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Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo.

Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B - J. Cell Biol. (2003)

Bottom Line: In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites.When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels.Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The alpha1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte-specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive beta-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2- mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

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RUNX2 binds to cis elements A and B within Col10a1 promoter region. (A) EMSA using His-tagged recombinant RUNX2 Runt polypeptide with cis element A or B as probe (Fig. 1 A). 32P-End–labeled wild-type (WT) probe A or B (Fig. 1) and the probe mutated outside of the core sequence (MO) exhibited binding (arrow), whereas a mutation in the core sequence (MI) abolished binding. (B) EMSA using nuclear extracts from hypertrophic MCT cells grown at 37°C with A or B probe. Both 32P-end–labeled wild-type probe A or B (lane 1) shows two specific DNA–protein complexes (arrows). These complexes diminish with competition with 100-fold cold probe (lane 2). Presence of Runx2 in the DNA–protein complex is demonstrated by the formation of a new low mobility complex (arrowhead) with the addition of anti-Runx2 antibody to the MCT cell nuclear extracts (lane 3) but not in the control with preimmune serum (lane 4). (C) Runx2 binds to the mouse A and B elements in hypertrophic MCT cells. A representative chromatin immunoprecipitation experiment with hypertrophic MCT cells is shown. Immunoprecipitation was performed with anti-Runx2 antibody (lanes 2, 6, and 10), preimmune antiserum (lanes 3, 7, and 11) or no antibody (lanes 4, 8, and 12). Following DNA purification, samples were subject to 35 cycles of PCR with primers designed for the A (lanes 1–4), B (lanes 5–8), and negative control elements (lane 9–12). A portion of input was used as positive control for PCR (lanes 1, 5, and 9). The results indicated that Runx2 binds to these putative Runx2 binding A and B elements within Col10a1 promoter in vivo (lanes 2 and 6).
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fig4: RUNX2 binds to cis elements A and B within Col10a1 promoter region. (A) EMSA using His-tagged recombinant RUNX2 Runt polypeptide with cis element A or B as probe (Fig. 1 A). 32P-End–labeled wild-type (WT) probe A or B (Fig. 1) and the probe mutated outside of the core sequence (MO) exhibited binding (arrow), whereas a mutation in the core sequence (MI) abolished binding. (B) EMSA using nuclear extracts from hypertrophic MCT cells grown at 37°C with A or B probe. Both 32P-end–labeled wild-type probe A or B (lane 1) shows two specific DNA–protein complexes (arrows). These complexes diminish with competition with 100-fold cold probe (lane 2). Presence of Runx2 in the DNA–protein complex is demonstrated by the formation of a new low mobility complex (arrowhead) with the addition of anti-Runx2 antibody to the MCT cell nuclear extracts (lane 3) but not in the control with preimmune serum (lane 4). (C) Runx2 binds to the mouse A and B elements in hypertrophic MCT cells. A representative chromatin immunoprecipitation experiment with hypertrophic MCT cells is shown. Immunoprecipitation was performed with anti-Runx2 antibody (lanes 2, 6, and 10), preimmune antiserum (lanes 3, 7, and 11) or no antibody (lanes 4, 8, and 12). Following DNA purification, samples were subject to 35 cycles of PCR with primers designed for the A (lanes 1–4), B (lanes 5–8), and negative control elements (lane 9–12). A portion of input was used as positive control for PCR (lanes 1, 5, and 9). The results indicated that Runx2 binds to these putative Runx2 binding A and B elements within Col10a1 promoter in vivo (lanes 2 and 6).

Mentions: Electrophoretic mobility shift assays (EMSA) showed that His-tagged recombinant DNA-binding RUNT domain polypeptide bound to each of the putative RUNX2 binding sites A and B found in the Col10a1 promoter (Fig. 4 A). As expected, mutations within the core binding sequences abolished binding, whereas mutations outside of the core sequence had no significant effect on formation of the DNA–protein complex (Fig. 4 A). EMSA, using nuclear extracts from hypertrophic MCT cells, showed DNA–protein complexes specific for each of these two cis elements (Fig. 4 B). These DNA–protein complexes were effectively competed for by unlabeled probe. Moreover, the addition of anti-Runx2 antibody resulted in the formation of a new low mobility complex, suggesting that Runx2 is able to bind the DNA elements and might be a component of the higher mobility complex(es) formed in the absence of antibody (Fig. 4 B). Interestingly, the EMSA with MCT nuclear extracts showed two complexes that were both effectively competed for by cold probe suggesting that different molecular complexes may form on these cis elements. The two additional putative Runx2 binding sites in the mouse 4-kb promoter bound RUNX2 weakly and the addition of antibody did not generate bands of delayed mobility (unpublished data).


Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocyte-specific expression in vivo.

Zheng Q, Zhou G, Morello R, Chen Y, Garcia-Rojas X, Lee B - J. Cell Biol. (2003)

RUNX2 binds to cis elements A and B within Col10a1 promoter region. (A) EMSA using His-tagged recombinant RUNX2 Runt polypeptide with cis element A or B as probe (Fig. 1 A). 32P-End–labeled wild-type (WT) probe A or B (Fig. 1) and the probe mutated outside of the core sequence (MO) exhibited binding (arrow), whereas a mutation in the core sequence (MI) abolished binding. (B) EMSA using nuclear extracts from hypertrophic MCT cells grown at 37°C with A or B probe. Both 32P-end–labeled wild-type probe A or B (lane 1) shows two specific DNA–protein complexes (arrows). These complexes diminish with competition with 100-fold cold probe (lane 2). Presence of Runx2 in the DNA–protein complex is demonstrated by the formation of a new low mobility complex (arrowhead) with the addition of anti-Runx2 antibody to the MCT cell nuclear extracts (lane 3) but not in the control with preimmune serum (lane 4). (C) Runx2 binds to the mouse A and B elements in hypertrophic MCT cells. A representative chromatin immunoprecipitation experiment with hypertrophic MCT cells is shown. Immunoprecipitation was performed with anti-Runx2 antibody (lanes 2, 6, and 10), preimmune antiserum (lanes 3, 7, and 11) or no antibody (lanes 4, 8, and 12). Following DNA purification, samples were subject to 35 cycles of PCR with primers designed for the A (lanes 1–4), B (lanes 5–8), and negative control elements (lane 9–12). A portion of input was used as positive control for PCR (lanes 1, 5, and 9). The results indicated that Runx2 binds to these putative Runx2 binding A and B elements within Col10a1 promoter in vivo (lanes 2 and 6).
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fig4: RUNX2 binds to cis elements A and B within Col10a1 promoter region. (A) EMSA using His-tagged recombinant RUNX2 Runt polypeptide with cis element A or B as probe (Fig. 1 A). 32P-End–labeled wild-type (WT) probe A or B (Fig. 1) and the probe mutated outside of the core sequence (MO) exhibited binding (arrow), whereas a mutation in the core sequence (MI) abolished binding. (B) EMSA using nuclear extracts from hypertrophic MCT cells grown at 37°C with A or B probe. Both 32P-end–labeled wild-type probe A or B (lane 1) shows two specific DNA–protein complexes (arrows). These complexes diminish with competition with 100-fold cold probe (lane 2). Presence of Runx2 in the DNA–protein complex is demonstrated by the formation of a new low mobility complex (arrowhead) with the addition of anti-Runx2 antibody to the MCT cell nuclear extracts (lane 3) but not in the control with preimmune serum (lane 4). (C) Runx2 binds to the mouse A and B elements in hypertrophic MCT cells. A representative chromatin immunoprecipitation experiment with hypertrophic MCT cells is shown. Immunoprecipitation was performed with anti-Runx2 antibody (lanes 2, 6, and 10), preimmune antiserum (lanes 3, 7, and 11) or no antibody (lanes 4, 8, and 12). Following DNA purification, samples were subject to 35 cycles of PCR with primers designed for the A (lanes 1–4), B (lanes 5–8), and negative control elements (lane 9–12). A portion of input was used as positive control for PCR (lanes 1, 5, and 9). The results indicated that Runx2 binds to these putative Runx2 binding A and B elements within Col10a1 promoter in vivo (lanes 2 and 6).
Mentions: Electrophoretic mobility shift assays (EMSA) showed that His-tagged recombinant DNA-binding RUNT domain polypeptide bound to each of the putative RUNX2 binding sites A and B found in the Col10a1 promoter (Fig. 4 A). As expected, mutations within the core binding sequences abolished binding, whereas mutations outside of the core sequence had no significant effect on formation of the DNA–protein complex (Fig. 4 A). EMSA, using nuclear extracts from hypertrophic MCT cells, showed DNA–protein complexes specific for each of these two cis elements (Fig. 4 B). These DNA–protein complexes were effectively competed for by unlabeled probe. Moreover, the addition of anti-Runx2 antibody resulted in the formation of a new low mobility complex, suggesting that Runx2 is able to bind the DNA elements and might be a component of the higher mobility complex(es) formed in the absence of antibody (Fig. 4 B). Interestingly, the EMSA with MCT nuclear extracts showed two complexes that were both effectively competed for by cold probe suggesting that different molecular complexes may form on these cis elements. The two additional putative Runx2 binding sites in the mouse 4-kb promoter bound RUNX2 weakly and the addition of antibody did not generate bands of delayed mobility (unpublished data).

Bottom Line: In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites.When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels.Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.

ABSTRACT
The alpha1(X) collagen gene (Col10a1) is the only known hypertrophic chondrocyte-specific molecular marker. Until recently, few transcriptional factors specifying its tissue-specific expression have been identified. We show here that a 4-kb murine Col10a1 promoter can drive beta-galactosidase expression in lower hypertrophic chondrocytes in transgenic mice. Comparative genomic analysis revealed multiple Runx2 (Runt domain transcription factor) binding sites within the proximal human, mouse, and chick Col10a1 promoters. In vitro transfection studies and chromatin immunoprecipitation analysis using hypertrophic MCT cells showed that Runx2 contributes to the transactivation of this promoter via its conserved Runx2 binding sites. When the 4-kb Col10a1 promoter transgene was bred onto a Runx2(+/-) background, the reporter was expressed at lower levels. Moreover, decreased Col10a1 expression and altered chondrocyte hypertrophy was also observed in Runx2 heterozygote mice, whereas Col10a1 was barely detectable in Runx2- mice. Together, these data suggest that Col10a1 is a direct transcriptional target of Runx2 during chondrogenesis.

Show MeSH
Related in: MedlinePlus