Several autoimmune diseases such as: rheumatoid arthritis, multiple sclerosis, and insulin- dependent diabetes mellitus are associated with specific alleles of particular class II major histocompatibility complex (MHC) molecules. The aberrant expression of the class II MHC molecules is implicated in the pathogenesis of certain autoimmune diseases and lack of expression of these molecules is the cause of the severe combined immunodeficiency disorder: Bare Lymphocyte Syndrome. Overexpression of class II molecules is also thought to contribute to the rapidity of transplantation rejection. It is therefore of broad interest to dissect the molecular basis of the transcriptional regulation of these genes. The long-term objectives of these investigations are to identify important transcriptional regulators of human class II MHC genes, to elucidate how they participate in this process, and to make use of these data to attempt to design novel approaches to affect expression of these genes in the context of immunologic disease. The applicant has focused on detailed studies of two transcription factors that he has identified and demonstrated to participate in the regulation of the human class II MHC gene, HLA-DRA. This proposal focuses on one of these proteins, NFX.1, which is a 1104 amino acid, cysteine-rich protein that interacts with the conserved X-box motif of all human class II MHC genes and which functions as a transcriptional repressor. This protein contains a reiterated LIM/Ring finger-like motif, and is overexpressed late after incubation of cells with gamma-interferon. Building upon completed structure/function studies, this research program will focus on four specific aims. (1) The expression pattern of the NFX.1 polypeptide will be assessed in a variety of cells (by Western blot analysis) and tissue sections (by immunohistochemistry) using two polyclonal antisera. Possible post-translational modifications will be assessed by immunoprecipitation analysis. (2) The mechanism of sequence-specific binding will be investigated using a combination of chemical cleavage and NMR studies. Attempts at cocrystallizing the protein with the binding site will also be initiated. (3) The mechanism of transcriptional repression will be investigated using both in vitro and in vivo approaches. (4) A recently isolated NFX.1 human genomic clone will be characterized with particular attention to its proximal promoter. It is hypothesized that this focused analysis of NFX.1 and its gene will contribute to our understanding of the biology of class II MHC gene regulation, and will form the foundation for future strategies to use NFX.1 in targeted immunomodulation.
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