p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network

Abstract

Summary We report that p73 is expressed in multiciliated cells (MCCs), is required for MCC differentiation, and directly regulates transcriptional modulators of multiciliogenesis. Loss of ciliary biogenesis provides a unifying mechanism for many phenotypes observed in p73 knockout mice including hydrocephalus, hippocampal dysgenesis, sterility and chronic inflammation/infection of lung, middle ear and sinus. Through p73 and p63 ChIP-seq using murine tracheal cells, we identified over 100 putative p73 target genes that regulate MCC differentiation and homeostasis. We validated Foxj1, a transcriptional regulator of multiciliogenesis, and many other cilia-associated genes as direct target genes of p73 and p63. We show p73 and p63 are co-expressed in a subset of basal cells, and suggest that p73 ‘marks’ these cells for MCC differentiation. In sum, p73 is essential for MCC differentiation, functions as a critical regulator of a transcriptome required for MCC differentiation and, like p63, has an essential role in development of tissues.

Mechanism of TAp73 inhibition by ΔNp63 and structural basis of p63/p73 hetero-tetramerization

“…This peak shows chemical shifts similar to K372 in the p63 2 /p73 2 hetero-tetramer obtained with the two wild-type domains (Supplementary Figure S2). Similar results were obtained by titrating unlabeled p73 TD to 15 N-lysine-labeled p63 TD (Figure 2d). These results suggested that indeed the mutations selectively stabilize the expected hetero-tetramer.…”

“…This observation is consistent with earlier reports of p73 expression in the skin 11,13,14 as well as in multiciliated cells. 15 To further investigate if cells exist that simultaneously express both p63 and p73, we used immunofluorescence (IF). This analysis showed that both in the basal layer of the skin as well as in the outer root sheath (ORS) of the hair follicle double-positive cells exist.…”

“…For labeled expression, cells were grown at 37°C in 2xYT medium and harvested when the optical density reached 0.4. After centrifugation, the medium was discarded and the cells were resuspended in M9 minimal medium supplemented with the appropriate isotopic labeling (1 g 15 NH 4 Cl and 4 g glucose for 15 N labeling; 1 g NH 4 Cl and 2 g 13 C-glucose for 13 C labeling and 1 g 15 NH 4 Cl and 2 g 13 Cglucose for double labeling, respectively). The cells were then grown to an OD of 0.6 or at least 1 h before induction with IPTG.…”

“…To test whether this potential interaction is indeed relevant for the increased stability of the hetero-tetramer, a L395A/L396A mutant of the otherwise wild-type p73 TD was expressed and titrated to 15 For clarity reasons, the flexible c-termini of the p63 chains after Q409 have been omitted. p63 chains (blue) and p73 chains (red) each form an independent dimer consisting of an N-terminal antiparallel β-sheet and one subsequent helix per monomer.…”

Unifying the p73 knockout phenotypes: TAp73 orchestrates multiciliogenesis

“…A direct link between p73 and Foxj1 was also recently published by the Pietenpol laboratory (Marshall et al 2016). They found defects in p73-deficient multiciliated cells in several organs and that p73 could only be detected in 50% of basal cells of the trachea, where it colocalized with its family member and basal cell marker p63.…”