Evolution of Enhancer Function in the Immunoglobulin Heavy Chain Gene

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9807531 Warr Transcriptional control is now recognized to be important for all aspects of immunoglobulin (Ig) gene expression: that is, enhancer function is important not just for driving functional expression of the gene, but it is also essential for the processes of V (D) J recombination, heavy chain class switching, and somatic hypermutation of the Ig genes to occur. The channel catfish provides a well-developed and unique model to study the function of Ig genes at the phylogenetic level of teleost fish: the immunobiology of this species has been extensively studied and well documented, and the Ig genes (for mu, delta, and L chains) have all been cloned. The overall objective of the project is to understand the function of the IgH enhancer of the channel catfish, which has been shown in prior studies to occupy an unusual position in the locus, and to possess an unusually diffuse structure. The following questions will be addressed in this project. 1) Which motifs in the catfish enhancer are important in driving transcription? 2) What is the role played by Oct1 in driving expression from the promoter-associated octamer found in all VH genes (including those of the catfish)? Is a co-activator such as Bob 1 involved in the function of Oct1 and Oct2 in the catfish? 3) What is the nature of the factors (other than octamer transcription factors) that may drive transcription from the catfish enhancer? The approaches to be employed include site-directed mutagenesis to delete individual or multiple motifs (such as octamer, muE3, muE5) from the enhancer to determine their contribution to function, and the cloning of transcription factors (such as Oct1) and the co-activator Bob-1 (a co-activator of Oct transcription factors) from the catfish, in order to define their roles in driving transcription of the Ig genes at this level of evolution. Antibodies have two polypeptide chains, a heavy chain and a light chain. The expression of heavy chain genes occurs largely at the level of transcript ion, the process by which information in DNA is copied into mRNA. Transcription is regulated by sequences in the DNA, called enhancers, which interact with protein transcription factors. This project is investigating the enhancers and transcription factors that regulate the expression of antibody heavy chains in catfish.