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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
First Independent Research Support & Transition (FIRST) Awards (R29)
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Immunobiology Study Section (IMB)
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Vanderbilt University Medical Center
Schools of Medicine
United States
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