Abstract

The multiciliated cell (MCC) is an evolutionarily conserved cell type, which in vertebrates functions to promote directional fluid flow across epithelial tissues. In the conducting airway, MCCs are generated by basal stem/progenitor cells and act in concert with secretory cells to perform mucociliary clearance to expel pathogens from the lung. Studies in multiple systems, including Xenopus laevis epidermis, murine trachea, and zebrafish kidney, have uncovered a transcriptional network that regulates multiple steps of multiciliogenesis, ultimately leading to an MCC with hundreds of motile cilia extended from their apical surface, which beat in a coordinated fashion. Here, we used a pool-based short hairpin RNA screening approach and identified TRRAP, an essential component of multiple histone acetyltransferase complexes, as a central regulator of MCC formation. Using a combination of immunofluorescence, signaling pathway modulation, and genomic approaches, we show that (a) TRRAP acts downstream of the Notch2-mediated basal progenitor cell fate decision and upstream of Multicilin to control MCC differentiation; and (b) TRRAP binds to the promoters and regulates the expression of a network of genes involved in MCC differentiation and function, including several genes associated with human ciliopathies.

Highlights

  • A key function of epithelial tissues is to act as protective barriers between the body and the environment

  • A pooled shRNA screen uncovers epigenetic regulators of airway epithelial cell fate To identify genes required for airway epithelial cell fate decisions, we established a pooled shRNA screening strategy in primary human airway basal cells, the progenitor cells in the conducting airway

  • The strategy used a lentiviral shRNA library of 4792 shRNAs targeting 281 genes encoding proteins involved in epigenetic regulation, or with domains known for this function (Hoffman et al, 2014; Table S1)

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Summary

Introduction

A key function of epithelial tissues is to act as protective barriers between the body and the environment. The flow cytometric analysis revealed a significant reduction in the ratio of ciliated to secretory cells by each of the TRRAP shRNA treatments (Fig. 2, B and C), consistent with the primary screening results.

Results
Conclusion
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