Fig. 2: Chimeric AGO2 eCLIP and transcriptomics identify let-7 targetome in AT2 cells.

a Schematic of experimental design for AGO2-eCLIP+let-7 analysis after bleomycin treatment. b Fractional representation of let-7:mRNA chimeric reads of let-7 members. c The let-7 binding motif is highly enriched by HOMER. d Total number of genes with let-7:mRNA chimeric peaks. e Sylamer shows overrepresentation of let-7 binding motifs in let-7afd^−/− AT2 cells. f Venn Diagram shows gene totals of let-7 AT2 cell targetome. g Heatmaps show relative expression of significantly upregulated and validated let-7 gene targets in let-7afd^−/− vs control Sftpc-tdT⁺ AT2 cells (n = 3 samples per group) or AT2 organoids (n = 2 mice per group) by bulk RNA-seq. Adjusted p value < 0.05 vs controls using a two-sided Wald test and BH correction. h KEGG pathway enrichment analysis for the let-7 targetome was computed with a one-sided hypergeometric test and FDR correction via Enrichr⁵⁹. i ChIP-Seq regulatory network analysis indicates significant TF footprints of hub targets of let-7 in let-7afd^−/− Sftpc-tdT⁺ cells and organoids. A two-tailed hypergeometric test was used as previously described⁶¹. j Functional pathway and protein interactome network shows significant connectivity of let-7 hub targets. TFs (blue), enzymes (orange). Network q value was calculated with EnrichmentMap in Cytoscape. k Schematic alignments of let-7 “seed” region with target mRNA sequences in mice and humans. l UCSC genome browser tracks from AGO2-eCLIP+let-7 indicates binding of let-7 to targets. Purple triangles indicate let-7 motifs. White asterisks indicate aligned site in (k). m QPCR analysis for indicated let-7 targets in let-7afd^−/− vs control Sftpc-tdT⁺ AT2 cells 14-days post-iTAM (samples per group: n = 3, Ezh2; n = 4, Bach1; n = 4 Myc). Data are mean ± s.e.m. P values by two-tailed unpaired Student’s t test. n Line plots show smoothed relative expression of hub genes across the ADI pseudotime trajectories after bleomycin injury in published dataset¹². Expression values were estimated by fitting a generalized additive model across pseudotime trajectory. o The expression pattern for 217 of 394 let-7 targets is based on inferred likelihood of detection. p Jaccard overlap scores were calculated between let-7 targetome and epithelial cells defined in GSE141259¹². Source data are provided as a Source Data file.