Activator protein transcription factors coordinate human IL-33 expression from non-canonical promoters in chronic airway disease

Interleukin-33 (IL-33) is a cytokine central to type-2 immune pathology in chronic airway disease. This cytokine is abundantly expressed in the respiratory epithelium and increased in disease, but how this is regulated is undefined. Here we show IL-33 is expressed from multiple promoters in human chronic obstructive pulmonary disease (COPD) and non-canonical promoters mediate production of alternatively spliced isoforms. Transposase-accessible chromatin sequencing (ATAC-seq) performed on cell lines with distinct IL-33 expression levels demonstrated chromatin accessibility as a control point for IL-33 expression. We further discovered that phorbol esters activate non-canonical IL-33 promoters through protein kinase C in airway cells. Transcription factor (TF) binding arrays combined with RNA interference revealed Activator Protein transcription factors as the downstream effectors of non-canonical IL-33 promoter activation. In support of a role for Activator Protein-2 family members in COPD pathogenesis, we found both TFAP2A and TFAP2C are upregulated in disease, with phosphorylated TFAP2C localized to IL-33-positive airway basal cells in COPD lung tissue. This study defines how pioneer transcription factors associated with stemness can drive non-canonical IL-33 promoters and alternative splicing patterns in airway epithelial progenitors. Our work reveals a novel approach for targeting the IL-33 pathway to promote the development of new therapeutics for COPD. Overall design: To address whether altered chromatin accessibility may be responsible for these different expression patterns of IL33 transcripts, we performed transposase-accessible chromatin sequencing (ATAC-seq) on the HBE-1, Beas2B, and 16HBE14o cell lines to identify peaks within the gene in proximity to the promoters. We compared this to whole lung ATAC-seq data and RNA annotation and mapping of promoters (RAMPAGE) data available through ENCODE.