Claspin haploinsufficiency leads to defects in fertility, hyperplasia and an increased oncogenic potential.

Claspin is an adaptor protein required for ATR-dependent phosphorylation of CHK1 during S-phase following DNA replication stress. Claspin expression is highly variable in cancer, with low levels frequently correlating with poor patient survival. To learn more about the biological consequences of reduced Claspin expression and its effects on tumorigenesis, we investigated mice with a heterozygous knockout of the Clspn gene. Claspin haploinsufficiency resulted in reduced female fertility and a maternally inherited defect in oocyte meiosis I cell cycle progression. Furthermore, aged Clspn+/- mice developed spontaneous lymphoid hyperplasia and increased susceptibility to non-alcoholic fatty liver disease. Importantly, we demonstrate a tumour suppressor role for Claspin. Reduced Claspin levels result in increased liver damage and tumourigenesis in the DEN model of hepatocellular carcinoma. These data reveal that Clspn haploinsufficiency has widespread unanticipated biological effects and establishes the importance of Claspin as a regulatory node controlling tumorigenesis and multiple disease aetiologies.

Temporal modulation of the NF-κB RelA network in response to different types of DNA damage.

Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-κB transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3ε binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA.