AT SPECIFIC DNA BINDING PROPERTIES OF A HUMAN PROTEIN

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DESCRIPTION: The HMG-I(Y) group of mammalian "high mobility group" (HMG) chromatin proteins are in vivo regulators of gene expression and founding members of a new class of nonhistone proteins called 'architectural transcription factors'. The investigator has demonstrated in human T cells that HMG-I(Y) proteins participate, along with transcription factors NF-kB, Elf-1 and SRF, in the initial in vivo transcription induction of the interleukin-2 receptor a-chain gene (IL-2Ra), an essential controlling step in mounting an effective immune reaction. The goals of the proposed research are to elucidate, at the molecular/mechanistic level, how different regions of the HMG-I(Y) protein function in the transcriptional regulation of the IL-2Ra gene promoter. The investigator has produced a large number of both variant HMG-I(Y) proteins and mutant IL-2Ra promoter DNAs for use in both in vitro and in vivo investigations of the protein-DNA complexes formed on the IL-2Ra promoter during transcriptional activation. The Specific Aims of the research are to use these mutant proteins and promoter DNAs to: 1) Determine the relative importance of different peptide domains of the HMG-I(Y) protein in the formation of a looped, stereospecific activation complex on the promoter of the human IL-2Ra gene and investigate the molecular events involved in such complex formation in vitro and in vivo. 2) Determine the effects of site-specific HMG-I(Y) protein phosphorylations on protein-protein and protein-DNA interactions during promoter complex formation in vitro and in vivo. 3) Investigate the role played by HMG-I(Y) protein-induced alterations of the nucleosomal chromatin structure of the IL-2Ra promoter region during gene transcriptional activation in vitro and in vivo. A variety of extremely sensitive and quantitative biochemical, biophysical, and biological techniques will be employed to analyze the ability of mutant HMG-I(Y) proteins to participate in IL-2Ra promoter complex formation and function. Results from these experiments will contribute significant new information concerning the regulated expression of the IL-2Ra gene, one of the rate limiting steps in T cell activation during induction of the immune response.