Investigation of molecular mechanisms of Sox9 transcriptional factory during enchandral ossification

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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.