Biological roles and Mediator-dependent transcription mechanisms of RNA polymerase II(G)

RNA聚合酶II(G)的生物学作用和介体依赖性转录机制

• 批准号: 9009066
• 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: 2015
• 资助国家: 美国
• 起止时间: 2015-12-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9009066
• 关键词: Ablation; Adult; Animals; Binding; Biochemical; Biological; Biological Assay; Biological Process; Breast Cancer cell line; Breast Carcinoma; Cancer cell line; Cell-Free System; Cells; ChIP-seq; Chemicals; Code; Complex; Cryoelectron Microscopy; DNA Polymerase II; Defect; Drosophila genus; Drosophila melanogaster; Embryo; Embryonic Development; Event; Family suidae; Fibroblasts; Gene Expression; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Hepatocyte; Human; In Vitro; Inhibition of Cell Proliferation; Injection of therapeutic agent; Knock-out; Knockout Mice; Liver; Malignant Neoplasms; Malignant neoplasm of liver; Maps; Mediating; Mediator of activation protein; Molecular; Mouse Cell Line; Mus; Oncogenic; Pathology; Peptide Initiation Factors; Physiological Processes; Play; Polymerase; Primary carcinoma of the liver cells; Promoter Regions; Protein Footprinting; Proteins; RNA Polymerase II; Recruitment Activity; Regulation; Repression; Role; Site; Stress; System; TP53 gene; Testing; Therapeutic; Thymus Gland; Transcription Coactivator; Transcriptional Activation; Transcriptional Regulation; Untranslated RNA; Validation; Xenograft procedure; Yeasts; acute liver injury; base; biological adaptation to stress; blastocyst; c-myc Genes; cancer cell; carcinogenesis; cell growth; crosslink; embryonic stem cell; genome-wide; human disease; in vivo; insight; knock-down; mouse model; mutant; novel; polypeptide; preimplantation; prevent; promoter; public health relevance; response; transcription factor TFIIF; transcriptome sequencing; tumorigenesis; vector; zygote

 DESCRIPTION (provided by applicant) RNA polymerase II (Pol II) plays a critical role in readout of the eukaryotic genome through its transcription of protein-coding and non-coding RNAs. 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 (co)factors that include the multi-subunit Mediator, which directly binds both Pol II and transcriptional activators to facilitate activation of essentially all genes. Pol II(G) is a ditinct, recently discovered (originally in calf thymus and pig liver) form of Pol II that contains the tighly associated polypeptide Gdown1 along with the normal 12 subunits of Pol II. In vitro transcription studies revealed that, in the absence of Mediator, the Pol II-associated Gdown1 represses transcription by preventing the association of initiation factor TFIIF with Pol II and thus inhibitng pre-initiation complex formation. Mediator reverses the repressed transcriptional capacity of Pol II(G), although the underlying mechanism remains unclear. While Mediator is conserved from yeast to human, the conservation of Gdown1 extends only to Drosophila melanogaster. Thus, the transcriptional regulatory system in fruit flies and higher metazoans has evolved through acquisition of an additional and functionally distinct Pol II, Pol II(G), although the specific rol(s) played by Pol II(G) in transcriptional regulation in higher metazoans remains unclear. Toward an understanding of the biological role(s) of Pol II(G), preliminary studies have demonstrated that Gdown1 ablation causes early embryonic lethality in both fruit flies and mice. This lethality may be explained in part by a severe inhibition of cell proliferation, as observed in Gdown1-deficient embryonic stem cell and cancer cell lines, and by dysregulation of gene expression, as observed in Gdown1-deficient hepatocytes. However, it seems unlikely that Pol II(G) is important primarily for cell growth since Gdown1 is not conserved in yeast and lower metazoans. With the general objective of understanding the in vivo roles and Mediator-dependent activation mechanisms for Pol II(G), our specific aims are: (i) to investigate the biological functions of Pol II(G) in embryonic and adult cells through determination of genes directly regulated by Pol II(G) -- by RNA-seq and ChIP-seq assays with Gdown1 null blastocysts, conditional Gdown1 knockout mouse, mouse embryonic stem cells, and Gdown1-null hepatocytes); (ii) to investigate the molecular mechanism of Mediator-dependent Pol II(G) transcription by genetic and biochemical determination of contact sites between Pol II, Gdown1 (wt and mutant) and Mediator and by CX-MS and cryoEM structural analyses of corresponding complexes; (iii) to examine the impact of Gdown1 ablation on tumorigenesis using mouse models that involve xenograft of human cancer cells and hydrodynamic injection of oncogenic vectors to generate liver cancer. Thus, in addition to revealing biological roles of Pol II(G) and the mechanism underlying its regulation by Mediator, these studies may reveal new targets for cancer therapeutics.

 描述（由申请人提供） RNA 聚合酶 II (Pol II) 通过蛋白质编码和非编码 RNA 的转录，在真核基因组的读出中发挥着关键作用。正常的生理过程依赖于精确的转录控制，而转录失调是许多病理学的基础，包括： Pol II 向特定启动子的募集受到多种（辅助）因子的调节，其中包括多亚基介体，它直接结合 Pol II 和转录激活因子，以促进基本上所有基因的激活。 II(G) 是最近发现的 Pol II 的一种独特形式（最初在小牛胸腺和猪肝中），包含紧密相关的多肽 Gdown1 以及 Pol II 的正常 12 个亚基。在 Mediator 中，与 Pol II 相关的 Gdown1 通过阻止起始因子 TFIIF 与 Pol II 的结合来抑制转录，从而抑制前起始复合物的形成，从而逆转被抑制的转录能力。 Pol II(G) 的转录调控机制尚不清楚，虽然 Mediator 从酵母到人类都是保守的，但 Gdown1 的保守性仅延伸到果蝇和高等后生动物中。另一个功能上不同的 Pol II，Pol II(G)，尽管 Pol II(G) 在高等后生动物转录调控中所发挥的具体作用仍不清楚。 II(G)，初步研究表明，Gdown1 消除会导致果蝇和小鼠的早期胚胎死亡，这种死亡的部分原因可能是细胞增殖的严重抑制，如在 Gdown1 缺陷的胚胎干细胞和癌细胞中观察到的那样。然而，Pol II(G) 似乎不太可能主要对细胞生长起重要作用，因为 Gdown1 在酵母和低等后生动物中并不保守。总体目标是了解 Pol II(G) 的体内作用和介体依赖性激活机制，我们的具体目标是： (i) 通过基因测定来研究 Pol II(G) 在胚胎和成体细胞中的生物学功能直接受 Pol II(G) 调控——通过使用 Gdown1 缺失囊胚、条件性 Gdown1 敲除小鼠、小鼠胚胎干细胞和 Gdown1 缺失肝细胞进行 RNA-seq 和 ChIP-seq 分析来进行研究；通过遗传和生化测定 Pol II、Gdown1（野生型和突变体）和介体之间的接触位点以及通过相应复合物的 CX-MS 和冷冻电镜结构分析，了解介体依赖性 Pol II(G) 转录的分子机制；使用小鼠模型检查 Gdown1 消融对肿瘤发生的影响，该模型涉及人类癌细胞的异种移植和致癌载体的流体动力学注射以产生肝癌，因此，除了揭示 Pol II(G) 和 Pol II(G) 的生物学作用之外。介体对其调节的机制，这些研究可能揭示癌症治疗的新靶点。