Medaka vasa gene has an exonic enhancer for germline expression

Enhancers are DNA sequences that enhance gene transcription in a position- and orientation-independent manner. Many enhancers controlling somatic gene expression have been described. Enhancers controlling germline expression have remained rare. Here we report the identification of V35 as a first exonic germline enhancer in vertebrates. V35 constitutes the first 35 bp of exon 1 of the medaka vasa gene. V35 is required for vasa promoter function and sufficient to increase transcriptional activity of a heterologous promoter by ~ 13 fold in either forward or reverse orientation. V35 contains CAGCAGCACGAG for two paired E box-like motifs. Upon incubation with nuclear extract from spermatogonial cells, V35 formed three DNA-protein complexes. We show that complex formation is inhibited partially by oligos containing an E box or E box-like motif but completely by V35 and oligos that contain overlapped E box and E box-like motifs. Most importantly, V35 is sufficient to drive transgene expression in germ cells of developing embryos. These results establish V35 as the first exonic germline enhancer in a lower vertebrate, and provide evidence for the importance of exonic sequences in controlling germ gene expression.