Mechanistic studies of transcription initiation and elongation functions of an RNA polymerase II variant, Pol II(G), that is implicated in development and cancer

真核细胞RNA聚合酶II(POL II)在转录过程中起着关键作用。正常生理过程 依赖于精确的转录控制，而转录失调是许多 包括癌症在内的病理学。POL II招募到特定推动者受到多个辅助因素的调节，这些辅助因素 包括直接与增强子/启动子结合的多亚基介体 转录激活剂和Pol II促进基因激活。在启动和启动子逃逸之后， POL II仍然受到多个伸长因素的调节，在POL II暂停释放或 富有成效的延伸步骤。POL II(G)是最近描述的POL II的形式，它包含 相关的后生动物特异性Gdown 1多肽以及正常的12个亚基。我们的基因- 对POL II(G)的基础研究表明，Gdown 1对早期胚胎发育是必不可少的 在静止的肝细胞中进行细胞特异性转录，其中大量定位于基因体。 高表达的肝脏特异性基因(例如，白蛋白)是延长功能的指标，在这种情况下，消融 导致肝脏特异性和脂代谢基因的下调，细胞周期重新进入和(在 P53缺失)是一种癌前类型的转化。关于肝癌和乳腺癌细胞的研究 也表明Gdown 1在细胞生长和脂代谢基因的表达中发挥关键作用， 通常对维持癌细胞的生长很重要。我们的生化研究显示 POL II相关的Gdown 1有条件地抑制基础(激活物和介体非依赖) 通过阻止TFIIB和TFIIF与POL II结合而启动转录，从而建立潜在的 检查点，并引发了对激活器绑定的调解人的强烈需求，以克服压制。我们的 结构研究已经确定了POL II上的Gdown 1相互作用位点，并提供了关于介体的线索 可能有助于其逆转Pol II(G)有条件抑制的启动能力的相互作用， 尽管潜在的机制尚不清楚。总的目标是理解 Pol II(G)作用的分子机制及其在乳腺癌和肝癌中的作用 细胞，特别是Gdown-1调控的细胞特异性和脂代谢基因，作为新的潜在基础 癌症治疗学，我们的具体目标是：(I)研究介体依赖的潜在机制 Pol II(G)的转录启动和启动后事件，包括伴随事件，新描述 与普通转录因子和延伸因子TFIIS的相互作用，使用强大的体外转录 和固定化模板分析以及相互作用络合物的CX-MS和冷冻-EM结构分析和(II) 研究Gdown 1在启动子-近端暂停、暂停释放和 使用(A)以细胞为基础的多组学方法结合急性降解的转录过程 Gdown 1和(B)生化(用纯化因子和重组人在体外重建这些过程 (染色质模板)和结构(CX-MS和冷冻-EM)分析POL II(G)延伸因子复合体。

Eukaryotic RNA polymerase II (Pol II) plays a pivotal role in transcription. Normal physiological processes depend upon precise transcriptional controls, whereas transcriptional dysregulation is the basis of numerous pathologies that include cancer. Pol II recruitment to specific promoters is regulated by multiple cofactors that include the multi-subunit Mediator, which directly binds both to enhancer/promoter-bound transcriptional activators and to Pol II to facilitate gene activation. Following initiation and promoter escape, Pol II remains subject to regulation by multiple elongation factors, acting either at Pol II pause-release or productive elongation steps. Pol II(G) is a recently described form of Pol II that contains the tightly associated, metazoan-specific Gdown1 polypeptide along with the normal 12 subunits. Our genetic- based studies of Pol II(G) have demonstrated that Gdown1 is essential for early embryonic development and for cell-specific transcription in quiescent hepatocytes, in which heavy localization to gene bodies of highly expressed liver-specific genes (e.g., albumin) is indicative of elongation functions and in which ablation leads to downregulation of both liver-specific and lipid metabolism genes, cell cycle re-entry and (in the absence of p53) a premalignant type of transformation. Studies in hepatocarcinoma and breast cancer cells have also indicated a key role for Gdown1 in cell growth and in expression of lipid metabolism genes, which are generally important for maintenance of cancer cell growth. Our biochemical studies have revealed that the Pol II-associated Gdown1 conditionally represses basal (activator- and Mediator-independent) transcription initiation by preventing association of TFIIB and TFIIF with Pol II, thereby establishing a potential checkpoint and eliciting a strong requirement for activator-bound Mediator to overcome repression. Our structural studies have defined Gdown1 interaction sites on Pol II and provided clues regarding Mediator interactions that might facilitate its reversal of the conditionally repressed initiation capacity of Pol II(G), although the underlying mechanism remains unclear. With the general objective of understanding the molecular mechanisms of action of Pol II(G) in conjunction with its roles in breast cancer and hepatocarcinoma cells, especially on Gdown1-regulated cell-specific and lipid metabolism genes, as a potential basis for new cancer therapeutics, our specific aims are: (i) to investigate the mechanisms underlying Mediator-dependent transcription initiation and post-initiation events by Pol II(G), including concomitant, newly described interactions with general transcription factors and elongation factor TFIIS, using powerful in vitro transcription and immobilized template assays and CX-MS and cryo-EM structural analyses of interacting complexes and (ii) to investigate Gdown1 functions in hepatocarcinoma cells in promoter-proximal pausing, pause release and transcriptional processivity using (a) a multiomics cell-based approach in conjunction with acute degradation of Gdown1 and (b) biochemical (in vitro reconstitution of these processes with purified factors and recombinant chromatin templates) and structural (CX-MS and cryo-EM) analyses of Pol II(G) elongation factor complexes.