Investigating Transcriptional Responses to the Environment

传统的基因调控模型认为Pol II在启动子上的募集既是基因表达的必要条件也是充分条件，而我们最近发现，暂停的Pol II从启动子-近端区域的释放对于大量基因的表达是限速的。我们最初的工作采用了全球定位分析(使用称为芯片和芯片序列的技术)和体内足迹分析相结合的方法，调查了暂停的POLII在果蝇中的流行情况。令人惊讶的是，这些数据显示Pol II停顿比之前认识到的要广泛得多，发生在全基因组的数千个启动子中。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)，表明暂停在高等生物体中流行的基因调控策略。此外，我们的结果显示，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，促进了对动态变化的环境的高效、综合的反应。 了解暂停的POL II的基本性质，以及控制启动子-近端POL II的维持和释放进入生产性延伸的因素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，这项工作还有望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和JunB等原癌基因的哺乳动物启动子以及HIV启动子中也观察到了类似的停滞的POL II。作为更好地确定暂停的机制的努力的一部分，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，并通过对单个RNA分子的大规模平行测序来分析它们。这一策略使我们能够在单核苷酸分辨率下准确地定位转录启动和暂停的位置。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序列在指定早期伸长效率方面的作用，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暂停的POL II，包括JunB和HIV启动子。为了在全球范围内识别NELF的靶点，我们使用RNA干扰对NELF缺失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。为了评估这一发现的生理学相关性，我们最近在果蝇脂肪体(主要的免疫反应组织)中进行了NELF耗尽，随后对RNA水平进行了微阵列分析，以确定NELF的靶基因。这项工作证实了NELF在体内调节先天性免疫系统各组成部分的表达方面发挥着关键作用。在细胞和果蝇中的后续研究表明，NELF介导的Pol II停滞对于细菌攻击的最佳免疫反应是必不可少的，并表明聚合酶停滞对于核因子-kB转录因子美味(REL)的结合或活性是必要的。这一结果与我们早先的建议是一致的，即POL II暂停有助于建立围绕基因启动子的核小体剥夺的、可访问的染色质结构，促进未来的激活。 除了我们在果蝇上的工作外，我们正在使用来自小鼠的原代巨噬细胞来研究聚合酶暂停在哺乳动物炎症反应中的作用。这些研究表明，许多即时早期反应基因，如肿瘤坏死因子-α和JunB，在静止的、未诱导的细胞中，在它们的启动子区域具有暂停的POLII等待。相反，晚期初级和次级反应基因通常在诱导前缺乏停滞的POLII和NELF。总体而言，我们发现在停滞的POLII的存在和免疫反应的动力学之间存在着有趣的关系，因为停滞的基因表现出比通过POLII招募调节的基因更多的瞬时转录活性爆发。这表明，基因调控策略(即转录周期中对基因表达起限速作用的步骤)与基因表达的动力学或大小之间可能存在根本联系。

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 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 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 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 stalling is essential for an optimal immune response to bacterial challenge 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 pausing helps establish a nucleosome-deprived, accessible chromatin architecture around gene promoters, facilitating future activation. 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 many immediate early response genes, like TNF-alpha and junB, possess paused Pol II waiting 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. Overall, we find that there is an interesting relationship between the presence of stalled Pol II and the kinetics of the immune response, in that paused genes exhibit much more transient bursts of transcription activity than do genes regulated through Pol II recruitment. 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.