Investigating Transcriptional Responses to the Environment

研究对环境的转录反应

• 批准号: 9143485
• 负责人: Karen L Adelman
• 金额: $ 171.43万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9143485
• 关键词: Acquired Immunodeficiency Syndrome; Biological Assay; Biological Models; Cells; ChIP-on-chip; ChIP-seq; Chronic; Complex; Cues; Data; Development; Dissection; Drosophila genus; Embryonic Development; Environment; Epigenetic Process; Equilibrium; FOS gene; Gene Activation; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Growth and Development function; HIV; Human; Immune; Immune response; Individual; Inflammatory; Inflammatory Response; Knockout Mice; Laboratories; Location; Maintenance; Malignant Neoplasms; Mediating; Molecular Probes; Mus; Nucleotides; Organism; Pathway interactions; Physiological; Play; Polymerase; Polymerase Gene; Prevalence; Process; Production; Property; Prothrombin; Proto-Oncogenes; RNA; RNA Polymerase II; RNA chemical synthesis; Recruitment Activity; Regulation; Regulator Genes; Research; Resolution; Rest; Role; Signal Pathway; Signal Transduction; Stimulus; Stress; System; Techniques; Transcript; Transcription Elongation; Transcription Initiation; Work; base; biological adaptation to stress; c-myc Genes; cytokine; extracellular; gene environment interaction; in vivo; insight; interest; macrophage; negative elongation factor; novel; promoter; response

Our laboratory has found that release of paused Pol II from the promoter-proximal region is essential, regulated step in the expression of most metazoan genes. Our initial work investigated the prevalence of paused Pol II in Drosophila, employing a combination of global location analysis (using techniques called ChIP-chip and ChIP-seq) as well as in vivo footprinting assays. These data showed that Pol II pausing is much more widespread than previously appreciated. We and others have extended these findings to mammalian systems (mouse and human), demonstrating that pausing a prevalent gene regulatory strategy in higher organisms. Moreover, our results reveal that Pol II is constitutively present at many genes in environmentally- or developmentally-responsive gene networks, suggesting that the presence of Pol II facilitates efficient, integrated responses to a dynamically changing environment. Understanding the fundamental properties of paused Pol II, and the factors that govern maintenance vs. release of promoter-proximal Pol II into productive elongation are specific aims of research in the Adelman laboratory. In addition to providing crucial insight into stress-responses, this work is anticipated to elucidate gene expression during the development of cancer and AIDS, since similarly paused Pol II are observed at the mammalian promoters of proto-oncogenes like c-myc, c-fos and junB, as well as at the HIV promoter. As part of our efforts to better define the mechanisms underlying pausing, we have recently developed a novel technique for isolating the short RNA transcripts generated by paused Pol II, and analyzed them through massively-parallel sequencing of individual RNA molecules. This strategy allowed us to pinpoint both the locations of transcription initiation and pausing, at single-nucleotide resolution. In probing the molecular mechanisms governing Pol II pausing, the Negative ELongation Factor, or NELF complex, is of particular interest to the laboratory. Mechanistically, we have shown that NELF is broadly important for the stable pausing of Pol II. To define its physiological role, we have developed conditional knockout mice. Studies in both cells and in vivo revealed that NELF-mediated Pol II pausing is essential for an optimal immune response to bacterial challenge and and for early embryonic development. Notably, in both systems we find that disruption of pausing dramatically impacts cellular responsiveness to environmental or extrinsic cues. Dissection of this effect has revealed that pausing plays a key role in determining the expression level of critical hubs of signal transduction machineries. In this way, pausing tunes signaling responses, and the consequences of signaling on gene expression. Taken together, the data suggest that pausing of Pol II allows for coordinated tuning of both basal gene expression and activation, to enable precise, balanced responses to environmental or developmental cues.

我们的实验室发现，在大多数后生动物基因的表达中，暂停的POL II从启动子-近端区域释放是必不可少的、受调控的步骤。我们最初的工作采用了全球定位分析(使用称为芯片和芯片序列的技术)和体内足迹分析相结合的方法，调查了暂停的POLII在果蝇中的流行情况。这些数据表明，波尔II停顿比之前认识到的要普遍得多。我们和其他人已经将这些发现扩展到哺乳动物系统(小鼠和人类)，表明暂停在高等生物体中流行的基因调控策略。此外，我们的结果显示，在环境或发育反应基因网络中，POL II存在于许多基因中，这表明POL II的存在有助于对动态变化的环境做出有效的、综合的反应。 了解暂停的POL II的基本性质，以及控制启动子-近端POL II的维持和释放进入生产性延伸的因素是阿德尔曼实验室研究的具体目标。除了提供对应激反应的重要洞察外，这项工作还有望阐明癌症和艾滋病发展过程中的基因表达，因为在c-myc、c-fos和JunB等原癌基因的哺乳动物启动子以及HIV启动子中也观察到了类似的停滞的POL II。作为更好地确定暂停的机制的努力的一部分，我们最近开发了一种新的技术，用于分离由暂停的Pol II产生的短RNA转录本，并通过对单个RNA分子的大规模平行测序来分析它们。这一策略使我们能够在单核苷酸分辨率下准确地定位转录启动和暂停的位置。 在探索控制POL II停顿的分子机制时，负延长因子或NELF复合体是实验室特别感兴趣的。从机制上讲，我们已经证明了NELF对于POL II的稳定暂停具有广泛的重要性。为了确定它的生理作用，我们建立了条件基因敲除小鼠。细胞和体内的研究表明，NELF介导的POL II暂停对于细菌攻击和早期胚胎发育的最佳免疫反应是必不可少的。值得注意的是，在这两个系统中，我们发现暂停的中断极大地影响了细胞对环境或外部线索的反应。对这一效应的剖析表明，停顿在决定信号转导机制的关键枢纽的表达水平方面起着关键作用。通过这种方式，暂停可以调整信号反应，以及信号对基因表达的影响。综上所述，这些数据表明，暂停POL II可以协调调整基础基因的表达和激活，从而能够对环境或发育线索做出准确、平衡的反应。