, four HIS3- and lacZ-positive clones were isolated and sequenced entirely. Two of them were found to encode the same gene and were further characterized. The DNA sequence analysis of cDNA clone B25 revealed an open reading frame with a 665-amino-acid polypeptide that was identical to the 3' portion (amino acid residues 2023 to 2688) of CRYBP1, a previously identified nuclear factor bound to the mouse A-crystallin promoter (7).

Inverse correlation of the expression pattern of CRYBP1 and Col2a1. The expression of CRYBP1 mRNA was analyzed in tissues from newborn mice and in cell lines by Northern blotting and in mouse embryos by in situ hybridization. Northern analysis of total RNA from newborn mice revealed that CRYBP1 mRNA was strongly expressed in the brain, thymus, heart, spleen, and rib cartilage, whereas little expression in liver, kidney, and skeletal muscle was found (Fig. 1A). When poly(A)⁺ RNA was analyzed by Northern blotting, CRYBP1 expression was also found in lung, intestine, liver, and testis (data not shown), consistent with previous observations (7, 33). CRYBP1 mRNA was highly expressed in BALB/c 3T3 and 10T1/2 cell lines and undifferentiated ATDC5, a chondrocytic cell line (Fig. 1B). However, CRYBP1 mRNA was not detected in RCS, a rat chondrosarcoma cell line. A low level of CRYBP1 mRNA was observed in muscle cell lines L6 and C2C12, an osteoblastic cell line (MC3T3), and an osteosarcoma cell line (ROS17/2.7). Although rib cartilage expressed CRYBP1 mRNA, RCS cells that synthesize Col2a1 showed little expression of CRYBP1.

View larger version (89K): [in this window] [in a new window]   FIG. 1.   mRNA and protein expression of CRYBP1 in various tissues and cell types. (A) Analysis of CRYBP1 expression in newborn mouse tissues by Northern blotting. For each lane, 20 µg of total RNA from various tissues was loaded, transferred to the nylon membrane, and hybridized with labeled CRYBP1 cDNA. CRYBP1 mRNA was strongly expressed in the brain, thymus, heart, spleen, and rib cartilage. Lane 1, brain; lane 2, thymus; lane 3, heart; lane 4, liver; lane 5, spleen; lane 6, kidney; lane 7, skeletal muscle; lane 8, rib cartilage. (B) Total RNA (20 µg/lane) extracted from various cells was analyzed by Northern blotting using the CRYBP1 cDNA probe. CRYBP1 mRNA was highly expressed in BALB/c 3T3 and 10T1/2 cell lines and in undifferentiated ATDC5, a chondrocytic cell line, whereas little expression in RCS, a rat chondrosarcoma cell line, was observed. Lane 1, BALB/c 3T3; lane 2, 10T1/2; lane 3, undifferentiated ATDC5; lane 4, RCS; lane 5, MC3T3; lane 6, ROS17/2.7; lane 7, L6; lane 8, C2C12. The lower sets of gels in panels A and B show ethidium bromide-stained 18S rRNA. (C) Western blot analysis of CRYBP1 protein expression in cell lines. Nuclear extracts (2 µg each) from cells were fractionated by SDS-polyacrylamide gel electrophoresis, blotted, and incubated with CRYBP1 antibodies. In vitro-translated CRYBP1 (C-ZF) (2 µg) was also subjected to Western blot analysis. Lane 1, RCS; lane 2, NIH 3T3; lane 3, undifferentiated ATDC5; lane 4, in vitro-translated CRYBP1. Protein standards are indicated on the left. (D) DNA binding of CRYBP1 in cells to the labeled wild-type Col2a1 enhancer. EMSAs were performed with nuclear extracts from RCS (lanes 1 and 2), NIH 3T3 (lanes 3 and 4), and undifferentiated ATDC5 (lanes 5 and 6) cells. The presence (+) or absence () of antibodies to CRYBP1 is indicated. The large arrows indicate CRYBP1 enhancer complexes (lanes 3 and 5). The small arrows mark supershifted CRYBP1 enhancer complexes by the anti-CRYBP1 antibodies (lanes 4 and 6). Arrowheads indicate an RCS cell-specific protein-enhancer complex (lanes 1 and 2). This complex is specific to RCS cells, is not found in either NIH 3T3 or undifferentiated ATDC5 cells, and is not supershifted by anti-CRYBP1 antibodies.

View larger version (139K): [in this window] [in a new window]   FIG. 2.   In situ hybridization of longitudinal sections of the radius in the forelimb of 16.5-day-old mouse embryos with antisense Col2a1, Col10a1, and CRYBP1 riboprobes labeled with digoxigenin-11-UTP. (A) Staining with hematoxylin and eosin. (B) Expression of Col2a1 in a semiserial section. Strong signals of Col2a1 were detected in the resting and proliferating (p) chondrocytes. (C) Expression of Col10a1 was observed in hypertrophic chondrocytes. (D) CRYBP1 mRNA was highly expressed in the hypertrophic zone; however, it was faint in the resting and proliferative zones. The localization of CRYBP1 mRNA was similar to that of Col10a1.

View larger version (122K): [in this window] [in a new window]   FIG. 3.   Expression of CRYBP1 in differentiating ATDC5 cells in vitro. ATDC5 cells were cultured with 10 µg of bovine insulin per ml for differentiation. The cells differentiated into the proliferative chondrocyte phenotype (lanes 2 and 3) and then into the hypertrophic chondrocyte phenotype (lanes 4 to 6) over time. Total RNA was isolated from the cells, which were cultured for 1 day (lane 1), 1 week (lane 2), 2 weeks (lane 3), 3 weeks (lane 4), 4 weeks (lane 5), and 5 weeks (lane 6). The total RNA (20 µg/lane) was electrophoresed in each lane, transferred to the nylon membrane, and hybridized with CRYBP1, Col2a1, and Col10a1 cDNA probes as indicated at the left of the panels. CRYBP1 mRNA was strongly detected in undifferentiated cells (lane 1), but the expression level was decreased when the cells differentiated into the proliferative chondrocyte and began to express Col2a1 (lanes 2 and 3). As the expression of the collagen types was switched from Col2a1 to Col10a1 in differentiating ATDC5 cells, CRYBP1 expression was induced again (lanes 4 to 6). The signal intensity of CRYBP1 transcripts was measured using densitometry, and the ratios of the level of CRYBP1 mRNA in the differentiated cells to that in the undifferentiated cells are as follows: lane 1, 1; lane 2, 0.52; lane 3, 0.28; lane 3, 1.08; lane 4, 1.34; lane 5, 1.60. The values are the means of three experiments. In order to show the switch in mRNA expression of Col2a1 and CRYBP1 more clearly, nodule- and non-nodule-forming cell populations were separated from 10-day culture cells, and total RNAs from the two cell populations were extracted and subjected to Northern blot analysis. Lane 7, non-nodule-forming cells; lane 8, nodule-forming cells. The lower panels show the blots hybridized with the glyceraldehyde-3-phosphate dehydrogenase probe and ethidium bromide staining.

View larger version (165K): [in this window] [in a new window]   FIG. 4.   In situ hybridization of axial sections of day 8.5 (A), day 9.5 (B), and day 12.5 (C and D) mouse embryos with antisense CRYBP1 and Col2a1 riboprobes labeled with digoxigenin-11-UTP. The signals for CRYBP1 mRNA were ubiquitous, but there was little signal in the neural tube and somites in the day 8.5 and day 9.5 mouse embryos (A and B). In the 12.5-day-old embryo, Col2a1 mRNA was strongly expressed at the mesenchymal condensations for rib and vertebral cartilage primordia (C), whereas CRYBP1 mRNA was not expressed in those locations (D). Nt, neural tube; r, rib cartilage primordia; s, somite; v, vertebral cartilage primordia.

CRYBP1 protein specifically binds to the Col2a1 enhancer sequence. The two Flag-tagged zinc finger domains of CRYBP1, N-ZF and C-ZF, were synthesized using an in vitro transcription-translation system and examined for their binding activity to the Col2a1 enhancer (Fig. 5). We found that C-ZF was able to bind to the double-stranded Col2a1 enhancer oligonucleotide (the WT probe), whereas N-ZF could not (Fig. 5A, lanes 2 and 9). Competition experiments with unlabeled oligonucleotides containing substitution mutations were performed to determine a target sequence within the enhancer for the C-ZF binding. An excess of the unlabeled WT probe abolished the binding of C-ZF to the Col2a1 enhancer (Fig. 5A, lane 3). Mutated oligonucleotides M1, M3, and M4 inhibited the binding, whereas M2 failed to block the C-ZF binding to the Col2a1 enhancer (Fig. 5A, lanes 4 to 7). These results suggest that the sequence within the enhancer used for the M2 substitution mutation contains the binding site for C-ZF. Further competition experiments were carried out to delineate more precisely a core-binding sequence of CRYBP1. Binding of C-ZF to the labeled WT was inhibited by excess of either unlabeled M5 or M9 (Fig. 5B, lanes 2 to 4 and 8) but not by M6, M7, or M8 oligonucleotide (Fig. 5B, lanes 5 to 7). These results indicate that the CRYBP1 protein binds specifically to the Col2a1 enhancer through its carboxyl-terminal zinc fingers and that the core-binding sequence in the enhancer is GAGAAAAGCC.

View larger version (40K): [in this window] [in a new window]   FIG. 5.   Specific DNA binding of CRYBP1 to the Col2a1 enhancer, analyzed by EMSA. (A) The upper panel shows the location of the 100-bp enhancer (positions +2146 to +2245) within the first intron. The coding strand sequences of oligonucleotide probes used in EMSA and substitution mutations as competitors (M1 to M4) are also indicated. Only mutated nucleotides are shown. In the lower left panel, lane 2 shows that the CRYBP1 carboxyl-terminal zinc finger domain (C-ZF) binds to the WT Col2a1 enhancer sequence. Lanes 3 to 7 show competitions between the labeled WT Col2a1 enhancer probe and a 50-fold molar excess of cold probes, as indicated above. Lane 1, no competitor. The DNA binding of C-ZF is inhibited by addition of the WT, M1, M3, or M4, whereas the M2 probe showed minimal inhibition. The lower right panel shows that the CRYBP1 amino-terminal zinc finger domain (N-ZF) failed to bind to the enhancer sequence (lanes 8 to 14). (B) The upper panel shows the WT sequence and substitution mutations as competitors. Only mutated nucleotides are indicated. In the lower panel, the DNA binding of C-ZF to the labeled WT (lane 2) is inhibited by unlabelled WT (lane 3), M5 (lane 4), and M9 (lane 8). M6, M7, and M8 do not affect the binding of C-ZF to the enhancer (lanes 5 to 7), indicating that the core-binding sequence of CRYBP1 in the enhancer is GAGAAAAGCC. Lane 1, no competitor.

CRYBP1 represses chondrocyte-specific Col2a1 enhancer activity by inhibiting Sox9 binding to the enhancer. The expression vector of the C-ZF domain of CRYBP1 (pCA1F-CZF) was constructed and examined for its inhibitory activity of the Col2a1 enhancer by cotransfection with the Col2a1 promoter-enhancer reporter gene constructs into RCS cells. The three reporter constructs, pKN185luc, pKN159luc, and pKN159Bx6luc, which contain the functional enhancer of different sizes, were active in RCS cells but inactive in BALB/c 3T3, 10T1/2, or NIH 3T3 cells, indicating cell type specificity of the enhancer activity (data not shown). When pKN185luc or pKN159luc was cotransfected with pCA1F-CZF into RCS cells, Col2a1 enhancer activity was reduced to more than half of that of the control in a dose-dependent manner (Fig. 6). The expression plasmid pCA1F-NZF, containing the amino-terminal zinc finger domain, had no significant effects on reporter gene activity in RCS cells (data not shown). The repression by pCA1F-CZF was not due to a general suppressive effect on transcriptional regulation, because it had no significant effects on the luciferase activity of the pGL3-Control plasmid driven by the simian virus 40 promoter (Fig. 6). The reporter gene activity of pKN159luc was also significantly inhibited by cotransfection with full-length PRDII-BF1, a human homologue of CRYBP1 (Fig. 6). In RCS cells, pKN159Mluc, which contains a substitution mutation in the Sox9 binding site, showed only weak activity, consistent with a previous report (23, 25). Neither C-ZF nor PRDII-BF1 modulated the luciferase activity of pKN159Mluc (Fig. 6). These results suggest that CRYBP1 specifically inhibits Col2a1 enhancer activity.

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Inhibition of Col2a1 enhancer activity by CRYBP1 in cotransfection assays. In the left panel, RCS cells were transiently transfected with 2 µg of the reporter plasmid (pGL3-Control or pKN185luc containing 640-bp Col2a1 enhancer sequences) along with 4 µg of the C-ZF expression vector or a control vector (pCA1F). C-ZF suppressed Col2a1 enhancer activity of pKN185luc to 45% of that of the control, whereas C-ZF did not affect luciferase activity of the pGL3-Control plasmid driven by a simian virus 40 promoter. In the right panel, a total of 4 µg of the C-ZF expression vector, a control vector (pCA1F), or the PRDII-BF1 (full-length human homologue of CRYBP1) expression vector was cotransfected with 2 µg of the pKN159luc or pKN159Mluc reporter plasmid containing the WT 100-bp Col2a1 enhancer or the mutant enhancer with a substitution mutation in the Sox9 binding site, respectively. C-ZF suppressed the luciferase activity of pKN159luc to 28% of that of the control in a dose-dependent manner. PRDII-BF1 also reduced the enhancer activity to 52% of that of the control. pKN159Mluc showed very weak activity in RCS cells. C-ZF and PRDII-BF1 did not modulate the luciferase activity of pKN159Mluc. A Renilla luciferase expression vector, pRL-SV40, was used as an internal control for transfection efficiency. The relative luciferase activities are average values ± the standard errors for three independent transfected cultures from two experiments.

View larger version (88K): [in this window] [in a new window]   FIG. 7.   Inhibition of Sox9 binding to the Col2a1 enhancer by C-ZF. The left panel shows the results of EMSA using the labeled WT Col2a1 enhancer and in vitro-translated C-ZF or Sox9. Two micrograms of the protein was added to the reactions. Arrows indicate C-ZF-enhancer complexes (lane 2). Solid arrowheads indicate Sox9-enhancer complexes (lane 3). The open arrowhead marks a supershifted Sox9-enhancer complex by anti-Sox9 antibodies (lane 4). Lane 1, lysate only. The middle panel shows dose-dependent inhibition of Sox9-enhancer complex formation by C-ZF. As the amounts of the C-ZF protein are increased, the Sox9-enhancer complex is decreased, whereas C-ZF-enhancer complex formation is inhibited (lanes 5 to 8). A constant amount (2 µg) of Sox9 was used in lanes 5 to 8, and various amounts of C-ZF were used in the reactions, as follows: lane 5, 0 µg; lane 6, 1 µg; lane 7, 2 µg; lane 8, 4 µg. The right panel shows dose-dependent inhibition of C-ZF-enhancer complex formation by Sox9. As the amount of Sox9 is increased, formation of C-ZF-enhancer complex is inhibited (lanes 9 to 12). Two micrograms of C-ZF protein was used in lanes 9 to 12. The amount of Sox9 in each lane is as follows: lane 9, 0 µg; lane 10, 1 µg; lane 11, 2 µg; lane 12, 4 µg.

View larger version (17K): [in this window] [in a new window]   FIG. 8.   Inhibition of Sox9-mediated activation of the Col2a1 enhancer by CRYBP1. RCS and NIH 3T3 cells were transiently transfected with 2 µg of the reporter construct pKN159Bx6luc containing six tandem copies of the Col2a1 enhancer sequences along with 2 µg of the C-ZF expression vector, Sox9 expression vector, or control vector (pCA1F). C-ZF suppressed the enhancer activity of pKN159Bx6luc to 32% of that of the control in RCS cells. pKN159Bx6 was not active in NIH 3T3 cells; however, expression of Sox9 resulted in significant activation of the Col2a1 enhancer in the nonchondrocytic cells. Cotransfection of increasing amounts of the C-ZF expression vector inhibits Sox9-mediated activation of the Col2a1 enhancer in a dose-dependent manner. A Renilla luciferase expression vector, pRL-SV40, was used as an internal control for transfection efficiency. The relative luciferase activities are average values ± the standard errors for three independent transfected cultures from two experiments.

	DISCUSSION

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Increasing evidence has demonstrated that transcriptional repression plays crucial roles in regulating cell type-specific gene expression and that gene regulation is mediated through a balance between activator and repressor factors. Several models of the negative regulatory mechanism have been proposed (e.g., competition, quenching, and direct repression of the transcription complex [13, 20]). However, compared to transcriptional activation, the precise mechanisms of transcriptional repression are poorly understood. In the present study, we have identified CRYBP1 as a Col2a1 enhancer binding protein and a suppressor for Col2a1 enhancer activity.

Competitive binding to DNA is one of the major mechanisms of controlling cell type-specific gene regulation (13, 20). Usually, a binding site of a repressor overlaps with that of an activator. The repressor locally blocks the binding of the activator and thus inhibits target gene expression (1, 12, 14, 19, 21, 35, 40). Repressors tend to be more widely expressed, spatially and temporally, than activators (13, 20). The expression pattern and binding site of CRYBP1 agree with these characteristics. The CRYBP1 binding site is located just 1 bp upstream from the Sox9 binding site. CRYBP1 competes for binding to the Col2a1 enhancer with Sox9 and inhibits transcriptional activation by Sox9. The yeast two-hybrid analysis using CRYBP1 as a bait and Sox9 as a target showed no signals for binding between CRYBP1 and Sox9 (data not shown). Recently, it was reported that the CRYBP1 binding site is also required for the chondrocyte-specific enhancer activity of Col2a1 (48) and that new members of the Sox family, L-Sox5 and Sox6, form a heterodimer and activate the Col2a1 enhancer cooperatively with Sox9 (26). CRYBP1 may also compete for binding to the same or overlapping sequence with such a complex and suppress the activity of the Col2a1 enhancer in vivo.

Northern blot and in situ hybridization analyses revealed that CRYBP1 mRNA is widely expressed. Although CRYBP1 was originally cloned as a murine nuclear factor bound to the A-crystallin gene promoter, its expression level in fibroblastic cells is higher than that in lens epithelial cells (22). Sox9 is expressed not only in the resting and proliferating zones in cartilage but also in several noncartilaginous tissues, including heart, lung, intestine, and testis (34), in which CRYBP1 is also expressed. It is possible that CRYBP1 could also inhibit Sox9 activity in these noncartilaginous tissues. However, the most prominent cell types where CRYBP1 is expressed are undifferentiated mesoderm cells and hypertrophic chondrocytes. We have demonstrated that CRYBP1 is strongly expressed in undifferentiated ATDC5 cells, whereas it is downregulated during differentiation of ATDC5 cells into the chondrocyte phenotype. Little expression of CRYBP1 was also observed in RCS cells, which express Col2a1, a characteristic marker gene for proliferative chondrocytes (Fig. 1 and 3).

When ATDC5 cells further differentiate into hypertrophic chondrocytes and express Col10a1, the level of expression of CRYBP1 is increased again (Fig. 3). These observations are consistent with the in situ hybridization data in which CRYBP1 is not or weakly expressed by resting and proliferating chondrocytes in developing mouse embryos but is strongly expressed by hypertrophic chondrocytes where Col2a1 is downregulated. Interestingly, CRYBP1 mRNA expression is faint in the somites and sclerotomes in 8.5- and 9.5-day-old mouse embryos (Fig. 4), suggesting that downregulation of CRYBP1 may occur during early mesoderm differentiation. The level of CRYBP1 expression is very low at the mesenchymal condensations in 12.5-day-old embryos, also indicating the inverse relationship of the expression patterns in vivo between CRYBP1 and Col2a1 (Fig. 4). These patterns of expression suggest that CRYBP1 may be involved in both the early and late stages of chondrocyte differentiation.

Molecular characterizations of CRYBP1 also support the hypothesis that CRYBP1 functions as a regulator for mesodermal development. CRYBP1 is a homologue of Drosophila Schnurri (2, 15, 41), C. elegans SEM-4 (4), rat AT-BP2 (30), and human PRDII-BF1/MBP1/HIV-EP1 (3, 10, 28). The vertebrate homologues have been isolated on the basis of their ability to bind to cis-regulatory elements of various genes, including rat 1-antitrypsin (30) and human interferon- (10) genes. However, the function of the vertebrate proteins for these genes is still not clear. Recent studies demonstrated the possible involvement of Schnurri and SEM-4 in the growth factor signaling pathway and mesoderm development. Schnurri is shown to be necessary for the signaling pathway of Drosophila decapentaplegic, a member of the transforming growth factor  superfamily most closely related to the vertebrate bone morphogenetic proteins BMP-2 and BMP-4 (2, 15, 17). Genetic analysis using C. elegans has demonstrated that SEM-4 is required for the development of neuronal cells and mesodermal cell lineage (4, 8). These studies suggest that a family of zinc finger transcription factors, including CRYBP1, play roles in the signal transduction pathway of growth factors and mesodermal cell development in vertebrates.

CRYBP1 has two widely separated C₂H₂-type zinc finger clusters, as does its human homologue PRDII-BF1 (10). The DNA binding domain specific to the Col2a1 enhancer sequence was shown to be the zinc finger domain at the carboxyl terminus. The amino-terminal zinc finger domain (N-ZF) failed to bind to the Col2a1 enhancer sequence, while a fragment of CRYBP1 carrying the carboxyl-terminal zinc fingers (C-ZF) could bind to the DNA sequence (Fig. 5). These results are consistent with a previous study in which each set of the zinc finger domain can independently recognize DNA sequences (10). It was also reported that alternative splicing of the PRDII-BF1 gene generates two proteins of 200 and 68 kDa, respectively, which contain either of the two zinc finger DNA binding domains (31). The proteins are potential repressors of human immunodeficiency virus type 1 gene expression (31, 38). Consistent with the human data, Western blot analysis using antibody against the carboxyl terminus of CRYBP1 indicated the presence of truncated forms of CRYBP1 containing C-ZF alone by alternative splicing or processing in mouse cell lines (31). These studies showed that truncated CRYBP1 proteins are approximately 60 and 90 kDa in size. We also detected variant forms of CRYBP1 proteins in NIH 3T3 and ATDC5 cells, whose sizes are similar to those reported previously (Fig. 1). These findings indicate that a processed form of CRYBP1 containing C-ZF can recognize and bind to the Col2a1 enhancer sequence. Further examination should be needed to elucidate this possibility. Although C-ZF is sufficient for the significant repression of the Col2a1 enhancer activity (Fig. 6), we also examined activity of the full-length protein carrying both sets of the zinc finger domains with PRDII-BF1. Consistent with the data of C-ZF, PRDII-BF1 also significantly inhibited Col2a1 enhancer activity (Fig. 6). In summary, we have identified for the first time the inhibitor protein that blocks the chondrocyte-specific activity of Col2a1 enhancer by competing with the activator protein for binding to the enhancer. These results suggest that Col2a1 expression is regulated by both positive and negative protein factors and that such a suppressor may play an important role in cartilage development.