A bioluminescence-based lentiviral approach to identify regulators of the transcription factor TP63 in airway basal cells.

In the human respiratory system, airway epithelial cells represent a key physical and immunological barrier. Maintenance of mucosal homeostasis is dependent on the regenerative capacity of basal stem cells. The transcription factor TP63 is a marker and known regulator of the stem cell phenotype of basal cells in multiple epithelial tissues, including the airway. However, the mechanisms that regulate the self-renewal and differentiation of airway basal cells are not fully understood, including the role of the TP63 gene regulatory network.

To screen for regulators of TP63 expression, we developed an editable lentiviral reporter vector to track the activity of promoter sequences of interest. The selected promoter drives expression of firefly luciferase, while a constitutively active CMV promoter drives expression of renilla luciferase and dsRed2, to enable normalization and cell sorting, respectively.

We generated lentiviral reporter vectors driven by promoter sequences from the TP63 (basal cells), MUC5AC (mucosecretory cells) and FOXJ1 (multiciliated cells) genes, and transduced both a human bronchial epithelial cell line (HBEC3-KT) and primary human airway basal cells. In organoid assays, changes in promoter activity reflected cellular differentiation, validating our approach.

Following this, we have optimised a screening protocol to identify cytokines and growth factors that regulate TP63 expression. We defined positive and negative control conditions for modulating TP63 promoter activity. The results of this screen could identify approaches to promote the stem cell phenotype of airway basal cells, which may represent future therapeutic strategies to regenerate airway tissue in bioengineering or chronic lung diseases.