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，我们已经发现，暂停Pol II从启动子近端区域的释放是限速的表达大量的基因。我们最初的工作调查了暂停Pol II在果蝇中的流行情况，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。令人惊讶的是，这些数据表明，Pol II暂停比以前认识到的更广泛，发生在数千个启动子基因组范围内。我们和其他人最近将这些发现扩展到哺乳动物系统（小鼠和人类），表明暂停高等生物中普遍存在的基因调控策略。此外，我们的研究结果表明，Pol II是组成性存在于许多基因在环境或发育反应的基因网络，这表明Pol II的存在有利于有效的，集成的响应动态变化的环境。 了解暂停的Pol II的基本特性，以及控制启动子近端Pol II维持与释放到生产性伸长的因素是Adelman实验室研究的具体目标。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，因为在哺乳动物原癌基因（如c-myc，c-fos和junB）的启动子以及HIV启动子处观察到类似的暂停Pol II。作为我们努力更好地定义暂停的机制的一部分，我们最近开发了一种新的技术，用于分离由暂停的Pol II产生的短RNA转录本，并通过单个RNA分子的并行测序对其进行分析。这种策略使我们能够以单核苷酸分辨率精确定位转录起始和暂停的位置。值得注意的是，这项令人兴奋的新技术揭示了最初转录区域内的DNA序列在指定早期延伸效率方面的作用，为为什么聚合酶暂停在某些基因上比在其他基因上更突出提供了见解。 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暂停的Pol II，包括junB和HIV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关键作用。在细胞和果蝇中的后续研究表明，NELF介导的Pol II暂停对于对细菌挑战的最佳免疫应答是必不可少的，并且表明聚合酶暂停调节关键免疫调节因子如NF-κ B转录因子Relish（Rel）的基础表达水平。 除了我们在果蝇中的工作，我们正在研究聚合酶暂停在哺乳动物炎症反应中的作用，使用来自小鼠的原代巨噬细胞。这些研究表明，在对细菌感染的反应中起关键作用的几个基因，如TNF-α和junB，具有暂停的Pol II，在静息的未诱导细胞中的启动子区域等待。然而，许多其他细菌诱导的基因并不具有暂停的Pol II，并且主要由Pol II在基因活化期间的募集来调节。有趣的是，我们发现，暂停Pol II的存在和免疫反应的动力学之间的关系，在暂停基因表现出更精细的调谐突发的转录活性比通过Pol II的招聘，这往往表现出更持久的反应调节基因。这表明基因调控策略（即转录周期中限制基因表达速率的步骤）与基因表达的动力学或幅度之间可能存在根本联系。通过这种方式，暂停可能允许对基础基因表达和激活进行微调，以实现对环境或发育线索的精确，平衡的反应。

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 found that release of paused Pol II from the promoter-proximal region is rate-limiting for expression of a large number of genes. Our initial work investigated the prevalence of paused Pol II in Drosophila, employing a combination of global location analysis (using techniques called ChIP-chip and ChIP-seq) as well as in vivo footprinting assays. Surprisingly, these data showed that Pol II pausing is much more widespread than previously appreciated, occurring at thousands of promoters genome-wide. We and others have recently extended these findings to mammalian systems (mouse and human), demonstrating that pausing a prevalent gene regulatory strategy in higher organisms. Moreover, our results reveal that Pol II is constitutively present at many genes in environmentally- or developmentally-responsive gene networks, suggesting that the presence of Pol II facilitates efficient, integrated responses to a dynamically changing environment. Understanding the fundamental properties of paused Pol II, and the factors that govern 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 stress-responses, this work is anticipated to elucidate gene expression during the development of cancer and AIDS, since similarly paused Pol II are observed at the mammalian promoters of proto-oncogenes like c-myc, c-fos and junB, as well as at the HIV promoter. As part of our efforts to better define the mechanisms underlying pausing, we have recently developed a novel technique for isolating the short RNA transcripts generated by paused Pol II, and analyzed them through massively-parallel sequencing of individual RNA molecules. This strategy allowed us to pinpoint both the locations of transcription initiation and pausing, at single-nucleotide resolution. Notably, this exciting new technique revealed a role for the DNA sequence within the initially transcribed region in specifying the efficiency of early elongation, providing insights into why polymerase pausing is more prominent at some genes than at others. 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 paused Pol II at several genes to date, including the junB and HIV promoters. 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 vivo in regulating expression of components of the innate immune system. Follow-up studies in both cells and flies revealed that NELF-mediated Pol II pausing is essential for an optimal immune response to bacterial challenge and indicated that polymerase pausing tunes the basal expression level of critical immune regulators such as the NF-kB transcription factor Relish (Rel). In addition to our work in Drosophila, we are studying the role of polymerase pausing in the mammalian inflammatory response, using primary macrophages derived from mouse. These investigations reveal that several genes that play critical roles in the response to bacterial infection, like TNF-alpha and junB, possess paused Pol II, waiting in their promoter regions in resting, uninduced cells. However, many other bacterially-induced genes do not harbor paused Pol II, and are regulated primarily by Pol II recruitment during gene activation. Interestingly, we find that there is a relationship between the presence of paused Pol II and the kinetics of the immune response, in that paused genes exhibit more finely-tuned bursts of transcription activity than do genes regulated through Pol II recruitment, which often exhibit a more sustained response. This suggests that there could be a fundamental link between gene regulatory strategy (i.e. the step in the transcription cycle that is rate-limiting for gene expression) and the kinetics or magnitude of gene expression. In this way, pausing might allow for fine tuning of both basal gene expression and activation, to enable precise, balanced responses to environmental or developmental cues.