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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=663463
• 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.**

调解人在发射后事件中的作用******调解人是通过真核生物高度保守的大型，多育和必不可少的转录共激活器。介体由组织为四个模块的25至30个亚基组成：头部，中间，尾部和激酶（也称为CKM）。介体与增强子的基因特异性转录因子以及RNA聚合酶II（RNAPII）的转录机制与启动子结合的转录机制相互作用，因此可以桥接增强子和核心启动子。在最近的两个出版物中，我们在酵母中描述了介体的这一方面，并​​表明介体组成在启动过程中是高度动态的：通过基因特异性转录因子募集介体后，CKM被弹出，允许介体在核心启动子处暂时与RNAPII相互作用。 ******在后生动物中，调解员的作用扩展到了发射后事件。值得注意的是，越来越多的证据表明，调解人调节启动子暂停。实际上，在许多基因上，RNAPII在开始部位后停止了30-60个核苷酸，并从此停顿中释放是限制速率（调节）步骤。此暂停释放受诸如P-TEFB或含P-TEFB的超级伸长络合物（SEC）等因素的募集来调节。 调解员在该过程中起着关键作用，但机制仍然不确定。值得注意的是，有人提出调解员通过其CKM招募P-TEFB，但我们以前的工作表明，在启动过程中，CKM从中介中弹出。这种明显的矛盾表明，不仅在我们之前所展示的启动期间，而且在随后的步骤中，不仅在启动期间，还具有动态性。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，调解员在与其他组件进行短暂接触时重新组织了其构象和组成。 ******在此应用程序中，我建议以酵母中的调解人的工作为基础，并将其扩展到果蝇，使我们能够研究发射后事件（在酵母中不存在启动子 - 抗毒素）。果蝇是启动子暂停的良好模型。调整一些在酵母中富有成果的策略，我们将在不同的转录步骤中剖析各种​​介体模块与基因的动态相互作用。这将由Chip-Nexus完成，Chip-Nexus是芯片序列的变体，允许以几乎碱基的分辨率绘制蛋白质-DNA相互作用的基因组。使用策略阻止或减慢转录过程的不同步骤，我们将能够强调沿该过程中调解人组成的变化，并表征其招募各种暂停释放因子的机制。通过这个项目，我们将能够对调解员在发射后事件中的作用进行更完整，更详细的描述。**