Abstract
Previously we have identified a cis-acting regulatory domain in the human type X collagen gene upstream of the transcription start site which acts as a strong enhancer in hypertrophic, but not in resting chondrocytes. Here we show that this enhancer is highly conserved also in the murine and bovine Col10a1 genes, but not found in the known promoter sequences of chicken Col10a1. It contains a functionally active AP-1 site (TPA Responsive Element, TRE) which is essential for the high transcriptional activity of the COL10A1 enhancer in transiently transfected hypertrophic chondrocytes. Gel-shift experiments with nuclear extracts of hypertrophic chondrocytes revealed FosB and Fra-1 as candidates regulating AP-1 factors binding to the TRE site. In fact, coexpression of FosB and Fra-1 in reporter gene assays greatly stimulated transcriptional activity of enhancer bearing reporter genes. Quantitative analysis of AP-1 factor mRNA levels in distinct fractions of fetal bovine epiphyseal chondrocytes by real-time PCR confirmed significant levels of FosB and Fra-1 mRNA besides other AP-1 factors in hypertrophic chondrocytes. A key role of the enhancer element in regulating tissue-specific expression of the Col10a1 gene was shown by establishing transgenic mouse lines with a reporter gene containing a 4.6 kb murine Col10a1 promoter fragment which included the enhancer, exon 1, part of exon 2 and the first intron. Reporter gene expression was seen exclusively in hypertrophic cartilages in the growth plates of long bones, ribs, vertebrae, sternum and mandibles of 17.5-18.5 dpc embryos, confirming that the 4.6 kb promoter is able to drive specific expression of Col10a1 in hypertrophic cartilage. These established transgenic lines should facilitate the genetic analysis of regulatory pathways of chondrocyte maturation and Col10a1 gene expression in the future.
| Original language | English |
|---|---|
| Pages (from-to) | 309-322 |
| Number of pages | 14 |
| Journal | Matrix Biology |
| Volume | 23 |
| Issue number | 5 |
| DOIs | |
| Publication status | Published - Aug 2004 |
| Externally published | Yes |
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Keywords
- Chondrocyte maturation
- Col10a1 gene expression
- Exon
ASJC Scopus subject areas
- Molecular Biology
Cite this
A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo. / Gebhard, Sonja; Pöschl, Ernst; Riemer, Silvia; Bauer, Eva; Hattori, Takako; Eberspaecher, Heidi; Zhang, Zhaoping; Lefebvre, Veronique; De Crombrugghe, Benoit; Von Der Mark, Klaus.
In: Matrix Biology, Vol. 23, No. 5, 08.2004, p. 309-322.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo
AU - Gebhard, Sonja
AU - Pöschl, Ernst
AU - Riemer, Silvia
AU - Bauer, Eva
AU - Hattori, Takako
AU - Eberspaecher, Heidi
AU - Zhang, Zhaoping
AU - Lefebvre, Veronique
AU - De Crombrugghe, Benoit
AU - Von Der Mark, Klaus
PY - 2004/8
Y1 - 2004/8
N2 - Previously we have identified a cis-acting regulatory domain in the human type X collagen gene upstream of the transcription start site which acts as a strong enhancer in hypertrophic, but not in resting chondrocytes. Here we show that this enhancer is highly conserved also in the murine and bovine Col10a1 genes, but not found in the known promoter sequences of chicken Col10a1. It contains a functionally active AP-1 site (TPA Responsive Element, TRE) which is essential for the high transcriptional activity of the COL10A1 enhancer in transiently transfected hypertrophic chondrocytes. Gel-shift experiments with nuclear extracts of hypertrophic chondrocytes revealed FosB and Fra-1 as candidates regulating AP-1 factors binding to the TRE site. In fact, coexpression of FosB and Fra-1 in reporter gene assays greatly stimulated transcriptional activity of enhancer bearing reporter genes. Quantitative analysis of AP-1 factor mRNA levels in distinct fractions of fetal bovine epiphyseal chondrocytes by real-time PCR confirmed significant levels of FosB and Fra-1 mRNA besides other AP-1 factors in hypertrophic chondrocytes. A key role of the enhancer element in regulating tissue-specific expression of the Col10a1 gene was shown by establishing transgenic mouse lines with a reporter gene containing a 4.6 kb murine Col10a1 promoter fragment which included the enhancer, exon 1, part of exon 2 and the first intron. Reporter gene expression was seen exclusively in hypertrophic cartilages in the growth plates of long bones, ribs, vertebrae, sternum and mandibles of 17.5-18.5 dpc embryos, confirming that the 4.6 kb promoter is able to drive specific expression of Col10a1 in hypertrophic cartilage. These established transgenic lines should facilitate the genetic analysis of regulatory pathways of chondrocyte maturation and Col10a1 gene expression in the future.
AB - Previously we have identified a cis-acting regulatory domain in the human type X collagen gene upstream of the transcription start site which acts as a strong enhancer in hypertrophic, but not in resting chondrocytes. Here we show that this enhancer is highly conserved also in the murine and bovine Col10a1 genes, but not found in the known promoter sequences of chicken Col10a1. It contains a functionally active AP-1 site (TPA Responsive Element, TRE) which is essential for the high transcriptional activity of the COL10A1 enhancer in transiently transfected hypertrophic chondrocytes. Gel-shift experiments with nuclear extracts of hypertrophic chondrocytes revealed FosB and Fra-1 as candidates regulating AP-1 factors binding to the TRE site. In fact, coexpression of FosB and Fra-1 in reporter gene assays greatly stimulated transcriptional activity of enhancer bearing reporter genes. Quantitative analysis of AP-1 factor mRNA levels in distinct fractions of fetal bovine epiphyseal chondrocytes by real-time PCR confirmed significant levels of FosB and Fra-1 mRNA besides other AP-1 factors in hypertrophic chondrocytes. A key role of the enhancer element in regulating tissue-specific expression of the Col10a1 gene was shown by establishing transgenic mouse lines with a reporter gene containing a 4.6 kb murine Col10a1 promoter fragment which included the enhancer, exon 1, part of exon 2 and the first intron. Reporter gene expression was seen exclusively in hypertrophic cartilages in the growth plates of long bones, ribs, vertebrae, sternum and mandibles of 17.5-18.5 dpc embryos, confirming that the 4.6 kb promoter is able to drive specific expression of Col10a1 in hypertrophic cartilage. These established transgenic lines should facilitate the genetic analysis of regulatory pathways of chondrocyte maturation and Col10a1 gene expression in the future.
KW - Chondrocyte maturation
KW - Col10a1 gene expression
KW - Exon
UR - http://www.scopus.com/inward/record.url?scp=4644372042&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=4644372042&partnerID=8YFLogxK
U2 - 10.1016/j.matbio.2004.05.010
DO - 10.1016/j.matbio.2004.05.010
M3 - Article
VL - 23
SP - 309
EP - 322
JO - Matrix Biology
JF - Matrix Biology
SN - 0945-053X
IS - 5
ER -