Investigating Transcriptional Responses to the Environment

研究对环境的转录反应

基本信息

批准号：
8553780
负责人：
Karen L Adelman
金额：
$ 171.57万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/8553780
关键词：
Acquired Immunodeficiency Syndrome Bacterial Infections Biological Assay Biological Models Biological Response Modifiers Cells ChIP-on-chip ChIP-seq Chromatin Chromatin Structure Complex Cues DNA Sequence Data Defect Development Drosophila genus Environment Epigenetic Process Equilibrium Exhibits FOS gene Fat Body Gene Activation Gene Expression Gene Expression Profile Gene Expression Regulation Gene Targeting Genes Genetic Transcription Goals Growth and Development function HIV Human Immune Immune response Immune system Individual Inflammatory Response Investigation Kinetics Laboratories Link Location Maintenance Malignant Neoplasms Mediating Microarray Analysis Modeling Molecular Probes Mus NF-kappa B Nucleotides Organism Pathway interactions Physiological Play Polymerase Polymerase Gene Prevalence Production Promoter Regions Property Prothrombin Proto-Oncogenes RNA RNA Interference RNA Polymerase II RNA chemical synthesis Recruitment Activity Regulator Genes Research Resolution Rest Role Signal Pathway Signal Transduction Specific qualifier value Stimulus Stress System Techniques Tissues Transcript Transcription Elongation Transcription Initiation Transcriptional Regulation Tumor Necrosis Factor-alpha Work base biological adaptation to stress c-myc Genes cytokine extracellular fly follow-up gene environment interaction genome-wide human TNF protein in vivo insight interest macrophage negative elongation factor novel promoter response

项目摘要

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 paused Pol II from the promoter-proximal region is rate-limiting for expression of a large number of 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. Surprisingly, these data showed that Pol II pausing is much more widespread than previously appreciated, occurring at thousands of promoters genome-wide. We and others have recently 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. Notably, this exciting new technique revealed a role for the DNA sequence within the initially transcribed region in specifying the efficiency of early elongation, providing insights into why polymerase pausing is more prominent at some genes than at others. 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 paused Pol II at several genes to date, including the junB and HIV promoters. 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 vivo in regulating expression of components of the innate immune system. Follow-up studies in both cells and flies revealed that NELF-mediated Pol II pausing is essential for an optimal immune response to bacterial challenge and indicated that polymerase pausing tunes the basal expression level of critical immune regulators such as the NF-kB transcription factor Relish (Rel). In addition to our work in Drosophila, we are studying the role of polymerase pausing in the mammalian inflammatory response, using primary macrophages derived from mouse. These investigations reveal that several genes that play critical roles in the response to bacterial infection, like TNF-alpha and junB, possess paused Pol II, waiting in their promoter regions in resting, uninduced cells. However, many other bacterially-induced genes do not harbor paused Pol II, and are regulated primarily by Pol II recruitment during gene activation. Interestingly, we find that there is a relationship between the presence of paused Pol II and the kinetics of the immune response, in that paused genes exhibit much more transient bursts of transcription activity than do genes regulated through Pol II recruitment, which often exhibit a more sustained response. This suggests that there could be a fundamental link between gene regulatory strategy (i.e. the step in the transcription cycle that is rate-limiting for gene expression) and the kinetics or magnitude of gene expression. In this way, pausing might allow for fine tuning of both basal gene expression and activation, to enable precise, balanced responses to environmental or developmental cues.

虽然传统的基因调控模型认为将 Pol II 募集到启动子对于基因表达来说既是必要的又是充分的，但我们最近发现，暂停的 Pol II 从启动子近端区域的释放对于大量基因的表达是限速的。基因。我们最初的工作结合了全球定位分析（使用称为 ChIP 芯片和 ChIP-seq 的技术）以及体内足迹分析，研究了果蝇中暂停 Pol II 的流行情况。令人惊讶的是，这些数据表明 Pol II 暂停比之前想象的要广泛得多，发生在全基因组的数千个启动子中。我们和其他人最近将这些发现扩展到哺乳动物系统（小鼠和人类），证明暂停高等生物体中普遍存在的基因调控策略。此外，我们的结果表明，Pol II 组成性地存在于环境或发育响应基因网络中的许多基因中，这表明 Pol II 的存在有助于对动态变化的环境做出有效、综合的响应。了解暂停的 Pol II 的基本特性，以及控制启动子近端 Pol II 的维持与释放以产生有效伸长的因素是 Adelman 实验室研究的具体目标。除了提供对压力反应的重要见解外，这项工作预计还将阐明癌症和艾滋病发展过程中的基因表达，因为在哺乳动物原癌基因（如 c-myc、c-fos）启动子处也观察到类似的 Pol II 暂停和junB，以及HIV启动子。作为我们更好地定义暂停机制的努力的一部分，我们最近开发了一种新技术，用于分离暂停的 Pol II 生成的短 RNA 转录本，并通过单个 RNA 分子的大规模并行测序来分析它们。这种策略使我们能够以单核苷酸分辨率精确定位转录起始和暂停的位置。值得注意的是，这项令人兴奋的新技术揭示了最初转录区域内的 DNA 序列在指定早期延伸效率中的作用，提供了关于为什么聚合酶暂停在某些基因上比其他基因更突出的见解。在探索控制 Pol II 失速的分子机制时，实验室对负伸长因子 (NELF) 复合体特别感兴趣。迄今为止，NELF 已被证明可以在多个基因上建立暂停的 Pol II，包括 junB 和 HIV 启动子。为了全面识别 NELF 的靶点，我们使用 RNA 干扰对 NELF 耗尽的果蝇细胞进行了微阵列分析。我们发现许多 NELF 靶基因参与刺激响应途径，特别丰富了先天免疫反应。为了评估这一发现的生理相关性，我们最近在果蝇脂肪体（主要免疫反应组织）中进行了 NELF 去除，然后对 RNA 水平进行微阵列分析，以确定 NELF 目标基因。这项工作证实了 NELF 在体内调节先天免疫系统成分的表达方面发挥着关键作用。对细胞和果蝇的后续研究表明，NELF 介导的 Pol II 暂停对于对细菌攻击的最佳免疫反应至关重要，并表明聚合酶暂停可调节关键免疫调节因子（例如 NF-kB 转录因子 Relish）的基础表达水平（相对）。除了在果蝇中的工作之外，我们还利用来自小鼠的原代巨噬细胞研究聚合酶暂停在哺乳动物炎症反应中的作用。这些研究表明，一些在细菌感染反应中发挥关键作用的基因，如 TNF-α 和 junB，具有暂停的 Pol II，在静息、未诱导的细胞中等待其启动子区域。然而，许多其他细菌诱导的基因不包含暂停的 Pol II，并且主要受基因激活期间 Pol II 募集的调节。有趣的是，我们发现暂停的 Pol II 的存在与免疫反应的动力学之间存在关系，因为暂停的基因比通过 Pol II 募集调节的基因表现出更多的瞬时转录活性爆发，后者通常表现出更多的转录活性。持续的反应。这表明基因调控策略（即转录周期中限制基因表达速率的步骤）与基因表达的动力学或幅度之间可能存在根本联系。通过这种方式，暂停可能允许对基础基因表达和激活进行微调，从而实现对环境或发育线索的精确、平衡的反应。