X-linked immunodeficient (xid) mice and humans with X-linked agammaglobulinemia (XLA), exhibit lower levels of serum immunoglobulin than normal individuals. Many patients lack detectable B lymphocytes. Bruton's tyrosine kinase (BTK) is mutated in both xid and XLA, but the mechanism by which mutations in this kinase cause B cell defects is unknown. Bright, (B cell regulator of immunoglobulin heavy chain transcription), is a 70 kDa DNA-binding protein expressed primarily in B lymphocytes. It binds to several regions within the murine immunoglobulin heavy chain locus, and has been associated with increases in immunoglobulin RNA levels. Bright expression and DNA-binding activity can be induced in normal adult spleen screens by a number of stimuli. Recent studies showed that stimulated lymphocytes from xid mice did not produce Bright DNA-binding activity; even though Bright protein was not present. Other experiments suggest that Bright and BTK interact directly in normal mice. Thus, Bright activity may require a functional BTK. The proposed studies will address the hypothesis that defective BTK leads to inactive forms of Bright that might at least partially explain the low serum immunoglobulin levels observed in xid, and by extension, XLA.
The specific aims are. 1) to investigate the importance of BTK for Bright DNA- binding activity by co-expression, immunoprecipitation, and identification of post-translational modifications, 2) to determine if functional Bright is present in B cells from XLA patients using mobility shift assays, 3) to determine the relationship of Bright to the xid phenotype by producing dominant negative forms of Bright and expressing then in transgenic mice, 4) to determine how Bright functions in the immunoglobulin locus using in vitro transcription and topoisomerase assays, and 5) to identify additional genes potentially regulated by Bright interactions by mobility shift assay and antibody facilitated cloning. These studies will provide important new insights into Bright's potential role in xid and the human immunodeficiency disease, XLA, and will contribute to our understanding of immunoglobulin gene regulation.
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