Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.
Article
Liu, Y, Li, H, Tanaka, K et al. (2000). Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.
. JOURNAL OF BIOLOGICAL CHEMISTRY, 275(17), 12712-12718. 10.1074/jbc.275.17.12712
Liu, Y, Li, H, Tanaka, K et al. (2000). Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.
. JOURNAL OF BIOLOGICAL CHEMISTRY, 275(17), 12712-12718. 10.1074/jbc.275.17.12712
Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.
