Investigating Transcriptional Responses to the Environment

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，但我们最近发现，在大量基因中，停滞的Pol II从启动子-近端区域的释放是限速的。我们的工作采用了全基因组定位分析(使用一种称为芯片的技术)以及体内足迹分析相结合的方法来探索停滞的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，这些结果表明，在许多对环境或发育刺激做出反应的基因中，POL II处于未诱导状态，这表明POL II的存在，准备逃逸到基因中，促进了对不断变化的环境的有效、综合的反应。 了解停滞的POL II的基本性质，以及维持和释放启动子-近端POL II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。 在探索控制POL II失速的分子机制时，负伸长因子或NELF复合体是实验室特别感兴趣的。到目前为止，NELF已经被证明在几个基因上建立了停滞的POL II，包括JunB和HIV启动子，以及几个已知含有停滞的POL II的果蝇启动子。为了全面了解NELF在体内的靶标，我们利用RNA干扰对已经耗尽NELF的果蝇细胞进行了微阵列分析。我们发现许多NELF靶基因都参与了刺激反应途径，尤其是在先天免疫反应中。令人惊讶的是，我们发现NELF不仅作为负延长因子发挥作用，而且还刺激许多基因的表达，包括那些由免疫挑战激活的基因。进一步的分析表明，NELF介导的POLII在这些基因的启动子-近端区域的停滞通过阻止核小体在启动子区域的组装来增强基因的表达。因此，通过发挥核小体排斥因子的作用，停滞的POL II标记了这些基因的进一步或未来的激活。我们在接下来的一年的目标是探索POL II和核小体之间的相互作用的动力学，以便更好地了解POL II如何决定核小体的定位，并防止在这些免疫反应基因上形成抑制性染色质结构。 除了研究Pol II停滞对免疫基因基础基因表达水平和染色质结构的影响外，我们目前正在使用基于细胞和整体的方法来研究Pol II停滞在诱导果蝇在败血症攻击时的先天免疫反应中的作用。我们已经开发出在果蝇免疫反应器官脂肪体中实现NELF蛋白组织特异性耗竭的方法，并正在积极研究这种耗尽对抗菌肽和其他天然免疫基因产生的影响。我们希望确定POL II停滞在免疫反应中的功能和生理作用，并评估其在使有机体从败血症损伤中恢复的重要性。

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 stalled Pol II from the promoter-proximal region is rate-limiting at a large number of genes. Our work 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 that Pol II is pre-loaded in the uninduced state at many genes that respond to environmental or developmental stimuli, suggesting that the presence of Pol II, poised for escape into the gene, facilitates efficient, integrated responses to a changing environment. Understanding the fundamental properties of stalled Pol II, and the mechanisms for 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 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. 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 stalled Pol II at several genes to date, including the junB and HIV promoters, as well as at several Drosophila promoters know to harbor stalled Pol II. To get a comprehensive picture of NELF targets in vivo, we performed a microarray analysis on Drosophila cells that had been 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. Surprisingly, we found that NELF does not only function as a negative elongation factor, but it also stimulates expression of a number of genes, including those activated by immune challenge. Further analysis has revealed that NELF-mediated stalling of Pol II in the promoter-proximal region of these genes enhances gene expression by blocking the assembly of nucleosomes in the promoter region. Thus, by functioning as a nucleosome exclusion factor, stalled Pol II marks these genes for further or future activation. Our goal in the upcoming year is to probe the dynamics of the interactions between Pol II and nucleosomes, in order to better understand how Pol II dictates nucleosome positioning and prevents the formation of repressive chromatin structure at these immune-responsive genes. In addition to investigating the effects of Pol II stalling on basal levels of gene expression and chromatin architecture at immunity genes, we are currently using both cell-based and whole organism approaches to investigate the role of Pol II stalling during induction of the Drosophila innate immune response upon septic challenge. We have developed ways in which to achieve tissue-specific depletion of the NELF protein in the Drosophila immune-responsive organ, the fat body, and are actively pursuing the effects of this depletion on the production of anti-microbial peptides and other innate immunity genes. We hope to determine the functional, physiological role of Pol II stalling during the immune response, and to asses its importance in allowing the organism to recover from septic injury.