Pro-inflammatory gene regulation in a native chromatin environment

天然染色质环境中的促炎基因调控

• 批准号: 8053398
• 负责人: Stephen T Smale
• 金额: $ 30.25万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8053398
• 关键词: Acute; Applications Grants; Atherosclerosis; Autoimmune Diseases; Automobile Driving; Binding Proteins; Biological Assay; CD8-Positive T-Lymphocytes; Cell Communication; Cell Nucleus; Cells; Chromatin; Conserved Sequence; CpG dinucleotide; DNA; DNA-Protein Interaction; Disease; Distant; Elements; Enhancers; Environment; Event; Evolution; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Genomics; Goals; Immune response; Immune system; Infectious Agent; Inflammation; Inflammatory; Interleukin-12; Knowledge; Lead; Link; Malignant Neoplasms; Modeling; Molecular; Monitor; Mus; Mutation; Natural Killer Cells; Plasmids; Play; Proteins; RNA Polymerase II; Regulation; Reporter; Research; Role; Series; Signal Pathway; Stimulus; Study models; T-Lymphocyte; Techniques; Testing; Tissues; Transcriptional Activation; Transfection; chromatin immunoprecipitation; comparative; cytokine; embryonic stem cell; extracellular; gain of function; homologous recombination; human disease; insight; interest; loss of function; macrophage; nuclease; promoter; public health relevance; research study; response; restriction enzyme; tumor

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.

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调节的机制，长期目标是揭示在正常和患病状态下调节免疫应答和炎症的策略。同样重要的目标是使用促炎基因作为模型，用于阐明哺乳动物RNA聚合酶II响应急性刺激而激活基因的基本机制。为了实现这些目标，我们将使用编码异源二聚体细胞因子IL-12的p40亚基的模型基因Il 12 b。II 12 b是许多促炎基因的代表，但它在连接先天性和适应性免疫系统方面发挥着异常重要的作用，是针对肿瘤和感染因子的免疫应答的关键调节因子。在过去的二十年里，主要通过对转染的启动子-报告质粒的研究，已经获得了对诱导基因调控的相当深入的了解。然而，对内源性染色质环境中的基因调控知之甚少。染色质免疫沉淀试验（ChIP）已经有可能检查内源性事件，但功能策略，以补充这一描述性技术已受到限制。我们假设，重要的内源性基因调控机制的新见解，可以通过引入一系列的突变直接进入一个内源性位点。为了实现我们的目标，我们将在内源性Il 12 b基因座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但在转染测定中没有贡献重要功能的DNA元件和区域。这些后者的突变将测试的假设，高度保守的序列通常是关键的转录在内源性设置。ChIP和限制性内切酶可及性将用于监测每种突变对导致Il 12 b转录的级联事件的影响。在最后的目标，我们将研究如何将Il 12 b基因座组装成染色质状态准备激活探索有趣的观察，诱导增强子已经标记在胚胎干细胞（ES）。我们将鉴定与ES细胞中的Il 12 b增强子和其他模型增强子相关的蛋白质，并询问这些相互作用是否对分化细胞中的转录至关重要。公共卫生相关性声明：促炎基因的异常表达在许多常见疾病中起主要作用，包括癌症、动脉粥样硬化和许多炎性自身免疫性疾病。本申请中提出的研究目的是增加我们对调节促炎基因表达的分子机制的理解。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。利用最新的技术进步和比较基因组分析获得的知识，我们建议研究促炎基因在其天然基因组环境中的调节机制。这项研究的长期目标是开发在人类疾病背景下选择性调节促炎基因的策略。