Vanderbilt Institute of Chemical Biology



Discovery at the VICB







Role for p73 in Ciliogenesis Affects Multiple Organ Systems


By: Carol A. Rouzer, VICB Communications
Published:  March 30, 2016



The seemingly unrelated aggregate of abnormalities observed in p73-null mice is due to the regulation of multiple genes required for ciliogenesis by p73.


The p53 protein family comprises p53, p63, and p73. These sequence-specific transcription factors play an important role in a broad range of cellular functions including cell cycle control, DNA repair, apoptosis, and differentiation. Much is known about the most widely studied member of the family, p53, whereas the exact functions of p63 and p73 remain less well understood. Each of the genes for both of the latter two proteins contains two promoters, P1 and P2. Transcription from P1 produces a full length protein (TAp63 and TAp73) that includes the transactivation domain and is active as a transcription factor. Transcription from P2 produces a truncated protein (ΔNp63 and ΔNp73) that acts as a dominant negative modulator of its full length counterpart. Mice lacking the gene for p63 suffer severe developmental defects and die shortly after birth. Their abnormalities indicate that p63 is required for the maintenance of epithelial progenitor cells. In contrast, although p73-null mice survive to adulthood, their seemingly unrelated array of defects, including runting, sterility, hypocampal dysgenesis, hydrocephalus, and chronic inflammation and/or infection of the airways, sinuses, and ears, has been difficult to explain. This led Vanderbilt Institute of Chemical Biology member Jennifer Pietenpol and her laboratory to take a closer look at p73-null mice. Their research led to the discovery that p73 is required for the differentiation of multiciliated cells (MCCs, Figure 1), which play a key role in the respiratory, reproductive, and nervous systems. This single mechanism can explain all of the abnormalities observed in mice lacking p73 [C. B. Marshall, et al. (2016) Cell Rep., published online March 2, DOI:10.1016/j.celrep.2016.02.035].

Figure 1
. Scanning electron micrograph of the tracheal epithelium of a wild-type mouse. The cilia are readily apparent. Figure from The National Institutes of Health National Heart, Lung, and Blood Institute Electron Microscopy Core, U.S. Government Public Domain.


For their studies, the Pietenpol laboratory generated a p73-null mouse model by flanking exons 7 through 9 of the p73 gene of mouse embryonic stem cells with loxP sites. They then used the cells to develop mice bearing the mutated allele and bred them with CMV-Cre mice. This approach produced mice (p73-/-) that were deficient in p73 in all tissues, though they expressed a truncated form of the protein comprising exons 1 through 6 followed by exon 10. The mice exhibited all of the abnormalities previously reported for p73-null animals. To better understand these abnormalities, the researchers performed a histological analysis of multiple tissues from the mice using a conventional hematoxylin and eosin (H & E) stain. They immediately observed a striking absence of ciliated cells in every tissue examined from the p73-/- mice. They confirmed these observations by staining the tissues using fluorescent antibodies against p73 and acetylated α-tubulin (α*-tubulin), a cilia marker (Figure 2). The bronchioles from p73-/- mice also exhibited a focal loss of epithelium interspersed with areas of hyperplasia. Hypertrophy and hyperplasia of cells expressing smooth muscle actin appeared in areas devoid of epithelium. Immunofluorescence staining of cells in the airways confirmed a 90% loss of MCCs in p73-/- mice as compared to their wild-type litter mates. A decrease of 35% in the basal cell population in the trachea was also evident. These cells were replaced by increased numbers of mucin producing, club, and neuroendocrine cells. Excess debris and mucus were present in the airways of p73-/- mice, consistent with the absence of cilia required to clear these materials, and evidence of pneumonia, otitis, and rhinitis was also clearly visible.




Figure 2. Hematoxylin and eosin (H & E) stained sections of epitheium of the indicated tissues from p73+/+ and p73-/- mice, and immunofluorescent staining of those same tissues to indicate the presence of cilia (α*-tubulin, green) and p73 (red). Figure reprinted under the CC BY-NC-ND license from C. B. Marshall, et al. (2016) Cell Rep., published online March 2, DOI:10.1016/j.celrep.2016.02.035.



To further explore the absence of cilia in the airways of p73+/+ mice, the investigators performed immunofluorescence staining for both p73 and Foxj1, a transcription factor that mediates multiple steps in cilia formation in MCCs. They discovered that all Foxj1-expressing cells in the trachea also expressed p73; however, not all p73-expressing cells also expressed Foxj1. Staining for p63 revealed the presence of basal cells, approximately 50% of which also expressed p73. They noted similar patterns of protein expression in human tracheal epithelium, although there were more ciliated cells and higher levels of p73 expression in the human than in the mouse.


These results suggested the hypothesis that development of cilia requires p73. To further test this hypothesis, the investigators isolated murine tracheal epithelial cels (MTECs) from both p73+/+ and p73-/- mice, and they grew the cells in both air-liquid interface or 2D submerged cultures. Once the cells were established, they transferred them to differentiation medium and then used immunofluorescence staining to search for the presence of α*-tubulin. They discovered that α*-tubulin appeared upon differentiatin of p73+/+ but not p73-/- MTECs. Expression of two small hairpin RNAs (shRNAs) targeting exon 6 and the 3′-UTR of p73 in both cell types resulted in a loss of expression of p73 or its truncated form in p73+/+ and p73-/- cells, respectively. The shRNA expression also eliminated α*-tubulin expression in the p73+/+ cultures, supporting the hypothesis that p73 expression is required for cilia formation.


To better understand the role of p73 in ciliogenesis, the investigators performed a ChIP-seq analysis on tracheal epithelium from p73+/+ mice. For this procedure, they treated the epithelium with formaldehyde immediately after the animals were sacrificed to cross-link protein and any closely associated DNA. They then isolated p73, p63, or RNA polymerase II (Pol II) along with cross-linked DNA by immunoprecipitation. Following cross-link reversal, they isolated the DNA and sheared it into ~300 bp fragments, for sequencing. Comparison of the resulting sequences to that of the mouse genome enabled them to identify 1,767, 3,861, and 53,684 binding sites for p73, p63, and Pol II, respectively. In all three cases, the regions identified were enriched for transcriptionally active areas of the genome, and parallel RNA sequencing of the tracheal epithelium demonstrated that genes containing a p73 binding site within 20,000 bp of a transcription start site were more likely to be expressed and expressed at a higher level than those that did not. The results also confirmed that the ChIP-seq data had identified well-known target genes for both p63 and p73.


Further analysis of the ChIP-seq data identified 1,011 genes from among 1,096 p73 binding sites that were within 25,000 bp of a transcription start site. Consistent with their hypothesis that p73 modulates ciliogenesis, the researchers discovered a highly significant overrepresentation of cilia-associated genes among those containing p73 binding sites. In all, 105 genes shared a p73 binding site and a role in ciliogenesis.


Based on their findings, the researchers infected MTECs from both p73+/+ and p73-/- mice with a lentivirus containing a TAp73β expression construct or an empty vector to serve as a control. They grew the infected cells under air-liquid interface conditions and then performed a global RNA sequencing analysis. Of 21,867 protein-coding genes examined, 3,300 were differentially expressed as a result of ectopic expression of p73, regardless of cell genotype. Among these genes were 49 of the 105 cilia-associated genes identified in the ChIP-seq experiments, including 14 known to have roles in MCC differentiation and homeostasis. In fact, 397 out of 1,398 genes in the total cilia-associated gene set were differentially expressed in response to p73 expression, despite the fact that many of these genes did not possess a nearby p73 binding site. These observations suggested that p73 triggers a cascade of signaling events that leads to upregulation of cilia-associated genes that are not directly under its control.


The gene that exhibited the greatest expression increase in response to ectopic p73 expression in p73-/- MTECs was Foxj1. The Foxj1 transcription factor is required for multiple aspects of ciliogenesis, including the docking of centrioles at the apical plasma membrane. Further experiments demonstrated that MTEC differentiation was not required to observe p73-dependent Foxj1 expression; however differentiation markedly increased both p73 and Foxj1 protein levels in p73+/+ cells. Also exhibiting highly p73-dependent expression was Traf3ip1, which encodes a protein that binds to the basal bodies of cilia and promotes acetylation of α-tubulin. Other p73-modulated genes of interest were Jag1, a Notch pathway regulator and Rfx3, a transcription factor, both of which are associated with ciliogenesis.


The investigators concluded that p73 is a key regulator of ciliogenesis, directly or indirectly modulating the expression of numerous genes required for MCC differentiation and homeostasis (Figure 3). They postulated that a p73-expressing subgroup of basal cells is destined to differentiate into MCCs, and that this process is triggered once the cells exit the cell cycle. They further hypothesized that p73 activity in the basal cells may be suppressed by the action of ΔNp63, expression of which decreases as differentiation progresses. These findings reveal important new mechanisms that control ciliogenesis, a process that is clearly highly important for the normal function of multiple organ systems as demonstrated by the widely varying abnormalities observed in p73-/- mice.



Figure 3. Diagrammatic representation of the regulation of MCC differentiation and homeostasis by p73. Expression of p73 in a select population of basal cells leads to expression of a series of transcription factors, including Myb, FoxJ1, and Traf3ip1, each of which promotes a key aspect of cilia development. Figure reprinted under the CC BY-NC-ND license from C. B. Marshall, et al. (2016) Cell Rep., published online March 2, DOI:10.1016/j.celrep.2016.02.035.




View Cell Rep. article: p73 Is Required for Multiciliogenesis and Regulates the Foxj1-Associated Gene Network








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