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Originally published In Press as doi:10.1074/jbc.M100110200 on May 25, 2001

J. Biol. Chem., Vol. 276, Issue 30, 27825-27830, July 27, 2001
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An IgH Enhancer That Drives Transcription through Basic Helix-Loop-Helix and Oct Transcription Factor Binding Motifs
FUNCTIONAL ANALYSIS OF THE Eµ3' ENHANCER OF THE CATFISH*

Christopher C. CioffiDagger , Darlene L. Middleton§, Melanie R. Wilson, Norman W. Miller, L. William Clem, and Gregory W. Warr§||

From the Departments of Dagger  Microbiology and Immunology and § Biochemistry and Molecular Biology, The Medical University of South Carolina, Charleston, South Carolina 29425 and the  Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi 39216

The transcriptional enhancer (Eµ3') of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian Eµ enhancer in both location and structure. It occurs between the µ and delta  genes and contains numerous transcription factor binding sites, predominantly octamer and µE5 motifs of consensus and variant sequences. It lacks the classical µA-µE3(CBF)-µB core array of binding motifs seen within mammalian IgH Eµ enhancers. To determine the functionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of consensus and variant octamer motifs (individually or in multiples) decreased enhancer function, mutation of a single consensus µE5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus µE5 site, combined with mutations of certain octamer sites, destroyed function in catfish B cells. Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal Eµ3' enhancer (a) contains a consensus µE5 site and two octamer sites, (b) is B cell-specific, and (c) is active across species. The dependence of an Ig enhancer on sites that bind basic helix-loop-helix and Oct transcription factors has not previously been observed and confirms large differences in structure and function between fish and mammalian IgH enhancers.

* This work was supported by awards from the National Science Foundation (MCB9807531) and the National Institutes of Health (R01-GM62317 and R37-AI-19530).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Medical University of South Carolina, Dept. of Biochemistry and Molecular Biology, 173 Ashley Ave., P. O. Box 250509, Charleston, SC 29425; Tel.: 843-792-4321; Fax: 843-792-4850; E-mail: warrgw@musc.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.