CIZ1 regulates G1 length and the CDK threshold for initiation of DNA replication to prevent DNA replication stress

Abstract

Eukaryotic cell division is regulated by oscillating CDK activity, which must reach critical CDK threshold activity levels to progress through cell cycle stages. In low mitogen, low CDK environments cells exit the cell cycle into a non-proliferative quiescent state, G0, that plays essential roles in stem cell maintenance and cellular homeostasis. CIZ1 regulates cell cycle and epigenetic programmes, and CIZ1 ablation enhances genomic instability after release from quiescence. Here, we determined the mechanisms that promote genome instability in CIZ1 ablated cells using a combination of Fucci(CA) live cell imaging, cell-free DNA replication assays and DNA combing. Cell cycle dynamics are unaffected in CIZ1-/- (CIZ1 KO) fibroblasts; however, a specific post-quiescent phenotype is observed resulting in a reduced G1 phase and cell cycle length. The reduction in G1 length in CIZ1 KO cells is associated with increased cyclin E1/E2 and A2 expression, and enhanced phosphorylation of Rb leading to early restriction point bypass. CIZ1-/- cells are deficient in cyclin A chromatin binding and required increased cyclin-CDK activity for the initiation of DNA replication, which is associated with DNA replication stress in vitro and in vivo. Significantly, the CDK threshold for initiation of DNA replication was 2-fold higher in CIZ1 KO nuclei than parental controls. Importantly, addition of recombinant CIZ1 in vitro and in vivo promotes recruitment of cyclin A to chromatin and reinstates the CDK threshold for initiation of DNA replication, reversing DNA replication stress and increasing replication fork rates. Loss of CIZ1 is associated with dysregulated cyclin-CDK signalling, resulting in reduced G1 length, an increased CDK activity threshold required to promote initiation of DNA replication that results in DNA replication stress. These data suggest that CIZ1 facilitates recruitment of cyclin-CDK complexes to chromatin and contributes to the mechanisms that determine the threshold CDK activity required for the G1/S transition in post-quiescent cells. Taken together the data support a role for CIZ1 in the prevention of DNA replication stress and maintenance of genome stability.