SOX9 Is an Activator and Repressor in Chondrocytes Figure 8. A model for SOX9 control of differentiation phase–specific gene expression in growth plate chondrocytes. As chondrocytes mature during endochondral bone formation, the concomitant transactivation and repression of transcription of differentiation stage-specific genes by SOX9 is exemplified in Col10a1 and Col2a1. Sox9, its transactivation target Col2a1, as well as GLI2 and GLI3 are preferentially expressed by non- hypertrophic chondrocytes. GLI3 is down-regulated when chondrocytes become prehypertrophic, while Sox9 and GLI2 expression is down-regulated when chondrocytes reach hypertrophy [8,9,29]. Regulation of both Col10a1 and Col2a1 involves dimeric binding of SOX9. The GLI factors, and possibly others, depending on the spatial context of their binding sites, are involved in the differential transcriptional control. MED12 and TRPS1 [25– 27] act as common mediators of transcriptional control. In proliferating chondrocytes, Col10a1 is repressed through binding of SOX9 and GLI2/3. SOX9 negatively regulates Runx2 expression via Bapx1 in proliferating chondrocytes [34,35]. When chondrocytes mature and become prehypertrophic, RUNX2 binding to SOX9 may reduce the amount available for Col10a1 [33] and, as GLI3 levels reduce, Col10a1 is partially released from repression. Col2a1 transcription may be maintained due to stabilization of SOX9 binding by SOX5/6 [15]. In hypertrophic chondrocytes, as SOX9-associated machinery diminishes, Col10a1 is completely de-repressed and is highly transactivated by factors such as RUNX2, MEF2C, AP-1. Concomitantly Col2a1 expression is markedly decreased. SOX9 therefore controls differentiation-phase specific gene expression in chondrocytes through direct and indirect mechanisms. PC, proliferating chondrocytes; PHC, prehypertrophic chondrocytes; HC, hypertrophic chondrocytes. doi:10.1371/journal.pgen.1002356.g008 ning the SOX9 and GLI sites was calculated based on the number of perfectly matched nucleotides among all the aligned species (mouse, human, chimpanzee, canine, bovine, and opossum). From the 32,120 genes in the mouse genome, the number of genes containing conserved linked SOX9-GLI sites was found and used as the reference frequency of such genes in the genome. Whether the frequency of the presence of conserved SOX9-GLI sites in HC or PC genes exceeded this reference frequency was assessed by the hypergeometric distribution. The difference in the frequency of the presence of SOX9-GLI sites in the HC and PC genes was assessed by the Fisher’s exact test. Cell culture and transfection Hypertrophic chondrocyte cell line MCTs (gift of Ve´ronique Lefebvre [62]) was transfected with pCol10Flag-E, pSG-Sox9 (gift of Peter Koopman), or pSG5 expression vector using Fugene 6 (Roche). Expression of the exogenous collagen X from pCol10Flag- E in MCTs cells was examined by immunohistochemistry using anti-Flag M2 antibody (Sigma). CCL (gift of James Kimura [63]), MCT3T3-E1, and COS-1 cells were cultured in DMEM (Invitrogen) containing 10% FCS (Wisent) at 37uC at 5% CO2. MCTs cells were normally cultured at 32uC at 5% CO2 for expansion. Prior to assays, MCTs cells were cultured at 37uC for 1 day to induce growth arrest [62]. DNase I footprinting A 300 bp DNA fragment within element A, corresponding to 24240 to 23935 bp of the mouse Col10a1, was used as probe. [c-32P]ATP-labeled probes were incubated with nuclear extracts from CCL, MCTs, MCT3T3-E1, or COS-1 cells in the presence PLoS Genetics | www.plosgenetics.org 13 November 2011 | Volume 7 | Issue 11 | e1002356