SOX9 Governs Differentiation Stage-Specific Gene Expression in Growth Plate Chondrocytes via Direct Concomitant Transactivation and Repression Victor Y. L. Leung1,2¤a, Bo Gao1¤b, Keith K. H. Leung1,2, Ian G. Melhado 1 , Sarah L. Wynn 1 , Tiffany Y. K. Au 1 , Nelson W. F. Dung1, James Y. B. Lau 1 , Angel C. Y. Mak1, Danny Chan1,2, Kathryn S. E. Cheah1,2* 1 Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China, 2 Centre for Reproduction, Development, and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China Abstract Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9–GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between these opposing modes of transcriptional control by SOX9 may be mediated by cooperation with different partners such as GLI factors. Citation: Leung VYL, Gao B, Leung KKH, Melhado IG, Wynn SL, et al. (2011) SOX9 Governs Differentiation Stage-Specific Gene Expression in Growth Plate Chondrocytes via Direct Concomitant Transactivation and Repression. PLoS Genet 7(11): e1002356. doi:10.1371/journal.pgen.1002356 Editor: Veronica van Heyningen, Medical Research Council Human Genetics Unit, United Kingdom Received June 20, 2010; Accepted September 10, 2011; Published November 3, 2011 Copyright: 2011 Leung et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Research Grant Council and University Grants Council of Hong Kong (HKU7222/97M, HKU2/02C, HKU 4/ 05C, AoE/M-04/04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kathycheah@hku.hk ¤a Current address: Department of Orthopaedics and Traumatology, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China ¤b Current address: Developmental Genetics Section, Genetic Disease Research Branch, National Human Genome Research Institute, Bethesda, Maryland, United States of America Introduction Chondrogenesis and the formation of bone by endochondral ossification depend on progressive steps of cell differentiation. Mesenchymal cells condense and differentiate into chondrocytes in a pattern that will define the eventual shape of the different skeletal elements. These chondrocytes proliferate, mature, exit the cell cycle and become prehypertrophic. The differentiation program culminates in the terminal differentiation and apoptosis of post-mitotic hypertrophic chondrocytes [1]. This differentiation program is controlled by members of the SOX and RUNX families of transcription factors and the integration of multiple signaling pathways mediated by Indian hedgehog (Ihh), parathyroid hormone-related protein (PTHrP), Wnts, BMPs, and Notch (reviewed in [2]). PTHrP and Ihh are two important players which interact to form a feedback loop that controls the pace of the differentiation program [3]. Sox9 is essential for chondrogenesis and chondrocyte differen- tiation [4–6]. It is essential for mesenchymal condensation prior to chondrogenesis, and in its absence chondrocyte differentiation fails. Inactivation of Sox9 in chondrocytes at different stages of differentiation suggests that its expression is essential for the survival of chondrocytes so that they can progress to hypertrophy [5–7]. Mutations in SOX9 are associated with the human skeletal malformation syndrome, campomelic dysplasia, in which skeletal abnormalities can be attributed to the disruption of the chondrogenic differentiation program due to failure to express SOX9 target genes. Upon hypertrophy, chondrocytes down- regulate Sox9 expression [8,9], which is believed to mark the end of SOX9 control in the growth plate. Despite the wealth of information about spatial and temporal gene expression patterns in the developing growth plate, it is not clear how transcriptional controls achieve appropriate and specific PLoS Genetics | www.plosgenetics.org 1 November 2011 | Volume 7 | Issue 11 | e1002356