SOX9 Is an Activator and Repressor in Chondrocytes positions of the chondrocyte-specific binding motifs H1–H4 and P1–P4 deduced from DNase I footprinting are indicated. The primer sequences used in the chromatin immunoprecipitation are indicated in blue boxes. Consensus GLI and TCF binding sites were identified near the SOX9/TG-rich motif. Sequences involved in mutagenesis are shaded in blue. Only the mismatched nucleotides are shown in the aligned sequence. doi:10.1371/journal.pgen.1002356.g001 Figure 3B, i) and in almost all the Sox9-expressing chondrocytes in the rest (Figure 4, e). Expansion of transgene expression in proliferating chondrocytes was also noted but was less marked when the TG-rich motif in element A was mutated (Col10Flag- ED2) (compare Figure 4, m and i with a and e). Mutation of either SOX9 or the TG-rich motif did not abrogate transgene expression in hypertrophic chondrocytes. Together, these obser- vations suggest that element A contains both positive and negative regulatory sequences, and that mutations in the SOX9/TG-rich motif in element A might disrupt SOX9- mediated repression in immature chondrocytes. SOX9 is a negative regulator of Col10a1 To test whether SOX9 negatively regulates Col10a1, we established a cell line from hypertrophic chondrocytes MCTs which expressed the Col10Flag-E transgene at the non-permissive (growth-arrest) temperature (Figure 5A). Similar to previous findings for dedifferentiated chondrocytic cells MC615, over- expression of SOX9 in MCTs did not transactivate endogenous Col2a1 [20]. However, SOX9 over-expression significantly down- regulated expression of both endogenous Col10a1 and the exogenous Col10Flag-E reporter (Figure 5B) supporting the notion that SOX9 is a negative regulator of Col10a1. To study the regulation in vivo, SOX9 was ectopically expressed in hypertro- phic chondrocytes in mice. Mice expressing Cre recombinase inserted into the endogenous Col10a1 gene (Col10a1-Cre) [21] were crossed with transgenic mice carrying a single copy of a Cre- inducible Sox9-IRES-EGFP expression construct (Z/Sox9) [22] (Figure 5C). In Col10a1-Cre;Z/Sox9 mice, Sox9 and the linked Egfp reporter gene were activated in the hypertrophic chondrocytes at E17.5 in the anterior ribs (Figure 5D, h, i). These mice displayed an expanded hypertrophic zone and reduced Col10a1 expression (Figure 5D, e, k). Interestingly, we also found that transcription of Cre from the Col10a1 locus was reduced in Col10a1-Cre;Z/Sox9 mice (Figure 5D, a, g). Col2a1 expression in Col10a1-Cre;Z/Sox9 mice hypertrophic chondrocytes was not up-regulated suggesting the reduction of Col10a1 expression in these cells was not because of a reversion to a more immature state (Figure 5D, d, j). In the ribs Runx2 was expressed predominantly in osteoblasts, the perichondrium flanking the hypertrophic chondrocytes, and in prehypertrophic chondrocytes (Figure 5D, f). Expression was low in hypertrophic chondrocytes. There was no significant change in Runx2 expression in prehypertrophic and hypertrophic chondro- cytes in Col10a1-Cre;Z/Sox9 mice (Figure 5D, l). Collectively, these data suggest that SOX9 negatively regulates Col10a1 gene expression independent of Runx2. GLI factors bind and regulate Col10a1 in proliferating chondrocytes The specificity of SOX protein action is known to be achieved through interaction with cell-specific partners [23,24]. We questioned whether concomitant transactivation of Col2a1 and repression of Col10a1 by SOX9 in proliferating chondrocytes could be mediated by different combinations of cofactors. ChIP assays in E13.5 mouse limb chondrocytes or CCL cells revealed similar interactions of TRAP230/MED12, a mediator of SOX9 activity [25], and of TRPS1, a GLI3-interacting repressor [26,27], with both the Col10a1 element A and the Col2a1 enhancer (Figure 6A, upper panel). On the other hand, the transcriptional co-repressor, histone deacetylase HDAC4 [28] immunoprecipitated neither element. GLI1, GLI2 and GLI3 are effectors of Hh signaling which controls chondrocyte proliferation and maturation [29]. GLI1 is a transactivator expressed in proliferating chondrocytes and perichondrial tissue flanking the prehypertrophic and hypertrophic zones [30] whereas GLI2 and GLI3 can act as repressors and are predominantly expressed in non-hypertrophic chondrocytes and are down-regulated in hypertrophic chondrocytes [29,31]. Since there is a conserved GLI-binding site near the SOX9/TG-rich motif in the same footprint block P3 (Figure 1C), we examined whether GLI1, GLI2 and GLI3 can interact with the element A. Strikingly, while SOX9 bound to both the Col10a1 element A and Col2a1 enhancer, GLI2 and GLI3 associated with only Col10a1 element A (Figure 6A, lower panel). GLI3 interacted the most with element A, while GLI1 interaction was much less. Quantitative ChIP assays confirmed the preferential interaction (Figure 6B). From these results we hypothesized that GLI proteins may repress Col10a1 expression. To test this in vivo, we examined the impact on transgene expression of mutating the GLI-binding site in element A (Col10Flag-ED3). Consistent with our hypothesis, the majority of fetuses (7 out of 10) expressing Col10Flag-ED3 showed distinct islands of transgene misexpression in non-hypertrophic chondrocytes (Figure 6C, e, f). Mutating all three sites (GLI, SOX9, TG-rich) in the transgene (Col10Flag-ED4) did not restrict the expansion of the expression domain to proliferating chondrocytes in all the expressing transgenic fetuses obtained (Figure 6C, i, j). Indeed in the majority of these expressing fetuses (3 out of 5), transgene expression extended throughout the entire cartilage zones. Thus mutation of the GLI site alone had a similar derepressing effect as mutating the SOX9/TG-rich motif and mutating all the motifs did not restrict expression but resulted in more extensive mis-expression. This is consistent with a model whereby SOX9 and GLI act cooperatively to repress Col10a1 transcription. To assess whether the cooperation of SOX9 and GLI2/3 is a potential common mechanism for restricted or preferential gene expression in hypertrophic chondrocytes, we searched in silico for this configuration of binding sites in genes, other than Col10a1, that have strong and specific up-regulation in hypertrophic chondrocytes (HC genes) in the growth plate. Six of 11 HC genes analyzed, namely Col10a1, Bmp2, Hdac4, Mef2c, Runx2, and Sox4, possess the linked SOX9 and GLI sites (,100 nt spacing) in the inter- or intragenic conserved non-coding regions (Figure 7A and Figure S3). In contrast, these sites were absent from most of the genes tested (12 out of 14) that were expressed in proliferating but not (or down-regulated) in hypertrophic chondrocytes (PC genes). These include known SOX9 targets: Col2a1, Col9a1, Col11a2, Acan, and Mia1 (see Figure 7A legend for all negative genes). The exceptions were Sox5 and Sox6 (Figure 7A and Figure S3). To investigate whether the over-representation of linked SOX9-GLI sites in the HC genes but not the PC genes occurs by chance, we performed a hypergeometric test to calculate the probability of finding 6 or more SOX9-GLI site-containing genes out of 11 genes randomly sampled from the mouse genome. For the HC genes, the results showed that the occurrence of 6 or more genes with associated conserved SOX9-GLI sites is unlikely to occur by PLoS Genetics | www.plosgenetics.org 4 November 2011 | Volume 7 | Issue 11 | e1002356