57Arg in the bHLH transcription factor DEC2 is essential for the suppression of CLOCK/BMAL2-mediated transactivation

Abstract

The basic helix-loop-helix (bHLH) transcription factors, DEC2 and DEC1, play critical roles in the circadian rhythm of the suprachiasmatic nucleus (SCN). It is known that mammalian circadian rhythms are regulated by molecular clockwork systems based on a negative-feedback loop, and CLOCK/BMAL1 and CLOCK/BMAL2 enhance DEC2 transcription via CACGTG E-boxes. To understand the role of arginine 57 (57Arg) within the basic region of DEC2, we examined the effect of substituting this residue into DEC2 on CLOCK/BMAL2-mediated transactivation. A luciferase assay showed that substituting 57Arg for Ala or Lys in DEC2 diminished the suppressive activity of wild-type DEC2 on CLOCK/ BMAL2-mediated transactivation, while substituting 48Pro for Ala in DEC2 did not alter it, and the same was true for wild-type DEC2. We also showed that proteins which were wildtype and substitution mutants of DEC2 were expressed at nearly equivalent levels by Western blotting. These findings demonstrate that 57Arg in the basic region of DEC2 is essential for its activity in suppressing CLOCK/BMAL2-mediated transactivation.