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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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Comparative genomic analysis of type X collagen gene promoter. (A) Schematic illustration of human, mouse and chicken Col10a1 promoter. Putative Runx2 binding sites within the promoter region are indicated by yellow triangle. The two conserved sites A and B between human and mouse are highlighted as red triangle. (B) Sequence and position of the potential Runx2 binding sites within 5-kb of the human, mouse, and chick Col10a1 promoter. The putative RUNX2 binding sequence and their position within the type X promoter are shown in the Table. There are 7, 4 and 5 potential RUNX2 binding sites within 5-kb of the human, mouse, and chicken Col10a1 promoters, respectively. (C) Sequence comparison between mouse and human A and B elements. The sequence including the mouse A and B elements and 10 bp of immediate flanking sequence show 60–70% homology to the corresponding human elements. No significant similarity was found among other putative RUNX2 binding sites (not depicted). Core sequences of the putative RUNX2 binding sites are underlined. Bars represent identical nucleotides and vertical dots represent conserved nucleotide base pairing.
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fig1: Comparative genomic analysis of type X collagen gene promoter. (A) Schematic illustration of human, mouse and chicken Col10a1 promoter. Putative Runx2 binding sites within the promoter region are indicated by yellow triangle. The two conserved sites A and B between human and mouse are highlighted as red triangle. (B) Sequence and position of the potential Runx2 binding sites within 5-kb of the human, mouse, and chick Col10a1 promoter. The putative RUNX2 binding sequence and their position within the type X promoter are shown in the Table. There are 7, 4 and 5 potential RUNX2 binding sites within 5-kb of the human, mouse, and chicken Col10a1 promoters, respectively. (C) Sequence comparison between mouse and human A and B elements. The sequence including the mouse A and B elements and 10 bp of immediate flanking sequence show 60–70% homology to the corresponding human elements. No significant similarity was found among other putative RUNX2 binding sites (not depicted). Core sequences of the putative RUNX2 binding sites are underlined. Bars represent identical nucleotides and vertical dots represent conserved nucleotide base pairing.

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)

Comparative genomic analysis of type X collagen gene promoter. (A) Schematic illustration of human, mouse and chicken Col10a1 promoter. Putative Runx2 binding sites within the promoter region are indicated by yellow triangle. The two conserved sites A and B between human and mouse are highlighted as red triangle. (B) Sequence and position of the potential Runx2 binding sites within 5-kb of the human, mouse, and chick Col10a1 promoter. The putative RUNX2 binding sequence and their position within the type X promoter are shown in the Table. There are 7, 4 and 5 potential RUNX2 binding sites within 5-kb of the human, mouse, and chicken Col10a1 promoters, respectively. (C) Sequence comparison between mouse and human A and B elements. The sequence including the mouse A and B elements and 10 bp of immediate flanking sequence show 60–70% homology to the corresponding human elements. No significant similarity was found among other putative RUNX2 binding sites (not depicted). Core sequences of the putative RUNX2 binding sites are underlined. Bars represent identical nucleotides and vertical dots represent conserved nucleotide base pairing.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172833&req=5

fig1: Comparative genomic analysis of type X collagen gene promoter. (A) Schematic illustration of human, mouse and chicken Col10a1 promoter. Putative Runx2 binding sites within the promoter region are indicated by yellow triangle. The two conserved sites A and B between human and mouse are highlighted as red triangle. (B) Sequence and position of the potential Runx2 binding sites within 5-kb of the human, mouse, and chick Col10a1 promoter. The putative RUNX2 binding sequence and their position within the type X promoter are shown in the Table. There are 7, 4 and 5 potential RUNX2 binding sites within 5-kb of the human, mouse, and chicken Col10a1 promoters, respectively. (C) Sequence comparison between mouse and human A and B elements. The sequence including the mouse A and B elements and 10 bp of immediate flanking sequence show 60–70% homology to the corresponding human elements. No significant similarity was found among other putative RUNX2 binding sites (not depicted). Core sequences of the putative RUNX2 binding sites are underlined. Bars represent identical nucleotides and vertical dots represent conserved nucleotide base pairing.
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