Regulation of G2 phase and long-term consequences of DNA damage
Cell proliferation requires the accurate replication of DNA and equal segregation of replicated genes, important for maintaining the integrity of newly formed cells. At the centre of this process is a series of coordinated events termed ‘the cell cycle’, which ensures cell proliferation proceeds with high fidelity. Cell cycle regulation is driven by the activity of cyclin-dependent kinases (Cdks), which require binding to their regulatory subunit cyclin to become activated. However, the activity of Cdk is regulated by several different mechanisms. Transcription and degradation control mechanisms indirectly affect Cdk activity by modulating the expression of several regulatory proteins, including cyclins, while regulatory phosphorylation and dephosphorylation of cyclin-Cdk complexes provide direct control of Cdk activity. Such posttranslational modifications are frequently part of feedback loops, which fine-tune Cdk activity. These mechanisms collectively modulate successive activation of Cdks, and is responsible for timely phosphorylation of Cdk substrates to complete different phases of the cell cycle. This thesis concerns the regulation of G2 phase in the cell cycle, in relation to: 1) the effect of cyclin A2 localisation in G2 phase, 2) the changes in G2 phase regulation in a genetic disorder, and 3) the long-term consequences if G2 phase regulators are completely suppressed. Although Cdk activity is required for well-delineated cell cycle phase transitions, the spatiotemporal regulation of cyclin is important, as it provides unique substrate specificity and accessibility to the Cdk. Nevertheless, the exact mechanisms underlying the activation of cyclin-Cdk complexes remain largely elusive. The first part of this thesis investigates unknown mechanisms of mitotic kinase activation in G2 phase, by assessing the spatio-temporal regulation of cyclin A2 and its function in G2 phase.
In paper I, we observe that nuclear cyclin A2 partially translocates to the cytoplasm at S/G2 phase transition. Interestingly, we reveal that cyclin A2-Cdk2 can initiate the activation of Plk1 through phosphorylation of Bora, but only cyclin A2 localised to the cytoplasm can interact with Bora and Plk1. We find no evidence that the change in localisation of cyclin A2 is involved in feedback loops in G2 phase. Thus, our study strongly supports the notion that cytoplasmic A2 functions as a trigger for the activation of mitotic kinases. Although the precise mechanism that changes the localisation of cyclin A2 to the cytoplasm requires further study, we show that cyclin A2 nuclear localisation until S/G2 phase transition is contributed, in part, by the association of cyclin A2 to chromatin during DNA replication. In addition, our work also reveals p21 can restrict cyclin A2 to the nucleus, especially after DNA damage. Together, paper I expands our understanding of the mechanisms of mitotic kinase activation in G2 phase, and identifies future areas of study to fill in our knowledge gaps of how cyclin A2 changes its cellular localisation. Cell cycle dysregulation has been implicated in many genetic diseases and disorders. This highlights the importance of understanding cell cycle regulation in certain disease settings. The second part of this thesis is dedicated to studying the role of a non-coding nuclear RNA gene, RMRP, that is mutated in the rare genetic disorder, cartilage-hair hypoplasia (CHH). CHH cells show proliferation defects, and studies on yeast suggest that RMRP could regulate the accumulation of cyclins. In paper II, we reveal RMRP has pleiotropic effects on several cell cycle regulatory genes, and the mutation of RMRP delays G2 phase progression to mitosis. Furthermore, our work finds evidence of possible impairment in the PI3K-Akt signalling pathway in CHH. These findings contribute to understanding the role of RMRP in cell cycle regulation, particularly in relation to CHH, and indicate a possible pathway for therapeutic interventions.
The uncontrolled proliferation of cells with genomic instability can lead to the development of cancer. The cell cycle checkpoint is a mechanism that can restrict cell cycle progression in response to DNA damage and replication blocks. When checkpoint kinases are activated, signals are transmitted to a network of regulatory proteins that increase the inhibitory force and delay cell cycle progression. In the case of persistent DNA damage in G2 phase, p53 and p21- dependent premature activation of APC/CCdh1 mediates cell cycle termination by degrading all cell cycle regulatory proteins. While all these processes ensure genomic integrity, the mechanisms that allow escape from a checkpoint have been the focus of many studies, but whether cell cycle termination in G2 phase can be reversed remains unclear. Therefore, the last part of this thesis investigates the long-term consequences of DNA damage-induced cell cycle termination in G2 phase. Paper III shows that cells can re-initiate S phase after terminating the cell cycle in G2 phase. Interestingly, expression of p21 persists until cells re-initiate DNA replication and increases further once DNA re-replication is complete. This finding supports our observation of repeated cell cycle termination of re-replicated cells. Furthermore, re-replicated cells can progress to mitosis, which creates a heterogenous cell population, and is linked to genomic instability. Thus, resumption of the cell cycle a long period after termination in G2 phase can give rise to multiple cell fates. This shifts our current perception of the long-term consequences of cell cycle termination in G2 phase, from a singular outcome of senescence to that of multiple cell fates, possibly alluding to a mechanism by which cells can undergo oncogenic transformation.
In summary, this thesis highlights the importance of the spatio-temporal regulation of cyclin A2 in modulating Cdk to initiate the mitotic entry network in G2 phase, ensuring welldelineated progression to mitosis. Identifying the function of RPRM in G2 phase adds to our limited understanding of cell cycle regulation in relation to CHH. Moreover, this thesis reveals that DNA damage-induced cell cycle termination in G2 phase can lead to cell fates other than senescence, an implication that could have relevance in tumourigenesis.
List of scientific papers
I. Silva Cascales H, Burdova K, Middleton A, Kuzin V, Müllers E, Stoy H, Baranello L, Macurek L, Lindqvist A. Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1. Life Sci Alliance. 2021 Jan 5;4(3):e202000980.
https://doi.org/10.26508/lsa.202000980
II. Vakkilainen S, Skoog T, Einarsdottir E, Middleton A, Pekkinen M, Öhman T, Katayama S, Krjutškov K, Kovanen PE, Varjosalo M, Lindqvist A, Kere J, Mäkitie O. The human long non-coding RNA gene RMRP has pleiotropic effects and regulates cell-cycle progression at G2. Sci Rep. 2019 Sep 24;9(1):13758.
https://doi.org/10.1038/s41598-019-50334-6
III. Middleton A, Suman R, O’Toole P, Akopyan K, Lindqvist A. p53-dependent polyploidisation after DNA damage in G2 phase. bioRxiv. 2020. Jun 9:141770. [Manuscript]
https://doi.org/10.1101/2020.06.09.141770
History
Defence date
2021-04-29Department
- Department of Cell and Molecular Biology
Publisher/Institution
Karolinska InstitutetMain supervisor
Lindqvist, ArneCo-supervisors
Björkegren, Camilla; Ström, Lena; Müllers, ErikPublication year
2021Thesis type
- Doctoral thesis
ISBN
978-91-8016-204-3Number of supporting papers
3Language
- eng