The DNA damage response pathway is required for multiciliated cell differentiation

Multiciliated cells (MCCs) lining the airways, reproductive tracts, and brain ventricles construct hundreds of motile cilia, each anchored by a centriole.¹ This necessitates the production of hundreds of centrioles in a post-mitotic state, yet centriole duplication is normally restricted to the S and G2 phases of the cell cycle.² During their differentiation, MCCs utilize an alternative cell cycle repurposing many of the Cyclin-CDKs used in a canonical cell cycle,^3,4 yet how this alternative cell cycle bypasses the numerical and temporal constraints governing centriole duplication remains unclear. DNA damage can result from external sources or occur during programmed genome rearrangements in processes like immunity or meiosis.⁵ To maintain genomic integrity, cells activate the DNA repair pathways that alter the cell cycle to prevent harmful outcomes such as cancer or immune dysfunction.⁶ Here, we uncover an unexpected role for DNA damage during the terminal differentiation of MCCs. We show that differentiating MCCs accumulate extensive double-strand DNA breaks during centriole amplification, with DNA damage levels scaling with centriole number. DNA damage response (DDR) kinases are required to support centriole biogenesis and ciliogenesis. Moreover, we find that the high transcriptional output needed to express centriole and cilia genes generates RNA-DNA hybrids (R-loops) that co-localize with the sites of DNA damage. These findings suggest that transcription-coupled DNA damage engages DDR signaling to permit centriole amplification in MCCs. Together, our findings reveal a developmental program that harnesses physiological DNA damage and DDR signaling to adaptively rewire the canonical cell cycle.