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 新建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，重点关注果蝇的应激反应。该模型系统非常适合探测应激信号对基因激活和抑制的直接影响；在经历热或氧化应激后几秒钟内，果蝇应激反应基因就会强烈上调（20 分钟内 mRNA 水平增加 100 至 1000 倍）。同时，这些基因周围的染色质急剧解压缩，管家基因被关闭。主要应激反应基因的快速、稳健激活以及其他细胞转录的伴随抑制允许对体内染色质结构和基因表达之间的联系进行详细的动力学检查。我们利用该系统解决了有关转录调控分子机制的几个关键问题，如下所述。 应激反应的一项显着特性是基因激活的速度。一个可能的原因是在激活之前应激反应基因的启动子处存在 RNA 聚合酶 II (Pol II)。该 Pol II 启动转录，合成短 RNA，然后停滞在启动子近端区域。应激引起的基因诱导会迅速将这种停滞的 Pol II 释放到基因中，从而在几秒钟内就可以在编码区内观察到第一波 Pol II。了解停滞的 Pol II 的基本特性，以及 Pol II 维持与释放成有效伸长的机制是我研究的具体目标。除了提供对应激反应的重要见解外，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 c-myc、c-fos、junB 和HIV启动子。 为了表征通过停滞的 Pol II 调节转录延伸的因素，我们建立了一种有效的遗传测定方法，利用 RNA 干扰来消除果蝇 S2 细胞中的特定蛋白质。我们筛选了大量假定的转录延伸和染色质修饰因子，了解它们对果蝇关键应激反应基因 Hsp70（热休克蛋白 70）RNA 产生的影响。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许多致癌物和诱变剂有反应。我们的基因筛查已经确定了一些可供进一步研究的候选者。其中，负伸长因子或 NELF 复合体特别令人感兴趣。我们通过微阵列分析表明，NELF 的缺失会增加许多 Hsp 基因的基础转录，并影响许多其他诱导基因，包括那些对氧化损伤、细菌病原体和细胞周期激酶敏感的基因。此外，染色质免疫沉淀 (ChIP) 测定表明，大多数 NELF 依赖性基因在其启动子附近具有接合的 Pol II，并且 NELF 通过这些基因的启动子近端区域控制转录效率。这项重要的工作证实了 NELF 和体内停滞的 Pol II 之间存在全局联系，并且 NELF 活性对于调节无数诱导基因的早期伸长至关重要。我们认为 NELF 作为分子开关发挥作用，在没有诱导的情况下抑制基因转录，但在基因激活时可以极快地解除抑制。鉴于最近的证据表明 NELF 在哺乳动物细胞中 junB 癌基因的转录中发挥着关键作用，这些数据特别有趣。 我们的微阵列分析鉴定出的大量 NELF 依赖性基因表明，操纵早期伸长的效率是比以前认识到的更为普遍的基因调控方法。为了评估这种可能性，我们正在果蝇中进行全基因组搜索，寻找停滞的 Pol II，采用 ChIP-on-chip 位置分析（与 MIT 的 Rick Young 实验室和 NIEHS 微阵列设施合作）。该技术涉及使用源自传统 ChIP 测定的 DNA 材料探测阵列，可以在整个果蝇基因组中以高分辨率绘制 Pol II 结合位点。 Pol II 占据率与 RNA 表达水平的比较使我们能够识别出一类独特的基因，这些基因在其启动子处具有结合的 Pol II，但不产生显着水平的 mRNA。果蝇中 Pol II 占据和基因子集的基因表达之间缺乏相关性，结合最近使用人成纤维细胞进行芯片上 ChIP 的数据，表明高等真核生物中很大一部分基因是在将 Pol II 招募到基因启动子上。引人注目的是，在果蝇和哺乳动物细胞中，大量具有看似无活性的 Pol II 的启动子可被环境（例如类固醇激素、应激）和细胞周期线索诱导。阐明这种控制基因表达的新范式肯定不仅会为果蝇中应激反应基因的激活提供新的见解，而且还会为与人类疾病有关的关键癌基因的激活提供新的见解。 通过利用胚胎细胞和发育中生物体中果蝇遗传学的力量，我们正在继续鉴定在这些不同基因上协调停滞 Pol II 的建立和释放的蛋白质，并确定利用这种新形式的基因的定义特征的基因调控。