SOX9 Is an Activator and Repressor in Chondrocytes Figure 3. Element A enhances Col10a1 transgene expression in the growth plate. (A) Diagram showing the Flag-tagged Col10a1 mini-genes for transgenesis. The constructs included all introns and a 59 flanking region up to 22.07 kb (green). At the 59 end, the constructs were combined with the 640 bp element A (orange) containing wild-type sequence (E). (B) The expression pattern of the Flag-tagged Col10a1 mini-genes (a,e,i) was compared with that of endogenous Col10a1 (b,f,j), Runx2 (c,g,k), and Sox9 (d,h,l) by in-situ hybridization of proximal humerus in E15.5 expressing fetuses. (a–d) Tissue-specific Col10a1 expression in hypertrophic zone of non-transgenic littermate (NT) was shown. (e–h) In the absence of element A, patchy expression of the mini-gene was observed in prehypertrophic and upper hypertrophic chondrocytes. (i–l) Addition of element A resulted in marked up-regulation of mini-gene expression in the whole hypertrophic zone (*). Since expression of Col10a1 was used as readout of derepression, Runx2 was used as an alternative marker of hypertrophic chondrocytes. In addition to hypertrophic chondrocytes, expression of Runx2 was noted in the trabecular bone and in the prehypertrophic chondrocytes. The prehypertrophic and hypertrophic zones (encircled) were identified based on Runx2 expression. PC, proliferating chondrocytes; PHC, prehypertrophic chondrocytes; HC, hypertrophic chondrocytes. doi:10.1371/journal.pgen.1002356.g003 Discussion The positive and negative mechanisms mediating the stage- specific transcription of genes within the growth plate are not well defined, partly because of the difficulty in distinguishing direct effects on transcription from the consequences of abnormal differentiation. In this study we have exploited the specificity of Col10a1 expression in hypertrophic chondrocytes and the fact that manipulating its expression in vivo has no overt effect on differentiation, to dissect these transcriptional controls. We provide new insight into how differentiation stage-specific gene expression is achieved in the growth plate, presenting in vitro and in vivo evidence that SOX9, in addition to its known role as a transactivator of many genes preferentially expressed in non- hypertrophic chondrocytes, such as Col2a1, directly represses expression of Col10a1 at a stage prior to the onset of hypertrophy and subsequently in proliferating chondrocytes. This discovery extends our understanding of the mecha- PLoS Genetics | www.plosgenetics.org 6 November 2011 | Volume 7 | Issue 11 | e1002356