The circadian clock is driven by cell-autonomous transcription͞ translation feedback loops. The BMAL1 transcription factor is an indispensable component of the positive arm of this molecular oscillator in mammals. Here, we present a molecular genetic screening assay for mutant circadian clock proteins that is based on real-time circadian rhythm monitoring in cultured fibroblasts. By using this assay, we identified a domain in the extreme C terminus of BMAL1 that plays an essential role in the rhythmic control of E-box-mediated circadian transcription. Remarkably, the last 43 aa of BMAL1 are required for transcriptional activation, as well as for association with the circadian transcriptional repressor CRYPTO-CHROME 1 (CRY1), depending on the coexistence of CLOCK protein. C-terminally truncated BMAL1 mutant proteins still associate with mPER2 (another protein of the negative feedback loop), suggesting that an additional repression mechanism may converge on the N terminus. Taken together, these results suggest that the C-terminal region of BMAL1 is involved in determining the balance between circadian transcriptional activation and suppression.circadian clock ͉ real-time monitor T he mammalian circadian clock is a highly dynamic system that generates periodic fluctuations in the mRNA expression levels of hundreds of genes to confer near 24-h rhythmicity to behavior, physiology, and metabolic processes, thereby allowing mammals to anticipate the momentum of the day (1). The master clock resides in the suprachiasmatic nuclei (SCN) of the brain and, in turn, synchronizes circadian clocks in peripheral tissues (2). Even fibroblasts in culture contain an active circadian clock that has the same genetic makeup of the central clock in the SCN (3-6). To keep pace with the day-night cycle, the SCN clock, but not peripheral clocks, are entrained by light.Circadian rhythms are generated by a molecular oscillator that consists of intertwined positive and negative transcription͞ translation feedback loops involving a set of clock genes (7) and clock-controlled output genes that link the oscillator to clockcontrolled processes (8). BMAL1 (MOP3) and CLOCK are basic helix-loop-helix PAS transcription factors that heterodimerize and (by means of binding to E-box promoter elements) transactivate the Period (Per1 and Per2) and Cryptochrome (Cry1 and Cry2) genes and an orphan nuclear receptor Rev-Erb␣ core oscillator gene. Subsequently, PER and CRY proteins act as negative elements by inhibiting the activity of the CLOCK͞BMAL1 heterodimer, whereas REV-ERB␣ negatively regulates Bmal1 gene expression (1, 9). The above feedback mechanism is supported by biochemical, molecular, and genetic evidence; however, formal proof of its requirement in the maintenance of circadian clock oscillations has not been shown thus far.Genetic ablation of mBmal1 results in complete disruption of the mammalian circadian clock at the behavioral and molecular levels (10). However, except for the PAS elements, which are required for association with CLOCK (11)...

Light is the most important factor controlling circadian systems in response to day-night cycles. In order to better understand the regulation of circadian rhythms by light in Synechococcus elongatus PCC 7942, we screened for mutants with defective phase shifting in response to dark pulses. Using a 5-h dark-pulse protocol, we identified a mutation in kaiC that we termed pr1, for phase response 1. In the pr1 mutant, a 5-h dark pulse failed to shift the phase of the circadian rhythm, while the same pulse caused a 10-h phase shift in wild-type cells. The rhythm in accumulation of KaiC was abolished in the pr1 mutant, and the rhythmicity of KaiC phosphorylation was reduced. Additionally, the pr1 mutant was defective in mediating the feedback inhibition of kaiBC. Finally, overexpression of mutant KaiC led to a reduced phase shift compared to that for wild-type KaiC. Thus, KaiC appears to play a role in resetting the cellular clock in addition to its documented role in the feedback regulation of circadian rhythms.

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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.