SOX9 Is an Activator and Repressor in Chondrocytes Figure 2. Interaction of SOX9 with a conserved upstream element of Col10a1. (A) A fragment of element A (24239 to 23933 bp) was digested with increasing concentrations of DNase I in the presence of extract buffer alone or nuclear extracts (NE) from COS-1 (Fib), MCTs (HC), MC3T3-E1 (Ost), or CCL cells (PC), or used to produce the G/A ladder (L). (B) Diagram of interaction between cell type-specific nuclear factors (ovals) and element A based on the footprints. A SOX9 binding site is located in P3. (C) Electromobility shift assay (EMSA) of the interaction of the SOX9/TG- rich motif in element A of Col10a1 (C10C1) with SOX9. The interaction involved dimeric SOX9 binding (C10C1-SOX92). Complexes formed between SOX9 and C10C1 were challenged with SOX9 antibody (lane 4) and competitors of wild-type or mutant sequence (lane 5–10). Mutation of the SOX9 site and the adjacent TG-rich motif reduced SOX9 binding. (D) In EMSA, RUNX2 interacted with the Bglap (Osteocalcin) enhancer probe (OSE2) containing the osteoblast-specific RUNX2 binding consensus motif. The interaction was challenged with RUNX2 antibody (lane 4) and competitors of wild-type or mutant OSE2 (lane 5–8), or C10C1 (lane 9–10), at 10- or 100-fold excess. (E) Chromatin immunoprecipitation (ChIP) assay demonstrating SOX9 interaction with the SOX9/TG-rich binding site. Real-time PCR was performed to amplify element A of Col10a1 as well as sequences from the Col2a1 enhancer and Crygb (c-b-crystallin) promoter. See also Figure S1. doi:10.1371/journal.pgen.1002356.g002 chance (p = 0.0000201) (Figure 7B). For the PC genes, the p-value was 0.17, which is comparable to random occurrence. Further- more the frequency of the presence of SOX9-GLI sites for HC genes (6/11) was significantly higher than that for PC genes (2/14) (Fisher’s test p = 0.043, one tailed). This suggests that the linked SOX9-GLI sites are preferentially associated with the HC genes. PLoS Genetics | www.plosgenetics.org 5 November 2011 | Volume 7 | Issue 11 | e1002356