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。正常生理过程 依赖于精确的转录控制，而转录失调是许多转录调控的基础。 包括癌症在内的疾病Pol II向特定启动子的募集受多种辅因子调节， 包括多亚基介体，其直接结合增强子/启动子结合的 转录激活因子和Pol II以促进基因激活。在起始和启动子逃逸之后， Pol II仍然受到多种延伸因子的调节，这些因子在Pol II暂停释放时起作用， 生产性延伸步骤。Pol II（G）是最近描述的Pol II的形式，其包含紧密的 相关的后生动物特异性Gdown 1多肽沿着正常的12个亚基。我们的基因- 基于Pol II（G）的研究表明，Gdown 1对早期胚胎发育至关重要 和细胞特异性转录在静止的肝细胞，其中重定位于基因体， 高度表达的肝特异性基因（例如，白蛋白）指示延长功能，并且其中消融 导致肝脏特异性基因和脂质代谢基因的下调，细胞周期重新进入， p53缺失）癌前转化类型。肝癌和乳腺癌细胞的研究 也表明Gdown 1在细胞生长和脂质代谢基因表达中的关键作用， 通常对维持癌细胞生长很重要。我们的生化研究表明， Pol II相关的Gdown 1有条件地抑制基础（激活剂和介体非依赖性） 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介体的强烈需求以克服阻遏。我们 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Mediator的线索 可能促进其逆转Pol II（G）的条件性抑制的起始能力的相互作用， 尽管潜在的机制仍不清楚。总的目标是了解 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。 癌症治疗学，我们的具体目标是：（i）研究介体依赖性的潜在机制 Pol II（G）的转录起始和起始后事件，包括伴随的、新描述的 与一般转录因子和延伸因子TFIIS的相互作用，使用强大的体外转录 和相互作用复合物的固定化模板测定和CX-MS和cryo-EM结构分析，以及（ii） 研究Gdown 1在肝癌细胞中启动子近端暂停、暂停释放和 使用（a）基于多组学细胞的方法结合急性降解 Gdown 1和（B）生物化学（用纯化的因子和重组体体外重建这些过程 染色质模板）和结构（CX-MS和cryo-EM）分析。