Investigating Transcriptional Responses to the Environment

研究对环境的转录反应

• 批准号: 7594020
• 负责人: Karen L Adelman
• 金额: $ 137.28万
• 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/7594020
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Biological Assay; Biological Models; Carcinogens; Cell Cycle; Cells; Chromatin; Complex; Data; Development; Drosophila genus; Drosophila inturned protein; Embryo; Environment; Epigenetic Process; FOS gene; Gene Activation; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Goals; Growth and Development function; HIV; Heat-Shock Proteins 70; Housekeeping Gene; Immune; Investigation; Laboratories; Light; Link; Location; Maintenance; Malignant Neoplasms; Mammalian Cell; Manuscripts; Microarray Analysis; Modeling; Molecular; Nature; Nucleotides; Numbers; Organism; Pattern; Phosphotransferases; Play; Polymerase; Prevalence; Production; Property; Proteins; Proto-Oncogenes; RNA; RNA Interference; RNA Polymerase II; RNA chemical synthesis; Rate; Recruitment Activity; Repression; Research; Role; Signal Transduction; Stimulus; Stress; Techniques; Transcription Elongation; Transcriptional Regulation; Work; base; biological adaptation to stress; c-myc Genes; chromatin immunoprecipitation; extracellular; gene environment interaction; in vivo; insight; interest; negative elongation factor; novel; pathogen; promoter; response

Recent work in the Adelman laboratory has employed a combination of genome-wide location analysis (using a technique called ChIP-chip) 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 nearly 20% of promoters. Moreover, these results reveal stalled Pol II at many genes that respond to environmental or developmental stimuli, suggesting that the presence of a poised Pol II facilitates efficient, integrated responses to a changing environment. Whereas traditional models for gene regulation posit that recruitment of Pol II to the promoter is both necessary and sufficient for gene expression, our exciting finding demonstrates that release of Pol II from the promoter-proximal region is rate-limiting at a large number of genes. A manuscript describing this work is currently in press at Nature Genetics. Understanding the fundamental properties of the stalled Pol II, and the mechanisms for maintenance vs. release of 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. To characterize the factors involved in regulating transcription elongation by the stalled Pol II, we have established an efficient genetic assay using RNA interference to deplete specific proteins in Drosophila S2 cells. We have screened a large number of putative transcription elongation and chromatin modifying factors for their effect on RNA production from a key Drosophila stress-responsive gene, Hsp70 (Heat shock protein 70). The Hsp genes in Drosophila represent a well-studied, highly inducible set of genes that are responsive to thermal, oxidative and ionic stress, as well as a number of carcinogens and mutagenic agents. Our genetic screen has identified a number of candidates for further investigation. Among them, the Negative ELongation Factor, or NELF complex, is of particular interest. We have shown by Microarray analysis that depletion of NELF increases basal transcription of a number of Hsp genes as well as affecting a number of other inducible genes, including those responsive to oxidative damage, bacterial pathogens, and cell cycle kinases. Moreover, Chromatin Immunoprecipitation (ChIP) assays have revealed that the majority of NELF-dependent genes possess engaged, stalled Pol II near their promoters and that NELF controls the efficiency of transcription through the promoter-proximal region of these genes. This important work established that there is a global link between NELF and stalled Pol II in vivo, and that NELF activity is pivotal for regulating early elongation at a myriad of inducible genes. We propose that NELF functions as a molecular switch to repress gene transcription in the absence of induction, yet allowing for extremely rapid de-repression upon gene activation. These data are particularly interesting in light of recent evidence that NELF plays a critical role in transcription of the junB proto-oncogene in mammalian cells. By leveraging the power of Drosophila genetics in both embryonic-derived cells and developing organisms, we are continuing to identify and characterize the proteins that coordinate the establishment and release of stalled Pol II at these various genes, and to determine the defining features of genes that utilize this novel form of gene regulation.

阿德尔曼实验室最近的工作采用了全基因组定位分析（使用一种称为ChIP-chip的技术）和体内足迹测定相结合的方法来探测果蝇中停滞的Pol II的流行程度。令人惊讶的是，这些数据表明，Pol II失速比之前认为的要普遍得多，发生在近20%的启动子中。此外，这些结果揭示了许多对环境或发育刺激做出反应的基因中停滞的Pol II，这表明平衡的Pol II的存在促进了对变化环境的有效、综合反应。传统的基因调控模型认为，启动子中Pol II的募集是基因表达的必要条件和充分条件，而我们令人兴奋的发现表明，在大量基因中，启动子近端区域Pol II的释放是限速的。描述这项工作的手稿目前在《自然遗传学》杂志上出版。