SOX9 Is an Activator and Repressor in Chondrocytes specific interaction of GLI with Col10a1 element A was validated in CCL cells by quantitative ChIP assay with real-time PCR. The degree of enrichment was calculated by normalization of readouts to that of IgG control. (C) The expression of Col10a1 mini-genes with mutation in the GLI site was examined by in-situ hybridization of proximal humeri in E15.5 fetuses. For transgene with GLI site mutation alone (Col10Flag-ED3), islands of misexpression (Flag and Col10a1, arrows) were detected in the non-hypertrophic chondrocytes in most (7/10) expressing fetuses (e–h), while the rest showed milder mis-expression (not shown). For the transgene with a mutation in both the GLI site and the SOX9/TG-rich motif (Col10Flag-ED4), the majority of expressing fetuses (3/5) showed strong and extensive mis-expression (*) (i–l), while the rest showed relatively little mis-expression. Non- transgenic littermate (NT) is shown as control (a–d). The prehypertrophic and hypertrophic zones are encircled. PC, proliferating chondrocytes; PHC, prehypertrophic chondrocytes; HC, hypertrophic chondrocytes. doi:10.1371/journal.pgen.1002356.g006 not in proliferating chondrocytes, suggests that SOX9 may use this partnership to repress transcription of several genes in other chondrocyte types. However this partnership may not be the exclusive mechanism by which SOX9 acts to repress expression in chondrocytes. Hattori et al. have recently shown that SOX9 directly represses Vegfa in cultured primary chondrocytes [44] by interacting with the 59 untranslated region of the gene. This agrees with our findings of a repressive role of SOX9, however, we found no linked SOX9- GLI binding sites near the Vegfa gene and a recent SOX9 ChIP- on-chip study reported no in vivo interaction in Vegfa exon 1 [54]. A different mode by which SOX9 may repress gene expression in chondrocytes has been proposed by Huang et al [55]. In their model, SOX9 negatively regulates Ccn2 expression in non- hypertrophic chondrocytes via binding to overlapping binding sites for SOX and TCF/LEF, thereby interfering with binding of a TCF/LEF/b-catenin transactivation complex. Reduction of SOX9 upon hypertrophy allows this TCF/LEF/b-catenin com- plex to activate Ccn2 expression [55]. However, Ccn2 is also expressed in resting zone chondrocytes in the epiphyses of the growth plate and it is not clear why SOX9 does not repress the gene in these cells. We identified a conserved TCF consensus site in Col10a1 element A, but this is unlikely to interfere with SOX9 binding since it is located 59 bp downstream of the functional SOX9-GLI motif, unlike in Ccn2 where the SOX and TCF/LEF sites overlap (Figure 1C). This suggests that the model proposed by Huang et al. does not apply to Col10a1 element A-mediated repression of transcription. It is also possible that SOX9 and GLI cooperate to activate or repress transcription depending on context. While our data implicate a cooperation of SOX9 with GLI factors in transcrip- tional repression, this association between SOX9 and GLI may not be restricted to negative regulation. Amano et al. have recently reported that GLI2 cooperates with SOX9 to transactivate the Pthlh gene (also known as PTHrP) in chondrocyte culture without direct binding to the gene [56]. However the expression patterns of Sox9 and Pthlh are mutually exclusive in the developing growth plate, Pthlh being expressed mainly in the perichondrium and only at extremely low levels in proliferating chondrocytes [57,58]. This contradiction may reflect differences between in vitro assays and regulation in vivo, and it is also unclear whether the expressed GLI2 was processed to a repressor form or not in these cells. The observed stimulation of Pthlh promoter activity in the cultured chondrocytes could therefore be attributable to over-expression of GLI2 which persisted largely as the activated form GLI2A. However this report does raise the possibility of a context dependent SOX9-GLI partnership that mediates either transacti- vation or repression. Sox9 has been suggested to act upstream of Sox5 and Sox6 in chondrogenesis [6,46]. The presence of conserved SOX9–GLI sites in the Sox5 and Sox6 genes suggests their expression in proliferating chondrocytes may be positively controlled via cooperation of SOX9 with GLIA or GLI1, an activator that reinforces GLIA function. Hence, the roles played by SOX9 in transcriptional regulation may be determined by context— partnering with GLIA/GLI1 favours transactivation, with GLIR favours repression. Alternatively, as discussed above, the mode of regulation might depend on whether intermediate factors are present to interfere with the SOX9-GLI interaction. Interestingly, while there is a linked SOX9-GLI motif in Sox6, a conserved TCF site occurs between the SOX9 and GLI sites (Figure S3). Cooperation between SOX9 and TCF/LEF/b-catenin might therefore abrogate cooperative repression by SOX9 and GLI and transactivate Sox6 in proliferating chondrocytes. Validation of these different modes of cooperative regulation by SOX9 and GLI factors in vivo would require the generation and analyses of compound or conditional knockout mutants; however, the consequent dysregulation of chondrogenesis and impact on cell survival would make it impossible to distinguish changes in transcriptional control from effects on differentiation. For example, Sox9 is essential for chondrogenesis and Sox9 conditional chondrocytes undergo apoptosis and as a consequence, hypertrophy with the characteristic activation of Col10a1 expression, fails to occur [4,7,59]. Because inactivation of Col10a1 does not disrupt the chondrogenic program, it provides an ideal system and tools to interrogate the transcriptional controls governing specificity of gene expression within the growth plate, independent of changes in chondrocyte differentiation. Important questions to be addressed in future are the identities and diversity of SOX9 partnerships and how the activity of SOX9 and its partners is modulated. In early myogenic differentiation, SOX9 and Smad3 have been reported to prevent premature expression of a-sarcoglycan gene expression by synergistic repression in a transforming growth factor b dependent manner [60] suggesting negative transcriptional regulation by SOX9 and its partners may be more common. The transactivation of Matn1 in chondrocytes by SOX9 can be modulated by combined action of L-SOX5, SOX6 and NFI factors [61]. Whether control of transcription by the SOX9-GLI partnership can be modulated by additional factors is an important question to be addressed in future. In summary, our study implicates a complex regulatory function for SOX9 whereby it acts with different partners to orchestrate activation and repression of transcription in the chondrogenic differentiation pathway. Mutations in human SOX9 cause the skeletal malformation syndrome campomelic dysplasia which is attributed to the disruption of the chondrogenic differentiation program because of failure to express SOX9 target genes. This interpretation may need to be revised to include inappropriate expression of genes normally repressed by SOX9. Materials and Methods Bioinformatics analysis COL10A1 sequences were aligned using LAGAN and PipMaker. The conserved SOX9 binding sites (COL2C1 and COL2C2) [10] were identified by rVISTA. The UCSC Mouse 30-Way Multiz Alignment data and a custom Perl script were used to identify human-mouse perfectly conserved SOX9, GLI, and TCF sites with a maximum spacing of 100 bp in the inter- and intra-genic non-coding regions. Conservation percentage of sequence span- PLoS Genetics | www.plosgenetics.org 11 November 2011 | Volume 7 | Issue 11 | e1002356