The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK–BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK–BMAL1 to close the negative feedback loop and generate 24-hour timing is not known. Here we show that CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK–BMAL1. Mutations within the TAD that alter affinities for coregulators change the balance of repression and activation to consequently change intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators and highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing.

SUMMARY The circadian clock orchestrates global changes in transcriptional regulation on a daily basis via the bHLH-PAS transcription factor CLOCK:BMAL1. Pathways driven by other bHLH-PAS transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for CLOCK:BMAL1. We show here that the cancer/testis antigen PASD1 fulfills this role to suppress circadian rhythms. PASD1 is evolutionarily related to CLOCK and interacts with the CLOCK:BMAL1 complex to repress transcriptional activation. Expression of PASD1 is restricted to germline tissues in healthy individuals, but can be induced in cells of somatic origin upon oncogenic transformation. Reducing PASD1 in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. Our results describe a function for a germline-specific protein in regulation of the circadian clock and provide a molecular link from oncogenic transformation to suppression of circadian rhythms.