<p>Control of E2F transcription factor activity, regulated by the action of the retinoblastoma tumor suppressor, is critical for determining cell cycle entry and cell proliferation. However, an understanding of the precise determinants of this control, including the role of other cell cycle regulatory activities, has not been clearly defined. </p><p>Recognizing that the contributions of individual regulatory components could be masked by heterogeneity in populations of cells, we made use of an integrated system to follow E2F transcriptional dynamics at the single cell level and in real time. We measured and characterized E2F temporal dynamics in the first cell cycle where cells enter the cell cycle after a period of quiescence. Quantitative analyses revealed that crossing a threshold of amplitude of E2F transcriptional activity serves as the critical determinant of cell-cycle commitment and division. </p><p>By using a developed ordinary differential equation model for Rb/E2F network, we performed simulations and predicted that Myc and cyclin D/E activities have distinct roles in modulating E2F transcriptional dynamics. Myc is critical in modulating the amplitude whereas cyclin D/E activities have little effect on the amplitude but do contribute to the modulation of duration of E2F transcriptional activation. These predictions were validated through the analysis of E2F dynamics in single cells under the conditions that cyclin D/E or Myc activities are perturbed by small molecule inhibitors or RNA interference. </p><p>In an ongoing study, we also measured E2F dynamics in cycling cells. We provide preliminary results showing robust oscillatory E2F expression at the single-cell level that aligns with the progression of continuous cell division. The temporal characteristics of the dynamics trajectories deserve further quantitative investigations.</p><p>Taken together, our results establish a strict relationship between E2F dynamics and cell fate decision at the single-cell level, providing a refined model for understanding the control logic of cell cycle entry.</p> / Dissertation

Quiescence is a non-proliferative cellular state that is critical to tissue repair and regeneration. Although often described as the G0 phase, quiescence is not a single homogeneous state. As cells remain quiescent for longer durations, they move progressively deeper and display a reduced sensitivity to growth signals. Deep quiescent cells, unlike senescent cells, can still re-enter the cell cycle under physiological conditions. Mechanisms controlling quiescence depth are poorly understood, representing a currently underappreciated layer of complexity in growth control. Here, we show that the activation threshold of a Retinoblastoma (Rb)-E2F network switch controls quiescence depth. Particularly, deeper quiescent cells feature a higher E2F-switching threshold and exhibit a delayed traverse through the restriction point (R-point). We further show that different components of the Rb-E2F network can be experimentally perturbed, following computer model predictions, to coarse-or fine-tune the E2F-switching threshold and drive cells into varying quiescence depths.