The transcriptional enhancer (Eμ3′) of the IgH locus of the channel catfish, Ictalurus punctatus, differs from enhancers of the mammalian IgH locus in terms of its position, structure, and function. Transcription factors binding to multiple octamer motifs and a single μE5 motif (an E-box site, consensus CANNTG) interact for its function. E-box binding transcription factors of the class I basic helix-loop-helix family were cloned from a catfish B cell cDNA library in this study, and homologs of TF12/HEB were identified as the most highly represented E-proteins. Two alternatively spliced forms of catfish TF12 (termed CFEB1 and -2) were identified and contained regions homologous to the basic helix-loop-helix and activation domains of other vertebrate E-proteins. CFEB message is widely expressed, with CFEB1 message predominating over that of CFEB2. Both CFEB1 and -2 strongly activated transcription from a μE5-dependent artificial promoter. In catfish B cells, CFEB1 and -2 also activated transcription from the core region of the catfish IgH enhancer (Eμ3′) in a manner dependent on the presence of the μE5 site. Both CFEB1 and -2 bound the μE5 motif, and formed both homo- and heterodimers. CFEB1 and -2 were weakly active or inactive (in a promoter-dependent fashion) in mammalian B-lineage cells. Although E-proteins have been highly conserved in vertebrate evolution, the present results indicate that, at the phylogenetic level of a teleost fish, the TF12/HEB homolog differs from that of mammals in terms of 1) its high level of expression and 2) the presence of isoforms generated by alternative RNA processing.

The transcriptional enhancer (E3) of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian E enhancer in both location and structure. It occurs between the and ␦ genes and contains numerous transcription factor binding sites, predominantly octamer and E5 motifs of consensus and variant sequences. It lacks the classical A-E3(CBF)-B core array of binding motifs seen within mammalian IgH E 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 E5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus E5 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 E3 enhancer (a) contains a consensus E5 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.Teleost (bony) fishes represent the evolutionary lineage that is most divergent from mammals yet still shares with them the organization of the IgH 1 locus. In both mammals and bony fish the V H , 1 D, J H , and C gene segments are in tandem arrays, also known as the translocon arrangement (1). Within the IgH locus of mammals, there are six described enhancers, required for the processes of V-D-J recombination, class switching, somatic hypermutation, and expression of the functionally rearranged locus (2-4). These enhancers are both internal to and 3Ј of the locus and are tightly regulated in function at different stages of B cell development (5, 6). The E enhancer, located in the J H -C intron, is arguably the most important; not only is it required for expression of the rearranged IgH locus, but it also plays an important role in the processes of V H -D-J H recombination and somatic hypermutation and is not deleted by any of the recombination processes that occur during B cell development. The IgH locus of the channel catfish, the best-established model for the teleost immune system, is less complex than that of mammals; only two classes of immunoglobulin (IgM and IgD) are produced through alternative processing of the primary transcript from the IgH locus (7,8). Class switching by chromosomal recombination does not occur in teleost fish. The single identified enhancer (E3Ј) driving IgH transcription is located in the -␦ intergenic region (9). The structure and organization of E and E3Ј differ greatly. Mammalian E is ϳ500 bp in length and contains several DNA binding mot...

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