RNA聚合酶II新亚基Gdown1调控转录延伸的分子机理

Gdown1是近年来新发现的哺乳动物RNA聚合酶II（Pol II）的亚当量组分。虽然体外转录实验显示Gdown1对转录的起始和延伸步骤都具有明显抑制效应，但是关于细胞内Gdown1调控转录的作用时相尚存争议。基于Gdown1与转录延伸因子存在互作的前期结果，本项目将为Gdown1参与转录延伸调控寻找更确凿的证据并解析该过程的分子机制。结合分子手段及组学方法我们将检测改变Gdown1自身水平或阻断与延伸调控因子的功能互作对其识别及调控靶基因转录延伸的影响，进而揭示Gdown1调控转录延伸的工作机制以及在干细胞自我更新及分化中发挥的生物学功能。此外前期结果意外发现Gdown1大量存在于胞质中且其入核可能受到调控。我们将整合分子及生化的技术手段探索控制Gdown1核质分配及入核结合Pol II调控转录的机制。所得研究结果将有助于揭示Gdown1参与执行转录延伸调控的分子机制及其生物学意义。

Gdown1 was recently identified as a substoichiometric subunit of the mammalian RNA Polymerase II (Pol II). Although it was demonstrated by in vitro transcription assays that Gdown1 had severe inhibitory effects on both transcription initiation and elongation, it has been controversial about the detailed transcriptional phase(s) during which Gdown1 functions in cells. Our pilot study demonstrated that Gdown1 interacted with known transcription elongation factors, supporting the roles of Gdown1 in the elongation control. In this study, we will gather convincing evidence to confirm the regulatory role of Gdown1 during the transcription elongation phase and further dissect the molecular mechanisms behind. Target genes of Gdown1 throughout the genome will be identified through combinatorial applications of -omics methodologies and changes in binding to and regulating transcription of these genes by Gdown1 will be monitored upon knockout of Gdown1 or destroy of its interactions with other elongation controlling factors. We will also uncover the roles Gdown1 plays in self renewal of mouse embryonic stem cells and during their differentiation. In addition, our preliminary results indicated that a significant amount of cellular Gdown1 was surprisingly present in the cytoplasm and the entrance of this part of Gdown1 into the nucleus appeared to be tightly regulated. Thus, Series of molecular and biochemical techniques will be applied to uncover the mechanisms about balancing the cytoplasm-nucleus distribution of Gdown1 and about controlling the specific interaction between Pol II and Gdown1 in the nucleus. Results from this study would help us elucidate the molecular mechanisms and biological significance of Gdown1-mediated transcription elongation control.