title: Investigating the dynamic rewiring of the p53 network
Creators: name: Mittermeier, Anna
Publisher: name: UNSPECIFIED
Year: 2021

description: A cell has to react to a multitude of different extrinsic and intrinsic stress signals on a daily basis. This challenging task is fulfilled by intracellular signaling pathways with pleiotropic functions. The dynamics of these signaling pathways contribute to information processing and determine cellular outcomes, therefore they are tightly regulated by complex molecular networks. In this study, the transcription factor p53 and its activity in response to DNA damage is used as a paradigm to investigate the underlying regulatory principles. The dynamic p53 response relies on a balance between repeated activation through DNA damage and the transcriptional induction of negative feedback regulators. Strikingly, a pulsatile p53 response results in activation of genes involved in cell cycle arrest and DNA repair, while sustained high p53 levels trigger transcriptional programs associated with apoptosis. This was demonstrated by pharmacological and genetic perturbation of direct p53 network members. However, p53 dynamics are determined by different layers of regulation including interaction with physiological signaling that can directly shape the dynamics of the p53 response and its posttranslational modifications, which are responsible for p53 target gene activation. Importantly, although individual interaction points with other signaling networks have been reported, a comprehensive understanding of how these pathways modulate p53 is lacking. For this reason, the present thesis aims to systematically identify further p53 modulators apart from direct network members and to evaluate their contribution in determining p53-associated cell fates. To this end, live-cell time-lapse microscopy was deployed in order to monitor the p53 response to ionizing radiation in single cells upon varying perturbations in cancer as well as non-transformed reporter cell lines. As part of a collaboration project, I revealed that upon perturbation of the cell survival pathway NF-κB, the p53 response is delayed and displays changes in distinct features. Complementing my single cell data with mathematical modeling, it was elucidated that three different processes are affected by NF-κB inhibition: The activation as well as degradation of p53 and degradation of Mdm2. Furthermore, in a small-scale screening approach, I identified positive modulators of the p53 response: The immune pathways STAT3 and STAT6. While the increase in p53 upon STAT3 activation can be linked to Mdm2, the effect of STAT6 is Mdm2-independent. Instead, the initial activation of p53 is necessary to observe the increase in the p53 response. Last, I established a large-scale screening approach to decipher how the activity of p53 in response to ionizing radiation is controlled by different epigenetic modifiers. Surprisingly, I found that inhibition of a series of deacetylases and acetyltransferases both lead to a reduction in p53 transcription. Despite that, the p53 target gene p21 is upregulated and p53-independent mechanism could be confirmed. My findings demonstrate the complexity of how p53 is modulated by numerous processes in the cell and suggest mechanisms how cellular pathways can interact in more detail. However, I found that the p53 network is surprisingly stable against single perturbations, especially concerning p53-driven cell fates. Hence, the present thesis sets the basis for large-scale screening experiments to systematically assess dynamic changes in signaling molecules, thereby providing large data sets with high resolution and reproducibility.