Dynamic approach to study the mechanisms of circadian gene expression in mammals: Development of cell culture-based real-time monitoring system

This thesis is concerned with the development of a real-time monitoring system in mammalian cell cultures for long-term assay of gene expression patterns with particular emphasis on the examination of fundamental properties associated with circadian gene expression.; Using Rat-1 fibroblast cells, I developed a cell culture-based real-time monitoring system to assay temporal patterns of the expression of "clock" genes. I found that Rat-1 cells stably transfected with a Pmper1::Luc construct exhibited a period of circa 24 hr (22.6 to 22.8 hr) and that it was temperature-compensated over a range of 36.5°C to 28.5°C, demonstrating that this system can be used as a model to study circadian rhythms in mammals.; I also established analytical methods to quantify rhythmic patterns that exhibit damping and unstable baseline shifting such as those observed in peripheral mammalian tissues. In particular, these new methods quantitatively determine the degree of rhythmicity and calculate oscillatory strength. Using these tools and Rat-1 cells stably transfected with a Pmper2::dLuc construct, I analyzed the effects of two stimulatory reagents, forskolin and dexamethasone. The results with forskolin versus dexamethasone suggested that there are at least two different mechanisms that can "initiate" rhythmicity in populations of Rat-1 cells.; Towards an in-depth study of the mechanisms of circadian gene expression, I made different clock gene promoter-reporter constructs and examined their temporal expression patterns. I also compared the sequence of each promoter, especially focusing on E-box elements. Based on these studies, I present the possibility that the determination of the critical "circadian E-box" must go beyond a simple identification of canonical CACGTG motifs; I hypothesized that non-canonical E-boxes might also play a key role in the interaction with CLOCK/BMAL1 complexes to produce dynamic oscillatory expression.; These studies demonstrated that the cell culture-based real-time monitoring assay is a non-invasive, precise, and useful system with significant potential towards the molecular genetic and biochemical dissection of molecular mechanisms that regulate circadian gene expression in mammals.