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反应需要先天免疫系统和获得性免疫系统的细胞之间广泛的沟通。巨噬细胞和先天免疫系统的其他细胞在调节免疫反应方面发挥着重要作用，它们在微生物产品或细胞因子的刺激下，以精心安排的转录级联方式表达数百个基因。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。这一特征在正常的免疫反应中通常是有益的，但巨噬细胞的长期激活或异常激活会在感染期间促进组织损伤，并与许多疾病密切相关，包括癌症、动脉粥样硬化和几种炎症性自身免疫性疾病。尽管有抗炎药物可用，但对个别基因或特定基因子集的选择性调节的额外策略的需求很大。为了开发选择性调节免疫和炎症反应的方法这一长期目标，当前的一个主要目标是揭示协调强大转录反应的分子机制和总体逻辑。我们根据启动子特性和对新蛋白质合成、SWI/SNF核小体重塑复合体和转录因子IRF3的要求，对几十个诱导基因进行了分类，从而对这一调控逻辑有了相当大的了解。我们的结果导致了一个模型，在这个模型中，一些基因包含染色质和启动子功能，允许它们被广泛的一系列刺激混杂激活，而其他基因包含染色质和启动子功能，促进通过有限数量的刺激进行高度选择性的激活。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的方法，用RNAseq分别分析新生、染色质相关转录本、核质转录本和细胞质转录本。这种新的方法，新生序列，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的视图，并使我们能够根据其新生转录动力学对诱导基因进行分类。在目标1中， 我们将进行一系列额外的初生序列实验，以合并和扩展我们对内毒素诱导基因进行分类的两个不同方案。在目标2中，我们将使用细菌人工染色体来确定CpG-Island启动子如何获得独特的特性，使它们能够以与低CpG启动子完全不同的方式进行调控。最后，在目标3中，我们将使用从基础的、基因组规模的巨噬细胞反应研究中获得的知识来检查抗炎细胞因子IL-10和内毒素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.