While the genetics of retinoblastoma and the implications of the retinoblastoma susceptibility gene, RB1, are well described, there is still scarce evidence to suggest why RB1 acts in such a cell-type specific manner. Using the murine cortex as a model, we examined the effects of RB1 deletion of cycling neural progenitors and post-mitotic neurons, in order to ascertain cell-type specific functions in the central nervous system. Using the previously identified cell-cycle independent role for Rb in tangential migration, we validated Rb/E2f regulation of neogenin and implicated it in this process. In quiescent cortical neurons, we identified a pivotal role for Rb in neuronal survival. Unlike in cycling progenitors, in post-mitotic neurons Rb specifically represses the expression of cell-cycle associated genes in an E2f-dependent manner. Finally, in cortical neurons in the absence of Rb, we observe an activation of chromatin at E2f associated promoters. To determine the role of direct interaction between Rb and chromatin modifying enzymes, we utilized an acute LXCXE-binding deficient mutant paradigm. We report that the LXCXE binding motif is dispensable in establishment and maintenance of cortical neuron quiescence and survival. The activation state of E2f-responsive promoters appears to be dependent on E2f-activity and not simply Rb-mediated repression. Taken as a whole, this thesis serves to support the hypothesis that Rb plays a diverse role in different cell-types by regulation of unique gene targets and regulatory mechanisms. Characterizing specific cancer-initiating populations and understanding the specific function of Rb will help in the treatment of many cancers resulting from RB1 mutation or mutation within the Rb/E2f pathway.

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.

While the genetics of retinoblastoma and the implications of the retinoblastoma susceptibility gene, RB1, are well described, there is still scarce evidence to suggest why RB1 acts in such a cell-type specific manner. Using the murine cortex as a model, we examined the effects of RB1 deletion of cycling neural progenitors and post-mitotic neurons, in order to ascertain cell-type specific functions in the central nervous system. Using the previously identified cell-cycle independent role for Rb in tangential migration, we validated Rb/E2f regulation of neogenin and implicated it in this process. In quiescent cortical neurons, we identified a pivotal role for Rb in neuronal survival. Unlike in cycling progenitors, in post-mitotic neurons Rb specifically represses the expression of cell-cycle associated genes in an E2f-dependent manner. Finally, in cortical neurons in the absence of Rb, we observe an activation of chromatin at E2f associated promoters. To determine the role of direct interaction between Rb and chromatin modifying enzymes, we utilized an acute LXCXE-binding deficient mutant paradigm. We report that the LXCXE binding motif is dispensable in establishment and maintenance of cortical neuron quiescence and survival. The activation state of E2f-responsive promoters appears to be dependent on E2f-activity and not simply Rb-mediated repression. Taken as a whole, this thesis serves to support the hypothesis that Rb plays a diverse role in different cell-types by regulation of unique gene targets and regulatory mechanisms. Characterizing specific cancer-initiating populations and understanding the specific function of Rb will help in the treatment of many cancers resulting from RB1 mutation or mutation within the Rb/E2f pathway.

No description available.

<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

No description available.

Recent advances in high throughput technology have opened the door to systematic studies of epigenetic mechanisms. One of the key components in the regulation of the cell cycle and differentiation is the retinoblastoma protein (pRB), a component of the RB/E2F tumor suppressor pathway that is frequently deregulated in cancer. The RBP2/KDM5A histone demethylase was shown to interact with pRB and regulate pRB function during differentiation. However, how precisely differentiation is coupled with halted cell cycle progression and whether an epigenetic mechanism is involved remain unknown. In the present study, I analyzed gene expression levels of human histone methyltransferases (HMT) and demethylases (HDM), as well as their targets in human cancers; and focused on RB/KDM5A connection in control of cell cycle and differentiation. In particular, I used Drosophila as a model to describe a novel mechanism where the RB/E2F pathway interacts with the Hippo tumor suppressor pathway to synergistically control cell cycle exit upon differentiation. Studying the role of miR-11, I found that the inhibition of dE2F1-induced cell death is its highly specialized function. Furthermore, I studied the induction of differentiation and apoptosis as the consequences of KDM5A deletion in cells derived from Rb knockout mice. I concluded that during differentiation, KDM5A plays a critical role at the enhancers of cell type-specific genes and at the promoters of E2F targets; in cooperation with other repressor complexes, it silences cell cycle genes. I found that KDM5A binds to transcription start sites of the majority of genes with H3K4 methylation. These are highly expressed genes, involved in certain biological processes, and occupied by KDM5A in an isoform-specific manner. KDM5A plays a unique and non-redundant role in histone demethylation and its promoter binding pattern highly overlaps with the opposing enzyme, MLL1. Finally, I found that HMT and HDM enzymes exhibit a distinct co-expression pattern in different cancer types, and this determines the level of expression of their target genes. / Los avances recientes en las tecnologías de alto flujo han abierto el camino a los estudios sistemáticos de los mecanismos epigenéticos. La proteína retinoblastoma (pRB), uno de los elementos de la ruta de supresión de tumores RB/E2F que se encuentra desregulado con frecuencia en el cáncer, es uno de los componentes esenciales de la regulación del ciclo celular y la diferenciación. Sin embargo, aún no se conoce de qué manera precisa la diferenciación se acopla a la detención del avance del ciclo celular y si hay algún mecanismo epigenético vinculado a este proceso. En este estudio, he analizado los niveles de expresión de histona metiltransferasas (HMT) y desmetilasas humanas (HDM), así como sus dianas en cánceres humanos, y me he centrado en la conexión de RB/KDM5A en el control del ciclo celular y la diferenciación. Específicamente, utilicé Drosophila como modelo para describir un mecanismo nuevo mediante el cual RB/E2F interactúa con la ruta Hippo de supresión de tumores para controlar de manera sinérgica la detención del ciclo celular relacionada con la diferenciación. Mediante la investigación del papel de miR-11, determiné que su función altamente especializada es la inhibición de la muerte celular inducida por dE2F1. Además, estudié la inducción de la diferenciación y la apoptosis como consecuencia de la pérdida de KDMA5 en células obtenidas a partir de ratones sin Rb. Extraje como conclusión que, durante la diferenciación, KDMA5 desempeña un papel esencial sobre los estimuladores de los genes específicos de los tipos celulares, así como en los promotores de las dianas de E2F; en cooperación con otros complejos represores silencia a los genes del ciclo celular. Investigué el mecanismo de reclutamiento de KDM5A y encontré que se une al sitio de inicio de la transcripción de la mayoría de los genes que poseen metilación en H3K4. Estos genes tienen elevados niveles de expresión, están involucrados en determinados procesos biológicos y están ocupados por diferentes isoformas de KDM5A. KDM5A desempeña un papel único y no redundante en la desmetilación de las histonas y que en gran medida se solapa con la enzima con la función opuesta, MLL1. Para terminar, encontré que las enzimas HMT y HDM muestran patrones de co-expresión distintos en diferentes tipos de cáncer, y que este hecho determina los niveles de expresión de sus genes diana.

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.