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

RNA 聚合酶 II 变体 Pol II(G) 的转录起始和延伸功能的机制研究，该变体与发育和癌症有关

• 批准号: 10670981
• 负责人: ROBERT G ROEDER
• 金额: $ 38万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-25 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10670981
• 关键词: Ablation; Acute; Albumins; Binding; Biochemical; Biological; Biological Assay; Breast Cancer Cell; Breast Carcinoma; Cancer Cell Growth; Carcinoma; Cell Cycle; Cell Differentiation process; Cell Line; Cells; ChIP-seq; Chromatin; Complement; Complex; Cryoelectron Microscopy; Development; Down-Regulation; Elongation Factor; Embryo; Embryonic Development; Enhancers; Event; Experimental Designs; Gene Activation; Genes; Genetic; Genetic Transcription; Genetic study; Growth; Hep3B; Hepatocarcinogenesis; Hepatocyte; Human; Immobilization; In Vitro; Individual; Intervention; Liver; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator; Modeling; Molecular Mechanisms of Action; Mus; Pathology; Peptide Initiation Factors; Physiological; Physiological Processes; Play; Polymerase; Primary carcinoma of the liver cells; Process; Productivity; Property; Proteins; RNA Polymerase II; Recombinants; Regulation; Repression; Role; Signal Pathway; Site; Structure; TP53 gene; Therapeutic; Tissues; Transcription Coactivator; Transcription Elongation; Transcription Factor TFIIB; Transcription Initiation; Transcriptional Elongation Factors; Transcriptional Regulation; Variant; Yeasts; cancer cell; cell growth; cofactor; embryonic stem cell; fly; gene repression; human disease; insight; lipid metabolism; liver development; malignant breast neoplasm; multiple omics; negative elongation factor; novel; p53 Signaling Pathway; pancreatic differentiation 2 protein; polypeptide; premalignant; prevent; programs; promoter; reconstitution; recruit; transcription factor; transcription factor S-II; transcription factor TFIIF

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.

真核RNA聚合酶II（POL II）在转录中起关键作用。正常的生理过程 依赖于精确的转录控制，而转录失调是众多的基础 包括癌症的病理。 Pol II招募特定启动子受多种辅助因子的调节 包括多支亚基介质，该介体直接结合了增强子/启动子结合 转录激活剂和Pol II以促进基因激活。启动和发起人逃脱， Pol II仍受到多个伸长因素的监管，在Pol II暂停释放或 生产伸长步骤。 Pol II（G）是Pol II的最近描述的形式，包含紧密的 相关的，特异性的GDown1多肽以及正常的12个亚基。我们的遗传 - 对POL II（G）的基于的研究表明，GDown1对于早期胚胎发育至关重要 对于静态肝细胞中细胞特异性转录 高表达肝特异性基因（例如白蛋白）表示伸长函数，其中消融 导致肝特异性和脂质代谢基因的下调，细胞周期重新进入和（在 缺乏p53）一种前转化类型。肝癌和乳腺癌细胞的研究 还表明了GDown1在细胞生长和脂质代谢基因表达中的关键作用，这些基因的表达 通常对于维持癌细胞生长很重要。我们的生化研究表明 POL II相关的GDown1有条件地压制基础（非激活因子和中介者） 通过防止TFIIB和TFIIF与Pol II的关联来引发转录，从而建立了潜力 检查点并提出了对激活剂结合的介体克服抑制的强大要求。我们的 结构研究已定义了POL II上的GDown1相互作用位点，并提供了有关调解员的线索 可能促进其有条件地抑制pol II（g）的有条件抑制的起始能力的相互作用 尽管基本机制尚不清楚。以理解的一般目标 Pol II（G）作用的分子机制以及其在乳腺癌和肝癌中的作用 细胞，特别是在GDown1调节的细胞特异性和脂质代谢基因上，作为新的潜在基础 癌症治疗剂，我们的具体目的是：（i）调查依赖介体的机制 POL II（G）的转录启动和发射后事件，包括伴随，新描述 使用强大的体外转录与一般转录因子和伸长因子TFII的相互作用 以及固定的模板分析，CX-MS以及相互作用复合物和（ii）的冷冻EM结构分析 研究GDown1在启动子暂停，暂停释放和 使用（a）基于多素细胞的​​方法与急性降解的转录加工性 GDown1和（b）生化（这些过程的体外重组具有纯化的因素和重组 染色质模板）和POL II（G）伸长因子复合物的结构（CX-MS和冷冻EM）分析。