Chereji Razvan V, Bharatula Vasudha, Elfving Nils, Blomberg Jeanette, Larsson Miriam, Morozov Alexandre V, Broach James R, Björklund Stefan
Division of Developmental Biology, Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, PA 17033, USA.
Nucleic Acids Res. 2017 Sep 6;45(15):8806-8821. doi: 10.1093/nar/gkx491.
Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization.
中介体是一种多亚基分子复合物，在真核生物中，它在将信号从转录调节因子传递到RNA聚合酶II的过程中发挥关键作用。我们将从染色质中对酿酒酵母中介体进行生化纯化与染色质免疫沉淀相结合，以揭示全基因组范围内中介体在DNA上的占据情况，并鉴定在染色质模板上与中介体特异性相互作用的蛋白质。对中介体复合物免疫沉淀中的蛋白质进行串联质谱分析，揭示了染色质中中介体与RSC、Arp2/Arp3、CPF、CF 1A和Lsm复合物之间的特异性相互作用。这些因子主要参与染色质重塑、肌动蛋白组装、mRNA 3'端加工、基因环化和mRNA降解，但它们也已被证明可进入细胞核并参与Pol II转录。此外，我们发现，中介体除了结合Pol II启动子外，还占据染色体相互作用结构域（CID）边界，并且染色质中的中介体与已被证明与CID边界相互作用的蛋白质相关联，如Sth1、Ssu72和组蛋白H4。这表明中介体在高阶基因组组织中发挥着重要作用。
