Zebrafish CRY represses transcription mediated by CLOCK-BMAL heterodimer without inhibiting its binding to DNA

Background: CLOCK and BMAL1 proteins, members of the basic helix-loop-helix PAS (PER-ARNT-SIM) superfamily of transcription factors which bind to the E-box DNA motif, are required for the high-level expression of the circadian clock genes period (per) and cryptochrome (cry). CRY inhibits transcriptional activity of the CLOCK-BMAL1 heterodimer, generating a negative-feedback loop that is the core element of the circadian oscillator. Results: We show that zebrafish CRY (zCRY1a) neither disrupts the association between zfCLOCK and zfBMAL nor inhibits binding of the zfCLOCK-zfBMAL heterodimer to an E-box-bearing DNA fragment. Instead it binds to the heterodimer to form a stable zCRY1a-zfCLOCK-zfBMAL-E-box complex. Another zebrafish CRY protein, zCRY4, does not have transcriptional inhibitor activity, whereas zCRY1a has strong activity. zCRY4 does not associate with zfCLOCK and zfBMAL. We also show that the presence of a chemical reductant in the reaction mixture is crucial for efficient binding of the CLOCK-BMAL heterodimer to E-box bearing DNA, which is indicative of the reduction/oxidation (redox)-sensitive character of the heterodimer. Conclusions: Our findings suggest that CRY represses CLOCK-BMAL-mediated transcription by interacting directly with the zfCLOCK-zfBMAL-E-box complex.