Limits...
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.

Show MeSH

Related in: MedlinePlus

Col10a1 promoter and its upregulation in MCT cells. (A) Restriction map of murine Col10a1. Col10a1 is composed of three exons numbered I, II and III. Position of TATA box in the minimal promoter, ATG start codon, and TGA stop codon are marked. Positions of the two putative Runx2 binding elements are highlighted as A and B. The 4-kb promoter used in transgenic mice studies is indicated below by arrows. E, EcoRI; Xh, XhoI; K, KpnI. (B) Endogenous Col10a1 mRNA and Col10a1 4-kb promoter activity are up-regulated in hypertrophic MCT cells. (left) Northern analysis of Col10a1 expression from MCT cells grown for 3 d at either 32°C or 37°C. Loading was normalized by hybridization with Gapdh. Col10a1 was expressed abundantly in hypertrophic MCT cells at 37°C but not in proliferating MCT cells at 32°C. (right) Col10a1-SAβgeobpA reporter plasmid was transfected into MCT cells grown at 32°C and then further cultured at either 32°C or 37°C. A RSV-luc luciferase expression plasmid was cotransfected as internal control for transfection efficiency. Bars represent the average ratios of β-galactosidase to luciferase activity. The standard deviations from three independent transfections of one representative experiment are indicated by the error bars.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172833&req=5

fig2: Col10a1 promoter and its upregulation in MCT cells. (A) Restriction map of murine Col10a1. Col10a1 is composed of three exons numbered I, II and III. Position of TATA box in the minimal promoter, ATG start codon, and TGA stop codon are marked. Positions of the two putative Runx2 binding elements are highlighted as A and B. The 4-kb promoter used in transgenic mice studies is indicated below by arrows. E, EcoRI; Xh, XhoI; K, KpnI. (B) Endogenous Col10a1 mRNA and Col10a1 4-kb promoter activity are up-regulated in hypertrophic MCT cells. (left) Northern analysis of Col10a1 expression from MCT cells grown for 3 d at either 32°C or 37°C. Loading was normalized by hybridization with Gapdh. Col10a1 was expressed abundantly in hypertrophic MCT cells at 37°C but not in proliferating MCT cells at 32°C. (right) Col10a1-SAβgeobpA reporter plasmid was transfected into MCT cells grown at 32°C and then further cultured at either 32°C or 37°C. A RSV-luc luciferase expression plasmid was cotransfected as internal control for transfection efficiency. Bars represent the average ratios of β-galactosidase to luciferase activity. The standard deviations from three independent transfections of one representative experiment are indicated by the error bars.

Mentions: We isolated a murine Col10a1 containing BAC clone. Sequence analysis on the 5′ promoter region revealed several putative Runx2 binding sites (PuACCPuCA or TGTGGT; Ducy et al., 1997; Jimenez et al., 1999). Then we performed comparative genome analysis of type X collagen gene promoter across species. Multiple potential Runx2 binding sites were also observed within 5 kb of the human and chicken Col10a1 promoters (Fig. 1, A and B). Interestingly, no significant conservation within long stretches of sequences flanking the Runx2 binding sites was observed between the human and mouse type X collagen genes. However, immediate flanking sequences, i.e., 10 bp on either side of sites −3485 and −2379 bp (A and B elements; Fig. 1 A and Fig. 2 A) in the mouse Col10a1 gene, showed 60–70% homology between the human and murine Col10a1 promoter region (Fig. 1 C).


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 and its upregulation in MCT cells. (A) Restriction map of murine Col10a1. Col10a1 is composed of three exons numbered I, II and III. Position of TATA box in the minimal promoter, ATG start codon, and TGA stop codon are marked. Positions of the two putative Runx2 binding elements are highlighted as A and B. The 4-kb promoter used in transgenic mice studies is indicated below by arrows. E, EcoRI; Xh, XhoI; K, KpnI. (B) Endogenous Col10a1 mRNA and Col10a1 4-kb promoter activity are up-regulated in hypertrophic MCT cells. (left) Northern analysis of Col10a1 expression from MCT cells grown for 3 d at either 32°C or 37°C. Loading was normalized by hybridization with Gapdh. Col10a1 was expressed abundantly in hypertrophic MCT cells at 37°C but not in proliferating MCT cells at 32°C. (right) Col10a1-SAβgeobpA reporter plasmid was transfected into MCT cells grown at 32°C and then further cultured at either 32°C or 37°C. A RSV-luc luciferase expression plasmid was cotransfected as internal control for transfection efficiency. Bars represent the average ratios of β-galactosidase to luciferase activity. The standard deviations from three independent transfections of one representative experiment are indicated by the error bars.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172833&req=5

fig2: Col10a1 promoter and its upregulation in MCT cells. (A) Restriction map of murine Col10a1. Col10a1 is composed of three exons numbered I, II and III. Position of TATA box in the minimal promoter, ATG start codon, and TGA stop codon are marked. Positions of the two putative Runx2 binding elements are highlighted as A and B. The 4-kb promoter used in transgenic mice studies is indicated below by arrows. E, EcoRI; Xh, XhoI; K, KpnI. (B) Endogenous Col10a1 mRNA and Col10a1 4-kb promoter activity are up-regulated in hypertrophic MCT cells. (left) Northern analysis of Col10a1 expression from MCT cells grown for 3 d at either 32°C or 37°C. Loading was normalized by hybridization with Gapdh. Col10a1 was expressed abundantly in hypertrophic MCT cells at 37°C but not in proliferating MCT cells at 32°C. (right) Col10a1-SAβgeobpA reporter plasmid was transfected into MCT cells grown at 32°C and then further cultured at either 32°C or 37°C. A RSV-luc luciferase expression plasmid was cotransfected as internal control for transfection efficiency. Bars represent the average ratios of β-galactosidase to luciferase activity. The standard deviations from three independent transfections of one representative experiment are indicated by the error bars.
Mentions: We isolated a murine Col10a1 containing BAC clone. Sequence analysis on the 5′ promoter region revealed several putative Runx2 binding sites (PuACCPuCA or TGTGGT; Ducy et al., 1997; Jimenez et al., 1999). Then we performed comparative genome analysis of type X collagen gene promoter across species. Multiple potential Runx2 binding sites were also observed within 5 kb of the human and chicken Col10a1 promoters (Fig. 1, A and B). Interestingly, no significant conservation within long stretches of sequences flanking the Runx2 binding sites was observed between the human and mouse type X collagen genes. However, immediate flanking sequences, i.e., 10 bp on either side of sites −3485 and −2379 bp (A and B elements; Fig. 1 A and Fig. 2 A) in the mouse Col10a1 gene, showed 60–70% homology between the human and murine Col10a1 promoter region (Fig. 1 C).

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