Intergenic region between TATA-box binding protein and proteasome subunit C3 genes of Medaka function as the bidirectional promoter in vitro and in vivo.

In the genome of eukaryotic organisms, each protein-coding gene has the unique promoter in the 5'-flanking region, and the direction of the promoter is usually controlled unidirectional. In this study, we revealed that the intergenic region between TATA-box binding protein (tbp) and proteasome subunit C3 (psmc3) genes in Medaka functions as bidirectional promoter in vitro and in vivo. The tbp and psmc3 genes were allocated as a head-to-head configuration with a 719bp intergenic region. A comparative analysis of gene arrangement surrounding loci of tbp in vertebrates also illustrated that it was unique in Acanthopterygii lineage. The transcription activities were about 1.2 times for tbp direction and 0.7 times for psmc3 direction against that of SV40 promoter in Medaka fibroblasts, respectively. A dual fluorescent reporter assay directly showed that the bidirectional promoter could express two divergent genes concurrently without disruption of RNA polymerase II elongation. In addition, an analysis of sequential deletion of this promoter suggested that the ETS binding site was necessary for maximum expression of downstream gene, and only the ETS binding site was shared from fish to mammals. In mammals, high correlation with CpG islands was observed in such bidirectional promoters, no association was found in the tbp/psmc3 bidirectional promoter in Medaka. These results suggest that molecular machineries of fish bidirectional promoter may be somehow different from those of mammals but the cis-acting element for binding ETS transcription factors is essential for divergent gene expression.

Characterization of a nervous system-specific promoter for growth-associated protein 43 gene in Medaka (Oryzias latipes).

Genes expressed by neurons are controlled by specific, interacting cis-regulatory elements and trans-acting factors within their promoters. In the present study, we asked whether the transcriptional machinery regulating neuron-specific gene expression was conserved in evolution. We identified a GAP-43 homolog in Medaka (Oryzias latipes), and analyzed its expression during various stages of development. Compared with the amino acid sequences of GAP-43 homologs in other vertebrates, the amino-terminus of GAP-43 was highly conserved evolutionarily, but the carboxy-terminus exhibited significant variability. Expression of GAP-43 predominantly occurred in cells of the central and peripheral nervous systems as determined by in situ hybridization and by RT-PCR. Expression of GAP-43 increased throughout development and significant levels continued to be expressed into adulthood. We also showed that a proximal approximately 2.0 kbp fragment in the 5'-flanking region had promoter activity as determined by in vivo reporter assays. Furthermore, based upon computational analysis of transcription factor binding sites and an in vivo reporter analysis using sequentially deleted promoters, we demonstrated that cis-regulatory elements for neuronal expression were widely distributed in this region. In mammals, a TATA-box, E-box and neuronal repressive elements have been thought to contribute to neuronal expression. However, these features were not found in the orthologous region of the Medaka GAP-43 promoter. Our results suggest that the arrangement of cis-regulatory elements of the GAP-43 ortholog in Medaka is different from that in mammals, yet maintains neuron-specific regulation.