Regulation of stochastic gene expression in single cells by the dynamic p53 response to genotoxic stress

• Applicant: Professor Dr. Alexander Loewer
• Subject Area: General Genetics and Functional Genome Biology ; Bioinformatics and Theoretical Biology ; Cell Biology
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 421980029

• Final Report Year: 2024

Final Report Abstract

The tumor suppressor p53 plays a crucial role in maintaining genome integrity in response to exogenous or endogenous stresses. The dynamics of p53 activation are stimulus and cell typedependent and regulate cell fate. Acting as a transcription factor, p53 induces the expression of target genes involved in apoptosis, cell cycle arrest and DNA repair. However, transcription is not a deterministic process, but rather occurs in bursts of activity and promoters switch stochastically between ON and OFF states, resulting in substantial cell-to-cell variability. We employed single molecule fluorescence in-situ hybridization (smFISH) to quantify the activation of target gene promoters at the single-cell and single-molecule level and demonstrated that upon induction of DNA double strand breaks, p53 mainly regulates how frequently promoters switch on. The rate of transcription, in contrast, remained largely the same. During early time points, all investigated targets genes reacted similarly. At later time points, we observed gene-specific patterns of regulation that were classified as “sustained”, “pulsatile”, and “transient”. These patterns were dependent on p53 dynamics and its posttranslational modifications. To further understand target gene regulation, we induced p53 by other types of cellular stress and performed smFISH. To analyze this comprehensive data set, we collaborated with the lab of Heinz Köppl at TU Darmstadt and developed a novel framework for describing stochastic gene expression based on Bayesian inference. Using this combined theoretical and experimental approach, we revealed additional stimulus-dependent expression patterns. For example, regulation of the transcription rate became more prominent for some target genes. Interestingly, we found that in many situations, transcriptional activity was uncoupled from the total amount of p53 and the fraction bound to DNA. This highlights that transcriptional regulation by p53 is a multi-dimensional process. To gain a higher temporal resolution, we quantified promoter activity in living cells using the MS2-MCP system. Interestingly, upon DNA damage we observed that initially promoter bursting was increased, consistent with our smFISH results, while it shows strong variation at later time periods. Taken together, our project provided insights into p53-mediated transcriptional regulation as an example of a transcription factor that shapes the cellular response to DNA damage.

Publications

• Stochastic transcription in the p53‐mediated response to DNA damage is modulated by burst frequency. Molecular Systems Biology, 15(12).
  Friedrich, Dhana; Friedel, Laura; Finzel, Ana; Herrmann, Andreas; Preibisch, Stephan & Loewer, Alexander
  
• The guardian's choice: how p53 enables context‐specific decision‐making in individual cells. The FEBS Journal, 289(1), 40-52.
  Friedel, Laura & Loewer, Alexander
  
• Super-promoters: Cooperativity between juxtaposed promoters. Springer Science and Business Media LLC.
  Wiechens, Elina; Vigliotti, Flavia; Riege, Konstantin; van Bömmel, Alena; Schwab, Katjana; Görlich, Ivonne; Bens, Martin; Schwarz, Robert; Sahm, Arne; Groth, Marco; Loewer, Alexander; Hoffmann, Steve & Fischer, Martin