Pro-inflammatory gene regulation in a native chromatin environment

描述（由申请人提供）：本申请的主要目标是阐明巨噬细胞中促炎基因的调节机制，长期目标是揭示正常和病变状态下调节免疫反应和炎症的策略。一个同样重要的目标是利用促炎基因作为模型来阐明哺乳动物RNA聚合酶II在急性刺激下基因激活的基本机制。为了实现这些目标，我们将使用编码异二聚体细胞因子IL-12的p40亚基的Il12b基因作为模型。II12b是许多促炎基因的代表，但它在连接先天和适应性免疫系统中起着异常重要的作用，是针对肿瘤和感染因子的免疫反应的关键调节因子。在过去的二十年中，主要通过对转染启动子报告质粒的研究，对诱导基因的调控有了相当深入的了解。然而，对内源性染色质环境中的基因调控知之甚少。染色质免疫沉淀测定（ChIP）已经使检测内源性事件成为可能，但补充这种描述性技术的功能策略受到限制。我们假设，通过将一系列突变直接引入内源基因座，可以获得对内源基因调控机制的重要新见解。为了实现我们的目标，我们将在内源性Il12b位点的启动子和增强子元件中引入突变。除了破坏已知的控制元件外，我们还将破坏在进化过程中高度保守的DNA元件和区域，但在转染试验中没有发挥重要作用。这些后一种突变将检验高度保守序列在内源性环境中通常对转录至关重要的假设。ChIP和限制性内切酶可及性将用于监测每个突变对导致Il12b转录的级联事件的影响。在最后的目标中，我们将通过探索在胚胎干细胞（ES）中已经标记了诱导增强子的有趣观察，来研究Il12b位点如何组装成准备激活的染色质状态。我们将在胚胎干细胞中鉴定与Il12b增强子和其他模型增强子相关的蛋白，并询问这些相互作用是否对分化细胞的转录至关重要。公共卫生相关声明：促炎基因的异常表达在许多常见疾病中起着重要作用，包括癌症、动脉粥样硬化和一些炎症性自身免疫性疾病。本研究的目的是增加我们对促炎基因表达调控的分子机制的理解。我们目前知识的一个主要缺陷是，大多数促炎基因表达的研究都依赖于人工实验方法，这只能导致对关键调控机制的部分看法。利用最近的技术进步和从比较基因组分析中获得的知识，我们建议研究促炎基因在其原生基因组环境中的调节机制。这项研究的长期目标是为人类疾病背景下的促炎基因的选择性调节制定策略。

DESCRIPTION (provided by applicant): A primary goal of this application is to elucidate the mechanisms by which pro-inflammatory genes are regulated in macrophages, with a long-term objective of uncovering strategies for modulating immune responses and inflammation in normal and diseased states. An equally important goal is to use a pro- inflammatory gene as a model for elucidating fundamental mechanisms of gene activation by mammalian RNA polymerase II in response to an acute stimulus. To achieve these goals, we will use as a model gene Il12b, which encodes the p40 subunit of the heterodimeric cytokine IL-12. II12b is representative of many pro- inflammatory genes, but it plays an unusually important role in bridging the innate and adaptive immune systems and is a key regulator of immune responses against tumors and infectious agents. Considerable insight into the regulation of inducible genes has been obtained over the past two decades, primarily through studies of transfected promoter-reporter plasmids. However, much less is known about gene regulation in an endogenous chromatin environment. The chromatin immunoprecipitation assay (ChIP) has made it possible to examine endogenous events, but functional strategies to compliment this descriptive technique have been limited. We hypothesize that important new insight into endogenous gene regulation mechanisms can be obtained by introducing a series of mutations directly into an endogenous locus. To accomplish our objectives, we will introduce mutations into promoter and enhancer elements at the endogenous Il12b locus. In addition to disrupting known control elements, we will disrupt DNA elements and regions that have been highly conserved through evolution, but did not contribute important functions in transfection assays. These latter mutations will test the hypothesis that highly conserved sequences are generally critical for transcription in an endogenous setting. ChIP and restriction enzyme accessibility will be used to monitor the effect of each mutation on the cascade of events leading to Il12b transcription. In the final aim, we will examine how the Il12b locus becomes assembled into a chromatin state poised for activation by exploring the intriguing observation that an inducible enhancer is already marked in embryonic stem (ES) cells. We will identify proteins that associate with the Il12b enhancer and other model enhancers in ES cells and ask whether these interactions are essential for transcription in differentiated cells. Public Health Relevance Statement: The aberrant expression of pro-inflammatory genes plays a major role in a number of common diseases, including cancer, atherosclerosis, and a number of inflammatory autoimmune disorders. The objective of the research proposed in this application is to increase our understanding of the molecular mechanisms regulating pro-inflammatory gene expression. A major deficiency in our current knowledge is that most studies of pro- inflammatory gene expression have relied, by necessity, on artificial experimental approaches that only lead to a partial view of key regulatory mechanisms. Using recent technological advances and knowledge gained from comparative genome analyses, we propose to study mechanisms regulating pro-inflammatory genes in their native genomic environment. The long-term goal of this research is to develop strategies for the selective modulation of pro-inflammatory genes in the context of human disease.