Regulation of human COL9A1 gene expression. Activation of the proximal promoter region by SOX9.
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.
Zhang
Ping
P
Department of Medicine, Division of Rheumatology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.
Jimenez
Sergio A
SA
Stokes
David G
DG
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019902:Chondrocytes / Q000502:physiology; D003094:Collagen / Q000235:genetics* / Q000378:metabolism; D024081:Collagen Type IX; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D006609:High Mobility Group Proteins / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
C439970:COL9A1 protein, human; D024081:Collagen Type IX; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D011993:Recombinant Fusion Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D003094:Collagen