Author Summary Chondrogenic differentiation is a key process in the formation of endochondral bone. Despite the wealth of information about gene expression patterns and signaling pathways important for this process, it is not clear how differentiation state-specificity of transcription is con- trolled. The transcription factor SOX9 regulates chondro- cyte differentiation, proliferation, and entry into hypertro- phy and is highly expressed in immature/proliferating chondrocytes. It directly transactivates Col2a1, enhancing this gene’s expression in immature/proliferating chondro- cytes. The Col10a1 gene is specifically expressed in hypertrophic chondrocytes in which Sox9 is downregulat- ed. How is differentiation phase-specific transcription of genes controlled in chondrocytes, particularly during hypertrophy? We found that SOX9 directly represses Col10a1 expression in immature/proliferating chondro- cytes of the growth plate, so that its expression is restricted to hypertrophic chondrocytes. Discrimination of this concomitant opposing transcriptional control may involve cooperation between SOX9 and different partners such as GLI factors (effectors of hedgehog signaling). SOX9 control of chondrocyte maturation therefore may be integrated with hedgehog signaling. Mutations in human SOX9 cause the skeletal malformation syndrome campo- melic dysplasia, which is attributed to the disruption of the chondrogenic differentiation program because of failure to express SOX9 target genes. This interpretation should be revised to include inappropriate expression of genes normally repressed by SOX9. gene expression during chondrocyte differentiation. SOX9 activates many genes expressed in proliferating chondrocytes, including the extracellular matrix (ECM) genes Col2a1, Col9a1, Col11a2, Acan (aggrecan) and Cd-rap/Mia1 [10–15]. For the Col2a1 gene, which is expressed most strongly in proliferating chondro- cytes, SOX9 directly transactivates the gene in vivo via a conserved enhancer sequence within the first intron [10,11]. The collagen X gene, Col10a1, is a hypertrophic chondrocyte specific marker. The specificity and reciprocity of Sox9 and Col10a1 expression epitomise the strict control of temporal and differentiation phase-specific gene expression in the growth plate. Col10a1 is ideal for studying transcriptional regulation because as well as its highly specific expression pattern, over-expression or loss-of-function does not disrupt chondrocyte differentiation. These properties simplify interpretation of changes in gene expression resulting from perturbing transcriptional control [16– 18]. Here, we examined the transcriptional controls that restrict Col10a1 expression to hypertrophic chondrocytes. We found that SOX9 coordinates gene expression during chondrocyte differen- tiation through both transcriptional activation and repression. Discrimination between these opposing actions is probably achieved by cooperation between SOX9 and different partners such as GLI factors. Results Proliferating and hypertrophic chondrocytes show overlapping and different protein binding domains in the Col10a1 enhancer Previous cell transfection studies identified an enhancer element upstream of human COL10A1 [19]. This element is highly conserved in mammals and corresponds to a 640 bp region between 24.3 and 23.6 kb of the mouse Col10a1 gene (designated SOX9 Is an Activator and Repressor in Chondrocytes element A) (Figure 1A and 1B). We used DNase I footprinting assays to test the configurations in which the element A sequences could be directly bound by nuclear factors derived from chondrocytes at different differentiation states (Figure 2A). Extracts from hypertrophic chondrocytes MCTs, but not fibroblasts COS-1 or osteoblasts MC3T3-E1, protected six blocks of sequence (H1– H6). Noticeably, four different blocks (P1–P4) that partially overlap H1–H4 were protected by extracts from the proliferating chondrocyte/chondrosarcoma cell line CCL (Figure 1C and Figure 2A). Since proliferating chondrocyte/chondrosarcoma cells do not express Col10a1 (Figure S1A), these results suggest that in these cells, the proteins that bind to element A may contribute to the repression of Col10a1. SOX9 binds to Col10a1 element A in proliferating chondrocytes We and others previously showed that SOX9 regulates COL2A1/Col2a1 gene via a functional in vivo binding site in the intron 1 enhancer element [10,11]. We identified the same SOX9-binding sequence within the Col10a1 element A (Figure 1C and Figure 2B). This site, COL2C1, lies on a region in block P3 that is not protected in hypertrophic chondrocytes, and is adjacent to a stretch of thymidine/guanine-rich (TG-rich) sequence. Electromobility shift assays revealed that SOX9 bound to this SOX9/TG-rich motif with a similar affinity as to the COL2A1 enhancer element (Figure S1C, S1E, S1F) [10] and the interaction involved dimeric binding (Figure 2C, cf. lane 3–4). SOX9 also interacted with the TG-rich motif but with a lower affinity than with the consensus SOX9 site. Mutation of the TG- rich motif reduced the overall SOX9 binding to the P3 element (Figure 2C, cf. lanes 6–10). The TG-rich motif resembles a RUNX binding consensus sequence, but we found that RUNX2 did not interact with this motif effectively compared with its binding to the RUNX site in the Bglap (osteocalcin) enhancer (Figure 2D). Chromatin immunoprecipitation (ChIP) assays using extracts from E13.5 mouse limb, a stage at which the cartilage anlagen is largely composed of immature chondrocytes, con- firmed specific SOX9 binding to the Col10a1 element A and the Col2a1 enhancer in vivo (Figure 2E). SOX9/TG-rich motif is required for appropriate Col10a1 expression The paired SOX9 binding sequences in element A are separated by 4 bp, a distance similar to that between the paired SOX-like consensus sequences in Col2a1, Col9a1, and Acan that mediate transactivation of expression [15]. We tested the in vivo role of element A and the effects of SOX9/TG-rich motif mutations on the expression of Col10a1 mini-genes (Figure 3A) in transgenic mice. We have previously shown that a Flag-tagged Col10a1 vector Col10Flag (formerly known as FColX) is expressed in P10 hypertrophic chondrocytes [18]. Here, we show that in E15.5 humeri, the Col10Flag transgene was expressed in islands in prehypertrophic and hypertrophic chondrocytes in the upper hypertrophic zone (Figure 3B, e). In two independent mouse lines, a transgene comprising element A fused to the Col10Flag (Col10Flag-E) was expressed in a similar pattern as Col10Flag, but was significantly more strongly expressed than Col10Flag in all hypertrophic chondrocytes (Figure 3B, i), reflecting the enhancer activity of element A (see also Figure S2). However, mutation of the SOX9 site in element A (Col10Flag-ED1) resulted in marked expansion of the expression domain of the transgene, extending from the hypertrophic zone to the proliferating zone in the majority of transgenic fetuses (71.4%) (compare Figure 4, a with PLoS Genetics | www.plosgenetics.org 2 November 2011 | Volume 7 | Issue 11 | e1002356