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: $ 6.12万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757560
• 关键词: role; Mediator; post; initiation; events

The role of Mediator in post-initiation eventsMediator 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.

介体在启动后事件中的作用介体是一种大型、多亚基和必需的转录共激活因子，在真核生物中高度保守。介体由 25 至 30 个亚基组成，分为四个模块：头、中、尾和激酶（也称为 CKM）。介体与增强子处的基因特异性转录因子以及与启动子处结合的 RNA 聚合酶 II (RNAPII) 转录机制相互作用，从而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 Mediator 的这一方面，并​​表明 Mediator 组成在启动过程中是高度动态的：在基因特异性转录因子招募 Mediator 后，CKM 被弹出，从而允许 Mediator 与核心启动子处的 RNAPII 短暂相互作用。在后生动物中，调解者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介体调节启动子近端暂停。事实上，在许多基因中，RNAPII 在起始位点后暂停 30-60 个核苷酸，从该暂停中释放是速率限制（调节）步骤。这种暂停释放是通过 P-TEFb 或含有 P-TEFb 的超伸长复合物 (SEC) 等因子的募集来调节的。 调解员被证明在这一过程中发挥着关键作用，但其机制仍然不明确。值得注意的是，有人提议 Mediator 通过其 CKM 招募 P-TEFb，但我们之前的工作表明 CKM 在启动过程中被从 Mediator 中弹出。这种明显的矛盾表明，中介者的组成是动态的，不仅在我们之前展示的启动过程中，而且在后续步骤中也是如此。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，介体会重组其构象和组成，同时与其他组件进行短暂接触。 在此应用中，我建议以我们在酵母介导方面的工作为基础，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）。果蝇是一种成熟的启动子近端暂停模型。采用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中介模块与基因的动态相互作用。这将通过 ChIP-nexus 来完成，ChIP-nexus 是 ChIP 测序的一种变体，允许以接近碱基对的分辨率绘制全基因组范围内的蛋白质-DNA 相互作用图谱。使用策略来阻止或减慢转录过程的不同步骤，我们将能够突出整个过程中中介体组成的变化，并表征它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、更详细地了解调解员在启动后事件中的作用。