Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) defeat immunogenic tumors and virus infections. Physiologically, most CTL reside in the body as pre-effectors (pre-CTL) that are unable to kill. When the immune system is confronted with intracellular pathogens, preCTL are expanded and armed. During this process many genes are newly expressed or upregulated in preCTL, leading to their proliferation and differentiation into effector CTL(eff CTL). One of these genes is perforin, a membrane """"""""perforating"""""""" killer molecule essential for the function of the adaptive immune system. In contrast, NK cells, perhaps best thought of as primordial CTL that lack receptors for a specific antigen, constitutively express perforin and kill without activation. Our experiments have shown that perforin gene expression is determined at the transcriptional level and that this process requires regulatory domains in addition to the promoter. This application will continue to identify and characterize these regulatory domains by elucidating constitutive and inducible nuclear events at the perforin gene locus in NK cells and CTL. Molecules involved in this process ultimately may be of therapeutical use or may become therapeutical targets. From the immunological and developmental point of view, the regulatory domains ultimately may shed light on transcription factors that control not only the perforin gene but also the commitment to and progression of the perforin expressing lineages during hematolymphopoiesis. This application specifically proposes to: 1) explore the presence of additional regulatory domains within the boundaries of an active perforin gene locus in tumor cell lines; 2) characterize the function of potential regulatory domains in tumor cell lines; and 3) demonstrate the function of the regulatory domains in normal cells. To achieve these aims we will: 1) perform a DNase I sensitivity and hypersensitivity analysis of the perforin gene locus; 2) analyze potential regulatory domains by reporter gene assays; and 3) generate and analyze mice that are transgenic for regulatory DNAs identified in the first two aims.
