Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector-based strategy

In mammals, the expression of 5-10 of genes occurs with circadian fluctuation in various organs and tissues. This cyclic transcription is thought to be directly or indirectly regulated through circadian transcriptional/translational feedback loops consisting of a set of clock genes. Among the clock genes in mammals, expression of the Dbp mRNA robustly oscillates both in vivo and in culture cells. Here, we present circadian enhancer detection strategy using prokaryotic transposon system. The mDbp promoter drives reporter gene expression in robust circadian cycles in rat-1 fibroblasts. To identify the circadian enhancer generating this robust rhythm, we developed a prokaryotic transposon-based enhancer detecting vector for in vitro transposition. Using this system, we identified a strong circadian enhancer region containing the CATGTG sequence in the 5' flanking region of the mDbp gene; this enhancer region is critical for the ability of the mDbp promoter to drive robust oscillation in living cells. This enhancer is classified as a CANNTG type non-canonical E-box. These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression. Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.