Journal
MOLECULAR IMMUNOLOGY
Volume 45, Issue 14, Pages 3786-3796Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.molimm.2008.05.018
Keywords
early B cell factor (EBF); mb-1 promoter; DNA-binding specificity; Vpreb 1 promoter; zinc-binding motif
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Funding
- National Health Service [R01 AI54661, R01 AI56322, P01 AI22295]
- Rocky Mountain Chapter of the Arthritis Foundation
- [5 T32 AI007405]
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Early B cell factor (EBF) is a critical regulator of B lymphocyte-specific gene transcription. EBF functions, in part, by binding to regulatory sites of genes required for the pre-B- and mature B cell receptors. These DNA targets include the promoters of the mb-1 and Vpreb1 genes that encode Ig-alpha and one of the components of surrogate light chain, respectively. The biochemical basis of DNA binding and gene activation by EBF is poorly under-stood. The DNA-binding domain (DBD) of EBF includes a putative zinc-binding motif (HX(3)CX(2)CX(5)C), which we have designated the 'Zn-knuckle'. The Zn-knuckle is required for binding of the mb-1 promoter site in EMSA, but it has not been demonstrated to be important for functional activities of EBF in B cells. Therefore, we expressed EBF with mutations in the Zn-knuckle motif or flanking sequences in plasmacytoma cells in which activation of endogenous mb-1 and Vpreb1 genes is dependent on EBF EBF with mutations that prevent zinc coordination by the Zn-knuckle did not activate transcription of either target gene. Other mutations affected the sequence preference of DNA binding and differentially inhibited activation of these genes. Our results demonstrate the importance of the Zn-knuckle motif in EBF. These experiments also confirm that EBF can re-activate multiple genes of the early B cell program in plasmacytoma cells, which provide a useful cell-based assay for dissecting mechanisms involving EBF. (C) 2008 Elsevier Ltd. All rights reserved.
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