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Decoding cell-to-cell variation in genome integrity maintenance

Periodic Reporting for period 3 - DiVineGenoMe (Decoding cell-to-cell variation in genome integrity maintenance)

Reporting period: 2020-04-01 to 2021-09-30

When DNA gets damaged, this can lead to mutations and cause disease. DNA repair mechanisms have thus evolved to sense and repair DNA lesions. These mechanisms are often subverted in human cancers, indicating that defective DNA damage recognition and repair is linked to the accumulation of cancer-driving mutations. It is becoming increasingly clear that individual cells do not always respond equally well to genotoxic stress and DNA damage. Such cell-to-cell variability may affect cancer development, disease progression and the response to therapy. Not much is known, however, about the cellular and molecular determinants of cell-to-cell variability in the DNA damage response. This project aims at identifying determinants of heterogeneity in the cellular response to genotoxic stress and at characterizing their molecular functions.
Since the beginning of this project, cellular heterogeneity in the response to genotoxic stress and DNA damage has been characterized and quantified at multiple levels of the DNA damage response, and new tools, in particular cell lines and cell-based assays, have been developed. Genome editing by CRISPR/Cas9 has been used to introduce sequences encoding for fluorescent proteins in frame with the endogenous gene loci of DNA damage and repair markers. This endogenous protein tagging now allows us to follow and quantify the recruitment of the genome caretaker protein 53BP1 to sites of DNA damage in live cell experiments without ectopic overexpression of the protein of interest. We have further optimized the live cell imaging conditions so that the comparably dim signals from the labeled endogenous proteins can be imaged for extended periods of time with minimal phototoxicity. In conjunction with these optimizations we have developed cell-tracking algorithms, which allow us to track individual cells over time and at each time point quantify the accumulation of 53BP1 into sub-nuclear foci marking DNA lesions. These systems have significantly aided a recent study by our group on 53BP1 behavior at sites of DNA damage across the cell cycle (Kilic et al. EMBO J. 2019 Aug 15;38(16):e101379; see also Piccinno et al. EMBO J. 2019 Aug 15;38(16):e102871). In addition, first pilot siRNA screens were performed as proof-of-principle experiments, and conditions have been established for using the endogenously tagged cell lines for large-scale phenotypic screens.
It is expected that this project will reveal new insights into the cell-to-cell variation of genotoxic stress responses. Newly developed, genetically engineered cell lines and quantitative high-content microscopy-based readouts in conjunction with custom-built computational tools for advanced image analysis provide the technological basis of this project. The experimental results and biological concepts derived from this project may help guide targeted cancer therapy.