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Principal Investigator: HUA HUANG
Organization: NATIONAL JEWISH HEALTH
Fiscal Year: 2024
Award: $251,972
Funding agency: National Institute of Allergy and Infectious Diseases
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.
Terms: <Affinity><Allergic inflammation><Antigens><Assay><Basal Transcription Factor><Basal transcription factor genes><Binding><Binding Sites><Bioassay><Biological Assay><Biotin><CRISPR><CRISPR approach><CRISPR based approach><CRISPR based therapeutics><CRISPR based treatment><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR therapeutics><CRISPR tools><CRISPR treatment><CRISPR-CAS-9><CRISPR-Cas based therapeutics><CRISPR-based disease therapeutics><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based therapy><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas system><CRISPR/Cas technology><CRISPR/Cas therapeutics><CRISPR/Cas9><CRISPR/Cas9 technology><CRISPR/Cas9 therapeutics><CRISPR/Cas9 therapy><CRISPR/Cas9 treatment><CRISPR/Cas9-based therapy><Cas nuclease technology><Cas9 based therapeutics><Cell Communication and Signaling><Cell Signaling><Chemotactic Cytokines><Chromatin><Clustered Regularly Interspaced Short Palindromic Repeats><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats based therapeutics><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Clustered Regularly Interspaced Short Palindromic Repeats therapeutics><Code><Coding System><Combining Site><Complex><Cytokine Gene><DNA><DNA-Protein Interaction><Data><Deoxyribonucleic Acid><Disease><Disorder><EGR2><EGR2 gene><Effector Cell><Enhancers><Family><Gene Transcription><General Transcription Factor Gene><General Transcription Factors><Genes><Genetic Transcription><Genomic approach><Goals><Health><Homeo Boxes><Homeobox><Homologous Chemotactic Cytokines><IL-13><IL-13 Gene><IL13><IL13 gene><IgE><Immunity><Immunoglobulin E><Inflammatory><Intercrines><Interleukin 13 Gene><Interleukin 13 Precursor Gene><Interleukin-13><Intracellular Communication and Signaling><KROX20><Label><Marrow Mast Cell><Mediating><Methods><Molecular Interaction><NC30 Gene><Output><P600 Gene><Parasitic infection><Pathway interactions><Proteins><Publishing><RNA Expression><Reactive Site><Receptor Protein><Reporter Genes><Rest><Role><SIS cytokines><Signal Transduction><Signal Transduction Systems><Signaling><Sum><Testing><Tissue Basophils><Transcription><Transcription Factor Proto-Oncogene><Transcription Regulation><Transcription factor genes><Transcriptional Control><Transcriptional Regulation><Vitamin H><Work><Zinc Finger Domain><Zinc Finger Motifs><Zinc Fingers><biological signal transduction><chemoattractant cytokine><chemokine><coenzyme R><crosslink><cytokine><detection method><detection procedure><detection technique><genomic effort><genomic strategy><immunogen><improved><insight><mast cell><mastocyte><mouse model><murine model><novel><parasite infection><pathway><promoter><promotor><receptor><response><social role><transcription factor>