Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
PLoS Genet. 2011 Nov;7(11):e1002356. doi: 10.1371/journal.pgen.1002356. Epub 2011 Nov 3.
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.
软骨和成骨的发育需要 SOX9 来调节软骨生成、软骨细胞增殖以及向非有丝分裂性肥大状态的转变。在肥大软骨细胞中受限和相互表达的胶原 X 基因 Col10a1 和不成熟软骨细胞中 Sox9 完美地体现了基因表达的精确时空调控，因为软骨细胞在分化阶段会经历多个阶段，但目前尚不清楚如何实现这种调控。在这里，我们鉴定了 Col10a1 上游的一个调控元件，该元件在体内增强了其在肥大软骨细胞中的表达。在不成熟的软骨细胞中，Col10a1 不表达，SOX9 与该元件内的保守序列相互作用，该序列类似于胶原 II 基因 Col2a1 内含子增强子中的序列，后者是 SOX9 的已知转录激活靶标。通过在转基因小鼠中分析一系列 Col10a1 报告基因，我们表明该元件中 SOX9 结合的保守序列对于抑制非肥大软骨细胞中转基因的表达是必需的。体外和体内过表达 Sox9 导致肥大软骨细胞中 Col10a1 的下调。在 Col10a1 调控元件中靠近 SOX9 结合位点的Gli 转录因子（印度刺猬信号的效应物）结合的共识基序的突变也会使非肥大软骨细胞中转基因的表达去抑制。GLI2 和 GLI3 结合到 Col10a1 调控元件，但不结合到 Col2a1 的增强子上。除了 Col10a1 之外，配对的 SOX9-GLI 结合基序也存在于几个在肥大软骨细胞中优先表达的基因的保守非编码区中，这种配对不太可能是偶然发生的。我们提出了一个调控范例，其中 SOX9 直接进行协同的阳性和阴性转录调控，以确保软骨细胞中分化阶段特异性基因表达。SOX9 对这些相反的转录调控模式的区分可能是通过与 GLI 因子等不同的合作伙伴合作来介导的。
