The role of Mediator in post-initiation events

调解员在发起后事件中的作用

• 批准号: RGPIN-2018-04519
• 负责人: Robert, François
• 金额: $ 3.06万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=688561
• 关键词: role; Mediator; post; initiation; events

The role of Mediator in post-initiation events******Mediator is a large, multisubunit and essential transcriptional co-activator highly conserved through eukaryotes. Mediator is composed of 25 to 30 subunits organized into four modules: Head, Middle, Tail and Kinase (also called CKM). Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters therefore bridging enhancers and core promoters. In two recent publications, we have described this aspect of Mediator in yeast and showed that Mediator composition is highly dynamic during initiation: after recruitment of Mediator by gene-specific transcription factors, CKM is ejected, allowing for Mediator to transiently interact with RNAPII at the core promoter. ******In metazoans, the role of Mediator extends to post-initiation events. Notably, mounting evidence suggests that Mediator regulates promoter-proximal pausing. Indeed, at many genes, RNAPII pauses 30-60 nucleotides after the initiation site and release from this pause is the rate limiting (regulatory) step. This pause release is regulated by the recruitment of factors such as P-TEFb or the P-TEFb-containing super elongation complex (SEC). Mediator was shown to play a key role in that process but the mechanism(s) remain ill-defined. Notably, it was proposed that Mediator recruits P-TEFb via its CKM but our previous work showed that CKM is ejected from Mediator during initiation. This apparent contradiction suggests that the composition of Mediator is dynamic, not only during initiation as we showed previously, but also during subsequent steps. Hence, our overarching hypothesis is that, in order to achieve this flexible role in different steps of the transcription process, Mediator reorganises its conformation and composition while making transient contacts with other components. ******In this application, I propose to build on our work on Mediator in yeast and extend it to Drosophila, allowing us to study post-initiation events (promoter-proximal pausing is absent in yeast). Drosophila is a well-established model for promoter-proximal pausing. Adapting some of the strategies that were fruitful in yeast, we will dissect the dynamic interactions of the various Mediator modules with genes during the different steps of transcription. This will be done by ChIP-nexus, a variant of ChIP-sequencing allowing to map protein-DNA interactions genome-wide at nearly base-pair resolution. Using strategies to block or slow down different steps of the transcription process, we shall be able to highlight changes in the composition of Mediator along the process and characterise the mechanisms through which it recruits various pause release factors. Through this project, we shall be able to draw a more complete and detailed picture of the role of Mediator in post-initiation events.**

**Mediator是一个大的，多亚基和必需的转录辅激活因子，在真核生物中高度保守。Mediator由25至30个亚基组成，分为四个模块：头部，中部，尾部和激酶（也称为CKM）。介体在增强子处与基因特异性转录因子相互作用，以及与结合在启动子处的RNA聚合酶II（RNAPII）转录机构相互作用，因此桥接增强子和核心启动子。在最近的两篇出版物中，我们描述了酵母中介体的这一方面，并表明介体组成在启动期间是高度动态的：在基因特异性转录因子招募介体后，CKM被排出，允许介体在核心启动子处与RNAPII瞬时相互作用。** 在后生动物中，调解人的作用延伸到启动后事件。值得注意的是，越来越多的证据表明，中介调节启动子近端暂停。事实上，在许多基因中，RNAPII在起始位点后30-60个核苷酸暂停，并且从该暂停释放是限速（调节）步骤。这种暂停释放受因子如P-TEFb或含P-TEFb的超延伸复合物（SEC）的募集调节。 调解人在这一进程中发挥了关键作用，但机制仍然不明确。值得注意的是，有人提出，调解人招募P-TEFb通过其CKM，但我们以前的工作表明，CKM是从调解人在启动期间弹出。这种明显的矛盾表明，中介的组成是动态的，不仅在我们前面展示的初始阶段，而且在随后的步骤中。因此，我们的总体假设是，为了在转录过程的不同步骤中实现这种灵活的作用，介体重组其构象和组成，同时与其他组分进行短暂接触。 ** 在本申请中，我建议建立在我们对酵母中介体的工作基础上，并将其扩展到果蝇，使我们能够研究启动后事件（酵母中不存在启动子近端暂停）。果蝇是启动子近端停顿的成熟模型。适应一些在酵母中卓有成效的策略，我们将剖析在转录的不同步骤中各种介体模块与基因的动态相互作用。这将通过ChIP-nexus完成，ChIP测序的变体允许以接近碱基对分辨率在全基因组范围内绘制蛋白质-DNA相互作用。使用阻断或减慢转录过程不同步骤的策略，我们将能够突出显示中介体沿着该过程的组成变化，并阐明其招募各种暂停释放因子的机制。通过这个项目，我们将能够更完整和详细地描绘调解人在启动后事件中的作用。