We are analyzing the regulation of cytokine and chemokine gene expression in lymphoid cells. We have chosen IFN-gamma gene expression as a model system for analysis of the control of gene expression in T cells and NK cells. We are continuing to dissect the regions of the human IFN-g genomic DNA to determine which regions enhance/repress gene transcription in response to extracellular signals. In particular, we are utilizing NK cell lines to elucidate the mechanisms, both transcriptional and post-transcriptional, by which interleukins 2,12,15,18 or activation of LY49 activating receptors (murine models) induce or inhibit IFN-g gene expression and effect NK cell biology. We are investigating the role of STAT, NFkB and T-bet proteins in regulating IFN-g expression and how a highly conserved element in the 3' untranslated region may affect IFN-g mRNA stability. We are also characterizing the biochemical pathways involved in the synergistic induction of IFN-g gene expression in response to PMA or bryostatin + IL-12, IL-2 + IL-12, IL-2 + IL-18 and antibodies to the LY49 activating receptors + cytokines by both microarray and proteomic approaches. As a second approach, we have target a 100 bp region of the murine IFN-g 3' untranslated region for deletion as this region is highly conserved upon evolution. The construct has been successfully made and preliminary data indicates that this mouse produces significantly more IFN-g upon treatment with IL-12 or IL-18. Furthermore T cell homeostasis has been disrupted as increased CD4+ and CD8+ T cells are present and the T reg cells in the mouse have more potent suppressor activity. We have also identified a possible RNA binding protein that interacts with this region and are beginning biochemical experiments to determine the specificity of this interaction. As a third approach towards understanding the regulation of IFN-g gene expression, we are investigating if microRNAs may target the IFN-g mRNA. Bioinformatics analysis strongly suggests that the IFN-g gene maybe a target for microRNAs. To address this possibility, we have generated a stable siRNA transfectant of the human NK cell line NK92 where dicer, an enzyme critical for microRNA processing, has been significantly reduced. Preliminary results indicate that IFN-g expression may be increased in response to specific stimuli, thus implicating microRNAs in the regulation of IFN-g expression. In summary, our approaches towards elucidating the multiple mechanisms involved in the regulation of IFN-g demonstrates the complexity by which gene expression is regulated in immune effector cells.
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