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）通过其蛋白质编码和非编码RNA的转录在真核基因组的读出中起关键作用。正常的生理过程依赖于精确的转录控制，而转录失调是包括癌症在内的许多病理学的基础。Pol II向特定启动子的募集受多个（共）因子调节，所述多个（共）因子包括多亚基介体，其直接结合Pol II和转录激活因子以促进基本上所有基因的激活。Pol II（G）是Pol II的一种独特的、最近发现的形式（最初在小牛胸腺和猪肝中），其包含紧密结合的多肽Gdown 1沿着Pol II的正常12个亚基。体外转录研究表明，在没有中介体的情况下，Pol II相关的Gdown 1通过阻止起始因子TFIIF与Pol II的结合从而抑制前起始复合物的形成来抑制转录。介体逆转Pol II（G）被抑制的转录能力，尽管潜在的机制仍不清楚。虽然Mediator从酵母到人类都是保守的，但Gdown 1的保守性仅限于果蝇。因此，果蝇和高等后生动物中的转录调控系统已经通过获得额外的和功能上不同的Pol II，Pol II（G）而进化，尽管Pol II（G）在高等后生动物中的转录调控中所起的特定作用仍不清楚。为了理解Pol II（G）的生物学作用，初步研究表明Gdown 1消融导致果蝇和小鼠的早期胚胎死亡。这种致死性可以部分解释为细胞增殖的严重抑制，如在Gdown 1缺陷的胚胎干细胞和癌细胞系中观察到的，以及基因表达的失调，如在Gdown 1缺陷的肝细胞中观察到的。然而，Pol II（G）似乎不太可能主要对细胞生长重要，因为Gdown 1在酵母和低等后生动物中不保守。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）通过测定由Pol II（G）直接调节的基因来研究Pol II（G）在胚胎和成体细胞中的生物学功能-通过RNA-seq和ChIP-seq测定，用Gdown 1无效胚泡、条件性Gdown 1敲除小鼠、小鼠胚胎干细胞，和Gdown 1-null肝细胞）;（ii）通过对Pol II、Gdown 1之间的接触位点的遗传和生物化学测定来研究介体依赖性Pol II（G）转录的分子机制（野生型和突变型）和介体，并通过相应复合物的CX-MS和cryoEM结构分析;（iii）使用小鼠模型检查Gdown 1消融对肿瘤发生的影响，所述小鼠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）的生物学作用和介体对其调控的机制外，这些研究还可能揭示癌症治疗的新靶点。