Regulation of human COL9A1 gene expression - Activation of the proximal promoter region by SOX9

Department of Medicine , Thomas Jefferson University, Filadelfia, Pennsylvania, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2003; 278(1):117-23. DOI: 10.1074/jbc.M208049200
Source: PubMed


The COL9A1 gene contains two promoter regions, one driving expression of a long alpha1(IX) chain in cartilage (upstream) and one driving expression of a shorter chain in the cornea and vitreous (downstream). To determine how the chondrocyte-specific expression of the COL9A1 gene is regulated, we have begun to characterize the upstream chondrocyte-specific promoter region of the human COL9A1 gene. Transient-transfection analyses performed in rat chondrosarcoma (RCS) cells, human chondrosarcoma (HTB) cells, and NIH/3T3 cells showed that the COL9A1 promoter was active in RCS cells but not HTB or NIH/3T3 cells. Inclusion of the first intron had no effect on promoter activity. In transient-transfection analyses with promoter deletion constructs, it was found that full promoter activity in RCS cells depended on the region from -560 bp to +130 bp relative to the transcriptional start site (+1). Sequence analysis of the region from -890 bp to the transcriptional start predicted five putative SOX/Sry-binding sites. Mutation analysis revealed that two of three putative SOX/Sry binding sites within the -560 to +130 bp region are responsible for most of the COL9A1 promoter activity in RCS cells. Co-transfection experiments with a SOX9 expression plasmid revealed that a construct containing the five putative SOX/Sry-binding sites was transactivated 20- to 30-fold in both HTB and NIH/3T3 cells. Further co-transfection experiments showed that two of the SOX/Sry-binding sites located within the -560 to +130 bp region were required for full transactivation. However, mutation and deletion analyses indicated that a region from -560 to -357 bp, which does not contain any other conspicuous SOX9 sites, is also important for full promoter activity. DNA-protein binding assays and super-shift analysis revealed that SOX9 can form a specific complex with one of the SOX/Sry-binding sites with in the -560 to +130 region.

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    • "Sox9 is a member of Sox (Sry-type HMG box) family genes and has been revealed to be expressed mainly in mesenchymal condensations and cartilage [3].Sox9 plays a crucial role as transcription factor for chondrogenesis and cartilage formation [3] [4]. It up-regulates the cartilage specific markers namely collagen II, IX and IX and aggrecan [5] [6] [7] [8]. A successful gene transfer requires the use of method capable to achieve high transfection efficiency and low toxicity [9]. "
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    ABSTRACT: Sox9 plays an important role as transcription factor for chondrogenesis; the formation of cartilage. This study aimed to identify the potential of the transiently overexpressed Sox9 gene in human chondrocytes differentiation and tissue engineered cartilage (TEC) formation in vitro. Articular cartilage samples were obtained from osteoarthritic patients who underwent joint replacement surgery. The isolated chondrocytes were cultured and transfected with pcDNA3-Sox9 using lipofection technique. The TEC constructs were formed by incorporating the transfected and the nontransfected cells onto poly(lactic-co-glycolic acid) (PLGA) scaffold with or without fibrin. This approach allows a comparison between four groups i.e. (1) transfected chondrocytes seeded on PLGA/fibrin PFTC], (2) nontransfected chondrocytes on PLGA/fibrin PFC], (3) transfected chondrocytes on PLGA PTC. and (4) non-transfected chondrocytes on PLGA PC]. All TEC constructs were cultured and evaluated at each time point of 1, 2 and 3 weeks in vitro. All TEC constructs were analysed for gross observation, histology, immunohistochemistry, cell proliferation activity, gene expression and sulphated glycosaminoglycan (sGAG) production assay. After 3 weeks, all PFTC and PFC showed higher cell viability, higher sGAG content, better histological features and distribution of extracellular matrix in concert with positive glycosaminoglycan (GAG) accumulation when compared to the PTC and PC. However, at week 3, the PFC and PC exhibited significantly higher sGAG production than PFTC and PTC. Chondrogenic properties of the constructs were evidenced by the expression of cartilage-specific markers; collagen II, collagen XI and aggrecan core protein. In this study, due to the nature of a new cartilage formation, the co-expression of collagen I in all constructs can be an indication of early cartilage development. Based on the outcomes, it is hoped that this study will provide a good ground for future tissue engineering application.
    Full-text · Article · Dec 2015
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    • "The human COL9A1 promoter (sparse CpG promoter) contains 8 CpG sites in the 1,000-bp sequence upstream of exon 1 (GenBank accession No. AF036110), relative to the transcriptional start site (+1) [22] (Figure 2C). In general, methylation levels in all CpG sites in the COL9A1 promoter region were inversely correlated with foot length and developmental age; i.e. the oldest fetal sample analysed displayed the lowest percentage of CpG site methylation (Figure 2D). "
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    ABSTRACT: Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1(+) marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.
    Full-text · Article · Apr 2013 · PLoS ONE
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    • "The effects on collagen type II and aggrecan expression could, at least partly, be the result of a primary antichondrogenic or dedifferentiating effect induced by IL-1β, as Sox9 expression was decreased and versican expression increased in most IL-1β-treated pellets. Sox9 acts as a transcriptional activator for several genes involved in the formation of extracellular cartilage matrix [38] [39] [40] [41] [42] and is needed for chondrocyte differentiation [43]. Suppression of Sox9 by IL-1β has previously been reported in murine chondrocytes [44]. "
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    ABSTRACT: The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.
    Full-text · Article · Nov 2010 · Connective tissue research
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