Development, tissue renewal and longevity of multi-cellular organisms require the ability to switch between a proliferative state and quiescence, a reversible arrest from the cell cycle. The balance of quiescence and proliferation underlies the fundamental feature of generating and maintaining the appropriate number of cells, which is essential for tissue architecture, regeneration, and function. Disruption of quiescence and proliferation balance leads to hypo- or hyper-proliferative diseases. To date, the regulatory mechanism of proliferation has been well established, while cellular quiescence has remained a phenotypic description without a clearly defined molecular control mechanism. Simply, quiescence has long been considered a passive counterpart to proliferation. However, recent findings have revealed that quiescence is an actively maintained state exhibiting a unique gene expression pattern. While quiescence has been traditionally considered as a state (namely G0) outside of the cell cycle, it is in fact a collection of heterogeneous states. In studies conducted in the 70's and 80's using fibroblasts and lymphocytes, it has been observed that the longer the cells were kept under quiescence inducing conditions such as contact inhibition, the deeper the cells moved into quiescence. Deep quiescent cells are still able to reenter the cell cycle upon growth stimulation but they exhibit a longer pre-DNA synthesis phase [1-4]. Shallow quiescent state has also been recently reported in muscle and neural stem cells termed GAlert and "prime" quiescent state, respectively. Heterogeneous quiescent depth entails that cells vary in their sensitivity to growth signals, representing an important yet underappreciated layer of complexity in cell growth control. The cellular mechanisms that control the depth of quiescence remains elusive. In my thesis work, I first investigate the strengths of serum stimulation required for cells to exit deep and shallow quiescence as a determinant of quiescence depth. Through model simulations and experimental measurements, I further demonstrate that various components of the Rb-E2F pathway control quiescence depth with varying efficacy. The Rb-E2F pathway interacts with diverse cellular pathways that respond to environmental signals to jointly modulate quiescence depth. Given that certain circadian clock genes (e.g., Cry) affect key components in the Rb-E2F pathway, I tested the effect of Cry activity on quiescence depth. I found that increased Cry activity resulted in deeper quiescence, contrary to our anticipation based on the literature. Next, we constructed a library of mathematical models that represent possible interactions between Cry and the Rb-E2F pathway. We computationally searched this model library for links that could explain the experimental observations. The modeling search suggested that Cry upregulation may lead to increased expression of cyclin dependent kinase inhibitor (e.g., p21), which in turn drives cells into deeper quiescence. This model prediction was confirmed by my follow-up experiments. Collectively, my thesis work establishes an integrated modeling and experimental framework that will help us to further investigate diverse cellular mechanisms controlling the heterogeneous quiescence depth.

<p>Cellular dynamics arise from intricate interactions among diverse components, such as metabolites, RNAs, and proteins. An in-depth understanding of these interactions requires an integrated approach to the investigation of biological systems. This task can benefit from a combination of mathematical modeling and experimental validations, which is becoming increasingly indispensable for basic and applied biological research. </p> <p>Utilizing a combination of modeling and experimentation, we investigate mammalian cell cycle entry. We begin our investigation by making predictions with a mathematical model, which is constructed based on the current knowledge of biology. To test these predictions, we develop experimental platforms for validations, which in turn can be used to further refine the model. Such iteration of model predictions and experimental validations has allowed us to gain an in-depth understanding of the cell cycle entry dynamics. </p> <p>In this dissertation, we have focused on the Myc-Rb-E2F signaling pathway and its associated pathways, dysregulation of which is associated with virtually all cancers. Our analyses of these signaling pathways provide insights into three questions in biology: 1) regulation of the restriction point (R-point) in cell cycle entry, 2) regulation of the temporal dynamics in cell cycle entry, and 3) post-translational regulation of Myc by its upstream signaling pathways. The well-studied pathways can serve as a foundation for perturbations and tight control of cell cycle entry dynamics, which may be useful in developing cancer therapeutics. </p> <p>We conclude by demonstrating how a combination of mathematical modeling and experimental validations provide mechanistic insights into the regulatory networks in 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.