Cytokine gene expression is one of the most important functions of helper T-cells and one of the most precisely regulated. While much is known about the mechanisms controlling cytokine gene expression in response to stimulation, the developmental aspects of this function have remained more obscure. The well-characterized enhancer of the IL-2 gene, for example, has been notoriously inefficient in regulating reporter gene expression in transgenic mice, suggesting that the regulatory sequences needed for correct initial opening of the chromatin in a developmental context might be separate from the known enhancer. Another mystery is the mechanism of the surprising """"""""monoallelic"""""""" expression that is seen in many IL-2 producing cells: does this reflect an epigenetically inherited chromatin difference between alleles, a natural version of position effect variegation? Or does this simply reflect the steeply cooperative nature of transcription factor binding to the IL-2 promoter in any given case of stimulation? The investigator has developed unique tools to address this problem in the last project period of this application. Using DNase hypersensitive site mapping, she has located a new series of candidate regulatory sequences upstream of all regions previously characterized. This analysis has guided her in generating two new transgenic constructs in which the IL-2 regulatory region drives expression of Enhanced Green Fluorescent Protein (GFP). The larger of these constructs gives cell-type-specific, activation-sensitive, approximately copy number-dependent expression in 4/4 well-studied transgenic lines and in 10/11 transgenic line overall. The fidelity of expression even at high copy number gives unparalleled sensitivity for detection of living cells that have activated IL-2 transcription factors. In the next project period, the investigator wishes to exploit the exciting opportunities that these mice give us to answer three questions: (10 what sequences are responsible for the earliest onset of IL-2 expression in primitive lymphoid precursors; (2) what new cis-elements in the IL-2 flanking sequence confer position-independent expression in a full range of appropriate cell types: and (3) whether early expression of a particular IL-2 regulatory sequence locus is associated with establishing a later preference for activation of that locus in responses to stimulation.
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