Article

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

ABSTRACT

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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    • "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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    • "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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