Transcriptional Co-activators CREB-binding Protein and p300 Regulate Chondrocyte-specific Gene Expression via Association with Sox9*

Chondrocytes are critical components for the precise patterning of a developing skeletal framework and articular joint formation. Sox9 is a key transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expressions; however, the precise transcriptional activation mechanism of Sox9 is not fully understood. Here we demonstrate that Sox9 utilizes a cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300 to exert its effects. Sox9 associates with CBP/p300 in the chondrosarcoma cell line SW1353 via its carboxyl termini activation domain in a cell type-specific manner. In promoter assays, CBP/p300 enhances Col2a1, which encodes cartilage-specific type II collagen gene promoter activity via Sox9. Chromatin immunoprecipitation shows that p300 is bound to the Col2a1 promoter region. Furthermore, the CBP/Sox9 complex disrupter peptide suppresses Col2a1 gene expression and chondrogenesis from mesenchymal stem cells. These data demonstrate that CBP and p300 function as co-activators of Sox9 for cartilage tissue-specific gene expression and chondrocyte

The Sox9, a high-mobility group (HMG) domain transcription factor, has been identified as a key molecule in chondrocyte differentiation, a multi-step pathway during which multipotential mesenchymal stem cells differentiate into chondrocytes (1)(2)(3). Expression of Sox9 shadows that of Col2a1, which encodes cartilage-specific type II collagen during chondrogenesis (3,4,27). Analysis of mouse chimaeras using Sox9 Ϫ/Ϫ embryonic stem cells showed that Sox9 Ϫ/Ϫ cells are excluded from cartilage tissues in viable animals and are unable to express chondrocyte-specific extracellular matrix genes such as Col2a1 (5,6,27). However, the precise transcriptional activation mechanism of Sox9 is not fully understood.
In addition to sequence-specific binding factors such as Sox9, various co-activators are involved in transcriptional activation (7)(8)(9). For example, the transcriptional co-activator CBP 1 and its paralog, p300, are recruited on promoter regions via direct interactions with various sequence-specific activators, including the cAMP-response element-binding protein (CREB), activator protein 1 (AP-1), signal transducers and activators of transcription (STATs), and nuclear hormone receptors (10). These co-activators facilitate transcription by promoting interactions between DNA-binding proteins and the RNA polymerase II transcriptional machinery to initiate transcription (7)(8)(9). Another function of CBP and p300 is the modification of chromatin structure (11)(12)(13)(14)(15)(16). During DNA assembly, DNA wraps twice around the histone octamer to form chromatin (17). Chromatin is not only a system to package genome but also a key player in regulating gene expression (17). It represses transcription by inhibiting the access of the transcriptional machinery to DNA. Specific modifications of chromatin such as phosphorylation and acetylation, which are thought to alter histone-DNA contacts, facilitate gene expression by allowing the recruitment of the transcriptional complex to the promoter (18,19).
In humans, loss of one CBP allele causes Rubinstein-Taybi syndrome, which is characterized by abnormal pattern formation and mental retardation (20). This phenotype was partially reproduced in hemizygous CBP ϩ/Ϫ mice with abnormal skeletal framework (21,22). These data prompted us to examine the possible role of CBP and p300 in accordance with Sox9, which is also critical for the developing skeletal framework.
Here we demonstrate that Sox9 utilizes CBP and p300 as transcriptional co-activators. The transcription factor Sox9 binds to CBP/p300 both in vitro and in vivo. In addition, CBP and p300 enhance Sox9-dependent Col2a1 promoter activity, and disrupting the CBP/Sox9 complex inhibits Col2a1 mRNA expression and mesenchymal stem cell (MSC) differentiation into chondrocyte. These results establish CBP and p300 as important cofactors in chondrocyte-specific gene expression via regulating Sox9 transcriptional activity.

EXPERIMENTAL PROCEDURES
Tissue Culture Methods and Transfections-Human chondrosarcoma cell line (1353) cells were grown in Dulbecco's modified Eagle's medium (Cellgro, Mediatech, Washington, DC) supplemented with 10% fetal calf serum and penicillin/streptomycin (Sigma). Cells were transfected by using FuGENE 6 (Roche Applied Science) as described by the manufacturer. The amounts of transfected plasmids for cotransfection assays were as follows. For Sox9-dependent activation, 50 ng of Gal-TK luc reporter, 50 ng of Gal4-Sox9, and 100 ng of hemagglutinin (HA)-tagged p300 were used. The Col2a1 promoter (pKN185) (3) was assayed by cotransfection assays using 50 ng of plasmid or empty plasmid control per well. Luciferase activity was assayed, and reporter activities were normalized to activity from a cotransfected Rous sarcoma virus (RSV)galactosidase expression plasmid as described previously (23). Comparable expression levels of Gal4-Sox9 and p300 wild type and mutant polypeptides were verified by Western blot assay.
Immunoprecipitations and Western Blotting-Cells were washed once in ice-cold phosphate-buffered saline before scraping them off at 4°C with 1 ml of phosphate-buffered saline. Cells were resuspended in radioimmune precipitation assay buffer (RIPA) buffer (50 mM Tris⅐HCl, pH 7.5, 200 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, and the phosphatase inhibitors P-5726 and P-2850 (Sigma)). Cell extract then was sonicated and centrifuged at 14,000 rpm for 10 min. Supernatants were used as crude extracts for immunoprecipitations. Nonspecific binding was reduced by preincubation of extracts with protein G-Sepharose (P-4691; Sigma) for 30 min. Pellets were discarded, and extracts were incubated with immune sera or controls for 2-4 h. Immunoprecipitations were performed with 5 l of monoclonal anti-HA (Y11; Santa Cruz Biotechnology) and anti-FLAG antibody (M2; Sigma).
Chromatin Immunoprecipitation-SW1353 cells were treated with formaldehyde to cross-link protein-DNA complexes (25). Immunoprecipitates of cross-linked complexes were prepared with control, CBP, or p300 antibody (C-20, Santa Cruz Biotechnology). Immunoprecipitates were treated with proteinase K for 2 h and then incubated at 65°C to release cross-links. DNA was purified by phenol-chloroform extraction and ethanol precipitation. DNA samples were then analyzed with 20 cycles of PCR to amplify human Col2a1 first intron 2151-2305, which contains the Sox9 DNA binging site and was analyzed by 2% agarose gel with ethidium bromide. Different cycle numbers were employed to ensure linearity of amplification.
Quantitative PCR-Poly(A) ϩ RNA and total RNA were extracted from homogenized mice livers using the Fast Track 2.0 (Invitrogen) or the RNeasy (Qiagen) kit. RNA samples were treated with DNase I (Promega), and RNA quality was assessed by gel electrophoresis. cDNA was prepared by reverse transcription of 500 ng of total RNA using the Superscript II enzyme and oligo(dT) primer (Invitrogen). The resulting cDNAs were amplified using the QuantiTect SYBR Green PCR kit (Qiagen) and the iCycler iQ Real Time PCR detection system (Bio-Rad).
All mRNA expression data from the quantitative PCR with reverse transcription were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in the corresponding sample.
Adenovirus Infection-Recombinant adenovirus vectors carrying mouse CBP amino acids 1805-1890 with the Gal4 DNA biding domain as a tag or control lacZ gene were constructed in pJM17 as described (26) (generous gifts from Dr. Montminy, Salk Institute, La Jolla, CA). Viruses were purified by the CsCl method, and titer was checked as described (26). The infection efficiency of this adenovirus in SW1353 cells was examined by immunocytochemistry using an anti-Gal antibody, which showed almost 100% infection efficiency with an m.o.i. of 8 (data not shown).
MSC Cells Culture and Chondrogenesis-Human mesenchymal stem cells were purchased from BioWhittaker (Walkersville, MD). To induce chondrogenesis, pellet cultures were prepared by gently centrifuging 2.5 ϫ 10 5 cells at 500 ϫ g in 15-ml polypropylene conical tubes. The culture media was Dulbecco's modified Eagle's medium, low glucose supplemented with ITS Premix (BD Biosciences) consisting of insulin, transferrin, selenic acid, bovine serum albumin, and linoleic acid, Sodium pyruvate (1 mM), ascorbate 2-phosphate (37.5 g/ml), and transforming growth factor ␤3 (TGF-␤3) (10 ng/ml). The pellet cultures were incubated at 37°C, 5% CO 2 . Cells form an essentially spherical aggregate that does not adhere to the walls of the tube. Medium changes were carried out at 2-3 day intervals, and pellets were harvested for analysis at time points up to 4 weeks. The pellets were embedded, fixed, and stained with a type II collagen antibody.

Transcriptional Co-activators CBP and p300
Associating with Sox9 -To test an interaction between CBP/p300 and Sox9 in intact cells, amino-terminally FLAG-tagged, full-length Sox9 was expressed in SW1353 alone or in the presence of HA-tagged p300 or CBP. Cell extracts were prepared, and immunoprecipitations were performed with anti-HA antibodies followed by Western blotting utilizing anti-FLAG antibodies. In the presence of coexpressed HA-CBP, Sox9 was co-immunoprecipitated (Fig. 1A). Similarly, we detected co-immunoprecipitation of Sox9 with HA-tagged p300 (Fig. 1A). These data show a physical association of Sox9 with the transcriptional co-activators p300 and CBP.
Mapping of Interaction Domains of CBP and Sox9 Complex-To determine the Sox9 interaction domain with CBP in SW1353 cells, FLAG-tagged Sox9 truncations were constructed (Fig. 1B). As shown in Fig. 1C, a carboxyl-terminal truncation   FIG. 1. Sox9 associates with CBP/ p300 via its C-terminal activation domain. A, lysates of SW1353 cells transfected with HA-tagged p300 or CBP and FLAG-tagged Sox9 were immunoprecipitated with anti-HA antibody. Precipitates (IP) and 10% on-puts (OP) were subjected to Western blotting with anti-FLAG antibody. B, schematic illustration of Sox9 deletion mutants. Various regions of Sox9 were cloned into pcDNA3. C, the carboxyl terminus of Sox9 associates with p300. FLAG-tagged Sox9 mutants, shown in panel A, were transfected into SW1353 cells together with HA-p300. The cell lysates were immunoprecipitated with anti-HA antibody. Precipitates (IP) and 10% on-puts (OP) were run by 10% SDS-PAGE and analyzed for the presence of Sox9 by Western blotting with the anti-FLAG antibody.
(FLAG-Sox9 amino acids 1-423 and 1-327) did not bind to p300. However, deletion of Sox9 amino-terminal amino acids (FLAG-Sox9 amino acids182-507 and 328 -507) led to the same high degree of interaction with p300 as observed with the full-length protein, indicating that the carboxyl terminus of Sox9 is critical for binding to CBP/p300 (Fig. 1C).
To define the CBP/p300 interaction domain within Sox9, an in vitro GST pull-down assay was performed using bacterially expressed GST-Sox9 and in vitro translated CBP fragments. GST-Sox9 specifically interacted with CBP 4 fragment (Fig. 2,  A and B), which is known as a binding site of various nuclear proteins, including E1A (28), ␤-catenin (29 -32), and p53 (33).
Consistent with their considerable size (265 kDa), p300 and CBP contain numerous interaction surfaces. CBP/p300 contain two transcriptional adapter zinc-binding (TAZ) motifs, called cysteine/histidine-rich domains 1 and 3 (CH1 and CH3), that function in protein recognition (34). To further determine the interaction domain of p300/CBP with Sox9 in intact cells, we transfected truncated p300 constructs with FLAG-Sox9 in SW1353 cells and examined their interaction by co-immunoprecipitation (Fig. 2C). Consistent with the proceeding GST pull-down assay, p300 ⌬CH1 was recovered from co-immunoprecipitate with FLAG-Sox9 as well as wild type p300. In contrast, p300 ⌬CH3, which has the 33-amino acid deletion in the CH3 region (25), did not bind to Sox9 as well, suggesting a critical role for CH3 domain in Sox9/p300 interaction (Fig. 2D).
p300/CBP Activates Sox9 Transcriptional Activity-We next investigated the functional significance of the interaction between CBP/p300 and Sox9 by transient transfection reporter assays. Using a luciferase reporter plasmid containing Col2a1 promoter elements, including the Sox9 binding domain (pKN185) (3), cotransfection of p300 increased luciferase activity. Compared with wild type p300, p300⌬CH3 (25) functioned as a dominant negative inhibitor of transcription by reducing promoter activity 4-fold. Interestingly, p300⌬CH1 also showed strong dominant negative effect on the Col2a1 promoter, suggesting that the CH1 domain might also play a role in this transcriptional activation (Figs. 2C and 3A).
To further verify the CBP/p300 dependence on Sox9-mediated transactivation, we applied the Gal4 fusion system. Sox9 was fused to the Gal4 DNA binding domain ( Fig. 2A), and its activity was analyzed with a reporter containing five Gal4 binding sites and the TK promoter (Gal4 TK). Transfection with Gal4-Sox9-(182-507) in SW1353 cells led to a 21-fold increase in luciferase activity compared with the Gal4 DNA binding domain (Fig 3B). However, the Gal4-Sox9 construct lacking a C-terminal activation domain, which was identified as a CBP interaction domain, did not show a strong transcription activity (Gal4-Sox9 amino acids 1-423, 1-327). The Gal4-Sox9-(182-507) transcriptional activity was further enhanced by cotransfection of p300 or CBP. We observed that the transfection of p300 in conjunction with Gal4-Sox9-(182-507) in SW1353 cells showed a 13-fold increase in luciferase activity compared with Gal4-Sox9-(182-507) alone. CBP had a synergistic effect similar to that of p300 (Fig. 3C). Collectively, these data support the notion that p300/CBP enhances Sox9 transcription activity.
Critical Role of CBP/Sox9 Association for Col2a1 Gene Expression-To determine whether CBP/p300 is bound to the Col2a1 promoter, we performed chromatin immunoprecipitation using CBP-and p300-specific antibodies (Fig. 4A). PCR amplification products from reactions with human Col2a1 first intron 2151-2305, which contains the Sox9 DNA binging site, were analyzed by 2% agarose gel. The Col2a1 intron DNA was efficiently recovered from immunoprecipitates of CBP but not control immunoglobulin (IgG) (Fig. 4B). Confirming the specificity of these antisera, no PCR product was obtained from CBP immunoprecipitates using 293T cells in which Sox9 is not expressed, and, thus, CBP should not be recruited to the Col2a1 promoter (Fig. 4B). To further examine the role of CBP/p300 and Sox9 interaction for Col2a1 gene expression, we used adenovirus expressing CH3 peptide (mouse CBP, amino acids 1805-1890 with Gal4 DNA binding domain as a tag) to disrupt CBP/p300 and Sox9 complex formation. Because the CH3 domain was determined as the Sox9 binding domain (Fig. 2, B and D), we hypothesized that overexpression of the CH3 peptide may block CBP/p300 and Sox9 interaction and act as a potential dominant negative tool for Sox9-CBP/p300-dependent transactivation (Fig. 5A). The infection efficiency of this adenovirus in SW1353 cells was examined by immunocytochemistry using anti-Gal4 antibodies and shows almost 100% infection efficiency with an m.o.i. of 8 (data not shown). After infection, we examined p300-Sox9 complex formation by co-immunoprecipitation. With an m.o.i. of 8, the adenovirus infection blocked p300/Sox9 interaction, probably by masking the Sox9-p300 binding domain, whereas a control adenovirus carrying the lacZ gene had no effect on complex formation (Fig. 5B). Next, we tested endogenous Col2a1 gene expression. Levels of Col2a1 mRNA were determined by quantitative PCR. Col2a1 mRNA levels were strongly reduced by overexpression of CH3 peptide (about 80% reduction with m.o.i. of 8), whereas there was no effect in control adenovirus infected cells (Fig. 5C). Taken together, these results suggest that the association of CBP/p300 with Sox9 regulates Col2a1 gene expression.
Critical Role of CBP/Sox9 Association for Chondrogenesis from MSC-To examine the function of p300 in the chondrogenesis, we infected human MSCs with an adenovirus expressing the CH3 domain of CBP and examined the effect of the disrupter peptide on chondrocyte differentiation (35)(36)(37). As a control, we used adenovirus expressing the KIX domain of CBP (mouse CBP amino acids 586 -672), which is also characterized as a protein interaction domain for several transcription factors, including CREB, c-Jun, and c-Myb (10,25). Following the infection of the adenovirus, MSC cells were transferred to micromass cultures and incubated in the presence of transforming growth factor ␤ to induce chondrogenesis. More than 95% of MSCs were co-expressing the green fluorescent protein (GFP) marker (not shown). Col2a1 expression, a marker for chondrogenesis, was not affected by control GFP or KIX peptide (Fig. 6, A and B). CH3 peptide infection, however, blocked Col2a1 expression (Fig. 6C). Staining with control rabbit serum was negative for each of the samples (data not shown). These results indicate that CBP/p300 also plays a critical role in MSC differentiation into chondrocyte as well as chondrocyte-specific gene expression.

DISCUSSION
Sox9 has been identified as a critical molecule in chondrocyte differentiation (2,4). Expression of Col2a1 shadows Sox9 expression. In the genital ridge, however, Col2a1 is not expressed despite a high level of Sox9 expression (4). This suggests the existence of molecular partners of Sox9 that are required for cartilage-specific Col2a1 gene expression.
Here we identified the interaction of Sox9 with CBP/p300. The paralogous proteins CBP and p300 were originally identified as interaction partners for CREB (38) and the adenoviral FIG. 3. p300 promotes Sox9-dependent transcriptional activity. A, wild type but not mutant p300 activates transcription regulated by Sox9 binding sites. The effects of p300 or its mutant were monitored in a transient transfection luciferase assay. SW1353 cells were transfected with reporter plasmids carrying Sox9 binding site upstream of the TK promoter together with expression plasmids for wild type or the mutant of p300. Fold induction represents the ratio of luciferase activity measured in the absence of p300 expression vectors and the activity in the presence of p300 proteins after normalization. B, carboxyl terminal of Sox9 has transcriptional activity. Various regions of Sox9 indicated in Fig. 2A were fused with the Gal DNA binding domain and expressed in SW1353 cells. Reporter gene expression regulated by Gal binding site was measured. C, p300/CBP enhances transcriptional activity of Sox9. Expression vectors of p300 or CBP were tranfected into SW1353 cells together with Gal-fused, amino-terminal truncated Sox9 (182-507). Reporter gene expression regulated by the Gal binding site was measured.

FIG. 4. CBP binding to Col2a1 promoter chromatin. A and B,
Col2a1 promoter DNA is recovered and amplified from immunoprecipitates with CBP antibodies from SW1353 cells (A) but not from 293T cells (B). PCR amplification products from reactions with human Col2a1 first intron 2151-2305, which contains the Sox9 DNA binging site, were analyzed by 2% agarose gel.
E1A protein (28), respectively. Subsequently, a variety of different DNA binding transcription factors and co-activators have been shown to rely on CBP/p300 for their function in transcription activation (39).
CBP/p300 contains two TAZ motifs, which function as the sites of interaction with numerous transcription factors and viral oncogenes (34,40). The TAZ1 motif corresponds to cysteine/histidine-rich domain 1, i.e. the CH1 domain; the TAZ2 domain and the zinc-binding domain together make up cysteine/histidine-rich domain 3, i.e. the CH3 domain) (34). In addition to the CH3 domain, which is identified as an interaction domain for Sox9 binding, we observed that the CH1 domain of p300 is also critical for co-activator function. The CH1 domain of CBP/p300 has been shown to interact with transcriptional factor Hif-1 (34,40). One of the CBP/p300 functions is to recruit RNA polymerase machinery. For example, in the case of CREB transcription, Montminy and co-workers showed that CH3 is critical for its recruitment of RNA polymerase II (41,42). Because the CH3 domain is occupied by Sox9 interaction, the CH1 domain may play a role in recruiting RNA polymerase II or other basic transcriptional machinery in Sox9-dependent transcriptional activation.
Recently, mutations in the gene encoding CBP were found to cause Rubinstein-Taybi syndrome (20). The CBP ϩ/Ϫ mice exhibited the clinical features of Rubinstein-Taybi syndrome, including skeletal abnormalities (21,22). This phenotype could be, at least in part, explained by our findings that demonstrate the critical role of CBP/p300 in transcriptional activity of Sox9. Sox9 has been shown to plays an important role for developing skeletal framework (5,6). The absence of CBP may reduce Sox9 activity and, hence, may indirectly affect skeletal development (22). Research on molecular function of co-activator CBP/p300 in accordance with Sox9 will advance our understanding of skeletal development and has the potential to identify new approaches to the treatment of skeletal and articular joint diseases (43).