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A1205 - Mechanisms of Airway Epithelial Cell Reprogramming in Asthma
Author Block: L. R. Bonser1, W. Eckalbar1, X. Zeng1, S. Siddiqui2, J. Pollack1, K. Koh1, L. Zlock3, W. E. Finkbeiner3, N. R. Bhakta2, P. Woodruff2, N. Ahituv1, D. J. Erle4; 1Lung Biology Center, University of California San Francisco, San Francisco, CA, United States, 2Division of Pulmonary and Critical Care Medicine, University of California San Francisco, San Francisco, CA, United States, 3Department of Pathology, University of California San Francisco, San Francisco, CA, United States, 4University of California San Francisco, San Francisco, CA, United States.
Rationale: The airway epithelium is essential in homeostasis; dysfunction contributes to chronic airway diseases including asthma. The transcriptional mechanisms which specify airway epithelial subpopulations (basal, ciliated, secretory) during differentiation and in disease, are incompletely understood. We sought to discover how the airway epithelium is reprogrammed in asthma, initially investigating the effect of IL-13 on airway epithelial cell subpopulation transcription and enhancer activity.
Methods: We used single cell RNA-seq (scRNA-seq), chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), and the assay for transposase accessible chromatin using sequencing (ATAC-seq) to examine changes in transcription and enhancer activity of primary human bronchial epithelial (HBE) cells cultured in the absence or presence of IL-13. We sorted goblet (MUC5AC+) cells by flow cytometry and performed ChIP-seq to identify goblet cell specific enhancers. We also applied scRNA-seq, ChIP-seq and ATAC-seq to freshly isolated bronchial epithelial cells from bronchoscopic brushings of healthy controls and subjects with asthma. We tested candidate enhancers using an enhancer reporter assay.
Results: We identified clusters representing ciliated, basal, and secretory cells in our scRNA-seq dataset; a fourth cluster representing an intermediate cell type was also identified. Cytokine stimulation affected the transcriptional programs of each cell types differently. H3K27ac ChIP-seq revealed ~105 DNA regions (peaks) representing candidate active enhancers; IL-13 enriched 332 peaks and depleted 67 peaks. We found strong enrichment for IL-13-enriched H3K27ac peaks in a region extending ~200kb upstream and downstream of the transcription start site (TSS) of IL-13-induced genes, including a region upstream of the MUC5AC TSS. Analysis of sorted IL-13-stimulated cells showed that this peak was enriched in goblet cells and in brushings from individuals with asthma. We also identified a candidate enhancer for SPDEF, a transcription factor involved in mucous metaplasia. In reporter assays, this region drove expression in an IL-13-dependent and goblet cell-selective fashion. H3K27ac ChIP-seq suggested that the overall pattern of enhancer activity in HBE cell cultures was quite similar to that seen in freshly harvested bronchial epithelial cells from bronchoscopic brushings.
Conclusions: scRNA-seq identified cell type-specific transcripts and cell type-specific responses to IL-13. H3K27ac ChIP-seq identified many candidate IL-13-regulated enhancers, including candidate MUC5AC and SPDEF enhancers. IL-13 dramatically affects airway epithelial transcription programs contributing to goblet cell production and airway obstruction in asthma. Ongoing research using the approaches developed here will provide new insights into tightly coupled transcriptional and epigenetic mechanisms underlying airway epithelial cell differentiation and cytokine responses, and airway epithelial reprogramming in asthma.