Gemc1 Governs Multiciliogenesis Through Direct Interaction with And
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© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 132, jcs228684. doi:10.1242/jcs.228684 RESEARCH ARTICLE GemC1 governs multiciliogenesis through direct interaction with and transcriptional regulation of p73 Maria-Eleni Lalioti1, Marina Arbi2, Ioannis Loukas2, Konstantina Kaplani1, Argyro Kalogeropoulou1, Georgia Lokka1, Christina Kyrousi1, Athanasia Mizi3,4, Theodore Georgomanolis3, Natasa Josipovic3,4, Dimitrios Gkikas5, Vladimir Benes6, Panagiotis K. Politis5, Argyris Papantonis3,4, Zoi Lygerou2 and Stavros Taraviras1,* ABSTRACT therefore, been speculated that formation of multiprotein complexes A distinct combination of transcription factors elicits the acquisition of a is the crucial event providing specificity during these processes. specific fate and the initiation of a differentiation program. Multiciliated Multiciliated cells (MCCs) comprise a specialized population of cells (MCCs) are a specialized type of epithelial cells that possess post-mitotic epithelial cells harboring dozens of motile cilia that dozens of motile cilia on their apical surface. Defects in cilia function have generate fluid flow in a variety of different tissues, including the been associated with ciliopathies that affect many organs, including brain and the airway epithelium. Through the synchronized beating brain and airway epithelium. Here we show that the geminin coiled-coil of their cilia, MCCs in the brain generate a laminar flow of – − domain-containing protein 1 GemC1 (also known as Lynkeas) regulates cerebrospinal fluid termed ependymal flow which is essential for the transcriptional activation of p73, a transcription factor central to brain homeostasis and neurogenesis (Spector et al., 2015; Stolp and multiciliogenesis. Moreover, we show that GemC1 acts in a trimeric Molnár, 2015). MCCs are also essential for development, complex with transcription factor E2F5 and tumor protein p73 (officially homeostasis and regeneration of the airway epithelium, and the known as TP73), and that this complex is important for the activation of synchronized beating of their cilia is crucial for mucus clearance. the p73 promoter. We also provide in vivo evidence that GemC1 is Defects in the generation or function of cilia cause a wide variety of necessary for p73 expression in different multiciliated epithelia. We diseases, known as ciliopathies, which include primary ciliary further show that GemC1 regulates multiciliogenesis through the control dyskinesia (PCD), mucociliary clearance-associated defects and of chromatin organization, and the epigenetic marks/tags of p73 and infertility. Moreover, a link between ependymal cilia malfunction Foxj1. Our results highlight novel signaling cues involved in the and hydrocephalus has become apparent in different animal models commitment program of MCCs across species and tissues. (Fliegauf et al., 2007; Spassky and Meunier, 2017). Recent findings have emphasized the role of the geminin family This article has an associated First Person interview with the first author members McIdas (also known as Idas) (Pefani et al., 2011) and of the paper. geminin coiled-coil domain-containing protein 1 (GMNC; also known as Lynkeas; hereafter referred to as GemC1) (Balestrini KEY WORDS: GemC1, Lynkeas, GMNC, Epigenetic regulation, et al., 2010), to multiciliate cell fate acquisition and differentiation Multiciliated cells, Transcriptional regulation, Multiciliogenesis (reviewed in Arbi et al., 2018; Kyrousi et al., 2016, 2017). GemC1 had initially been identified as a cell cycle regulator, essential for the INTRODUCTION initiation of DNA replication during the transition from G1 to S The acquisition of a functionally specialized cellular phenotype is phase (Balestrini et al., 2010) but was later shown to constitute a key an organized process that requires restriction of stem and progenitor molecule in the early steps of multiciliate cell differentiation. More fate competence. To accomplish this, the chromatin landscape of specifically, GemC1 is essential for the commitment and generation individual stem and progenitor cells and expression of a distinct of MCCs in the mouse brain (Kyrousi et al., 2015) and airway transcription factor set orchestrate gene expression to specifically epithelium (Arbi et al., 2016; Terré et al., 2016) as well as in the instruct the acquisition of a specific cell fate. General transcription oviduct, affecting female fertility (Terré et al., 2016). The role of factors act in concert with tissue-specific proteins to regulate cell GemC1 in multiciliate cell differentiation is well conserved – it has fate decisions and differentiation in multiple cell types. It has, been shown to be essential for multiciliogenesis in both zebrafish and Xenopus skin (Zhou et al., 2015). McIdas has been shown to act in a complex with E2F4 and/or 1Department of Physiology, School of Medicine, University of Patras, 26504 Patras, E2F5 and to activate the transcription of genes involved in centriole Greece. 2Department of General Biology, School of Medicine, University of Patras, amplification (Ma et al., 2014; Stubbs et al., 2012). Mutations in the Patras 26504, Greece. 3Center for Molecular Medicine Cologne, University of C-terminal domain of human McIdas, responsible for the interaction Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany. 4Department of Pathology, University Medical Center Göttingen, Robert-Koch-Str. 40, 37075 with the E2F family members, results in mucociliary clearance Göttingen, Germany. 5Department of Center for Basic Research, Biomedical disorders (Boon et al., 2014). Similarly, GemC1 has been shown to Research Foundation of the Academy of Athens, 4 Soranou Efesiou Street, regulate transcription by interacting with E2F4 and E2F5 through a 115 27 Athens, Greece. 6European Molecular Biology Laboratory (EMBL), Core Facilities and Services, Meyerhofstraße 1, Heidelberg 69117, Germany. C-terminal domain homologous to the McIdas C-terminus (Arbi et al., 2016; Kyrousi et al., 2015; Terré et al., 2016). McIdas and *Author for correspondence ([email protected]) GemC1 regulate the transcriptional activation of Myb and Foxj1, S.T., 0000-0002-7455-647X genes essential for centriole amplification and basal body docking, respectively, as well as the regulation of a broad gene expression Received 17 January 2019; Accepted 16 April 2019 program linked with the fully differentiated phenotype of MCCs. Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 132, jcs228684. doi:10.1242/jcs.228684 The tumor protein p73 (officially known as TP73) has recently been the additional level of specificity needed for the regulation of the identified as a central regulator of multiciliogenesis; it is expressed distinct gene targets required for MCC differentiation. in MCCs of various tissues, and is required for MCC differentiation First, we examined whether GemC1 interacts with p73. and for the direct regulation of a plethora of transcriptional modulators Immunoprecipitation experiments performed in HEK293T cells of multiciliogenesis (Marshall et al., 2016; Nemajerova et al., revealed that pull down of HA-tagged p73 specifically precipitated 2016). At the same time, p73 was found to be an essential regulator GFP-tagged GemC1 (GFP-GemC1; see Fig. 1A). To further for the generation of mature ependymal cells in the adult mouse brain, determine the domain of GemC1 responsible for this interaction, we indicating a global role in the regulation of multiciliogenesis performed immunoprecipitations by using truncated GemC1 forms (Gonzalez-Cano et al., 2016; Medina-Bolívar et al., 2014). lacking the coiled-coil region (GemC1Δcc), previously shown to be Moreover, p73 has a central role in orchestrating multiciliogenesis required for interactions with geminin family members (namely through the transcriptional activation of a network of key ciliogenic geminin and McIdas) (Caillat et al., 2015) or the C-terminal domain of factors (namely Foxj1, Rfx2 and Rfx3) and through post- GemC1 (GemC1Δct), as well as a combination of both deletions transcriptional processing by miR-34b and/or miR-34c (Marshall (GemC1ΔΔ). When both regions were deleted, the observed et al., 2016; Nemajerova et al., 2016). Finally, ChIP-seq analysis in interaction was completely lost, showing that these two regions are murine tracheal cells revealed a plethora of p73-target genes, including essential for GemC1 interactions with p73 (Fig. 1B). Notably, only a Foxj1 and Myb (Marshall et al., 2016; Nemajerova et al., 2016), that small decrease in interaction affinity was observed with the are also transcriptionally regulated by GemC1 in cooperation with GemC1Δcc and GemC1Δct single mutants, indicating that both of E2F5 (Arbi et al., 2016; Kyrousi et al., 2015; Terré et al., 2016). these regions can mediate interactions with p73. Even though several factors have been implicated in the activation Second, we asked whether GemC1, p73 or E2F5 could be of the multiciliogenesis program, it remains unclear how the detected as part of the same complex. We first verified that we can combination of general E2F family transcription factors and of detect specific interactions of GemC1 with E2F5, as conflicting data tissue-specific factors, like GemC1 and p73, cooperate to instruct had been reported in the literature (Terré et al., 2016; Zhou et al., acquisition of a MCC fate. Here, we show that GemC1 physically 2015). To that end, hemagglutinin (HA)-tagged E2F5, FLAG interacts with p73 and that it is essential for the recruitment