Investigating Transcriptional Responses to the Environment

我们的实验室已经发现，从启动子近端区域释放暂停的Pol II是大多数后生动物基因表达中必不可少的调节步骤。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。这些数据表明，Pol II暂停比以前认识到的更广泛。我们和其他人已经将这些发现扩展到哺乳动物系统（小鼠和人类），证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。 在探索控制Pol II暂停的分子机制时，负延伸因子或NELF复合物对实验室特别感兴趣。从机制上讲，我们已经表明，NELF是广泛的稳定暂停的Pol II的重要。为了确定其生理作用，我们开发了条件性基因敲除小鼠。在细胞和体内的研究表明，NELF介导的Pol II暂停是必不可少的最佳免疫应答细菌的挑战和早期胚胎发育。值得注意的是，在这两个系统中，我们发现中断暂停显着影响细胞对环境或外在线索的反应。对这种效应的剖析表明，停顿在决定信号转导机制的关键枢纽的表达水平方面起着关键作用。通过这种方式，暂停调节信号反应，以及信号对基因表达的影响。总之，数据表明，Pol II的暂停允许基础基因表达和激活的协调调节，以实现对环境或发育线索的精确、平衡的响应。

Our laboratory has found that release of paused Pol II from the promoter-proximal region is essential, regulated step in the expression of most metazoan 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. These data showed that Pol II pausing is much more widespread than previously appreciated. We and others have 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. In probing the molecular mechanisms governing Pol II pausing, the Negative ELongation Factor, or NELF complex, is of particular interest to the laboratory. Mechanistically, we have shown that NELF is broadly important for the stable pausing of Pol II. To define its physiological role, we have developed conditional knockout mice. Studies in both cells and in vivo revealed that NELF-mediated Pol II pausing is essential for an optimal immune response to bacterial challenge and and for early embryonic development. Notably, in both systems we find that disruption of pausing dramatically impacts cellular responsiveness to environmental or extrinsic cues. Dissection of this effect has revealed that pausing plays a key role in determining the expression level of critical hubs of signal transduction machineries. In this way, pausing tunes signaling responses, and the consequences of signaling on gene expression. Taken together, the data suggest that pausing of Pol II allows for coordinated tuning of both basal gene expression and activation, to enable precise, balanced responses to environmental or developmental cues.