An integrated approach to study chromatin and transcriptional dynamics throughout the cell cycle without pharmacological inhibition

The cellular and nuclear states are constantly changing and closely linked to DNA replication and cell division, defining cell cycle phases. Pharmacological inhibition is a widely used strategy to enrich for specific phases to study them in isolation. However, chemical-induced cell cycle synchronization may influence chromatin openness and gene expression compared to unperturbed cell cycle states. Here, we use different synchronization methods to obtain G1, S and G2/M phases from mouse embryonic stem cells (mESCs) and profile their chromatin accessibility and post- translational modifications as well as steady and nascent transcripts levels. We demonstrate that synchronization with thymidine and nocodazole leads to dramatic changes in chromatin compaction and gene expression, which only partially resemble bona fide cell cycle-dependent differences. Blocking with CDK1 inhibitor results in efficient synchronization and evokes moderate changes in chromatin accessibility, resembling G2/M phase. Correlative analysis suggests that cell cycle-specific variations in chromatin openness in unperturbed mESCs are mediated by CDK1 substrates and CTCF. This integrated strategy can be applied to most cultured cell lines under any conditions and holds the potential to further our understanding of cell cycle- dependent susceptibility to external cues and chromatin regulation.