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.
The studies proposed in this application will contribute to an understanding of the mechanisms by which immune and inflammatory responses are orchestrated in cells of the innate immune system. The knowledge gained from these studies is likely to suggest strategies for the selective manipulation of immune and inflammatory responses, towards the goals of enhancing anti-microbial immunity and suppressing inflammatory autoimmune disorders.
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