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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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Col10a1 promoter activity is up-regulated via RUNX2 binding elements in vitro. (A) Transactivation of Col10a1 via RUNX2-binding A and B elements. The RUNX2 expression plasmid (RUNX2) was cotransfected in COS7 cells with reporter plasmids Min-Col10a1-pA or 8xA/B-Min-Col10a1-pA. Transfection of Min-Col10a1-pA with or without RUNX2 expression plasmid produced no transactivation (lanes 2 and 3). Transfections of 8xA/B-Min-Col10a1-pA alone also produced no transactivation (lanes 4 and 6). However, the addition of the RUNX2 expression plasmid resulted in strong transactivation of 8xA-Min-Col10a1-PA and 8xB-Min-Col10a1-pA reporter plasmids, respectively (lanes 5 and 7). A SV2βgal plasmid was cotransfected as an internal control for transfection efficiency. Representative data are presented as fold activation relative to the activity obtained with pcDNA3.1 empty vector plasmid (lane 1). Each transfection experiment was performed in triplicate and the standard deviations are shown by the error bars. (B) Contribution of RUNX2 binding sites to 4-kb Col10a1 promoter activity. (left) Overexpression of RUNX2 in MCT cells further upregulates the 4-kb Col10a1 promoter. MCT cells were transfected only at the nonpermissive temperature (37°C) with the reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβgeobpA alone, and 4-kb Col10a1-SAβ-geobpA along with the Runx2 expression plasmid. A RSV-luc plasmid was cotransfected as internal control for transfection efficiency. The endogenous activity of the 4-kb promoter was 10-fold higher than that of the basal promoter, whereas over-expression of RUNX2 further increased the promoter activity. (right) RUNX2 binding sites contribute to 4-kb Col10a1 promoter activity in MCT cells. MCT cells were transfected only at nonpermissive temperature (37°C) with reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβ-geobpA, and mut 4-kb Col10a1-SAβ-geobpA. When the two Runx2 binding sites were mutated, reporter activity was decreased by 35% compared to that of the wild-type promoter.
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fig5: Col10a1 promoter activity is up-regulated via RUNX2 binding elements in vitro. (A) Transactivation of Col10a1 via RUNX2-binding A and B elements. The RUNX2 expression plasmid (RUNX2) was cotransfected in COS7 cells with reporter plasmids Min-Col10a1-pA or 8xA/B-Min-Col10a1-pA. Transfection of Min-Col10a1-pA with or without RUNX2 expression plasmid produced no transactivation (lanes 2 and 3). Transfections of 8xA/B-Min-Col10a1-pA alone also produced no transactivation (lanes 4 and 6). However, the addition of the RUNX2 expression plasmid resulted in strong transactivation of 8xA-Min-Col10a1-PA and 8xB-Min-Col10a1-pA reporter plasmids, respectively (lanes 5 and 7). A SV2βgal plasmid was cotransfected as an internal control for transfection efficiency. Representative data are presented as fold activation relative to the activity obtained with pcDNA3.1 empty vector plasmid (lane 1). Each transfection experiment was performed in triplicate and the standard deviations are shown by the error bars. (B) Contribution of RUNX2 binding sites to 4-kb Col10a1 promoter activity. (left) Overexpression of RUNX2 in MCT cells further upregulates the 4-kb Col10a1 promoter. MCT cells were transfected only at the nonpermissive temperature (37°C) with the reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβgeobpA alone, and 4-kb Col10a1-SAβ-geobpA along with the Runx2 expression plasmid. A RSV-luc plasmid was cotransfected as internal control for transfection efficiency. The endogenous activity of the 4-kb promoter was 10-fold higher than that of the basal promoter, whereas over-expression of RUNX2 further increased the promoter activity. (right) RUNX2 binding sites contribute to 4-kb Col10a1 promoter activity in MCT cells. MCT cells were transfected only at nonpermissive temperature (37°C) with reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβ-geobpA, and mut 4-kb Col10a1-SAβ-geobpA. When the two Runx2 binding sites were mutated, reporter activity was decreased by 35% compared to that of the wild-type promoter.

Mentions: In cotransfection studies in COS7 cells, RUNX2 was able to transactivate reporter constructs containing eight copies of either of these RUNX2-binding elements upstream of a 44-bp Col10a1 minimal promoter and the luciferase reporter gene (Fig. 5 A). Promoter constructs containing the A element or B element were transactivated >20- and 40-fold above baseline, respectively (Fig. 5 A). These data show that RUNX2 can bind to the distal sequences of the 4-kb Col10a1 promoter and transactivate a Col10a1 minimal promoter via these sequences. We also transfected the Col10a1 4-kb promoter-β-galactosidase reporter plasmid with or without the RUNX2 expression plasmid into hypertrophic MCT cells. The endogenous activity of the 4-kb promoter was 10-fold greater than that of the basal promoter (Fig. 5 B, left). In addition, when RUNX2 is over-expressed in these cells, reporter activity is further upregulated more than two-fold above the endogenous activity of this promoter (Fig. 5 B, left). In converse, when the two RUNX2 binding sites are mutated in the 4-kb promoter, reporter activity is decreased by 35% compared to the wild-type promoter (Fig. 5 B, right). Together, these data show that the 4-kb Col10a1 promoter is up-regulated when MCT chondrocytes hypertrophy in culture and that RUNX2 binding contributes to this transactivation.


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)

Col10a1 promoter activity is up-regulated via RUNX2 binding elements in vitro. (A) Transactivation of Col10a1 via RUNX2-binding A and B elements. The RUNX2 expression plasmid (RUNX2) was cotransfected in COS7 cells with reporter plasmids Min-Col10a1-pA or 8xA/B-Min-Col10a1-pA. Transfection of Min-Col10a1-pA with or without RUNX2 expression plasmid produced no transactivation (lanes 2 and 3). Transfections of 8xA/B-Min-Col10a1-pA alone also produced no transactivation (lanes 4 and 6). However, the addition of the RUNX2 expression plasmid resulted in strong transactivation of 8xA-Min-Col10a1-PA and 8xB-Min-Col10a1-pA reporter plasmids, respectively (lanes 5 and 7). A SV2βgal plasmid was cotransfected as an internal control for transfection efficiency. Representative data are presented as fold activation relative to the activity obtained with pcDNA3.1 empty vector plasmid (lane 1). Each transfection experiment was performed in triplicate and the standard deviations are shown by the error bars. (B) Contribution of RUNX2 binding sites to 4-kb Col10a1 promoter activity. (left) Overexpression of RUNX2 in MCT cells further upregulates the 4-kb Col10a1 promoter. MCT cells were transfected only at the nonpermissive temperature (37°C) with the reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβgeobpA alone, and 4-kb Col10a1-SAβ-geobpA along with the Runx2 expression plasmid. A RSV-luc plasmid was cotransfected as internal control for transfection efficiency. The endogenous activity of the 4-kb promoter was 10-fold higher than that of the basal promoter, whereas over-expression of RUNX2 further increased the promoter activity. (right) RUNX2 binding sites contribute to 4-kb Col10a1 promoter activity in MCT cells. MCT cells were transfected only at nonpermissive temperature (37°C) with reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβ-geobpA, and mut 4-kb Col10a1-SAβ-geobpA. When the two Runx2 binding sites were mutated, reporter activity was decreased by 35% compared to that of the wild-type promoter.
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fig5: Col10a1 promoter activity is up-regulated via RUNX2 binding elements in vitro. (A) Transactivation of Col10a1 via RUNX2-binding A and B elements. The RUNX2 expression plasmid (RUNX2) was cotransfected in COS7 cells with reporter plasmids Min-Col10a1-pA or 8xA/B-Min-Col10a1-pA. Transfection of Min-Col10a1-pA with or without RUNX2 expression plasmid produced no transactivation (lanes 2 and 3). Transfections of 8xA/B-Min-Col10a1-pA alone also produced no transactivation (lanes 4 and 6). However, the addition of the RUNX2 expression plasmid resulted in strong transactivation of 8xA-Min-Col10a1-PA and 8xB-Min-Col10a1-pA reporter plasmids, respectively (lanes 5 and 7). A SV2βgal plasmid was cotransfected as an internal control for transfection efficiency. Representative data are presented as fold activation relative to the activity obtained with pcDNA3.1 empty vector plasmid (lane 1). Each transfection experiment was performed in triplicate and the standard deviations are shown by the error bars. (B) Contribution of RUNX2 binding sites to 4-kb Col10a1 promoter activity. (left) Overexpression of RUNX2 in MCT cells further upregulates the 4-kb Col10a1 promoter. MCT cells were transfected only at the nonpermissive temperature (37°C) with the reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβgeobpA alone, and 4-kb Col10a1-SAβ-geobpA along with the Runx2 expression plasmid. A RSV-luc plasmid was cotransfected as internal control for transfection efficiency. The endogenous activity of the 4-kb promoter was 10-fold higher than that of the basal promoter, whereas over-expression of RUNX2 further increased the promoter activity. (right) RUNX2 binding sites contribute to 4-kb Col10a1 promoter activity in MCT cells. MCT cells were transfected only at nonpermissive temperature (37°C) with reporter plasmids basCol10a1-pSAβ-geobpA, 4-kb Col10a1-SAβ-geobpA, and mut 4-kb Col10a1-SAβ-geobpA. When the two Runx2 binding sites were mutated, reporter activity was decreased by 35% compared to that of the wild-type promoter.
Mentions: In cotransfection studies in COS7 cells, RUNX2 was able to transactivate reporter constructs containing eight copies of either of these RUNX2-binding elements upstream of a 44-bp Col10a1 minimal promoter and the luciferase reporter gene (Fig. 5 A). Promoter constructs containing the A element or B element were transactivated >20- and 40-fold above baseline, respectively (Fig. 5 A). These data show that RUNX2 can bind to the distal sequences of the 4-kb Col10a1 promoter and transactivate a Col10a1 minimal promoter via these sequences. We also transfected the Col10a1 4-kb promoter-β-galactosidase reporter plasmid with or without the RUNX2 expression plasmid into hypertrophic MCT cells. The endogenous activity of the 4-kb promoter was 10-fold greater than that of the basal promoter (Fig. 5 B, left). In addition, when RUNX2 is over-expressed in these cells, reporter activity is further upregulated more than two-fold above the endogenous activity of this promoter (Fig. 5 B, left). In converse, when the two RUNX2 binding sites are mutated in the 4-kb promoter, reporter activity is decreased by 35% compared to the wild-type promoter (Fig. 5 B, right). Together, these data show that the 4-kb Col10a1 promoter is up-regulated when MCT chondrocytes hypertrophy in culture and that RUNX2 binding contributes to this transactivation.

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