E2F1 induces a G0-G1 re-entry transcriptional program without changing chromatin accessibility [RNA-seq]
Many cells in our body reside in a reversible nondividing state termed G0 or quiescence. Precise regulation of entry and exit from G0 is critical for maintaining tissue homeostasis. G0 entry can occur when cells do not receive the required signals for cell cycle progression, leading to active repression of cell cycle genes through several mechanisms, including formation of repressor complexes to restrict chromatin accessibility for transcriptional activators. G0 cells can re-enter the G1 phase of the cell cycle upon receiving growth factor signals, resulting in activation of the Mitogen-Activated Protein Kinase pathway, converging on E2F transcription factors. Ectopic expression of E2F1 alone, a transcriptional activator, is sufficient to promote G0 exit without requiring the upstream signals, but it is unclear how E2F1 can overcome the chromatin lockdown in G0 cells to activate cell cycle genes. To better understand this mechanism, we compared dynamics, gene regulation and chromatin accessibility state between E2F1-mediated and growth factor (serum)-induced cell cycle reentry. Using live cell single cell tracking, we observe that E2F1 induction leads to accelerated cell cycle progression compared to regular serum-induced cell cycle re-entry. This accelerated cell cycle re-entry by E2F1 is accompanied by faster upregulation of cell cycle genes. Interestingly, we find that E2F1 preferentially binds to gene promoters that remain in a poised chromatin state after G0 induction, suggesting that G0 cells are prepared for cell cycle re-entry via regulating chromatin states in essential cell cycle genes. Overall design: Constucts for doxycycline inducible expression of E2F1 or empty-vector (EV) control were stably integrated into 3T3 cells. The E2F1 and EV 3T3 cells were serum starved for 48 hours to induce a G0-like state, followed by E2F1 induction, EV induction or 10% serum addition, to induce G1/S phase reentry. Samples were collected at 0, 2, 6 and 12 hours after induction or serum addition. RNA was extracted for RNA-sequencing and nuclei were extracted for ATAC-Seq. In addition, cells were prepared for E2F1 ChIP-Seq at the 12 hour time point only. Together the RNA-Seq, ATAC-Seq and ChIP-Seq were used to identify E2F1 gene regulatory network involved in cell cycle reentry from G0.