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. ? ? ?
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