Investigating Transcriptional Responses to the Environme

研究对环境的转录反应

• 批准号: 7330699
• 负责人: Karen L Adelman
• 金额: --
• 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/7330699
• 关键词: Investigating; Transcriptional; Responses; Environme

My newly established laboratory at the NIEHS investigates the changes in chromatin architecture and gene activity that results from stimuli from the external environment, focusing on the stress response in Drosophila melanogaster. This model system is ideal for probing the direct, immediate effects of a stress signal on gene activation and repression; within seconds of experiencing a thermal or oxidative stress, Drosophila stress-responsive genes are strongly up-regulated (100 to 1000-fold increase in mRNA levels within 20 minutes). Simultaneously, the chromatin surrounding these genes is dramatically decondensed, and housekeeping genes are shut off. The rapid, robust activation of the major stress-responsive genes and concomitant repression of other cellular transcription allows for a detailed kinetic examination of the links between chromatin structure and gene expression in vivo. We have made use of this system to address several key questions concerning the molecular mechanisms of transcription regulation, as outlined below. One remarkable property of the stress-repsonse is the speed of gene activation. One proposed reason for this is the presence of RNA polymerase II (Pol II) at the promoter of stress-responsive genes prior to activation. This Pol II has initiated transcription, synthesized a short RNA, and then stalled within the promoter-proximal region. Gene induction by stress rapidly releases this stalled Pol II into the gene, allowing the first wave of Pol II to be observed within the coding regions in seconds. 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 my research. 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 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 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 oncogene in mammalian cells. The large number of NELF-dependent genes identified by our Microarray analysis suggests that manipulating the efficiency of early elongation is much more prevalent method of gene regulation than previously appreciated. To evaluate this possibility, we are pursuing a genome-wide search for stalled Pol II in Drosophila, employing ChIP-on-chip location analysis (in collaboration with Rick Young?s laboratory at MIT and the NIEHS microarray facility). This technique, which involves probing an array with DNA material derived from a traditional ChIP assay, permits the mapping of Pol II binding sites at high resolution throughout the Drosophila genome. Comparison of Pol II occupancy with RNA expression levels has allowed us to identify a distinct class of genes that posses bound Pol II at their promoters but do not produce significant levels of mRNA. The lack of correlation between Pol II occupancy and gene expression at a subset of genes in Drosophila, in conjunction with recent data from ChIP-on-chip using human fibroblasts, indicates that a significant percentage of genes in higher eukaryotes are regulated at a step after the recruitment of Pol II to the gene promoter. Strikingly, in both Drosophila and mammalian cells, a large number of the promoters that possess a seemingly inactive Pol II are inducible by environmental (e.g steroid hormones, stress) and cell cycle cues. Elucidating this novel paradigm for controlling gene expression will surely provide fresh insights into the activation of not only stress-responsive genes in Drosophila, but also pivotal oncogenes involved in human disease. By leveraging the power of Drosophila genetics in both embryonic cells and developing organisms, we are continuing to identify 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.

我在NIEHS新成立的实验室研究外部环境刺激引起的染色质结构和基因活性的变化，重点是果蝇的应激反应。这个模型系统是探索压力信号对基因激活和抑制的直接、即时影响的理想选择；在经历热或氧化压力的几秒钟内，果蝇的压力反应基因就会强烈上调(20分钟内mRNA水平增加100到1000倍)。与此同时，这些基因周围的染色质急剧解聚，管家基因被关闭。主要应激反应基因的快速、强有力的激活以及伴随而来的其他细胞转录的抑制，使得对染色质结构和体内基因表达之间的联系进行了详细的动力学检查。我们利用这个系统来解决与转录调控的分子机制有关的几个关键问题，如下所述。 应激反应的一个显著特征是基因激活的速度。其中一个原因被认为是在应激反应基因的启动子激活之前存在RNA聚合酶II(POL II)。这个POL II启动了转录，合成了一个短的RNA，然后停滞在启动子近端区域。应激诱导的基因迅速释放这种停滞不前的POLII进入基因，从而在几秒钟内就能在编码区观察到POLII的第一波。了解停滞不前的POL II的基本属性，以及POL II维持和释放进入生产性延伸的机制是我研究的具体目的。除了提供对应激反应的重要洞察外，这项工作还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c-myc、c-fos、JunB和HIV启动子中也被观察到。 为了确定参与调控Pol II转录延长的因素，我们建立了一种使用RNA干扰来耗尽果蝇S2细胞中特定蛋白质的有效遗传分析方法。我们已经筛选了大量可能的转录延长和染色质修饰因子，以研究它们对果蝇应激反应基因Hsp70(热休克蛋白70)的RNA生产的影响。果蝇中的HSP基因代表了一组经过充分研究、高度诱导的基因，这些基因对热、氧化和离子应激以及一些致癌物和诱变剂做出反应。我们的基因筛查已经确定了一些待进一步研究的候选对象。其中，负伸长因子或NELF复合体尤其令人感兴趣。我们通过微阵列分析表明，NELF的缺失增加了一些HSP基因的基础转录，并影响了一些其他诱导基因，包括那些对氧化损伤、细菌病原体和细胞周期蛋白激酶的反应。此外，染色质免疫沉淀(ChIP)分析表明，大多数NELF依赖的基因在其启动子附近都有Pol II参与，NELF通过这些基因的启动子-近端区域控制转录效率。这项重要的工作确立了NELF和体内停滞的POL II之间存在全球联系，并且NELF活性对于调节无数可诱导基因的早期伸长起着关键作用。我们认为，在没有诱导的情况下，NELF作为一个分子开关来抑制基因转录，但允许极快地抑制基因激活。鉴于最近有证据表明NELF在哺乳动物细胞JunB癌基因的转录中发挥关键作用，这些数据特别有趣。 我们的微阵列分析发现了大量依赖NELF的基因，这表明操纵早期伸长的效率是比以前所认识的更普遍的基因调控方法。为了评估这种可能性，我们正在进行全基因组搜索果蝇中停滞的POL II，使用芯片上的位置分析(与麻省理工学院的里克·杨？S实验室和NIEHS微阵列设施合作)。这项技术包括用来自传统芯片分析的DNA材料探测阵列，允许在整个果蝇基因组中以高分辨率绘制Pol II结合位点的图谱。通过比较Pol II的占有率和RNA的表达水平，我们可以确定一类不同的基因，这些基因在启动子上与Pol II结合，但不产生显著的mRNA水平。在果蝇中，Pol II的占有率与基因亚集上的基因表达之间缺乏相关性，结合最近使用人类成纤维细胞的芯片上的数据，表明高等真核生物中相当大比例的基因在Pol II被招募到基因启动子之后的一个步骤中受到调控。值得注意的是，在果蝇和哺乳动物细胞中，大量看似无效的POLII启动子可以被环境(如类固醇激素、压力)和细胞周期信号诱导。阐明这一控制基因表达的新范式，不仅将为果蝇应激反应基因的激活提供新的见解，还将为参与人类疾病的关键癌基因的激活提供新的见解。 通过在胚胎细胞和发育中的生物体中利用果蝇遗传学的力量，我们正在继续识别协调这些不同基因上停滞的POL II建立和释放的蛋白质，并确定利用这种新的基因调控形式的基因的定义特征。