DNA replication determines timing of mitosis by restricting CDK1 and PLK1 activation

To maintain genome stability, cells need to replicate their DNA before dividing. The kinases CDK1 and PLK1 drive mitotic entry and become active when bulk DNA synthesis is completed at the S/G2 transition. Here, we have tested the hypothesis that DNA replication controls activation of mitotic kinases. Using an optimized double-degron system, we find that human cells unable to initiate DNA replication in S-phase promptly activate CDK1 and PLK1 and prematurely enter mitosis. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of CDK1 and PLK1. While CDK2 activity promotes DNA replication, activation of CDK1 in S-phase induces severe replication stress. We propose that mitotic kinase activation is governed by a CDK2- and DNA replication-dependent feed-forward loop that ensures timely cell division while preserving genome stability. DNA replication thus functions as a break that coordinates cell cycle activities and determines cell cycle duration

Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition

The ultimate aim of the cell cycle is to create an identical daughter cell. Therefore, correct progression through the different phases of the cell cycle is crucial to ensure faithful cell division. Successful execution of the different processes in the cell cycle is achieved by the coordinated action of a complex network of protein kinases and phosphatases at the centre of which stand Cyclin-Cdk complexes. Human cells possess a variety of cyclins and Cdks, which form complexes that regulate cell cycle transitions. In an unperturbed cell cycle, preparing a cell for mitosis requires faithful DNA replication and reorganisation of the cell’s structures and organelles. In this scenario, cells initiate successive waves of Cdk activity that orchestrate the timely and spatially controlled phosphorylation of a multitude of targets. In contrast, upon DNA damage cells must halt cell cycle progression in order to prevent mitotic entry of damaged cells and subsequently avoid potential propagation of mutations. Strict control of Cyclin-Cdk complexes is, therefore, essential both for correct cell division and to maintain genome integrity. However, the exact mechanisms underlying the activation of Cyclin- Cdk complexes in these different scenarios remain largely unknown. In this thesis, I have investigated several aspects of the regulation of Cdk activity both in the unperturbed cell cycle and during a DNA damage response. To address Cdk activity in the unperturbed cell cycle we established a novel quantitative immunofluorescence method and assessed the dynamics of cyclin accumulation and Cdk target phosphorylation in the unperturbed cell cycle. We found that the mitotic entry network first becomes activated at the S/G2 transition. This finding shifts the classical view of an abrupt Cdk activation at mitotic entry to an earlier and more gradual activation. Furthermore, it provides a potential link between S phase and mitosis, suggesting the existence of a mechanism that maintains pro-mitotic activities under a certain threshold until DNA replication is completed (Paper I). Interestingly, in parallel to an increase of pro-mitotic activities at the S/G2 transition, we observed a change in the localisation of Cyclin A2. Using genome-edited cell lines that express endogenous Cyclin A2-eYFP we were able to determine the cell cycle-dependent localisation of Cyclin A2 to the cytoplasm. Interestingly, despite coinciding with an increase of Cdk activity in the cell cycle we found that the cytoplasmic accumulation Cyclin A2 is modulated by p21 and the presence rather than activity of Cdk1. These findings suggest that complex formation and interaction with Cdk inhibitor proteins (CKI) might regulate Cyclin A2 localisation throughout the cell cycle (Paper IV). Despite not having uncovered a role for cytoplasmic Cyclin A2, we hypothesise that the cell cycle-dependent localisation of cyclins may be an important step to regulate Cdk activity. In order to understand how cells modulate Cdk activity upon DNA damage we made use of endogenously tagged cell lines expressing Cyclin B1-eYFP. We found that upon DNA damage cells continue to accumulate Cyclin B1 until reaching levels that are normally present in G2 phase. At this point, cells translocate Cyclin B1 to the nucleus in a p21 and p53- dependent manner where it is degraded by APC/CCdh1. We identified nuclear translocation and degradation of Cyclin B1 as a restriction point in the cell cycle when cells irreversibly exit the cell cycle and become senescent (Paper II). Senescence is regarded as an early barrier for tumorigenesis as it prevents the propagation of cells with damaged DNA. Our findings in Paper II suggested a link between mitotic inducers and the induction of senescence; therefore we decided to investigate the role of Cdk activity in terminal cell cycle exit. We found that upon DNA damage cells preserve low levels of Cdk activity to ensure that damaged cells continue to progress through the cell cycle until they reach a point where they can be forced into senescence. In this context, we found that Cdk activity induces p21 expression in a p53-independent manner to promote nuclear translocation and degradation of Cyclin B1 and other mitotic inducers (Paper III). Altogether, the data presented in this thesis points towards the existence of a link between the mitotic entry network and the DNA damage response to modulate the activity of Cyclin-Cdk complexes in time and space to trigger ensure correct progression to mitosis or, when needed, to trigger senescence

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade

An experimental assay of the interactions of amino acids from orthologous sequences shaping a complex fitness landscape

Characterizing the fitness landscape, a representation of fitness for a large set of genotypes, is key to understanding how genetic information is interpreted to create functional organisms. Here we determined the evolutionarily-relevant segment of the fitness landscape of His3, a gene coding for an enzyme in the histidine synthesis pathway, focusing on combinations of amino acid states found at orthologous sites of extant species. Just 15% of amino acids found in yeast His3 orthologues were always neutral while the impact on fitness of the remaining 85% depended on the genetic background. Furthermore, at 67% of sites, amino acid replacements were under sign epistasis, having both strongly positive and negative effect in different genetic backgrounds. 46% of sites were under reciprocal sign epistasis. The fitness impact of amino acid replacements was influenced by only a few genetic backgrounds but involved interaction of multiple sites, shaping a rugged fitness landscape in which many of the shortest paths between highly fit genotypes are inaccessible.The work was supported by HHMI International Early Career Scientist Program (55007424), the MINECO (BFU2012-31329, BFU2012-37168, BFU2015-68351-P and BFU2015-68723-P), Spanish Ministry of Economy and Competitiveness Centro de Excelencia Severo Ochoa 2013-2017 grant (SEV-2012-0208), the Unidad de Excelencia María de Maeztu funded by the MINECO (MDM-2014-0370), Secretaria d'Universitats i Recerca del Departament d'Economia i Coneixement de la Generalitat AGAUR program (2014 SGR 0974), the CERCA Programme of the Generalitat de Catalunya, Russian Foundation for Basic Research grant (18-04-01173), the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie programme (665385) and the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013, ERC grant agreement 335980_EinME and Synergy Grant 609989). KSS was supported by EMBO long-term fellowship (ALTF 107-2016). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript