RNA聚合酶II新亚基Gdown1调控转录延伸的分子机理

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.

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最初作为与哺乳动物RNA聚合酶II（Pol II）直接结合因子被发现。它与转录起始所必需的因子TFIIF竞争结合Pol II，体外转录实验和转录组学分析数据支持Gdown1可能作为转录起始检查点关键分子发挥作用。有趣的是，果蝇中Gdown1在合子基因组激活时从细胞核转移到细胞质，此后在成体细胞中均定位于细胞质中；Gdown1在哺乳动物组织中广谱表达，而其亚细胞定位及调控机制完全未知。在这里，我们利用遗传学和基于显微镜的多种实验方法来检测了多种哺乳动物细胞系中Gdown1的亚细胞定位，发现其严格定位于所有被测的体细胞系的细胞质中；有趣的是，它对外加的不同类型的细胞核定位驱动力表现出非常不寻常的抵抗力。通过突变体筛选我们在人源Gdown1中鉴定出两类不同的定位调控元件，一类是典型的CRM1依赖性核输出信号（NES），另一类是不依赖于CRM1的新型调控元件，我们将其命名为细胞质锚定信号（CAS）。通过突变解除CAS的作用可将Gdown1有效地从完全不入核转变为常规的CRM1依赖性的核质穿梭蛋白。我们发现从果蝇到人类Gdown1，这两种相互关联的定位调节序列及其功能机制均相对保守。将以上Gdown1定位元件全部突变得到能够在细胞核中积累的Gdown1突变体，测试发现该突变体的核易位直接导致细胞整体Pol II转录水平的急剧下降，而该突变体过量的核积累在短时间内会触发细胞死亡。最后，我们发现内源性Gdown1响应细胞胁迫处理（如氧化应激）发生核易位，且敲除Gdown1的细胞抵御胁迫的能力下降。综上所述，本研究证实了Pol II结合因子Gdown1在哺乳动物体细胞中主要定位于细胞质中但具备核质穿梭潜能且鉴定到该蛋白序列中包含两类（共3个）定位调控元件。我们的研究结果揭示了控制Gdown1入核是赋予其参与核内转录调控能力的有效策略。本研究为进一步阐明通过定位调控Gdown1来动态改变细胞转录水平、调节细胞逆境适应能力的分子机理以及将该研究成果运用于靶向调控目标细胞活性的转化应用打下了坚实基础。