Functional analysis of the basic helix‐loop‐helix transcription factor DEC1 in circadian regulation

Abstract

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...

57Arg in the bHLH transcription factor DEC2 is essential for the suppression of CLOCK/BMAL2-mediated transactivation

“…Mutation analysis demonstrated that the basic region in DEC2 essential for the suppressive activity coincided with the region in DEC1 as described previously (10). The amino acid residue, 57 Arg, in the basic region of DEC2 is conserved among the group B bHLH proteins, including USF, c-Myc, MAX and MAD (16), which can bind to the CACGTG E-box (9,17) and some other transcription factors (6,18).…”

“…We recently demonstrated that Dec2 (Sharp-1) and Dec1 (Sharp-2/Stra13/ Clast5) were new regulators of the mammalian molecular clock which caused CLOCK/BMAL1-mediated transactivation (6). DEC2 and DEC1 are basic helix-loop-helix (bHLH) transcription factors (7,8) which bind to CACGTG E-boxes and BMAL1 to suppress transcription from target genes (6,9,10). The expression of DEC2 and DEC1 shows circadian rhythms in most organs, including the SCN (6,12), and the phases of the circadian rhythms for Dec2 and Dec1 were similar to those for Per1, Per2, Per3 (13), Rev-Erb · (14) and were somewhat similar to Cry1 (15).…”

Human mismatch repair gene, MLH1, is transcriptionally repressed by the hypoxia-inducible transcription factors, DEC1 and DEC2

“…As histone deacetylase (HDAC)-dependent mechanisms had been previously suggested (Sun and Taneja, 2000), trichostatin A (TSA) treatments remarkably canceled the repression of MLH1 promoter activity by DEC in a treatment dose-dependent manner without any detectable cytotoxicity (Figure 2d). Moreover, mutant type of DEC1 which had DNA-binding domain but lacked most of functional domains (Li et al, 2003;Sato et al, 2004) failed to repress MLH1 promoter activities, even enhanced them, suggesting that just a competitive occupancy on the promoter was not sufficient to explain the repression (Figure 2e). …”

“…Base-exchanged mutants of putative E-box sites in pGL-MLH1 Pro0.27 were generated by PCR-based sitedirected mutagenesis as previously reported (Tanimoto et al, 2003). Details of expression plasmid vectors of DEC1 (pcDNA-DEC1, p3xFLAG-CMV-DEC1 or pcDNA-DEC1 1-139) and DEC2 (pcDNA-DEC2) were previously described Sato et al, 2004). pcDNA-FLAG-DEC2 was constructed by swapping DEC2 cDNA fragment of pcDNA-DEC2 with the pcDNA-FLAG (kindly provided by Dr Igarashi).…”

<b>Circadian Expression of Differentiated Embryonic Chondrocytes Expressed Genes 1 </b><b>and 2 in Human Oral Squamous Cell Carcinoma HSC-3 Cells </b>

“…The transcription factor DEC2 also plays an important role in the circadian rhythm (20). Previous studies showed a rhythmic expression of DEC1 and DEC2 mRNA in the suprachiasmatic nucleus, and peripheral tissues (21)(22)(23)(24)(25). DEC1 and were harvested and sub-cultured.…”