Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation
Differential transactivation of the upstream aggrecan enhancer regulated by PAX1/9 depends on SOX9-driven transactivation
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DOI：
10.1038/s41598-019-40810-4
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发表时间：
2019-03
影响因子：
4.6
通讯作者：
A. Takimoto;C. Kokubu;Hitomi Watanabe;Tetsushi Sakuma;Takashi Yamamoto;G. Kondoh;Y. Hiraki;C. Shukunami
中科院分区：
文献类型：
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作者：
A. Takimoto;C. Kokubu;Hitomi Watanabe;Tetsushi Sakuma;Takashi Yamamoto;G. Kondoh;Y. Hiraki;C. Shukunami
先前发现的aggrecan （Acan）基因（UE）上游10kb的增强子可以在体内驱动软骨特异性报告基因的表达。在这里，我们报告了同源转录因子PAX1和PAX9的差异驱动，取决于是否存在sox9驱动的转激活。在发育中的脊柱中，PAX1/9的表达与acan9的表达呈负相关。此外，PAX1/9与SOX9/5/6在椎间质和纤维内环（AF）共表达，与SOX9在纤维外环共表达。pax1沉默的AF细胞在软骨化过程中观察到acan显著上调，而pax1在软骨中的持续表达则显著下调acan。使用CRISPR/Cas9删除fue1分别导致软骨和AF中facan表达减少30%和40%。在ue中，PAX1/9通过与sox9结合位点部分重叠的PAX1/9结合位点作为弱转录激活因子。在SOX9存在的情况下，PAX1/9与SOX5/6竞争占据结合位点，导致facan的转激活降低。共免疫沉淀显示Pax1与SOX9的物理相互作用。因此，通过PAX1/9、SOX9和SOX5/6的协同作用，以上下文依赖的方式对uei的交互激活进行差异调节。
A previously identified enhancer 10 kb upstream of theAggrecan(Acan) gene (UE) can drive cartilage specific reporter expressionin vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially driveUE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated withAcanexpression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. SignificantAcanupregulation was observed during chondrification ofPax1-silenced AF cells, while,Acanwas significantly downregulated by persistent expression ofPax1in cartilage. Deletion ofUEusing CRISPR/Cas9 resulted in ~30% and ~40% reduction ofAcanexpression in cartilage and the AF, respectively. In theUE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robustAcanexpression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation ofAcan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of theUEis differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.