Investigating Transcriptional Responses to the Environment

而传统的基因调控模型认为，招聘的启动子的Pol II是必要的和足够的基因表达，我们最近发现，从启动子近端区域的停滞Pol II的释放是限速在大量的基因。我们的工作采用了全球定位分析（使用称为ChIP-chip和ChIP-seq的技术）以及体内足迹分析的组合来探测果蝇中停滞的Pol II的流行。令人惊讶的是，这些数据表明，Pol II失速比以前认识到的更普遍，发生在数千个启动子基因组范围内。此外，这些结果表明，Pol II在许多对环境或发育刺激作出反应的基因中以未诱导状态预加载，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的综合反应。 了解停滞的Pol II的基本特性，以及启动子近端Pol II维持与释放到生产性伸长的机制是Adelman实验室研究的具体目标。除了对应激反应提供重要的见解外，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，因为在哺乳动物c-myc、c-fos、junB和HIV启动子的启动子处观察到类似的停滞Pol II。 在探索控制Pol II失速的分子机制时，负延伸因子或NELF复合物对实验室特别感兴趣。迄今为止，NELF已被证明在几个基因上建立停滞的Pol II，包括junB和HIV启动子，以及已知含有停滞的Pol II的果蝇启动子。为了在全球范围内确定NELF的靶点，我们对使用RNA干扰去除NELF的果蝇细胞进行了微阵列分析。我们发现许多NELF靶基因参与刺激应答途径，特别是在先天免疫应答中富集。为了评估这一发现的生理相关性，我们最近在果蝇脂肪体（主要的免疫应答组织）中进行了NELF耗竭，随后进行了RNA水平的微阵列分析以鉴定NELF靶基因。这项工作证实了NELF在体内调节先天免疫系统组分的表达中起着关键作用。在细胞中的后续研究显示，NELF介导的Pol II停滞对于最佳免疫应答是必不可少的，并且表明聚合酶停滞对于NF-kB转录因子Relish（Rel）的结合或活性是必需的。这个结果与我们早期的建议一致，即Pol II停滞有助于在基因启动子周围建立核小体缺失的、可接近的染色质结构，促进未来的激活。在NELF耗尽的果蝇中正在进行进一步的实验，以评估NELF在允许生物体从脓毒性损伤中恢复的重要性。 除了我们在果蝇中的工作，我们已经开始研究聚合酶停滞在哺乳动物免疫反应中的作用，使用来自小鼠的原代巨噬细胞。这些正在进行的研究表明，许多立即早期反应基因，如TNF-α，TTP，junB和tnfaip 3，在静止的，未诱导的细胞中，在其启动子区具有停滞的Pol II和NELF。相比之下，晚期初级和次级应答基因在诱导之前通常缺乏停滞的Pol II和NELF。我们正在研究停滞的Pol II的存在与免疫反应动力学之间的关系，以及评估NELF在这一过程中的作用。

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 global location analysis (using technique called ChIP-chip and ChIP-seq) 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 thousands of promoters genome-wide. 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 Drosophila promoters know to harbor stalled Pol II. To globally identify targets of NELF, we have performed a microarray analysis on Drosophila cells that were 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. To evaluate the physiological relevance of this finding, we have recently performed NELF depletion in the Drosophila fat body (the main immune responsive tissue), followed by microarray analysis of RNA levels to identify NELF target genes. This work confirms that NELF plays a key role in regulating expression of components of the innate immune system in vivo. Follow-up studies in cells revealed that NELF-mediated Pol II stalling is essential for an optimal immune response and indicated that polymerase stalling is necessary for either the binding or activity of the NF-kB transcription factor Relish (Rel). this result is consistent with our earlier suggestion that Pol II stalling helps establish a nucleosome-deprived, accessible chromatin architecture around gene promoters, facilitating future activation. Further experiments are underway in NELF-depleted flies to assess the importance of NELF in allowing the organism to recover from septic injury. In addition to our work in Drosophila, we have begun to study the role of polymerase stalling in the mammalian immune response, using primary macrophages derived from mouse. These ongoing investigations reveal that many immediate early response genes, like TNF-alpha, TTP, junB and tnfaip3, possess stalled Pol II and NELF in their promoter regions in resting, uninduced cells. In contrast, late primary and secondary response genes generally lack stalled Pol II and NELF prior to induction. We are pursuing the relationship between the presence of stalled Pol II and the kinetics of the immune response, as well as evaluating the role of NELF in this process.