As part of a search for genes expressed late (3-5 days) after T cell activation, our group identified, cloned, and partially characterized two genes, granulysin and RANTES, that function as immunoregulatory molecules. Granulysin is a cytolytic molecule produced by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells with activity against a variety of microbes and tumors. It also functions as a chemoattractant and activating molecule for a variety of lymphoid cells, including memory T cells, NK cells, and immature dendritic cells. Granulysin kills human tumor cells by initiating a cascade of intracellular events that involve the influx of calcium, efflux of potassium, damage to mitochondria, and apoptosis. The mechanisms by which granulysin kills microbes have not yet been elucidated. We prepared a panel of synthetic peptides based on the primary sequence of granulysin. Some of these are potent antimicrobial agents while others selectively kill tumor cells. Since granulysin is not found in mice, we generated mice expressing the human granulysin gene to evaluate its effects in vivo. RANTES is a chemokine expressed 3-5 days after T cell activation. Our analysis of the RANTES promoter revealed that the transcription factor KLF13 is the major activator of RANTES expression in T cells, while rel proteins are the major inducers of the immediate increase in RANTES observed in other cell types such as fibroblasts and monocytes. Kinetic analysis of the RANTES promoter for 7 days after T cell activation showed that a variety of transcription factors move on and off the promoter over time and that phosphorylation, acetylation, and deacetylation are all important for RANTES transcription. To study more closely the role of KLF13 in vivo, mice in which the RANTES gene was disrupted were generated. Using DNA microarrays comparing wild type and KLF13 knockout animals, we found that KLF13 is involved in the transcriptional regulation of many genes. For example KLF13 is a negative regulator of BCLXL, a potent regulator of apoptosis. In August 2007, my group relocated from Stanford University to the NCI. Thus, our efforts have mainly focused on equipping the laboratory and hiring personnel. We began experiments in October and are actively pursuing studies in the following areas: 1. Use the KLF13 knockout mice to identify other genes regulated by KLF13 and to evaluate these animals in animal models of human disease. 2. Identify promoters associated with KLF13 in human lymphocytes using chromatin immunoprecipitation assays. 3. Determine the mechanisms by which granulysin and the peptide derivatives kill bacteria and yeast. 4. Investigate the roles of perforin and granzyme b in granulysin-mediated killing of target cells using granulysin transgenic mice bred onto perforin and/or granzyme b knockout mice. These studies are aimed at designing new therapeutics for human diseases as diverse as cancer, tuberculosis, malaria, AIDS, and autoimmune diseases including diabetes, rheumatoid arthritis, and mutliple sclerosis.
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