Transcriptional regulation of a gene is controlled by nuclear factors (proteins) that bind to cis-active regulatory DNA sequences. One model for such gene regulation in the immune system is the control of class II MHC gene expression in B lymphocytes, which is developmentally stage-specific and responds to a number of well-defined stimuli, such as the cytokine interleukin 4 (IL- 4). We have identified a nuclear protein called NF-BRE (Nuclear Factor binding to a B-cell Regulatory Element). This protein binds to a regulatory region upstream from the murine class II MHC gene Aa. One hypothesis of this proposal is that NF-BRE is an important transcription factor because: (1) it is present in B cell lines that constitutively express Aa but undetectable in myeloma cell lines that lack class II gene expression, and (2) it is induced by IL-4, which is known to enhance class II expression. An analysis of the structure, expression, and mechanisms of action of NF-BRE will enhance our understanding of how class II genes are regulated during immune responses. Because of the pleiotropic effects of transcription factors, the potential role of NF-BRE as a mediator of other specific responses to IL-4 will also be investigated. The objective of this proposal is to examine the structure and function of this DNA-binding protein, NF-BRE. We will first establish the effects of NF-BRE on the transcriptional activity of class II regulatory elements and on promoters from other genes. This analysis will be performed using transfection assays with a reporter gene, CAT (choramphenicol acetyl transferase). Such assays will be performed in B lineage cells to define the role of NF-BRE in the developmental stage-specific control of class II MHC gene expression. A second goal of this proposal is to determine whether NF-BRE is present in T cells, which also respond to IL-4, and to characterize the effect of interferon gamma (INF-gamma) on NF-BRE, since INF-gamma is known to block IL-4 induction of B-cell class II MHC expression. Detailed analyses of the role of this protein in transcription control will benefit from the availability of cDNA clones that encode NF-BRE. A major goal of the proposal is to isolate full-length cDNA clones that encode NF-BRE, using methods that have been successful in cloning DNA-binding proteins in our work and with other binding proteins. Transfection experiments with NF-BRE cDNA and myeloma cell lines will be used to demonstrate directly that NF-BRE is a transcription factor. We will further test the hypothesis that NF-BRE is present in non-B cells by examining the constitutive and IL-4 inducible tissue distribution of NF-BRE expression with cloned cDNA probes. Structural analyses of NF-BRE protein in those cell types that contain NF-BRE RNA will be performed using antisera directed against cloned NF-BRE. We will test whether altered forms of NF- BRE cDNA can block transcription of class II MHC and other IL-4 inducible genes. These studies will provide insights into the mechanism of transcriptional regulation of immunologically important genes such as those of the MHC.
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