The cell cycle, by governing cell division and fate, is fundamental to tissue development, organ formation, and the progression of disease. Thymidine analogues are indispensable tools for studying DNA replication and cell proliferation. However, the cytotoxic potential of these analogues and the precise molecular mechanisms underlying this toxicity remain incompletely understood.
To elucidate the genotoxic impact of thymidine analogues, we employed a comprehensive, time-resolved multi-omics approach in human induced pluripotent stem cells (iPSCs). Our investigation integrated 3D imaging, bulk and single-cell RNA sequencing, MNase-seq for nucleosome occupancy, untargeted proteomics (LC-MS/MS), and phospho-proteomic analysis. We specifically examined the consequences of thymidine analogue (EdU) incorporation on DNA replication stress, cell cycle dynamics, and cell identity, with a particular focus on the interplay with chromatin architecture and dynamics.
Our findings reveal that EdU induces robust replication stress, marked by H2A.XS139 phosphorylation and activation of the ATM/ATR-mediated DNA damage response (DDR) and DNA repair pathways, alongside Checkpoint Kinase 2. This EdU-induced stress correlates with time-dependent disruptions in cell-cycle progression and mitotic timing, accompanied by increased nuclear size. Furthermore, our integrative analysis demonstrates that genotoxic stress triggers premature exit from pluripotency and stochastic differentiation into all three germ layers. These lineage transitions are characterized by enhanced expression of lineage-specific genes, diminished Polycomb Repressive Complex binding to chromatin, compromised nucleosome assembly, and impaired nucleolar ribogenesis. Notably, our study uncovered hundreds of previously uncharacterized proteins associated with these stress-induced cellular processes.
In conclusion, we have delineated novel molecular mechanisms by which thymidine analogues induce genomic and epigenomic instability, thereby perturbing cell cycle progression and chromatin dynamics. These findings have broad implications for understanding developmental and acquired disorders where replicative stress, whether genetic or environmentally induced, disrupts cell cycle control, organogenesis, and cell identity.