Wednesday, May 1, 2024
5/1/2024
PROJECT SUMMARY: Interleukin-13 (IL-13) is a key effector cytokine in causing allergic inflammation and in immunity against certain parasitic infections. The Il13 gene, like many cytokine and chemokine genes, is transcribed at low levels in resting effector cells and is dramatically upregulated in response to external stimulations. However, mechanisms by which innate effector cells rapidly and robustly transcribe the Il13 gene are still poorly understood. Our long-term goal is to understand transcriptional mechanisms that regulate cytokine and chemokine gene transcription in health and diseases. Our immediate goal is to investigate how Il13 enhancers can rapidly and robustly assemble transcriptional complexes to promote Il13 gene transcription in mast cells in response to antigenic stimulation. In our published and preliminary studies, we have identified enhancer candidates using a genomic approach. Enhancers can contribute to gene transcription additively or cooperatively. Additive enhancers contribute to less than 50% of gene transcription, while cooperative enhancers contribute to more than 50% of gene transcription. Using our improved CRISPR method, we identify several Il13 cooperative enhancers that contribute to more than 50% of Il13 gene transcription in response to antigenic stimulation. In addition, we found that signal-dependent transcription factors (SDTFs) bound to two of the Il13 cooperative enhancers. Deleting SDTFs EGR1 or EGR2 resulted in a profound reduction in Il13 gene transcription in response to IgECL. Based on these data, we hypothesize that the Il13 cooperative enhancers detect signals triggered by antigenic stimulation and cooperate to form an enhancer hub, looping in the Il13 promoter to transcribe the Il13 gene in a SWITCH-LIKE fashion robustly. Aim 1 will decipher the transcriptional codes in the Il13 cooperative enhancers in MCs. We will (1) identify Il13 cooperative enhancers unbiasedly and in a locus-wide manner using our improved CRISPR deletion method; (2) validate the function of the Il13 cooperative enhancers in MCs in mouse models of parasitic infections; and 3) decipher the transcriptional codes in the Il13 cooperative enhancers by experimentally determining the sum of the numbers of TF binding sites, types of TF binding sites and the proximity of TF binding sites. Aim 2 will investigate mechanisms by which TFs read the transcriptional codes in the Il13 cooperative enhancers to rapidly assemble functional Il13 transcriptional complex. We will use a novel proximity biotin-labeling method that can detect Il13 cooperative enhancer-specific DNA/TFs interactions to elucidate how SDTFs EGR1 and EGR2 assemble a functional Il13 transcriptional complex to drive rapid and robust Il13 gene transcription in response to antigenic stimulation, revealing a novel mechanistic insight into how enhancers cooperate to transcribe the Il13 gene rapidly and robustly. Our proposed work should also help us understand how other cytokine genes can be rapidly switched on in response to external stimulations and identify targets for CRISPR-based therapies to disrupt cytokine and chemokine-mediated inflammatory pathways.
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