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.

基因调节的传统模型认为，将POL II募集到启动子是基因表达既是必要且足够的，但我们最近发现，从启动子促销区域中释放停滞的Pol II是在大量基因上限制的速率。我们的工作结合了全球位置分析（使用称为Chip-Chip和Chip-Seq的技术）以及体内足迹测定法，以探测果蝇停滞的Pol II的患病率。令人惊讶的是，这些数据表明，在数千个启动子基因组全基因组中，Pol II停滞比以前所欣赏的要广泛得多。此外，这些结果表明，POL II是在许多基因的未诱导状态中预载的，这些基因对环境或发育刺激有反应，这表明POL II的存在有望逃脱到该基因中，促进了对不断变化环境的有效，综合的反应。 了解停滞的pol II的基本特性，以及维护机制与释放启动子pol II进入生产性伸长率是Adelman实验室研究的特定目的。除了提供对应激反应的关键见解外，这项工作还可以预计在癌症和艾滋病的发展过程中阐明基因表达，因为在C-Myc，C-FOS，JUNB，JUNB和HIV启动子的哺乳动物启动子上也观察到类似停滞的Pol II。 在探测有关Pol II停滞的分子机制时，负伸长因子或NELF复合物对实验室特别感兴趣。 NELF已被证明可以在迄今为止的几个基因上建立停滞的pol II，包括Junb和HIV启动子以及果蝇启动子，已知可以承受停滞的pol II。为了全球识别NELF的靶标，我们对使用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靶基因都参与刺激响应途径，具有先天免疫反应的特殊富集。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耗竭，然后进行了RNA水平的微阵列分析以鉴定NELF靶基因。这项工作证实，NELF在调节体内先天免疫系统组成部分的表达方面起着关键作用。细胞中的后续研究表明，NELF介导的POL II失速对于最佳免疫反应至关重要，并表明聚合酶停滞对于NF-KB转录因子的结合或活性是必要的。该结果与我们较早的建议一致，即Pol II失速有助于建立围绕基因启动子的核小体污染，可访问的染色质结构，从而促进未来的激活。进一步的实验正在耗尽NELF的果蝇中，以评估NELF在允许生物体中恢复败血症损伤中的重要性。 除了在果蝇中的工作外，我们还开始研究使用源自小鼠的一级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，许多立即的早期反应基因，例如TNF-Alpha，TTP，JUNB和TNFAIP3，在其启动子区域中具有停滞的POL II和NELF，在静止的，未诱导的细胞中。相反，晚期原发性和次要反应基因在诱导前通常缺乏停滞的pol II和NELF。我们正在追求停滞的Pol II与免疫反应的动力学之间的关系，并评估NELF在此过程中的作用。