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J Biol Chem, Vol. 273, Issue 29, 18647-18655, July 17, 1998

The Transcription Factor NF-B/p50 Interacts with the blk Gene during B Cell Activation

Patty Zwollo, Sulekha Rao^¶, Jeffrey J. Wallin^¶, Edwin R. Gackstetter^¶, and Marian E. Koshland^¶

From the  Department of Biology, The College of William and Mary, Williamsburg, Virginia 23187 and the ^¶ Department of Molecular and Cellular Biology, the University of California, Berkeley, California 94720

The B cell-specific transcription factor Pax-5 has been shown previously to interact with the promoter of the blk gene in vitro. blk encodes a tyrosine kinase associated with the B cell receptor, which is expressed during the early but not the final stages of B cell development. To investigate whether Pax-5 regulates expression of the blk gene in vivo during B cell development and/or activation, Pax-5a was overexpressed in B cell lines. Increases in blk promoter activity using a chloramphenicol acetyltransferase reporter gene system suggested a role for Pax-5a as a transcriptional activator. Subsequent site-specific mutagenesis studies showed that mutations of the Pax-5 binding site on blk significantly alter promoter activity, although results suggested that other factors could bind to this region as well. Using mobility shift assays, we detected an inducible transcription factor that interacts strongly with a sequence overlapping the Pax-5 site on the blk promoter and identified this as a homodimer of NF-B/p50, a member of the NF-B/Rel family of transcription factors. This factor was present at high levels in lipopolysaccharide-activated normal B cells and in plasma cell lines but either at low levels or undetectable levels in resting normal B cells or pre-B or mature B cell lines. In contrast, lipopolysaccharide induction of a pre-B cell line (703/Z) induced a complex that contained both NF-B/p50 and p65. These studies suggest that different NF-B complexes are able to interact with a sequence overlapping the Pax-5 site on the blk promoter and that the relative levels of "bound" factor influence levels of blk expression. Since p50 homodimers and p50/p65 heterodimers of the NF-B complex should have opposing effects on blk transcription, this could provide a mechanism to differentially regulate blk expression during B cell development and activation.

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