1) Novel technology was developed to monitor site specific binding of nuclear receptors to response elements with intact cells. Using GFP-tagged receptors and a cell line with amplified gene arrays, relative receptor binding could be correlated with gene output by measurement of the RNA FISH signal. Images were analyzed with a novel automated computer algorithm, allowing acquisition of data from large numbers of cells. 2) Although gene regulation often appears deterministic in the average cell population, transcription was found to be highly stochastic process at the single cell level. We find that regulatory proteins associate with target chromatin via a probabilistic mechanism that produces cell to cell variability in steady-state binding. This highly dynamic and probabilistic behavior of multiple regulators at target chromatin supports the return to template transcription model. 3) NF-kB can control its own activity by inducing inhibitor genes, producing negative feedback loops. We monitored endogenous NF-kB behavior using a GFP knock-in system. Oscillations in nuclear NF-kB were found to be sustained, with several cycles of transient nuclear translocation. Inhibition of negative feedback disrupted the oscillations and genome-scanning activities. This work contradicts widely held assumptions regarding NF-kB function. 4) Circulating glucocorticoids have been shown to be released from the adrenal gland in a highly pulsatile, or ultradian, secretion mode. The dynamics of GR interaction with regulatory elements during these cycles was found to fluctuate in concert with the changes in extracellular hormone concentration. Transcription rates for GR target genes (determined by nascent RNA) were shown to vary markedly between ultradian and constant ligand treatment regimens, leading to the postulate that the ultradian mode of hormone secretion is required for biologically accurate regulation of gene targets by GR.
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