Importancia de la fase G1 del ciclo celular en medios pobres en nitrógeno en Schizosaccharomyces pombe

Abstract

[EN] In fission yeast, the cell size at division depends on the growth conditions (Fantes and Nurse, 1977). Cells growing in nitrogen-rich media divide with a large cell size. On the contrary, cells growing in nitrogen-poor media divide with a small cell size. Recently, our group has described that the Greatwall-Endosulfine pathway (Ppk18- Igo1) couples cell growth and the nutritional environment to the cell cycle machinery by modulating the activity of PP2A/B55 protein phosphatase, that counteracts Cdk1/cyclinB substrate phosphorylation at the onset of mitosis (Chica et al., 2016). In nitrogen-rich medium, fully active TORC1 inhibits Greatwall leading to high PP2A/B55 activity. As a consequence, cells spend a long time in G2 before they initiate mitosis. In nitrogen poor media, TORC1 activity is reduced leading to the activation of Greatwall and phosphorylation of its main target Endosulfine. Phosphorylated Endosulfine is a potent and specific inhibitor of PP2A/B55. Low activity of PP2A/B55 allows entry into mitosis with reduced Cdk/CyclinB activity and a small cell size. After division, these cells delay the cell cycle in G1 because the newly born cells are too small to initiate S-phase, as a consequence a population of cells with 1C peak is detected by flow cytometry (Carlson et al., 1999). The timing and orderly progression through the different cell cycle events are regulated by cyclin-dependent kinase (CDK) activity oscillations (Coudreuse and Nurse, 2010). Rum1 and Ste9 are both G1 negative regulators, which maintain a low CDK activity during G1 until entry into S-phase. Rum1 is a CDK inhibitor (CKI) of Cdc2-Cig2 and Cdc2-Cdc13 complexes (Moreno and Nurse, 1994; Correa-Bordes et al, 1997; Benito et al, 1998), whereas Ste9 is an activator of Anaphase Promoting Complex (APC) that promotes the degradation of Cig1, Cig2 and Cdc13 cyclins in G1 (Kitamura et al., 1998; Blanco et al., 2000; Yamano, 2000, 2004). Rum1 and Ste9 have been described as necessary for G1 arrest in minimal medium without nitrogen and for mating (Moreno and Nurse, 1994; Stern and Nurse, 1998; Kominami et al., 1998). The double mutant rum1Δ ste9Δ does not show mitotic defects although it shows higher Cdc13 levels than wild-type cells (Blanco et al., 2000). Thus, both rum1+ and ste9+ single deletion mutants do not show any phenotypes in rich media apart from being sterile and unable to arrest in G1 in minimal medium without nitrogen (MMN). In this work, we have characterized the phenotype of the double mutant rum1Δ ste9Δ in poor nitrogen medium, where the cells grow with a reduced cell size and they have to extend the G1-phase before they undergo S-phase. We also show that Rum1 and Ste9 are required to prevent genomic instability in MMPhe, where they are up regulated. In rich media, there is no apparent defect of rum1+ and ste9+ deletions, although the double mutant shows HU sensitivity. However, when the mutant cells are grown in nitrogen poor medium, they show a slow S-phase, high levels of DNA damage and cell cycle delay in G2 promoted by the activation of the DNA damage checkpoint. This phenotype is rescued by extending the G2-phase or by reducing the CDK activity. It has been previously described that Sic1, the budding yeast orthologue of Rum1, promotes origin licensing G1 by inhibiting CDK activity and that sic1Δ cells accumulate double strand breaks (DSB) and genomic instability (Lengronne and Schwob, 2002). Also Cdh1, the budding yeast Ste9 orthologue, together with Sic1 are required to prevent chromosome instability by promoting efficient origin firing (Ayuda-Duran et al., 2014). Moreover, in mouse primary MEFs, acute depletion or permanent ablation of Cdh1 caused genomic instability by slowing down DNA replication fork movement and increased origin activity. Partial inhibition of origin firing did not accelerate replication forks, suggesting that fork progression is intrinsically limited in the absence of Cdh1 (García-Higuera et al., 2008; Garzón et al., in press). Since budding yeast and mammalian cells have a long G1 phase, it makes sense that the fission yeast rum1Δ ste9Δ cells only shows endogenous DNA damage in poor-nitrogen medium, where the G1-phase is extended. To promote the G1/S transition, the CDK activates the MluI cell cycle box-binding factor (MBF) complex (Reymond et al., 1993; Banyai et al., 2016), the fission yeast functional orthologue of mammalian E2F, that activates the expression of genes involved in the initiation of DNA replication, nucleotide biogenesis and the regulation of S-phase (Bahler, 2005). The MBF complex is bound to its target promoters throughout the cell cycle and it is tightly regulated by positive and negative regulators that restrict MBF activation to the G1/S transition (Wuarin et al., 2002). Rep2 is a MBF transcriptional activator, whereas Nrm1 and Yox1 are repressors of MBF during Sphase and G2 (Baum et al., 1997; Nakashima et al., 1995; De Bruin et al., 2006; Aligianni et al., 2009). In S-phase, MBF could be activated during replication stress by down-regulation of Nrm1 (de Bruin et al., 2008) and Yox1 after phosphorylation by Cds1 (Ivanova et al., 2011, Aligianni et al., 2009, Purtill et al., 2011). By contrast, when the DNA damage checkpoint is active, Chk1 phosphorylates Cdc10, one of the subunits of MBF, and promotes its release from some target promoters repressing MBF-dependent transcription (Ivanova et al., 2013).