Investigating Transcriptional Responses to the Environment

研究对环境的转录反应

• 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
• 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

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.

尽管传统的基因调控模型假设启动子募集Pol II对于基因表达是必要和充分的，但我们最近发现，从启动子近端区域释放暂停的Pol II对于大量基因的表达是限速的。我们的初步工作调查了暂停型Pol II在果蝇中的流行，采用了全球定位分析（使用ChIP-chip和ChIP-seq技术）以及体内足迹分析的组合。令人惊讶的是，这些数据表明，Pol II暂停比之前认为的要广泛得多，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），证明暂停高等生物中普遍存在的基因调控策略。此外，我们的研究结果表明，在环境或发育响应基因网络中，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进行有效的综合响应。