Investigating Transcriptional Responses to the Environment

阿德尔曼实验室最近的工作结合了全基因组定位分析(使用一种称为芯片的技术)以及体内足迹分析来探索停滞的POLII在果蝇中的流行情况。令人惊讶的是，这些数据显示，Pol II拖延的现象比之前认识到的要普遍得多，在近20%的推动者中发生。此外，这些结果揭示了POLII在许多对环境或发育刺激做出反应的基因上处于停滞状态，这表明稳定的POLII的存在有助于对不断变化的环境做出有效的、综合的反应。虽然传统的基因调控模型认为Pol II在启动子上的募集是基因表达的必要条件和充分条件，但我们令人兴奋的发现表明，在大量基因中，Pol II从启动子近端区域的释放是限速的。描述这项工作的手稿目前正在《自然遗传学》杂志上出版。 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阿德尔曼实验室研究的具体目标。除了提供对应激反应的重要洞察外，这项工作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在哺乳动物的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，大多数依赖NELF的基因在其启动子附近具有参与的、停滞的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在哺乳动物细胞中JunB原癌基因的转录中发挥关键作用，这些数据特别有趣。 通过在胚胎来源的细胞和发育中的生物体中利用果蝇遗传学的力量，我们正在继续识别和表征在这些不同的基因上协调停滞的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。

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.