Periodic Reporting for period 4 - DiVineGenoMe (Decoding cell-to-cell variation in genome integrity maintenance)
Reporting period: 2021-10-01 to 2022-09-30
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. The overall objectives of the project were to identifying determinants of heterogeneity in the cellular response to genotoxic stress and to characterize their molecular functions. Through multidimensional quantitative image-based cytometry, cellular responses to DNA lesions were evaluated at single cell and cell population level. In addition, CRISPR/Cas9 genome editing was used to tag endogenous DNA replication and repair proteins and follow their activities in living cells through the cell cycle and across cell generations, and a genome-wide screen was performed to identify cellular factors impacting heterogeneity in the cellular response to DNA damage. Collectively, the results of the project suggest that single cell heterogeneity in the DNA damage response arises due to multiple distinct events that can be traced back in the cellular history and include replication stress at specific genomic loci, cell cycle dependent regulation of repair factor condensation around DNA lesions, metabolic state, and redox homeostasis.
From the beginning of the 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 coding for fluorescent proteins in frame with the endogenous gene loci of DNA damage and repair markers. This endogenous protein tagging allows following and quantifying the recruitment of the genome caretaker protein 53BP1 to and its condensation around sites of DNA damage in live cell experiments without ectopic overexpression. 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 cells over time and across cell generations and at each time point quantify the accumulation of DNA repair factors into sub-nuclear foci marking DNA lesions. Several targeted gene perturbation screens and an arrayed genome-wide screen have been conducted, using endogenously tagged cell lines, to identify regulators of heterogeneity in the DNA damage response. The results have provided new insights into the causes and consequences of cell-to-cell heterogeneity, including the identification and characterization of a new type of replication stress-induced heritable DNA lesion in cancer cells, the cell cycle dependent regulation of DNA damage-induced condensation of the repair factor 53BP1, and the condensation properties of the single-stranded DNA binding protein RPA. The main results have been presented and discussed at international seminars and conferences, including Keystone symposia and EMBO conferences, and have been published open access in international peer-reviewed journals (e.g. Kilic et al. EMBO J. 2019 Aug 15;38(16):e101379; Lezaja et al. Nat Commun. 2021 Jun 22;12(1):3827; Michelena et al. Life Sci Alliance. 2021 Apr 2;4(6):e202101023). They have also provided the basis for topical reviews in international peer-reviewed journals (e.g. Lezaja et al. Curr Opin Cell Biol. 2021 Jun;70:27-36; Panagopoulos et al. Trends Biochem Sci. 2021 Apr;46(4):301-314; Spegg et al. DNA Repair (Amst). 2021 Oct;106:103179) and for fruitful collaborations on related research topics (e.g. Groelly et al. Mol Cell. 2022 Sep 15;82(18):3382-3397.e7; Porro et al. Sci Adv. 2021 Jul 30;7(31):eabf7906; Ghodke et al. Mol Cell. 2021 Jun 17;81(12):2596-2610.e7).
The project provided 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 further our understanding of the cellular processes that deal with replication stress and DNA damage and may help to guide targeted cancer therapy.