Investigating Transcriptional Responses to the Environment

研究对环境的转录反应

• 批准号: 7968206
• 负责人: Karen L Adelman
• 金额: $ 195.48万
• 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/7968206
• 关键词: Acquired Immunodeficiency Syndrome; Architecture; Binding; Biological Assay; Biological Models; Cells; Chromatin; Complex; Cues; DNA Damage; Data; Defect; Development; Drosophila genus; ERG gene; Environment; Epigenetic Process; FOS gene; Fat Body; Follow-Up Studies; Future; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Goals; Growth and Development function; HIV; Housekeeping Gene; Immune; Immune response; Immune system; Injury; Investigation; Kinetics; Laboratories; Location; Maintenance; Malignant Neoplasms; Mediating; Microarray Analysis; Modeling; Molecular Probes; Mus; NF-kappa B; Nucleosomes; Nucleotides; Organism; Pathway interactions; Pattern; Physiological; Play; Polymerase; Prevalence; Process; Promoter Regions; Property; RNA; RNA Interference; RNA Polymerase II; RNA chemical synthesis; Recruitment Activity; Research; Rest; Role; Signal Transduction; Stimulus; Stress; Suggestion; Techniques; Tissues; Transcription Elongation; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Work; alpha-tocopherol transfer protein; base; biological adaptation to stress; c-myc Genes; extracellular; fly; gene environment interaction; genome-wide; human TNF protein; in vivo; insight; interest; macrophage; negative elongation factor; novel; promoter; research study; response; septic

Whereas traditional models for gene regulation posit that recruitment of Pol II to the promoter is both necessary and sufficient for gene expression, we have recently found that release of stalled Pol II from the promoter-proximal region is rate-limiting at a large number of genes. Our work employed a combination of global location analysis (using technique called ChIP-chip and ChIP-seq) as well as in vivo footprinting assays to probe the prevalence of stalled Pol II in Drosophila. Surprisingly, these data show that Pol II stalling is much more widespread than previously appreciated, occurring at thousands of promoters genome-wide. Moreover, these results reveal that Pol II is pre-loaded in the uninduced state at many genes that respond to environmental or developmental stimuli, suggesting that the presence of Pol II, poised for escape into the gene, facilitates efficient, integrated responses to a changing environment. Understanding the fundamental properties of stalled Pol II, and the mechanisms for 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 the stress-response, this work is anticipated to elucidate gene expression during the development of cancer and AIDS, since similarly stalled Pol II are observed at the mammalian promoters of c-myc, c-fos, junB and the HIV promoter. In probing the molecular mechanisms governing Pol II stalling, the Negative ELongation Factor, or NELF complex, is of particular interest to the laboratory. NELF has been shown to establish stalled Pol II at several genes to date, including the junB and HIV promoters, as well as at Drosophila promoters know to harbor stalled Pol II. To globally identify targets of NELF, we have performed a microarray analysis on Drosophila cells that were depleted of NELF using RNA interference. We found that many NELF target genes are involved in stimulus-responsive pathways, with a particular enrichment in the innate immune response. To evaluate the physiological relevance of this finding, we have recently performed NELF depletion in the Drosophila fat body (the main immune responsive tissue), followed by microarray analysis of RNA levels to identify NELF target genes. This work confirms that NELF plays a key role in regulating expression of components of the innate immune system in vivo. Follow-up studies in cells revealed that NELF-mediated Pol II stalling is essential for an optimal immune response and indicated that polymerase stalling is necessary for either the binding or activity of the NF-kB transcription factor Relish (Rel). this result is consistent with our earlier suggestion that Pol II stalling helps establish a nucleosome-deprived, accessible chromatin architecture around gene promoters, facilitating future activation. Further experiments are underway in NELF-depleted flies to assess the importance of NELF in allowing the organism to recover from septic injury. In addition to our work in Drosophila, we have begun to study the role of polymerase stalling in the mammalian immune response, using primary macrophages derived from mouse. These ongoing investigations reveal that many immediate early response genes, like TNF-alpha, TTP, junB and tnfaip3, possess stalled Pol II and NELF in their promoter regions in resting, uninduced cells. In contrast, late primary and secondary response genes generally lack stalled Pol II and NELF prior to induction. We are pursuing the relationship between the presence of stalled Pol II and the kinetics of the immune response, as well as evaluating the role of NELF in this process.

虽然传统的基因调控模型认为Pol II在启动子上的募集是基因表达的必要条件和充分条件，但我们最近发现，在大量基因中，停滞的Pol II从启动子-近端区域的释放是限速的。我们的工作采用了全球定位分析(使用称为CHIP-CHIP和CHIP-SEQ的技术)以及体内足迹分析相结合的方法来探索停滞的POLII在果蝇中的流行情况。令人惊讶的是，这些数据显示，POLII停滞比之前认识到的要广泛得多，发生在全基因组的数千个启动子中。此外，这些结果表明，在许多对环境或发育刺激做出反应的基因中，POL II处于未诱导状态，这表明POL II的存在，准备逃逸到基因中，促进了对不断变化的环境的有效、综合的反应。 了解停滞的POL II的基本性质，以及维持和释放启动子-近端POL II进入生产性延伸的机制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，这项工作有望阐明癌症和艾滋病发展过程中的基因表达，因为类似停滞的POL II在哺乳动物的c-myc、c-fos、JunB和HIV启动子中也被观察到。 在探索控制POL II失速的分子机制时，负伸长因子或NELF复合体是实验室特别感兴趣的。到目前为止，NELF已经被证明在几个基因上建立了停滞的POL II，包括JunB和HIV启动子，以及已知含有停滞的POL II的果蝇启动子。为了在全球范围内识别NELF的靶点，我们利用RNA干扰对NELF缺失的果蝇细胞进行了微阵列分析。我们发现许多NELF靶基因都参与了刺激反应途径，尤其是在先天免疫反应中。为了评估这一发现的生理学相关性，我们最近在果蝇脂肪体(主要的免疫反应组织)中进行了NELF耗尽，随后对RNA水平进行了微阵列分析，以确定NELF的靶基因。这项工作证实了NELF在调节体内天然免疫系统各组成部分的表达方面发挥了关键作用。在细胞中的后续研究表明，NELF介导的POL II停滞对于最佳免疫反应是必不可少的，并表明聚合酶停滞对于核因子-kB转录因子美味(REL)的结合或活性是必要的。这一结果与我们早先的建议是一致的，即POL II停滞有助于在基因启动子周围建立一个核小体被剥夺的、可获得的染色质结构，从而促进未来的激活。在NELF耗竭的果蝇中正在进行进一步的实验，以评估NELF在使有机体从败血症损伤中恢复的重要性。 除了我们在果蝇上的工作外，我们已经开始使用来自小鼠的原始巨噬细胞来研究聚合酶停滞在哺乳动物免疫反应中的作用。这些正在进行的研究表明，许多即时早期反应基因，如TNF-α、TTP、JunB和TNFAIP3，在静息、未诱导的细胞中的启动子区域具有停滞的POL II和NELF。相反，晚期初级和次级反应基因通常在诱导前缺乏停滞的POLII和NELF。我们正在研究停滞的POL II与免疫反应动力学之间的关系，以及评估NELF在这一过程中的作用。