Investigation of molecular mechanisms of Sox9 transcriptional factory during enchandral ossification

为了研究Sox9在软骨细胞分化中发挥重要作用的分子机制，我们建立了Sox9转录工厂成员的基因筛选系统。利用该系统，我们发现钙离子通道TRPV_4是Sox9转录工厂的成员，并发现TRPV4上调Sox9的表达和功能，从而刺激软骨细胞分化。我们进一步筛选从软骨细胞ATDC5中产生的cDNA文库，并分离出Znf219 cDNA。全原位和RT-PCR分析表明，Znf219在E12.5胚胎小鼠肢芽和软骨细胞中特异性表达。共免疫沉淀实验表明Znf219与Sox9有物理相互作用。此外，我们发现Znf219在细胞核中与Sox9共定位。这些结果表明Znf219在细胞核内形成转录工厂。为了了解Znf219在软骨细胞分化中的功能作用，我们接下来研究了过表达Zof219对软骨细胞分化的影响。我们发现Znf219的过表达刺激了Sox9的软骨生成活性。相反，过表达显性阴性Znf219显著抑制Sox9诱导的软骨细胞分化。此外，利用微RNA系统敲除Znf219可抑制软骨细胞分化。总之，我们的结果表明Znf219可能在sox9调节的软骨细胞分化中发挥重要作用。

Tb investigate the molecular mechanism by which Sox9 plays an essential role in chondrocyte differentiation, we established a gene screening system for identifying the members of Sox9 transcriptional factory. Using this system, we could identify a calcium ion channel, TRPV_4, as a member of Sox9 transcriptional factory, and found that TRPV4 upregulated expression and function of Sox9, thereby stimulating chondrocyte differentiation. We further screened cDNA library generated from chondrogenic cell line, ATDC5, and isolated the Znf219 cDNA. Whole mount in situ and RT-PCR analyses indicated that Znf219 is specifically expressed in limb bud of E12.5 embryo mice and chondrocytes. Co-immunoprecipitation experiments indicated that Znf219 physically interacts with Sox9. Moreover, we found that Znf219 co-localized with Sox9 in the nucleus. These results indicated that Znf219 forms transcriptional factory in the nucleus. To understand the functional role of Znf219 m chondrocyte differentiation, we next examined the effect of overexpression of Zof219 on chondrocyte differentiation. We found that overexpression of Znf219 stimulated the chondrogenic activity of Sox9. In contrast, overexpression of a dominant negative Znf219 markedly inhibited chondrocyte differentiation induced by Sox9. Furthermore, knockdown of Znf219 using micro RNA system suppressed chondrocyte differentiation. Collectively, our results demonstrated that Znf219 might play an important role in Sox9-regulated chondrocyte differentiation.