An IgH enhancer that drives transcription through basic helix-loop-helix and oct transcription factor binding motifs - Functional analysis of the E mu 3 ' enhancer of the catfish

The transcriptional enhancer (E mu3') of the IgH locus of the channel catfi sh, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian E mu enhancer in both location and structure. It occurs between the mu and delta genes and contains numerous transcription factor b inding sites, predominantly octamer and mu E5 motifs of consensus and varia nt sequences. It lacks the classical muA-mu E3(CBF)-mu beta core array of b inding motifs seen within mammalian IgH E mu enhancers. To determine the fu nctionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of conse nsus and variant octamer motifs (individually or in multiples) decreased en hancer function, mutation of a single consensus mu E5 motif destroyed the f unction of this enhancer in mammalian plasmacytomas. Mutation of this conse nsus mu E5 site, combined with mutations of certain octamer sites, destroye d function in catfish B cells. Experiments using artificial enhancers conta ining multimers of motifs or short regions of the native enhancer suggested that the minimal E mu3' enhancer (a) contains a consensus mu E5 site and t wo 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 confirm s large differences in structure and function between fish and mammalian Ig H enhancers.