ABSTRACT Multiciliated cells (MCCs) occur in airways, ependyma and oviduct and are crucial for respiration, neurogenesis and fertility. MCCs nucleate up to 300 motile cilia at their apical surface to generate directional fluid flow. Res- piratory MCCs are responsible for constantly cleaning airways from inhaled pollutants and pathogens to maintain pulmonary health (mucociliary clearance). Notably, airway ciliated cells are the primary targets for human SARS- CoV-2 infections, and impaired clearance by defective MCCs, acquired or inherited as genetic ciliopathies, is associated with chronic airway infections, COPD and asthma. Motile ciliogenesis is directed by > 600 ciliary genes via sequential and hierarchically organized activity of MCC transcription factors. However, transcriptional regulation of multiciliogenesis is only partly deciphered, with major knowledge gaps in the early phases of differ- entiation which establish MCC fate and orchestrate centriolar amplification. E2f transcription factors, evolution- arily conserved from plants to mammals, are major regulators of cell cycle. The canonical E2f1-6 regulate ex- pression of genes controlling DNA replication and the mitotic machinery. In contrast, only a few physiological scenarios are known where the atypical repressors E2f7 and E2f8 are essential. We recently identified an unex- pected major new role of E2f7 and E2f8 in motile multiciliogenesis of airways, ependyma and oviducts. We find E2f7 and E2f8 exclusively expressed in two cell populations of airway epithelium, the cycling basal and the earliest MCCs progenitors. Notably, ablation of E2f7 in mice causes a profound reduction in basal bodies and cilia numbers in airways, ependyma and oviduct. Additional loss of E2f8 further exacerbates the phenotype, indicating physiological cooperation. We will test our hypothesis that E2f7 and E2f8 act as the switch from proliferating uncommitted progenitors to differentiating MCC progenitors which fundamentally requires overcom- ing the strict once-per-cell-cycle centriole duplication program of dividing cells. We postulate that E2f7 and E2f78 are critical for decoupling centrosome duplication from cell division. E2f7 and E2f78 achieve this by cooperatively blocking DNA replication and inducing the alternate S*/G2M*-like phases which enables centriolar biogenesis. This is followed by mainly E2f7 regulating and balancing the early MCC transcriptional program, thereby safe- guarding centriolar multiplication. This proposal will comprehensively and systematically identify mechanisms, molecular partners and their place in the transcriptional MCC hierarchy that underlie this new ciliary function of E2f7/f8. Aim 1 performs single cell mechanistic and functional analyses of E2f7 and E2f8 in MCCs in primary organotypic cultures. Aim 2 maps E2f7 and E2f8 within the MCC hierarchy and the alternate cell cycle in early ciliogenesis. Aim 3 identifies the proximal functional protein interactome of E2f7 and E2f8 ...