Cyclin-Dependent Kinase 8 Positively Cooperates with Mediator To Promote Thyroid Hormone Receptor-Dependent Transcriptional Activation
Cyclin-Dependent Kinase 8 Positively Cooperates with Mediator To Promote Thyroid Hormone Receptor-Dependent Transcriptional Activation
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DOI：
10.1128/mcb.01541-09
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发表时间：
2010-05-15
影响因子：
5.3
通讯作者：
Fondell, Joseph D.
中科院分区：
文献类型：
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作者：
Belakavadi, Madesh;Fondell, Joseph D.
介体是一种蛋白质的多亚基组合，最初在人类中被确定为与甲状腺激素受体(TRs)结合的辅助激活因子，对于甲状腺激素(T3)依赖的转录是必不可少的。细胞周期蛋白依赖性激酶8(CDK8)、细胞周期蛋白C、MED12和MED13形成一个可变相关的介体亚复合体(称为CDK8模块)，其在转录调节因子依赖的转录中的作用尚不清楚。使用体外和细胞方法，我们在这里展示了含有CDK8模块的介体复合体以依赖于tr和t3的方式被特异性地招募到TR靶基因I型脱碘酶(DioI)和RNA聚合酶II(POLII)的预起始复合体中。我们发现CDK8对于依赖T3的Dio1转录是必不可少的，并且通过RNA干扰敲除CDK8减少了Pol II的占有率，也减少了Pol II激酶CDK9在DioI启动子的募集。染色质免疫沉淀显示CDK8占据在DioI启动子上，同时激活转录，从而提示CDK8参与了转录的重新启动。突变分析表明，CDK8的活性是完全依赖于T3的DioI激活所必需的，而体外的激酶研究表明，CDK8可能有助于Pol II的磷酸化。总之，我们的数据表明，CDK8通过促进Pol II在tr靶基因启动子上的招募和激活，在tr依赖的转录中发挥重要的辅助激活作用。
Mediator is a multisubunit assemblage of proteins originally identified in humans as a coactivator bound to thyroid hormone receptors (TRs) and essential for thyroid hormone (T3)-dependent transcription. Cyclin-dependent kinase 8 (CDK8), cyclin C, MED12, and MED13 form a variably associated Mediator subcomplex (termed the CDK8 module) whose functional role in TR-dependent transcription remains unclear. Using in vitro and cellular approaches, we show here that Mediator complexes containing the CDK8 module are specifically recruited into preinitiation complexes at the TR target gene type I deiodinase (DioI) together with RNA polymerase II (Pol II) in a TR- and T3-dependent manner. We found that CDK8 is essential for robust T3-dependent Dio1 transcription and that CDK8 knockdown via RNA interference decreased Pol II occupancy, and also the recruitment of the Pol II kinase CDK9, at the DioI promoter. Chromatin immunoprecipitation revealed CDK8 occupancy at the DioI promoter concurrent with active transcription, thus suggesting CDK8 involvement in transcriptional reinitiation. Mutagenesis assays showed that CDK8 kinase activity is necessary for full T3-dependent DioI activation, whereas in vitro kinase studies indicated that CDK8 may contribute to Pol II phosphorylation. Collectively, our data suggest CDK8 plays an important coactivator role in TR-dependent transcription by promoting Pol II recruitment and activation at TR target gene promoters.