SOX9 Is an Activator and Repressor in Chondrocytes construct consists of a LacZ and Sox9-EGFP expression cassette attached to a chicken b-Actin promoter (Pb-Actin)/CMV enhancer (Enh). The Sox9-EGFP cassette is expressed after Cre-mediated recombination. (D) Cre-activated ectopic Sox9 expression in the hypertrophic zone of costal cartilage in Col10a1-Cre;Z/Sox9 compound mutants at E17.5. Ectopic Sox9 expression (i, encircled) down-regulated Col10a1 (k, encircled) and Cre (g) in hypertrophic chondrocytes, especially in the extended hypertrophic zone. The reduction of Col10a1 is not associated with change in Runx2 expression (l) in hypertrophic chondrocytes. PC, proliferating chondrocytes; PHC, prehypertrophic chondrocytes; HC, hypertrophic chondrocytes. doi:10.1371/journal.pgen.1002356.g005 nisms by which SOX9 controls chondrocyte differentiation phase-specific gene expression. We have identified a conserved regulatory sequence, element A, that acts as an enhancer of Col10a1 expression in both cultured cells and in vivo. This element contains a SOX9 binding sequence that, when bound by SOX9, represses Col10a1 expression in immature and proliferating chondrocytes. Since Sox9 is expressed in non-hypertrophic chondrocytes but not in hypertrophic chondrocytes, this repressive action of SOX9 restricts Col10a1 expression to hypertrophic chondrocytes. SOX9 has been proposed to direct chondrogenic fate in osteo- chondroprogenitor cells in part by interacting with RUNX2 [32,33]. SOX9 may inhibit chondrocyte hypertrophy in part via activation of Bapx1 which represses Runx2 [34,35]. Previous in vitro and in vivo studies suggest that Col10a1 expression is regulated positively by Mef2c, Runx2/Cbfa1, and AP-1 members, which are expressed in hypertrophic chondrocytes [19,36–38]. RUNX2 has been shown to directly regulate the expression of Col10a1 [37]. The element A that we identified contains no conserved consensus RUNX site. The RUNX2 site revealed by Zheng et al. [37] is located within a poorly conserved region outside the element. Our data showed that the ectopic expression of Col10a1 transgene in non-hypertrophic chondrocytes does not require co-expression of Runx2. In addition, RUNX2 is not expressed in the costal hypertrophic chondrocytes and cultured hypertrophic chondro- cytes MCTs (which is derived from costal cartilage), where Col10a1 expression is strong. Although real-time PCR showed levels of Col10a1 was markedly reduced in P1 Runx2-null mice [37], hypertrophic chondrocytes with strong Col10a1 expression do develop in many cartilages in Runx2 fetuses [39,40]. Collectively existing data suggest that RUNX2 together with other factors regulate Col10a1 in vivo via promoting chondrocyte hypertrophy or otherwise functions to initiate a cascade of regulatory pathways that sustain Col10a1 expression in hypertro- phic chondrocytes. Previous in vitro studies in chicken have suggested that a combined action of positive and negative DNA elements may contribute to the hypertrophic chondrocyte-specific expression of Col10a1 [41,42]; however, these chick Col10a1 elements are not conserved in mammals. The enhancer element we identified is highly conserved in mammals, but not in chicken, which agrees with previous data [43]. This suggests that in both mammals and chicken, Col10a1 transcription is restricted to hypertrophic chondrocytes by repression, though by different cis-acting elements. In the chicken, this repression may extend to non- chondrogenic cell types [41]. We found no evidence to support such a mechanism in the mouse since when we abolished the interaction of SOX9 with the repressive element, we observed no ectopic Col10a1 expression in non-chondrogenic cells. Consistent with a role for SOX9 in repressing Col10a1 in vivo, we have shown in Col10a1-Cre;Z-Sox9 mice, that activation of Sox9 expression in hypertrophic chondrocytes in costal cartilage caused down-regulation of Col10a1. It is also notable that in a recent report where Sox9 was over-expressed in hypertrophic chondro- cytes in transgenic mice, expression of Col10a1 and the BAC-Col10- Sox9 transgene appeared reduced in the hypertrophic chondro- cytes [44]. In the same report Sox9 knockdown in cultured chondrocytes did not affect Col10a1 expression [44]. By contrast Yamashita et al. showed that shRNA knockdown of Sox9 can up- regulate Col10a1 expression in primary costal chondrocyte culture, and that over-expression of Sox9 can down-regulate it [35]. These contradictory results may be related to the incomplete elimination of SOX9 protein and the known dosage dependent requirement for SOX9 action. A role for SOX9 as transcriptional repressor of Col10a1 in non-hypertrophic chondrocytes is consistent with the observation that COL10A1 expression was up-regulated in cartilage isolated from SOX9 haploinsufficient campomelic dys- plasia patients [33]. How may SOX9 act both as a transactivator and a repressor in non-hypertrophic chondrocytes? It has been proposed that interactions between specific partner factors stabilize SOX protein binding to DNA and hence regulate target selection [24], thereby determining cell specification, as exemplified by SOX2 in embryonic stem cells and other systems [45]. Such selective cooperation of protein binding partners may mediate the concomitant positive and negative regulation of SOX9 target genes in the same cell that contributes to specification of the differentiation state (Figure 8). As illustrated in the schematic (Figure 8), we propose that SOX9 mediates repression of Col10a1 in proliferating chondrocytes by selective cooperation with GLI factors. Together with its role in activating Col2a1 and other matrix genes, SOX9 therefore plays an important role in maintaining chondrocytes in an immature non-hypertrophic state. RUNX2 by contrast promotes chondrocyte hypertrophy. SOX9 cannot regulate chondrocyte differentiation appropri- ately without sonic hedgehog (Shh), which mediates the generation of chondrogenic precursor cells [46], and Indian hedgehog (Ihh), which regulates their proliferation and maturation [47]. GLI proteins are the effectors of Hh signaling. Double knockout mutants indicate that GLI2 has overlapping functions with GLI1 and GLI3 in skeletal and CNS development [48,49]. Binding of Hh to its receptor, Patched, blocks the proteolytic processing of the GLI transcription factors from active (GLIA) to repressive (GLIR) forms, and the balance between these forms modulates hedgehog target gene expression [50]. In the growth plate, GLI2A can positively regulate chondrocyte hypertrophy and control vascular- ization of the hypertrophic cartilage in endochondral ossification [29,51]. GLI3, which acts mainly as a repressor, has been suggested to inhibit chondrocyte hypertrophy [29,52] and it is interesting that the highest interaction of element A was with GLI3. Mau et al. reported that Gli2/3 mutations altered the expression domain of collagen X, but it was not possible to distinguish whether this was due to a direct effect on Col10a1 transcription or more general perturbation of hypertrophy [29]. How the GLI factors interact with other regulatory factors or genes in the chondrocyte differentiation program is not clear. Our results are consistent with cooperation between SOX9 and the Hh signaling pathway and suggest that SOX9 acts in synergy with GLI2 and GLI3, probably their repressive forms GLIR, to repress transcription in chondrocytes. Thus, reduced synergy between GLIR and SOX9 may explain the accelerated chondrocyte hypertrophy seen when Ptch1 is inactivated [53]. Over-represen- tation of the SOX9-GLI paired consensus in a number of genes that are preferentially expressed in hypertrophic chondrocytes and PLoS Genetics | www.plosgenetics.org 9 November 2011 | Volume 7 | Issue 11 | e1002356