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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 in transgenic reporter mice. (A and B) X-gal stained E15.5 (left) and postnatal day 1 (P1; right) transgenic mice line 41 showed blue staining in the chondroosseus junction (arrows) of mouse limbs and ribs but not in the wild-type littermate controls. Background staining probably due to endogenous β-galactosidase activity was also observed in craniofacial region both in transgenic and control P1 mice. (C and E) Sagittal sections of proximal humerus and femur from P1 transgenic mouse line 41 shows blue staining in lower hypertrophic zone. Although strongest staining is observed in the lower hypertrophic zone, weak staining is also occasionally present in the bone trabeculae. (D and F) Comparative sagittal sections from wild-type littermates control show no staining. Tg, transgenic mice; Ctrl, wild-type littermate control; HZ, hypertrophic zone of growth plate.
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fig3: Col10a1 promoter activity in transgenic reporter mice. (A and B) X-gal stained E15.5 (left) and postnatal day 1 (P1; right) transgenic mice line 41 showed blue staining in the chondroosseus junction (arrows) of mouse limbs and ribs but not in the wild-type littermate controls. Background staining probably due to endogenous β-galactosidase activity was also observed in craniofacial region both in transgenic and control P1 mice. (C and E) Sagittal sections of proximal humerus and femur from P1 transgenic mouse line 41 shows blue staining in lower hypertrophic zone. Although strongest staining is observed in the lower hypertrophic zone, weak staining is also occasionally present in the bone trabeculae. (D and F) Comparative sagittal sections from wild-type littermates control show no staining. Tg, transgenic mice; Ctrl, wild-type littermate control; HZ, hypertrophic zone of growth plate.

Mentions: To determine the in vivo relevance of these data, we generated four independent transgenic mouse lines harboring the 4-kb proximal Col10a1 promoter upstream of the β-galactosidase reporter gene. Three lines of mice exhibited similar X-gal staining, whereas the fourth one did not show any staining. We performed whole embryo staining of E15.5 mouse embryos and blue staining was noted only at the ends of long bones (Fig. 3 A). No blue staining was observed in any other tissues when analyzing sagittal sections of the whole embryos featuring a variety of tissues (unpublished data). Although some background staining in craniofacial region probably due to endogenous β-galactosidase, activity was observed both in transgenic and control postnatal day 1 (P1) mice, specific blue staining was observed only in the chondro-osseus junction of limbs, ribs and also in the nasal cartilage of transgenic mice (Fig 3 B). Indeed, histological analysis confirmed that β-galactosidase expression was observed in the lower zone of hypertrophy of rib sections and in the long bone sections of the limbs including proximal humerus and proximal femur at P1 stage (Fig. 3, C and E, and not depicted). Although some weak staining was also present in bone marrow along trabeculae presumably in osteoblasts, no blue staining was observed in other tissues including perichondrium, resting or proliferating chondrocytes, muscle fibers, or adhering connective tissues (Fig. 3 B and not depicted). These data show that the proximal 4-kb Col10a1 promoter was able to direct hypertrophic chondrocyte expression of the β-galactosidase reporter in vivo and, therefore, contributes to Col10a1 expression in these cells. Because a 1.7-kb mouse promoter was previously reported to be unable to direct expression of β-galactosidase to hypertrophic chondrocytes (Eerola et al., 1996), we hypothesized that the positive regulatory elements in this 4-kb construct most likely reside in the 5′ portion where the two conserved Runx2 binding sites A and B are found.


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 in transgenic reporter mice. (A and B) X-gal stained E15.5 (left) and postnatal day 1 (P1; right) transgenic mice line 41 showed blue staining in the chondroosseus junction (arrows) of mouse limbs and ribs but not in the wild-type littermate controls. Background staining probably due to endogenous β-galactosidase activity was also observed in craniofacial region both in transgenic and control P1 mice. (C and E) Sagittal sections of proximal humerus and femur from P1 transgenic mouse line 41 shows blue staining in lower hypertrophic zone. Although strongest staining is observed in the lower hypertrophic zone, weak staining is also occasionally present in the bone trabeculae. (D and F) Comparative sagittal sections from wild-type littermates control show no staining. Tg, transgenic mice; Ctrl, wild-type littermate control; HZ, hypertrophic zone of growth plate.
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fig3: Col10a1 promoter activity in transgenic reporter mice. (A and B) X-gal stained E15.5 (left) and postnatal day 1 (P1; right) transgenic mice line 41 showed blue staining in the chondroosseus junction (arrows) of mouse limbs and ribs but not in the wild-type littermate controls. Background staining probably due to endogenous β-galactosidase activity was also observed in craniofacial region both in transgenic and control P1 mice. (C and E) Sagittal sections of proximal humerus and femur from P1 transgenic mouse line 41 shows blue staining in lower hypertrophic zone. Although strongest staining is observed in the lower hypertrophic zone, weak staining is also occasionally present in the bone trabeculae. (D and F) Comparative sagittal sections from wild-type littermates control show no staining. Tg, transgenic mice; Ctrl, wild-type littermate control; HZ, hypertrophic zone of growth plate.
Mentions: To determine the in vivo relevance of these data, we generated four independent transgenic mouse lines harboring the 4-kb proximal Col10a1 promoter upstream of the β-galactosidase reporter gene. Three lines of mice exhibited similar X-gal staining, whereas the fourth one did not show any staining. We performed whole embryo staining of E15.5 mouse embryos and blue staining was noted only at the ends of long bones (Fig. 3 A). No blue staining was observed in any other tissues when analyzing sagittal sections of the whole embryos featuring a variety of tissues (unpublished data). Although some background staining in craniofacial region probably due to endogenous β-galactosidase, activity was observed both in transgenic and control postnatal day 1 (P1) mice, specific blue staining was observed only in the chondro-osseus junction of limbs, ribs and also in the nasal cartilage of transgenic mice (Fig 3 B). Indeed, histological analysis confirmed that β-galactosidase expression was observed in the lower zone of hypertrophy of rib sections and in the long bone sections of the limbs including proximal humerus and proximal femur at P1 stage (Fig. 3, C and E, and not depicted). Although some weak staining was also present in bone marrow along trabeculae presumably in osteoblasts, no blue staining was observed in other tissues including perichondrium, resting or proliferating chondrocytes, muscle fibers, or adhering connective tissues (Fig. 3 B and not depicted). These data show that the proximal 4-kb Col10a1 promoter was able to direct hypertrophic chondrocyte expression of the β-galactosidase reporter in vivo and, therefore, contributes to Col10a1 expression in these cells. Because a 1.7-kb mouse promoter was previously reported to be unable to direct expression of β-galactosidase to hypertrophic chondrocytes (Eerola et al., 1996), we hypothesized that the positive regulatory elements in this 4-kb construct most likely reside in the 5′ portion where the two conserved Runx2 binding sites A and B are found.

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