Regulated expression of the interleukin-2 (IL2) gene is critical for T cell proliferation in response to antigenic challenge. Considerable recent evidence suggests that sophisticated regulation of the expression of genes is dependent not only on positive transcription factors, but negatively acting ones as well. While several IL2 promoter positive regulatory sequences and binding proteins important for stimulated IL2 expression have been defined, little is known of the nuclear events responsible for the rapid shut-off of IL2 gene expression following activation. Furthermore, the mechanisms responsible for restriction of IL2 gene expression to activated T cells are poorly understood. We have screened an expression library derived from a human T cell leukemia line to identity a cDNA encoding a previously unknown DNA binding protein with zinc finger motifs which appears to be a negative regulatory factor (NRF-1). NRF-1 is a T cell nuclear protein which specifically binds to a negative regulatory element (NRE-A) within the interleukin-2 (IL2) gene promoter. Mutation of NRE-A and a related IL2 promoter domain 400 bp upstream (NRE-B) leads to 9 fold over-expression of transfected IL2 promoter-reporter gene constructs in stimulated Jurkat T cells. Our hypothesis is that NRF-1 is a transcription factor which plays a major role in the negative control of IL2 gene expression. We will identity the genomic DNA for NRF-1, characterize its structure, and study its regulated expression with RNA gel blots. Functional assays to define the role of this gene's encoded protein in the regulation of IL-2 gene expression will be conducted in T cells and non-T cells stably and transiently transfected with NRF-1 expression constructs. In vitro DNA binding assays and in vivo transient expression experiments will enable us to map DNA-binding and transcription regulatory domains of NRF-1 and to compare the functional relationships between NRE-A and NRE-B and positive IL2 promoter regulatory domains. Detailed knowledge of the function of nuclear factors such as NRF-1 which inhibit IL2 gene expression will enhance our understanding of normal IL2 gene regulation and may aid in the design of therapeutic strategies aimed at augmenting or suppressing immune function in disease states.
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