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bioRxiv · 10.64898/2026.03.13.711474

In vivo interrogation of transcriptional and epigenetic regulators of lung epithelial regeneration

Sun, D.·Hoagland, D. A.·Strebinger, D.·Hu, C.·Orr, B.·Begum, S.·Marrero, G.·Weir, J.·Franklin, R. A.·Chen, F.
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Abstract

Effective alveolar repair after viral lung injury requires precise coordination of alveolar type 2 cell (AT2) proliferation and differentiation to restore lung function. To uncover causal regulators of this process in the native tissue environment, we developed SAGE (Stable Adeno-Associated Virus Genomic IntEgration), an engineered AAV system that enables high-throughput in vivo genetic interrogation. SAGE supports both bulk phenotypic screening (SAGE-Perturb) and single-cell transcriptomic profiling (SAGE-Perturb-seq). Using this approach, we identified lysine acetyltransferase 8 (Kat8) as essential for epithelial repair following viral infection through the Non-Specific-Lethal (NSL) complex, and generated a time-resolved, high-resolution functional map of transcription factor knockouts during alveolar repair, revealing transcription factor dependences for distinct alveolar epithelial repair trajectories. This map further defined two independent AT2-derived transitional states: a reparative state, and a pathological state that is transcriptionally similar to the basaloid population observed in human pulmonary fibrosis. Disruption of transcription factors in the NF-{kappa}B pathway prevented the emergence of the pathological transitional state, linking inflammation and maladaptive epithelial remodeling. SAGE represents a versatile platform for functional genomics in vivo, with applications extending across respiratory biology and disease.

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Sun, D., Hoagland, D. A., Strebinger, D., Hu, C., Orr, B., Begum, S., Marrero, G., Weir, J., Franklin, R. A., Chen, F.. 2026-03-17. In vivo interrogation of transcriptional and epigenetic regulators of lung epithelial regeneration. https://doi.org/10.64898/2026.03.13.711474

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