Identification of an Enhancer Sequence within the First Intron Required for Cartilage-specific Transcription of the α2(XI) Collagen Gene

Abstract

Type XI collagen, a heterotrimer composed of ␣1(XI), ␣2(XI) and ␣3(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 ␣2(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 ␤-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the fulllength intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CT-CAAAG, 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 cartilagespecific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.Cartilage is a highly specialized tissue important for bearing compression loads in joints and also serves as the template for most developing bones. Cartilage contains unique extracellular matrix proteins produced by chondrocytes. The collagen network provides the scaffolding of the cartilage matrix and confers tensile strength important for resisting compression in cartilage. Collagen fibrils in cartilage consist of the major collagen, type II, and minor collagens, type IX and type XI. Type XI collagen, consisting of ␣1(XI), ␣2(XI), and ␣3(XI) chains, co-assembles with type II collagen to form cartilage collagen fibrils, whereas type IX collagen is associated with the surface of these fibrils (1, 2). The ␣1(XI) and ␣2(XI) chains are distinct gene products (3, 4), whereas the ␣3(XI) chain is a post-translational variant of the ␣1(II) chain (5). It has been postulated that type IX and type XI collagens regulate the collagen network by determining the diameter of cartilage collagen fibrils and their interactions with other matrix components (6 -9).Mutations in the type XI collagen genes have been identified and found to cause chondrodysplasia in both humans and mice. For example, mutations in the ␣2(XI) collagen gene (Col11a2) were identified in patients with Stickler syndrome and otospondylo-megaepiphyseal dysplasia (10). A null mutation in the ␣1(XI) gene of cho (chondrodysplasia) mice causes dwarfism with reduced matrix an...

Regulation of Human COL9A1 Gene Expression

“…Other work has implicated a 60-bp segment within the first intron that is important for COL11A2 gene expression. This segment was also shown to interact with SOX9 and promote cartilage-specific expression of reporter genes in transgenic mice (28). Interestingly, the SOX/Sry sites located in the COL11A2 proximal promoter region differ from the sites studied in the present work in that they are much closer to one another.…”

“…As mentioned above, the arrangement of the SOX/Sry-binding sites within the COL9A1 proximal-promoter region differ from those found in the COL11A2 promoter and also from the SOX/Sry-binding sites located in the COL2A1 intronic enhancer (5)(6)(7)(8)(27)(28)(29)(30). In the case of the COL11A2 gene, the five SOX/Sry-binding sites are found within a region of ϳ130 bp (27).…”

SOX9-dependent and -independent Transcriptional Regulation of Human Cartilage Link Protein

“…Supershift experiments were performed with purified SOX9 antibody (provided by Drs. Tomoatsu Kimura, Toyama Medical and Pharmaceutical University, and Yoshihiko Yamada, NIDCR, National Institutes of Health) (22). Competition experiments were performed by preincubating in vitro translated SOX9 or nuclear extract with excess unlabeled oligonucleotides prior to adding labeled oligonucleotides.…”

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation

“…14 Sox9 regulates the expression of several cartilage-specific matrix components like collagen II, IX, XI and aggrecan. [15][16][17] During transition to the hypertrophic phase, Sox9 expression is downregulated. A heterozygous mutation of the Sox9 gene in humans leads to embryonic dysplasia in particular of the skeleton, known as campomelic syndrome, caused by a reduced Sox9 dose.…”