Structure, Function, and Evolution of E-Protein Transcription Factors Driving Transcription of the Immunoglobulin Heavy Chain Locus Enhancer in Teleost Fish

Jun-ichi Hikima¹, Mara L. Lennard¹, Melanie R. Wilson², Norman W. Miller², L. William Clem², and Gregory W. Warr¹

 ¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

and Hollings Marine Laboratory, Charleston, SC

² University of Mississippi Medical Center, Jackson, MS

E-proteins of the class I basic helix-loop-helix (bHLH) family (E2A, HEB, E2-2), are of wide tissue distribution in mammals, typically exert their effects by binding to mE5 sites, and are involved in the development of the immune system. We have investigated the evolution of transcriptional control in the immunoglobulin heavy chain (IgH) locus of vertebrates, using as a model the transcriptional enhancer, Em3’, of the IgH locus of the channel catfish, Ictalurus punctatus. This enhancer functions through the interaction of transcription factors binding to multiple octamer motifs and to a single consensus mE5 site. Two catfish E-protein homologs, related to E2A and HEB were identified as highly expressed in a catfish B cell cDNA library. Regions homologous to the bHLH and activation domains of other vertebrate E-proteins were readily identified in these sequences. The catfish E-protein messages are ubiquitously expressed, being readily detected in catfish B cells, T cells, kidney, spleen, brain, and muscle. The catfish E-proteins strongly activated transcription of a mE5-dependent construct in catfish B cells, and also stimulated transcriptional activation from the core region of the catfish Em3’ enhancer. All E-protein homologues were capable of binding the mE5 motif, and of forming both homo- and heterodimers with other E-proteins. The ability of catfish E2A to homodimerize distinguishes it from its most closely-related mammalian homolog, E12. Comparative analysis of E-protein genes between Fugu and human revealed evolutionary differences. Two E2A genes were identified in Fugu; one of these showed the alternative processing of RNA transcripts and was identified as the ortholog of the human E2A (E12/E47) gene products, whereas the second Fugu E2A gene had no inferred alternative splice products. Overall, our results indicate that, although E-proteins have been highly conserved in vertebrate evolution, a detailed examination of structure/function relationships (as exemplified in the case of E2A) reveals significant evolutionary divergence within the vertebrates. Supported by awards R01 GM62317 and R01 AI19530 from the NIH.