Characterization and functional analysis of the Oct1 and BOB.1 transcription factors in the channel catfish (Ictalurus punctatus)

The intronic enhancer, Emu3' has previously been identified within the immunoglobulin heavy chain locus of the channel catfish (Ictalurus punctatus). Within the numerous potential cis-regulatory elements of this enhancer, are included two variant octamer motifs (ATGtAAAT) and a muE5 site which make up the functional core region necessary to drive transcription from this locus. The reliance on octamer motifs and a muE5 site to drive transcription represents a functional shift from that of higher vertebrates which rely primarily on Ets transcription factor binding sites and a muE3 site to drive transcription. This suggests that Oct transcription factors that bind to the octamer motif play an important role in immunoglobulin gene transcription in the channel catfish. The catfish Oct2 transcription factor was previously cloned and shown to activate transcription from the variant octamer motifs found within this enhancer. The goal of this study was to clone orthologues of the Oct1 transcription factor and the coactivator BOB.1 in the channel catfish and characterize their role in driving transcription of the immunoglobulin heavy chain locus. A combination of molecular techniques and comparative genomics were used to achieve this goal.; Catfish Oct1 was cloned and the overall gene structure was found to be highly conserved, particularly within the bipartite DNA binding domain, whose identity at the amino acid level to mouse Oct1 was 96% within the POU specific domain and 78% within the POU homeodomain. Catfish Oct1 was found to be widely expressed in all tissues and cell types tested. Surprisingly, catfish Oct1 failed to drive transcription from a number of octamer-containing reporter constructs including the core region of the Emu3' enhancer, but was capable of binding to both consensus and variant octamer motifs. The failure of catfish Oct1 to drive transcription was due to a lack of functional activation domains within either the N or C terminal regions of the protein. Catfish Oct1 acts as a dominant negative transcription factor over catfish Oct2, most likely through competition for DNA binding sites. Thus, catfish Oct1 appears to be a negative regulator of immunoglobulin gene transcription within the channel catfish.