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: 10503451
• 负责人: ROBERT G ROEDER
• 金额: $ 38.77万
• 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/10503451
• 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; DNA Polymerase II; DNA-Directed RNA Polymerase; Development; Down-Regulation; Elongation Factor; Embryo; Embryonic Development; Enhancers; Event; Experimental Designs; GTP-Binding Protein alpha Subunits, Gs; 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 of activation protein; Metabolism; Modeling; Molecular Mechanisms of Action; Mus; Pathology; Peptide Initiation Factors; Physiological; Physiological Processes; Play; Primary carcinoma of the liver cells; Process; Property; Proteins; Prothrombin; 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; base; 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; 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) 在转录中发挥着关键作用。正常生理过程 依赖于精确的转录控制，而转录失调是许多疾病的基础 包括癌症在内的病理。 Pol II 向特定启动子的招募受到多种辅因子的调节 包括多亚基介体，它直接与增强子/启动子结合 转录激活剂和 Pol II 以促进基因激活。在启动和启动子逃逸之后， Pol II 仍然受到多个伸长因子的调节，在 Pol II 暂停-释放或 生产性伸长步骤。 Pol II(G) 是最近描述的 Pol II 形式，其中包含紧密的 相关的后生动物特异性 Gdown1 多肽以及正常的 12 个亚基。我们的基因—— 基于 Pol II(G) 的研究表明 Gdown1 对于早期胚胎发育至关重要 以及静止肝细胞中的细胞特异性转录，其中大量定位于 高表达的肝脏特异性基因（例如白蛋白）表明了伸长功能，其中消融 导致肝脏特异性基因和脂质代谢基因、细胞周期重新进入和（在 p53 缺失）是一种癌前类型的转化。肝癌和乳腺癌细胞的研究 还表明 Gdown1 在细胞生长和脂质代谢基因表达中发挥关键作用， 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 有条件地抑制 basal（与激活剂和介体无关） 通过阻止 TFIIB 和 TFIIF 与 Pol II 的结合来启动转录，从而建立潜在的 检查点并引发对激活剂结合调解员克服抑制的强烈要求。我们的 结构研究确定了 Pol II 上的 Gdown1 相互作用位点，并提供了有关 Mediator 的线索 可能促进其逆转 Pol II(G) 条件抑制起始能力的相互作用， 尽管根本机制仍不清楚。总体目标是了解 Pol II(G) 的分子作用机制及其在乳腺癌和肝癌中的作用 细胞，特别是 Gdown1 调节的细胞特异性和脂质代谢基因，作为新的潜在基础 癌症治疗，我们的具体目标是：（i）研究介导依赖性的机制 Pol II(G) 的转录起始和起始后事件，包括伴随的、新描述的 使用强大的体外转录与一般转录因子和延伸因子 TFIIS 相互作用 和固定化模板测定以及相互作用复合物的 CX-MS 和冷冻电镜结构分析，以及 (ii) 研究肝癌细胞中 Gdown1 在启动子近端暂停、暂停释放和 转录持续性使用（a）基于多组学细胞的方法结合急性降解 Gdown1 和 (b) 生化（用纯化因子和重组体体外重建这些过程） Pol II(G) 延伸因子复合物的染色质模板）和结构（CX-MS 和冷冻电镜）分析。