The circadian rhythms in mammals are regulated by a pacemaker located in the suprachiasmatic nucleus of the hypothalamus. Four clock-gene families have been found to be involved in a transcription-translation feedback loop that generates the circadian rhythm at the intracellular level. The proteins Clock and Bmal1 form a heterodimer which activates the transcription of the Per gene from the E-box elements in its promoter region. Protein products of Per act together with Cry proteins to inhibit Per transcription, thus closing the autoregulatory feedback loop. We found that Dec1 and Dec2, basic helix-loop-helix transcription factors, repressed Clock/Bmal1-induced transactivation of the mouse Per1 promoter through direct protein-protein interactions with Bmal1 and/or competition for E-box elements. Dec1 and Dec2 are expressed in the suprachiasmic nucleus in a circadian fashion, with a peak in the subjective day. A brief light pulse induced Dec1 but not Dec2 expression in the suprachiasmic nucleus in a phase-dependent manner. Dec1 and Dec2 are regulators of the mammalian molecular clock, and form a fifth clock-gene family.

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DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1 ؊/؊ mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.The mammalian molecular clock system consists of various clock genes and their protein products involved in interlocked feedback loops of transcriptional and translational regulation through clock elements such as CACGTG E-box, D-box, and ROR/REV-ERB binding elements (RORE) (18,24). Among these regulatory sequences, the E box is thought to be the most important element in the molecular oscillatory system, since it is the binding site for the CLOCK/BMAL1 heterodimer, which up-regulates various clock genes, including Dec1, Dec2, Per1, Dbp, and Rev-erb␣. In this regulatory system, PER, CRY, and DEC serve as negative factors for transcription from E-boxdriven promoters, and the E-box-like element EЈ box (CAC GTT) was recently shown to be involved in the direct regulation of Per2 and Cry1 genes by CLOCK/BMAL1 (1, 31, 34). RORE, on the other hand, is a clock element to which the transcriptional activators-ROR␣/ROR␤/ROR␥-and repressors-REV-ERB␣/REV-ERB␤-bind, and ROR and REV-ERB regulate the circadian expression of Bmal1, Clock, Npas2, and Cry1 via RORE (31).In the mammalian clock system, DEC1 (also known as BHLHB2, STRA13, or SHARP2) and DEC2 (BHLHB3 or SHARP1) serve as transcriptional repressors for CLOCK/ BMAL1-enhanced promoter activity, through binding to E boxes or interaction with BMAL1 (12,14,18,26). Among suppressive factors for E boxes, DEC1 and DEC2 can bind directly to E boxes through their basic helix-loop-helix DNA binding domains (18,26), although it remains unclear whether DEC1 and DEC2 also bind to the EЈ boxes. In contrast, PER and CRY interact with the CLOCK/BMAL1 heterodimer but cannot bind directly to E/EЈ boxes, since they have no DNA binding domain.Dec1 expression shows...

The basic helix-loop-helix transcription factor DEC1 is expressed in a circadian manner in the suprachiasmatic nucleus where it seems to play a role in regulating the mammalian circadian rhythm by suppressing the CLOCK/BMAL1-activated promoter. The interaction of DEC1 with BMAL1 has been suggested as one of the molecular mechanisms of the suppression [Honma, S., Kawamoto, T., Takagi, Y., Fujimoto, K., Sato, F., Noshiro, M., Kato, Y. & Honma, K. (2002) Nature 419, 841-844]. Deletion analysis of DEC1 demonstrated that its N-terminal region, which includes the basic helix-loophelix domain, was essential for both the suppressive activity and the interaction with BMAL1, as DEC1 lacking the basic region did not show any suppression or interaction. Furthermore, we found that Arg65 in the basic region, which is conserved among group B basic helix-loop-helix proteins, was responsible for the suppression, for the interaction with BMAL1 and for its binding to CACGTG E-boxes. However, substitution of His57 for Ala significantly reduced the E-box binding activity of DEC1, although it did not affect the interaction with BMAL1 or suppression of CLOCK/BMAL1-induced transcription. On the other hand, the basic region-deleted DEC1 acted in a dominant-negative manner for DEC1 activity, indicating that the basic region was not required for homodimer formation of DEC1. Moreover, mutant DEC1 also counteracted DEC2-mediated suppressive activity in a dominant-negative manner. The heterodimer formation of DEC1 and DEC2 was confirmed by pull-down assay. These findings suggest that the basic region of DEC1 participates in the transcriptional regulation through a protein-protein interaction with BMAL1 and DNA binding to the E-box.Keywords: DEC1; DEC2; BMAL1; circadian rhythm; clock.Circadian rhythms are regulated by a molecular clock(s), which has an endogenous period of 24 h and synchronizes to the 24 h period after light entrainment. In mammals, the clock genes Clock, Bmal1, Per and Cry, and their protein products, comprise a molecular feedback loop in which a CLOCK/BMAL1 heterodimer binds to a CACGTG E-box and activates transcription of Per and Cry [1,2]; protein products of Per and Cry in turn suppress the transactivation by CLOCK/BMAL1 [3,4]. This core feedback loop apparently generates a 24 h period in the molecular oscillator. Furthermore, another feedback loop has been reported to control the rhythmic expression of Bmal1: expression of Rev-Erba is inducible by the CLOCK/BMAL1 heterodimer, and its protein product suppresses the expression of Bmal1 [5,6]. These two feedback loops may be interlocked to stabilize the circadian core loop system. DEC1 (bhlhb2) and DEC2 (bhlhb3) are basic helix-loophelix (bHLH) transcription factors which bind to CAC-GTG E-boxes and suppress transcription from target genes [7][8][9][10][11][12]. Expression of DEC1 and DEC2 showed circadian rhythms in most organs, including the suprachiasmatic nucleus (SCN) [7,13], and Dec1 expression in the SCN was enhanced by a light pulse in a phase-dependent manner sim...