P73 - a novel central regulator of m ulticiliogenesis

We identified TAp73 as a central regulator of airway multiciliogenesis (Marshall et al., 2016;

Nemajerova et al., 2016). In this chapter, background information regarding the structure of the transformation-related protein 73 (Trp73) gene as well as a phenotypic description of different p73 KO mouse models are listed. Regulation of multiciliogenesis by TAp73 is described in the preliminary results section 0.

1.3.4.2.1 Structure of the Trp73 gene

P73 belongs to the p53 tumor suppressor family of transcription factors, along with p63. The Trp73 gene encodes two different p73 isoform classes, TAp73 and DNp73, each arising from a different promotor and exerting opposing cellular functions (Figure 8) (Melino et al., 2002).

Figure 8.

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Figure 8: Gene structure of murine Trp73.

The Trp73 genes encodes two main different isoforms, TAp73 and DNp73, each transcribed from a different promotor. Alternative splicing of products generated from the TA-promotor give rise to distinct N-terminal truncated p73 isoforms (dEx2, dEx2/3, and dN′). Alternative splicing at the 3`-end generates different transcripts, while only the most relevant ones are depicted (α and β). Exons are displayed as boxes and colored according to their different domains. Adapted from (Tomasini et al., 2008).

TAp73 is transcribed from the extrinsic promotor P1 and acts as the transcriptional active form, since it contains the N-terminal transactivation (TA) domain. DNp73 representing the N-terminal truncated isoform is transcribed from the intrinsic promotor P2 and lacks the TA domain. In addition, other N-terminal truncated isoforms are generated by alternative splicing of the transcript generated from the TA-promotor (dEx2, dEx2/3, and dN′) (Melino et al., 2002).

p73 isoforms act as tetramers, which consist either of homo- or heterodimers. Heterodimers are generated either with other p73 splice variants, p63 or to a lesser extent with p53.The TAp73 homotetramer transcriptionally activates proapoptotic genes and thereby acts as a tumor suppressor, while the DNp73 isoforms are incapable of triggering gene expression directly, as this isoform lacks the TA domain. However, DNp73 exhibits a dominant negative effect on TAp73, which leads to the inhibition of the proapoptotic activity of TAp73. Thus, DNp73 indirectly inhibits apoptosis and acts as an oncogene. The inhibitory function of DNp73 is mediated either by the formation of heterodimers with TAp73 and thereby sequestering TAp73 or by competing with the proapoptotic protein p53 for the same promotor binding sites. Therefore, DNp73 regulates the activity of both proapoptotic proteins (TAp73, p53). Moreover, TAp73 induces DNp73 expression, which creates a dominant negative feedback loop (Melino et al., 2002).

Besides the N-terminal p73 isoform classes generated by alternative promoters (TAp73, DNp73), alternative C-terminal (3’-end) splicing also occurs yielding at least seven distinct C-terminal isoforms (α, β, γ, δ, ε, ζ, and η) (Figure 8). p73α is the most abundant isoform under physiological conditions and represents the longest isoform containing a fully functional sterile alpha motif (SAM) domain. All the other C-terminal isoforms lack the SAM domain. Current data suggest that the SAM domain contributes to the stability of p73 tetramer, thereby influencing p73 transcriptional activity. In contrast, it has been shown that p73β is the strongest proapoptotic transcriptional activator (Vikhreva et al., 2018).

1.3.4.2.2 Mouse models of p73

For the identification of the function of p73 during development, different mouse models have been generated: global p73 KO mice and isoform specific KO strains for TAp73 and DNp73. P73 KO mice deficient for TAp73 as well as DNp73 possess a deletion of exon 5 and 6 in the DNA binding domain, yielding a non-functional protein (Yang et al., 2000).

TAp73 KO mice lack exon 2 and 3, which encode the TA domain, while DNp73 isoforms

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can still be expressed using the intrinsic P2 promotor (Tomasini et al., 2008). In DNp73 KO mice, the alternative 3’ exon is depleted from the gene locus (Tissir et al., 2009; Wilhelm et al., 2010).

Analyses of the different p73 KO mouse models showed that the functions of p73 are complex. Therefore, p73 deficiency results in various phenotypes characterized by infertility as well as neurological and immunological defects (Yang et al., 2000; Tomasini et al., 2008;

Tomasini et al., 2009; Wilhelm et al., 2010; Holembowski et al., 2014; Inoue et al., 2014).

P73 and TAp73 KO mice are infertile (Yang et al., 2000; Tomasini et al., 2008), while DNp73 KO mice are fertile (Wilhelm et al., 2010). Hence, the infertility phenotype is attributed primarily to the loss of the TAp73 isoform. Poor quality of eggs caused by impaired meiosis contributes to female infertility in TAp73 KO mice. Moreover, oocytes from TAp73 KO mice did not reach the FT, since they were trapped in the bursa (Tomasini et al., 2008; Tomasini et al., 2009). Male infertility in TAp73 KO mice is associated with defective spermatogenesis, as TAp73 KO mice possess an increased apoptotic rate in spermatogonia, reduced adhesion of germ cells to Sertoli cells, and malformed spermatids.

Consequently, the seminiferous tubules of the testes are nearly empty. However, a few spermatozoa can be found in the testes (Holembowski et al., 2014; Inoue et al., 2014). In this study, we identified a new additional mechanism contributing the infertility phenotype in male and female TAp73 KO mice (described in 4.1.2 and 4.1.3).

The neuronal phenotype differs between the three p73 KO mouse models. P73 KO mice develop the strongest phenotype, including a variety of defects such as neuronal degeneration (cortical hypoplasia), hippocampal dysgenesis, and hydrocephalus. The neuronal degeneration phenotype has been primarily attributed to the loss of DNp73 due to two reasons. Firstly, a similar neurodegeneration pattern was observed in DNp73 and p73 KO mice but not in TAp73 KO mice (Tissir et al., 2009; Wilhelm et al., 2010). Secondly, it has been shown that DNp73 promotes the survival of mature neurons by counteracting TAp73-mediated apoptosis (Pozniak, 2000; Pozniak et al., 2002; Talos et al., 2010). The hippocampal dysgenesis defects of p73 KO mice are also observed in TAp73 KO mice but not in DNp73 KO mice, implicating that TAp73 is the essential isoform for hippocampal development (Tomasini et al., 2008). Of note, severe hydrocephalus is only observed in p73 KO mice (Yang et al., 2000). In addition, p73 KO mice display ependymal denudation and loss of ependymal cilia. Thus, p73 ensures ependyma maintenance and thereby helps in regulating cerebrospinal fluid homeostasis (Medina-Bolívar et al., 2014). In sum, both p73 isoforms contribute to different steps in the neuronal development as phenotypes from the isoform-specific KOs are clearly different. However, TAp73 and DNp73 may also possess some overlapping functions, since the p73 KO phenotype is much stronger than the phenotype of the isoform-specific KOs.

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Besides infertility and impaired neuronal development, p73 KO mice are susceptible to infections that lead to chronic inflammation due to defective airway multiciliogenesis (described in 1.6). Due to all these impairments, most of the p73 KO mice die within the first month (Yang et al., 2000). By contrast, TAp73 KO mice have a only slightly reduced life span, while the DNp73 KO mice possess a normal life span (Tomasini et al., 2008; Wilhelm et al., 2010).

1.3.4.3 Downstream effectors of multiciliogenesis