We determined that two mouse cryptochrome genes, mCry1 and mCry2, act in the negative limb of the clock feedback loop. In cell lines, mPER proteins (alone or in combination) have modest effects on their cellular location and ability to inhibit CLOCK:BMAL1 -mediated transcription. This suggested cryptochrome involvement in the negative limb of the feedback loop. Indeed, mCry1 and mCry2 RNA levels are reduced in the central and peripheral clocks of Clock/Clock mutant mice. mCRY1 and mCRY2 are nuclear proteins that interact with each of the mPER proteins, translocate each mPER protein from cytoplasm to nucleus, and are rhythmically expressed in the suprachiasmatic circadian clock. Luciferase reporter gene assays show that mCRY1 or mCRY2 alone abrogates CLOCK:BMAL1-E box-mediated transcription. The mPER and mCRY proteins appear to inhibit the transcriptional complex differentially.

We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

We examined the transcriptional regulation of the clock-controlled arginine vasopressin gene in the suprachiasmatic nuclei (SCN). A core clock mechanism in mouse SCN appears to involve a transcriptional feedback loop in which CLOCK and BMAL1 are positive regulators and three mPeriod (mPer) genes are involved in negative feedback. We show that the RNA rhythm of each mPer gene is severely blunted in Clock/Clock mice. The vasopressin RNA rhythm is abolished in the SCN of Clock/Clock animals, leading to markedly decreased peptide levels. Luciferase reporter gene assays show that CLOCK-BMAL1 heterodimers act through an E box enhancer in the vasopressin gene to activate transcription; this activation can be inhibited by the mPER and mTIM proteins. These data indicate that the transcriptional machinery of the core clockwork directly regulates a clock-controlled output rhythm.