title: From acute to persisting damage: CHK2 is crucial for sustaining the p53 response
Creators: name: Friedel, Laura
Publisher: name: UNSPECIFIED
Year: 2022

description: The choice between life and death is one of the most vital decisions a cell has to make when challenged with different sources of stress. This choice is confounded by variable cellular states, which change throughout the stress response due to both, input from the environment and countermeasures initiated by the cell. The transcription factor p53 is a crucial player orchestrating the cellular response to DNA damage. It not only decides whether a cell should live or die, but it also arrests the cell cycle to provide time for the DNA repair machinery to restore the integrity of the genome. In response to DNA double-strand breaks, p53 shows a series of uniform pulses of accumulation lasting over several hours. In this time, the status of the genome is changing significantly: While during the first pulse the cell is challenged with newly emerged breaks, during the second and the following pulses many breaks have been repaired with only complex breaks and lesions in heterochromatic regions remaining. In this thesis, I investigated whether this change in the status of the genome is reflected in the p53 response. Using live-cell microscopy and pharmacological inhibitors, I provide evidence that the immediate response to acute damage and the sustained response to persisting damage are mediated by two different though interconnected upstream signals: The kinase ATM is crucial to initiate the p53 response and to respond to new DNA damage detected by the cell. Furthermore, active ATM is essential for the initial activation of the checkpoint kinase CHK2. However, sustained activity of ATM is dispensable for both, sustained oscillations of p53 and maintaining CHK2 activity. In contrast, continuous input by CHK2 is crucial for sustaining the p53 response. I could provide first indications that CHK2 modulates p53 levels by inducing the degradation of the negative regulator MDMX thereby destabilizing MDM2. To investigate the impact of these different upstream networks on p53’s function as a transcription factor, the posttranslational modification (PTM) state, kinetics of promoter binding and target gene transcription were assessed for the immediate response (1st pulse) and the sustained response (2nd pulse). I could show that p53’s PTM state changes between the first and second pulse. Comparing the binding of p53 across different loci, no gene-specific patterns of promoter binding were observed. However, we observed gene-specific patterns of transcription which varied particularly for the second pulse. By altering the time-varying PTM state of p53, we were able to modulate both promoter binding and gene-specific patterns of target gene transcription. All in all, I provide evidence that the immediate and the sustained p53 response are mediated by different upstream signals inducing time-varying posttranslational modifications that in turn modulate target-specific gene transcription. Consequently, my work provides first indications that different molecular barcodes might allow differentiating between acute and persisting breaks.