Kyung Duk Koh, Luke R Bonser, Walter L Eckalbar, Ofer Yizhar-Barnea, Jiangshan Shen et al.
Epithelial responses to the cytokine interleukin-13 (IL-13) cause airway obstruction in asthma. Here we utilized multiple genomic techniques to identify IL-13-responsive regulatory elements in bronchial epithelial cells and used these data to develop a CRISPR interference (CRISPRi)-based therapeutic approach to downregulate airway obstruction-inducing genes in a cell type- and IL-13-specific manner. Using single-cell RNA sequencing (scRNA-seq) and acetylated lysine 27 on histone 3 (H3K27ac) chromatin immunoprecipitation sequencing (ChIP-seq) in primary human bronchial epithelial cells, we identified IL-13-responsive genes and regulatory elements. These sequences were functionally validated and optimized via massively parallel reporter assays (MPRAs) for IL-13-inducible activity. The top secretory cell-selective sequence from the MPRA, a novel, distal enhancer of the sterile alpha motif pointed domain containing E-26 transformation-specific transcription factor (SPDEF) gene, was utilized to drive CRISPRi and knock down SPDEF or mucin 5AC (MUC5AC), both involved in pathologic mucus production in asthma. Our work provides a catalog of cell type-specific genes and regulatory elements involved in IL-13 bronchial epithelial response and showcases their use for therapeutic purposes.
Kyung Duk Koh, Luke R Bonser, Walter L Eckalbar, Ofer Yizhar-Barnea, Jiangshan Shen, Xiaoning Zeng, Kirsten L Hargett, Dingyuan I Sun, Lorna T Zlock, Walter E Finkbeiner, Nadav Ahituv, David J Erle. Genomic characterization and therapeutic utilization of IL-13-responsive sequences in asthma. 2023-01-11. https://doi.org/10.1016/j.xgen.2022.100229
A lentiviral MPRA tested all 600 possible single-nucleotide substitutions across bases 1-200 of the 589-bp SPDEFe enhancer in primary human bronchial epithelial cells from two donors. Cells were cultured at an air-liquid interface with or without IL-13, and the table reports pooled variant effects relative to the wild-type sequence.
A lentiviral MPRA tested 1,904 candidate cis-regulatory sequences selected from IL-13-responsive H3K27ac peaks in primary human bronchial epithelial cells from two donors. Cells were cultured at an air-liquid interface with or without IL-13, and the table reports pooled and donor-level RNA/DNA activity measurements and the authors' IL-13 response calls.
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