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BMC Molecular Biology Biomed Central BMC Molecular Biology BioMed Central Research article Open Access A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes Yasukazu Nakahata1, Mayumi Yoshida1,2, Atsuko Takano1, Haruhiko Soma3, Takuro Yamamoto3, Akio Yasuda3, Toru Nakatsu4 and Toru Takumi*1,5 Address: 1Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan, 2Kyoto University Graduate School of Biostudies, Sakyo, Kyoto 606-8501, Japan, 3Life Science Laboratory, Material Laboratories, Sony Corporation, Shinagawa, Tokyo 144-0001, Japan, 4Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan and 5Department of Molecular Neuroscience, Kyoto University Graduate School of Medicine, Sakyo, Kyoto 606-8501, Japan Email: Yasukazu Nakahata - [email protected]; Mayumi Yoshida - [email protected]; Atsuko Takano - [email protected]; Haruhiko Soma - [email protected]; Takuro Yamamoto - [email protected]; Akio Yasuda - [email protected]; Toru Nakatsu - [email protected]; Toru Takumi* - [email protected] * Corresponding author Published: 4 January 2008 Received: 12 July 2007 Accepted: 4 January 2008 BMC Molecular Biology 2008, 9:1 doi:10.1186/1471-2199-9-1 This article is available from: http://www.biomedcentral.com/1471-2199/9/1 © 2008 Nakahata et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two basic helix-loop-helix (bHLH) PAS (for Period-Arnt-Sim) domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxes or E-box-like sequences have also been reported to be necessary for circadian oscillation. Results: We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E- box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E- elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region. Conclusion: We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell- autonomous transcriptional oscillation of clock and clock-controlled genes. Background components: input, pacemaker, and output. The endog- Circadian rhythms, periodicities with an approximate 24- enous period length is not exactly 24 h and must be h length, are essential physiological functions in almost entrained by light. In mammals, other entrainment cues all organisms on the earth and are generated by endog- such as food intake or temperature, even social cues, can enous biological clocks. The biological clocks consist of 3 reset the circadian rhythm. The central oscillator in mam- Page 1 of 11 (page number not for citation purposes) BMC Molecular Biology 2008, 9:1 http://www.biomedcentral.com/1471-2199/9/1 mals is located in the suprachiasmatic nuclei (SCN) in the tial for transcriptional oscillation of Per2, a crucial compo- anteroventral hypothalamus. Recent studies revealed that nent of molecular clocks [26]. Comparing with other the peripheral clocks reside not only in other brain mammalian genome sequences, within the conserved regions but also in peripheral organs and even in cultured regions among mammalian (human, rhesus, rat, mouse, cells [1-4]. The output of the biological clocks includes and opossum) genomes, we found that an E-box-like broad physiological phenomena such as locomotor activ- sequence followed by another non-canonical E-box with ity, sleep-wake cycle, hormonal secretion, cardiovascular 6-base pairs (bp) between the two (Fig. 1A). In addition condition, bowel movement, and even mental states. The to Per2, we found a similar E-box or E-box-like sequence disturbance of the biological clocks may cause many dys- followed by another E-box-like sequence with an inter- functions from routinely experienced jet-lag or work-shift vening space of 6 bp in Per1 and Per3, which are other problems to sleep disturbance and cardiovascular, meta- mammalian period homologues, in their upstream pro- bolic or mental diseases [2,5]. moter region close to the transcription start site (TSS). The E-box or E-box-like sequences distal and proximal to TSS Recent molecular biological studies have revealed that the were dubbed E1 and E2, respectively, and collectively as molecular mechanism of the biological clocks is based on the "EE-element" (Fig. 1A). These sequences are well con- the interlocked loops of transcriptional and translational served among diverse mammals, such as human, rhesus, feedback [6-8]. Among the transcriptional controls in rat, and mouse, and even the opossum which is evolution- mammals, 3 major clock components for canonical clock ally located at the farthest branch from humans among genes, i.e., E-box, RORE, and DBPE, have been reported so the mammals. To examine whether these EE-elements are far [3,9]. The E-box (CACGTG), for example, in the Per1 involved in transcriptional oscillation, we performed the promoter, is the most well-known regulatory element. It is IV-ROMS established in our previous study by using the considered the binding site for the heterocomplex of following constructs: The oligonucleotides of 3 (hPer1, CLOCK/BMAL1, which is a positive regulatory element. hPer2, and hPer3) EE-elements were inserted upstream of Negative regulatory elements, PERs and CRYs, restrain the the SV40 promoter in a luciferase (luc) plasmid, and the transcriptional activity [10-12]. It is widely known that constructed plasmids were used to transfect mouse not only other clock genes are regulated by the E-box but NIH3T3 cells. All 3 EE-elements gave significant oscilla- also output regulators or clock-controlled genes are regu- tion of luciferase activity but the amplitudes were differ- lated by it as well [13-17]. On the other hand, Bmal1, ent among the genes (Fig. 1B). The hPer2 EE-element whose circadian expression exhibits a sort of anti-phase to displayed the most robust oscillation, whereas the hPer1 Per2 expression, is positively regulated by Rorα and nega- EE-element gave the lowest amplitude, which correlated tively regulated by Rev-erbα, through RORE [18-21]. with the patterns derived from luc-constructs containing the longer promoter regions [26]. The amplitude in circa- Per2 was literally cloned as a second mammalian period dian oscillation seemed to correlate with the number of gene [22,23], however, gene-knockout analysis revealed a mismatches in the E2 sequence. In fact, a mutant hPer3 more prominent role for mPER2 in the mammalian clock EE-element, in which the GAG at the end of the E2 than PER1 [24]. Using the in vitro rhythm oscillation sequence was replaced with canonical GTG, exhibited a monitoring system (IV-ROMS) [3,25], instead of the higher amplitude than the wild-type hPer3 EE-element canonical E-box (CACGTG), we previously identified an (see Additional file 1). E-box-like sequence (CACGTT) in the Per2 promoter region in vitro [26], which is essential for the cell-autono- To investigate whether both E1 and E2 were necessary for mous transcriptional oscillation of Per2 as well as for Per2 generating the cell-autonomous oscillation, we again per- circadian oscillations in vivo [27]. For the cell-autono- formed IV-ROMS with the use of the mutant hPer2 EE-ele- mous transcriptional oscillation of Per2, not only this E- ment constructs. Not only the double E-box-like box-like sequence but also the downstream region is mutations (E1mE2m) but also the single E-box-like muta- involved in the circadian oscillation of Per2 [26]. Here, we tion (E1m or E2m) resulted in drastically reduced circa- identified closely spaced E-box or E-box-like elements in dian transcription or led to an almost complete loss of the regulatory regions of canonical clock genes including transactivation (Fig. 2). A longer Per2 promoter (~2 kb) Per2. Our comprehensive approach raised the possibility with double E-element mutants showed that the ampli- that not just a single E-box but a direct repeat of E-box-like tude was heavily reduced, with circadian transcription elements is required for circadian oscillation of core clock hardly being observed (see Additional file 2). These and clock-controlled genes. results are similar to those obtained with mutations of 2 ROREs in the Bmal1 promoter [21] and suggest that these Results 2 E-box-like elements function in Per2 transactivation. Our previous study demonstrated that an E-box-like sequence (CACGTT) and its downstream region are essen- Page 2 of 11 (page number not for citation purposes) BMC Molecular Biology 2008, 9:1 http://www.biomedcentral.com/1471-2199/9/1 TheFigure "EE-element" 1 possesses transcriptional activity displaying a circadian rhythm The "EE-element" possesses transcriptional activity displaying a circadian rhythm. (A) Sequence alignments of the promoter region, containing each EE-element of Per1, Per2 and Per3 gene, among human, rhesus, rat, mouse and opossum (The NCBI accession numbers are indicated in Additional file 6).
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