Selective Regulation of Pro-Inflammatory Genes in Macrophages

描述（由申请人提供）：对微生物病原体的有效免疫应答需要先天性和适应性免疫系统细胞之间的广泛沟通。先天免疫系统的巨噬细胞和其他细胞在受到微生物产物或细胞因子刺激后，通过以精心编排的转录级联表达数百个基因，在调节免疫应答中发挥重要作用。转录级联的一个重要特征是它通常是针对刺激和特定的生理环境而定制的。这一特征在正常免疫应答期间通常是有益的，但是延长的巨噬细胞活化或异常活化可以在感染期间促进组织损伤，并且与许多疾病密切相关，包括癌症、动脉粥样硬化和几种炎性自身免疫性疾病。虽然抗炎药物是可用的，但用于选择性调节单个基因或选择基因子集的额外策略是非常需要的。为了实现开发用于选择性调节免疫和炎症反应的方法的长期目标，当前的主要目标是揭示分子机制和整体逻辑，通过该分子机制和整体逻辑编排了稳健的转录反应。我们已经获得了相当深入的了解，这种监管逻辑的基础上，他们的启动子特性和新的蛋白质合成的要求，SWI/SNF核小体重塑复合物，和转录因子IRF 3的诱导基因进行分类。我们的研究结果导致了一个模型，其中一些基因含有染色质和启动子功能，使它们能够被广泛的刺激混杂激活，而其他基因含有染色质和启动子功能，促进高度选择性激活有限数量的刺激。最近，我们利用最先进的RNAseq方法在全基因组范围内检查转录级联。我们还开发了一种新方法，其中通过RNAseq分别分析新生的染色质相关转录物、核质转录物和细胞质转录物。这种新的方法，Nascent-Seq，使我们能够获得迄今为止对炎症刺激的转录反应的最高分辨率视图，并使我们能够根据其新生转录动力学对诱导型基因进行分类。在目标1中， 我们将进行大量的附加的Nascent-Seq实验，以合并和扩展我们的两个不同的用于分类LPS诱导的基因的方案。在目标2中，我们将使用细菌人工染色体来确定CpG岛启动子如何获得独特的特性，使它们能够以与低CpG启动子根本不同的方式进行调控。最后，在目标3中，我们将使用从我们的基础，基因组规模的巨噬细胞反应的研究中获得的知识，以检查通过抗炎细胞因子IL-10和LPS耐受诱导选择性抑制反应的分子机制。

DESCRIPTION (provided by applicant): An effective immune response to a microbial pathogen requires extensive communication between cells of the innate and adaptive immune systems. Macrophages and other cells of the innate immune system play a major role in regulating an immune response by expressing hundreds of genes in a well-orchestrated transcriptional cascade after they are stimulated by microbial products or cytokines. An important feature of the transcriptional cascade is that it generally is tailored to the stimulus ad the specific physiological setting. This feature is often beneficial during a normal immune response, but prolonged macrophage activation or aberrant activation can promote tissue damage during infection and has been closely linked to numerous diseases, including cancer, atherosclerosis, and several inflammatory autoimmune diseases. Although anti-inflammatory drugs are available, additional strategies for the selective modulation of individual genes or select subsets of genes are in great demand. Towards the long-term goal of developing methods for the selective modulation of immune and inflammatory responses, a major current objective is to uncover the molecular mechanisms and overall logic by which a robust transcriptional response is orchestrated. We have obtained considerable insight into this regulatory logic by classifying several dozen inducible genes on the basis of their promoter properties and their requirements for new protein synthesis, for the SWI/SNF nucleosome remodeling complex, and for the transcription factor IRF3. Our results led to a model by which some genes contain chromatin and promoter features that allow them to be promiscuously activated by a broad array of stimuli, whereas others contain chromatin and promoter features that facilitate highly selective activation by a limited number of stimuli. More recently, we have taken advantage of state-of-the-art RNAseq methodologies to examine transcriptional cascades at a genome-wide scale. We have also developed a new method in which nascent, chromatin- associated transcripts, nucleoplasmic transcripts, and cytoplasmic transcripts are analyzed separately by RNAseq. This novel method, Nascent-Seq, has allowed us to obtain the highest-resolution view to date of the transcriptional response to an inflammatory stimulus and has allowed us to classify inducible genes on the basis of their nascent transcript kinetics. In Aim 1, we will perform a large series of additional Nascent-Seq experiments to merge and extend our two distinct schemes for classifying LPS-induced genes. In Aim 2, we will use bacterial artificial chromosomes to determine how CpG-island promoters acquire unique properties that allow them to be regulated in a fundamentally different manner than low CpG promoters. Finally, in Aim 3, we will use the knowledge acquired from our fundamental, genome-scale studies of the macrophage response to examine the molecular mechanisms by which the response is selectively suppressed by the anti-inflammatory cytokine IL-10 and by LPS tolerance induction.