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CLOCK and HES1
Gene 510 (2012) 118–125
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Identification of a new clock-related element EL-box involved in circadian regulation by BMAL1/CLOCK and HES1 Taichi Ueshima a, Takeshi Kawamoto a,⁎, Kiyomasa K. Honda a, Mitsuhide Noshiro a, Katsumi Fujimoto a, Sanae Nakao b, Natsuhiro Ichinose b, Seiichi Hashimoto c, Osamu Gotoh b, Yukio Kato a, d,⁎⁎ a
Department of Dental and Medical Biochemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734‐8553, Japan Department of Intelligence Science and Technology, Graduate School of Informatics, Kyoto University, Sakyo-ku, Kyoto 606‐8501, Japan Molecular Medicine Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki 305‐8585, Japan d Japan Science and Technology Agency, JST Innovation Plaza Hiroshima, Higashi-Hiroshima 739‐0046, Japan b c
a r t i c l e
i n f o
Article history: Accepted 16 August 2012 Available online 29 August 2012 Keywords: Circadian rhythms Transcriptional regulation Cartilage Sequence elements Clock Transcription factor
a b s t r a c t Several cis-acting elements play critical roles in maintaining circadian expression of clock and clock-controlled genes. Using in silico analysis, we identified 10 sequence motifs that are correlated with the circadian phases of gene expression in the cartilage. One of these motifs, an E-box-like clock-related element (EL-box; GGCACGAGGC), can mediate BMAL1/CLOCK-induced transcription, which is typically regulated through an E-box or E′-box. Expression of EL-box-containing genes, including Ank, Dbp, and Nr1d1 (Rev-erbα), was induced by BMAL1/CLOCK or BMAL1/NPAS2. Compared with the E-box, the EL-box elements had distinct responsiveness to DEC1, DEC2, and HES1: suppressive actions of DEC1 and DEC2 on the EL-box were less potent than those on the E-box. HES1, which is known to bind to the N-box (CACNAG), suppressed enhancer activity of the EL-box, but not the E-box. In the Dbp promoter, an EL-box worked cooperatively with a noncanonical (NC) E-box to mediate BMAL1/CLOCK actions. These findings suggest that in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Circadian rhythms are one of the most critical biorhythms that are conserved among various species (Pittendrigh, 1993). In mammals, the central pacemaker in the suprachiasmatic nucleus (SCN) regulates diurnal rhythms of various physiological functions such as behavior, feeding, blood pressure, and hormonal secretion, whereas peripheral clocks synchronize various cellular activities, including metabolism and cell cycles, in a tissue-specific manner (Reppert and Weaver, 2002). About 5–9% of the transcriptomes in the liver, heart, and SCN are under circadian control (Hastings et al., 2003). Rhythmicity of gene expression in the SCN and peripheral tissues is thought to be tightly regulated by the molecular clock system, which consists of transcription factors and their modulators. Three clock elements, E/E′-box, D-box, and RORE, are pivotal in circadian expression of clock and clockcontrolled genes (Gekakis et al., 1998; Mitsui et al., 2001; Preitner et al., Abbreviations: SCN, suprachiasmatic nucleus; RORE, ROR-responsive element; ANOVA, analysis of variance; LD, light–dark; DD, constant darkness; NC E-box, noncanonical E-box; EL, E-box-like element; TSS, transcriptional start site; TRED, transcriptional regulatory database; ZT, Zeitgeber time; IC50, 50% inhibitory concentration; DNAP, DNA affinity precipitation; ChIP, chromatin immunoprecipitation. ⁎ Corresponding author. ⁎⁎ Correspondence to: Y. Kato, Japan Science and Technology Agency, JST Innovation Plaza Hiroshima, Higashi-Hiroshima 739‐0046, Japan. E-mail addresses: [email protected] (T. Kawamoto), [email protected] (Y. Kato). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.08.022
2002; Ueda et al., 2005; Yoo et al., 2005). Transcription factors that bind to these elements have the ability to induce or maintain circadian expression of their target genes. These promoter elements are also involved in determination of circadian phase. In the clock system, the heterodimer of BMAL1 and CLOCK plays a major role by binding to CACGTG E-box and CATGTT E′-box sequences and inducing circadian oscillation of its target genes (Gekakis et al., 1998; Ueda et al., 2005; Yoo et al., 2005). NPAS2 also forms a heterodimer with BMAL1 and activates the mRNA expression of E/E′-box-containing genes (Reick et al., 2001; Ueda et al., 2005). Recently, another noncanonical (NC) E-box (CATGTG) was found to be involved in circadian regulation (Kiyohara et al., 2008). Accordingly, various sequences similar to E-boxes may be responsible for circadian regulation via interaction with BMAL1/CLOCK or BMAL1/NPAS2. In addition, negative regulators such as PER, CRY, and DEC are crucial for producing circadian rhythmicity of E/E'-box-containing clock genes. One of negative regulators, DEC1, can directly bind to the same E/E′-boxes as BMAL1/CLOCK binds, and its binding affinity for E-box is much stronger than that for E'-box (Nakashima et al., 2008). Overexpression of DEC1 causes phase shifts of expression profiles of E-box-containing clock genes, but not those of E′-box-containing genes. Although these clock genes and clock elements are responsible for both central and peripheral autonomous oscillation, it is not known whether all kinds of circadian rhythms in various tissues can be accounted for by them. In addition, less than 10% of oscillated genes
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are common to any two tissues examined (Hastings et al., 2003). Zvonic et al. (2007) also reported that more than 26% of the calvarial transcriptome exhibits circadian rhythms, but only 3% of them are shared among bone, brown and white adipose tissues, and liver. These findings suggest the existence of another mechanism by which the circadian gene expression is regulated in a tissue-specific manner. In the previous studies, Simmons (1966) and Simmons et al. (1983) reported that incorporation of [ 35S]sulfate into chondroitin sulfate in rat growth plate cartilage was high in daytime and low at night, and alkaline phosphatase activity was high at night, suggesting the importance of circadian rhythmicity in growth and development of bones. We recently identified 277 genes that are expressed in growth plate cartilage in a circadian oscillated manner (Honda et al., submitted for publication). Most of these oscillating genes have not been shown to be expressed circadian-dependently in other tissues. In the present study, we attempted to identify new clock elements that produce circadian rhythms of these genes in the cartilage using in silico analysis. Here, we show 10 sequence motifs that may be responsible for circadian regulation of gene expression in the cartilage in a phase-dependent manner. Among these motifs, an E-box-like element (EL-box) showed similar properties to E-boxes, but had different responsiveness to DEC1 and HES1. These motifs may provide new insights into the circadian regulation of gene expression. 2. Materials and methods 2.1. Animals, RNA extraction and DNA microarray analysis Six-week-old male Sprague–Dawley rats (Crea Japan, Tokyo) were housed under a 12:12 h light–dark (LD) cycle for 14 days. Following this, rats of each group were housed under LD conditions or constant darkness (DD), and three of the rats were sacrificed every 4 h (n = 3) for 48 h. Total RNA was extracted from rib growth plates and resting cartilage. Biotinylated cRNA prepared from the RNA was subjected to DNA microarray analysis using GeneChip Rat Expression Array 230A/ B (Affimetrix, Santa Clara, CA). Details of the analysis will be described elsewhere (Honda, KK et al., submitted). Briefly, analysis was performed as described previously (Ueda et al., 2002). The time course for the expression profile obtained with each probe set was depicted, and any similarity between the profiles for the first and last 24 h was examined. In addition, any similarity between these profiles and that of a standard cosine curve, and between the profiles for 48 h under LD and DD conditions, was examined. Using these criteria, we selected candidates of oscillating genes. The rhythms of gene expression in DNA microarray analysis were evaluated by one-way ANOVA using the expression levels (n = 4) at ZT/CT 2, 6, 10, 14, 18, and 22, which were obtained regardless of whether they were in the first or second 24 h, and regardless of LD or DD. All procedures were performed in compliance with standard principles and guidelines for the care and use of laboratory animals, Hiroshima University Graduate School of Biomedical Sciences. 2.2. Isolation of clock elements Analyzing the microarray data, we identified 254 genes that showed statistically significant (Pb 0.05) circadian expression (Honda et al., submitted). The promoter of a gene is defined as the region 1000 bp upstream from the transcriptional start site (TSS) to 200 bp downstream from the TSS. We selected 54 genes for further analysis, because half of identified genes were unknown genes at the time we started the analysis and some of promoter regions were not conserved among mice, rats, and humans. We consulted transcriptional regulatory database (TRED) (Zhao et al., 2005) to retrieve reliable TSSs, and chose only the promoters categorized into levels 1–3 in TRED (Supplemental Table 1).
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To reduce noisy outcomes of motif finding programs, we first obtained a multiple sequence alignment of the promoter regions that were orthologous among rat, mouse and human genes. For alignment, we progressively applied Aln (Gotoh, 1999) in the semi-global mode. We then scanned the alignment columns with a window of a fixed size of five. If four or all of the five positions in a window are completely conserved among the three species, we regarded the sites within the window as “informative” and other sites as “non-informative”. We assigned ʻN ’ to a non-informative site, the most frequent nucleotide to a completely or partially conserved site, and the nucleotide of the rat genome to a completely divergent site. We finally obtained 54 promoter sequences that fulfilled all the criteria mentioned above. We then categorized the genes with these promoter sequences into six groups according to the peak times {Zeitgeber time (ZT) 0–4, 4–8, 8–12. 12–16, 16–20, 20–24} and then individually applied a motif finding program Weeder (Pavesi et al., 2004) to each group of the masked promoter sequences. We used the “large” and “zoop” options of Weeder for the analysis, which can detect motifs of sizes of 6, 8, 10, or 12. Then, we used Weeder's Motif Locator tool to search for instances of individual motifs thus found in all the 54 promoter sequences allowing for up to two mismatches compared with the consensus sequence. To test whether a motif thus found is correlated with a specific phase of circadian expression profile, we conducted a χ2 or Fisher's exact test with a 2 ×2 contingency table. Each element of the table represents the number of promoters conditioned by the presence or absence of the relevant motif, and the membership of the corresponding gene in the specific category of expression phase. The candidate motifs were sorted by the P-values of this test, and most significantly correlated motifs were selected. 2.3. Plasmid constructions Promoter regions of mouse Ank (−1107 to +293), Dbp (−1289 to +196) and Nr1d1 (−780 to +470) were amplified by PCR from mouse (C57BL/6J) genomic DNA and subcloned into pGL3-Basic (Promega). Sequences of primers used in this study are shown in Supplemental Table 2. Parts of Dbp promoter regions (−734 to +196 and −394 to +196) were also amplified and subcloned into pGL3-Basic. Mouse Bmal1, Clock, Npas2, Hes1, and Hes2 cDNAs were obtained by RT-PCR from mouse liver and subcloned into pcDNA4-V5-HisA (Invitrogen) or p3xFLAG-26 (Sigma). Oligonucleotides of three tandem sequences containing mouse Ank, Dbp, or Nr1d1 EL-box with 4-bp of flanking sequences (Supplemental Table 3) were subcloned into pGL3-TK. To construct a series of mutants of Ank EL-box, oligonucleotides of three tandem sequences containing mutated Ank EL-boxes with 4-bp of flanking sequence (Supplemental Table 3) were subcloned into pGL3-TK. Expression vectors for Bmal1 and Dec1 have been described previously (Kawamoto et al., 2004). Cry1 expression vector was generously supplied by M. Ikeda, Saitama Medical School. To prepare probes for DNAP assay, phosphorylated oligonucleotides of three tandem sequences containing human Dec1 E-box (CACGTG) with 6-bp of flanking sequence or mouse Ank or Dbp EL-box with 4-bp of flanking sequence with sticky ends were self-ligated using ligation high (Toyobo) and subjected to 8% acrylamide gel electrophoresis. DNA elements around 300 bp long were extracted from the gel and subcloned into pEGFP-N1 (Clontech) and resulting plasmids were named pEGFPDec1-E-boxX10, pEGFP-Ank-EL-boxX8 and pEGFP-Dbp-E-boxX7. All constructed described above were validated by nucleotide sequencing. 2.4. Transient transfection and luciferase reporter assay NIH3T3 cells were seeded at 1 × 10 4 cells per well in a 96-well plate 24 h before transfection. Luciferase reporter plasmid (4 ng per well) and 0.05 ng of pRL-SV40 (Promega), as an internal standard, were cotransfected with mouse Bmal1, Flag-Clock, and Flag-Npas2 expression vectors (each 60 ng per well) using Lipofectamine 2000. The total concentration of DNA was adjusted to 124.05 ng per well with
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an empty vector (pcDNA4-V5-HisA). The cells were incubated for 48 h and subjected to luciferase reporter assays using Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were normalized by internal control activities. The values represent the mean±SEM for three wells. Statistical significance was analyzed by Student t test (Fig. 5) or one-way analysis of variance (ANOVA) followed by Turkey's multiple comparison test (Figs. 3, 6, and 7). Various amounts of expression vectors for Cry1, Dec1, Dec2, Hes1, and Hes2 were added to determine the dose-dependent inhibition of the BMAL1/CLOCK activity. The amounts of each expression vector that resulted in the half-maximal activity were determined as 50% inhibitory concentration (IC50) by manual plotting.
time range of circadian expression and the existence of short sequence motifs in conserved regulatory regions in the promoters among humans, mice and rats. From this analysis, we found 10 sequence motifs commonly present in a group of genes, the circadian phases of which were significantly associated with the same peak
2.5. DNA affinity precipitation (DNAP) assay To examine the direct binding of BMAL1/CLOCK or BMAL1/NPAS2 to EL-boxes, DNAP assay was performed as described previously (Suzuki et al., 1993). Expression vectors (9 μg each) for BMAL1 and FLAG-tagged CLOCK, BMAL1 and FLAG-tagged NPAS2, or the same amounts of an empty vector were transfected into COS7 cells seeded in 10 cm dishes, and nuclear extracts were prepared from the transfected cells using Nuclear Extract Kit (Active motif) and dialyzed against DNAP buffer, pH7.9 (20 mM HEPES–KOH, 80 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 0.5 mM, DTT, 10% glycerol, 0.1% tritonX-100 and 0.1 mM PMSF). For preparation of biotinylated probes, multicopies of Dec1 E-box, Ank EL-box and Dbp EL-box were amplified by PCR from pEGFP-Dec1-E-boxX10, pEGFPAnk-EL-boxX8 and pEGFP-Dbp-E-boxX7, respectively, using biotinylated primers. As a negative control, a part of the luc2P gene (273 bp) was also amplified from pGL4.11 (Promega). The biotinylated DNA probes (1 μg) were incubated with the dialyzed nuculear extracts (100 μg) in 500 μl of DNAP buffer containing poly(dI–dC) (15 μg) at 4 °C for 2 h. Then, streptavidin beads (Dynabeads M-280, Dynal) were added to the mixture and mixed by rotation for 30 min. The Dynabeads were collected with a magnet and washed 5 times with DNAP buffer. The trapped proteins were analyzed by SDS-PAGE followed by immunoblotting with anti-FLAG antibodies (Sigma). The experiments were repeated 3 times and similar results were obtained. 2.6. Chromatin immunoprecipitation (ChIP) assay ChIP assays for Ank and Dbp EL-boxes were performed using Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. Forty-eight hours before the assays, expression plasmids (12 μg each) for BMAL1 and FLAG-tagged CLOCK or FLAG-tagged NPAS2 were transfected into ATDC5 cells. Extracts prepared from the cells were subjected to precipitation with anti-FLAG antibodies or control IgG. The precipitated DNA samples were subjected to PCR using gene specific primers (Supplemental Table 2). The PCR was carried out under the following condition: denaturation at 98 °C for 10 s, annealing at 60 °C for 15 s, and extension at 68 °C for 60 s using PrimeSTAR GXL DNA Polymerase (Takara). After 30 cycles of PCR, obtained products were analyzed by agarose gel electrophoresis. The experiments were repeated 3 times and similar results were obtained. 3. Results 3.1. Sequence motifs correlated with the circadian phases of rhythmic gene expression in growth plate cartilage Using microarray analysis we identified various genes, the expression of which showed circadian rhythmicity in the growth plate of rat rib (Honda et al., submitted for publication). To determine promoter elements that govern circadian expression of these rhythmic genes, we subjected 54 gene promoters, which had been well characterized among three species (up to 1 kb from the transcription initiation site; Supplemental Table 1), into correlation analysis between the peak
Fig. 1. Sequence motifs correlated with the circadian phases of rhythmic gene expression in growth plate cartilage. Sequence motifs were extracted from each promoter group with a specific range of peak time, and then their locations in all the 54 promoter sequences were searched for by a motif locator. The fourth column lists the genes that harbor the relevant motif and the fifth column indicates their peak time. Ten sequence motifs showed significant correlation with the peak times as evaluated by t-test or Fisher's exact test. Some genes listed are out of the peak time range, even though all genes carrying the motifs are listed here.
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time classification (Fig. 1). In this study, we focused on the EL-box that had a GGCACGAGGC consensus sequence (Fig. 1). 3.2. A new clock element (EL-box) responsible for induction by BMAL1/ CLOCK The genes containing the EL-box showed clear circadian rhythms (Supplemental Fig. 1). The existence of the EL-box was significantly correlated with the peak times of ZT 8–12 (Fig. 2; P b 0.001): Nr1d1, Slc20a1, Angptl2, Emp3, and Dbp showed the peak times between ZT 8 and ZT 12. In addition, Tef, Klf9, Ank, and Klf15 had similar peak times (ZT 12.0, ZT 12.4, ZT 12.8, and ZT 13.2). Since the EL-boxes contained a sequence, CACGAG, similar to the E-box (CACGTG) element, we hypothesized that EL-boxes may mediate circadian regulation through interaction with BMAL1/CLOCK or BMAL1/ NPAS2. To test this hypothesis, we examined whether EL-box elements have enhancer activities responsive to these transcription factors. ELboxes of Ank, Dbp, and Nr1d1 were subcloned into a luciferase reporter plasmid and co-transfected into NIH/3T3 cells with expression vectors for BMAL1 and CLOCK or BMAL1 and NPAS2. The results demonstrated that BMAL1/CLOCK or BMAL1/NPAS2 markedly increased expression levels of reporter genes carrying EL-box elements (Fig. 3A), whereas BMAL1 or CLOCK alone had no activity on the E- or EL-box-mediated transcription (Supplemental Fig. 2). The native promoters of these genes also showed similar responsiveness to BMAL1/CLOCK or BMAL1/ NPAS2 (Fig. 3B).
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NPAS2 were co-expressed in COS7 cells. Nuclear extracts were prepared from these cells and incubated with biotinylated DNA probes of Ank EL-box, Dbp EL-box and Dec1 E-box elements, and protein-DNA complexes were precipitated with streptavidin beads. As shown in Fig. 4A, binding of BMAL1/CLOCK and BMAL1/NPAS2 to oligonucleotides containing EL-boxes and Dec1 E-box was observed, whereas their binding to a control luc2P gene fragment was not detected. To confirm that BMAL1/CLOCK or BMAL1/NPAS2 actually binds to EL-box-containing promoters in living cells, ChIP assay was performed. Both BMAL1/CLOCK and BMAL1/NPAS2 heterodimers bound to the Dbp and Ank promoters (Fig. 4B). These findings in DNAP and ChIP assays, taken together, revealed that BMAL1/CLOCK and BMAL1/NPAS2 can bind to EL-boxes in oscillating genes. 3.4. Mutational analysis of EL-box elements In addition to E-box, E′-box, and NC E-box elements, the EL-box element may provide a new option for BMAL1/CLOCK action. For further characterization of the EL-box ability, we examined which nucleotides in Ank EL-box are essential for induction by BMAL1 and CLOCK. For this purpose, we generated a series of mutant constructs and performed luciferase reporter assays using these constructs together with expression vectors for BMAL1and CLOCK. All one or two point mutations,
3.3. Binding of BMAL1/CLOCK and BMAL1/NPAS2 to EL-box elements Since the BMAL1/CLOCK heterodimer and BMAL1/NPAS2 heterodimer enhanced the expression of EL-box-containing reporter genes, we performed DNAP (DNA affinity precipitation) assay to show their binding to EL-box elements. BMAL1 and FLAG-tagged CLOCK or FLAG-tagged
Fig. 2. Sequence comparison of newly identified clock elements (designated EL-box elements). Identified 54 genes, expression of which showed circadian rhythmicity, were categorized into six groups according to the peak times (ZT 0–4, 4–8, 8–12, 12–16, 16–20, and 20–24). EL-box elements exist in the promoter regions of 11 genes out of 54 oscillated genes and expression levels of 5 genes were peaked between ZT 8 and ZT 12. The DNA sequence logo describing a graphical representation of the EL-box consensus was generated using WebLogo (http://weblogo.berkeley.edu/).
Fig. 3. EL-box elements are able to mediate the induction by BMAL1/CLOCK or BMAL1/ NPAS2. (A) Activities of Ank, Dbp, and Nr1d1 EL-boxes were examined by luciferase assay. Reporter plasmids carrying three copies of EL-box connected to the TK promoter were cotransfected with expression vectors for BMAL1 and CLOCK or NPAS2 into NIH3T3 cells, and luciferase activities were measured. **, Pb 0.01; ***, Pb 0.001. (B) Promoter constructs of mouse Ank (−1107 to +293), Dbp (−1289 to +196), and Nr1d1 (−780 to +470) were also used for reporters to examine the induction by BMAL1/CLOCK or BMAL1/NPAS2.
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Fig. 4. BMAL1/CLOCK or BMAL1/NPAS2 heterodimers bind to EL-box elements. (A) DNA affinity precipitation (DNAP) assay. Expression plasmids for BMAL1 and FLAG-tagged CLOCK or FLAG-tagged NPAS2 were transfected into COS7 cells. Nuclear extracts prepared from the transfected cells were incubated with biotinylated probes of Dec1 E-box, Ank EL-box and Dbp EL-box. As a negative control, the luc2P gene was used. Proteins bound to the probes were precipitated with streptavidin beads and analyzed by SDS-PAGE followed by immunoblotting using anti-FLAG antibodies. (B) Chromatin immunoprecipitation (ChIP) assay. ATDC5 cells were transfected with expression vectors for BMAL1 and FLAG-tagged CLOCK or FLAG-tagged NPAS2. DNA samples precipitated from the cells with anti-FLAG antibodies or control IgG were subjected to PCR using gene specific primers for Ank and Dbp. The amplified regions by PCR were schematically shown in the bottom of the figure.
except for the substitution of CG in the first two nucleotides to TT (M1), caused significant reduction in the BMAL1/CLOCK-induced activation of reporter genes (Fig. 5 and Supplemental Fig. 4). However, the effect of M5 mutation was modest as compared with almost complete abolishment with M2, M6, M7, and M8 mutations. In addition, M5 mutation had lesser effect than M3 and M4 mutations. M5 mutant still showed more than 3-fold induction by BMAL1/CLOCK, which is only 25% less than about 4-fold induction in the wild-type construct. Thus, the 6-bp sequence (CACGAG) from the EL-box consensus, which is similar to
the E-box consensus, is critical, although its surrounding sequences have some activities. 3.5. Role of EL-box element in the Dbp promoter The transcriptional activity of the Dbp promoter has been reported to be inducible by the BMAL1/CLOCK heterodimer, which binds to a NC E-box element (CATGTG) in the promoter (Kiyohara et al., 2008). In this study, we examined whether the EL-box in the Dbp promoter actually responds to BMAL1/CLOCK. As shown in Fig. 6A, luciferase activities of the Dbp promoter constructs were enhanced by BMAL1/CLOCK or BMAL1/ NPAS2; the activity was markedly reduced by the deletion of the −734 to −394 region, which contained the NC E-box. However, significant induction of the promoter activity by BMAL1/CLOCK or BMAL1/NPAS2 was still observed even with a minimal promoter construct with the EL-box without the NC E-box. To further confirm the involvement of the Dbp EL-box in BMAL1/CLOCK or BMAL1/NPAS2 induction, we made another Dbp promoter construct that contained a mutated EL-box sequence (Fig. 6B). This substitution markedly reduced BMAL1/CLOCK- or BMAL1/ NPAS2-mediated transcription. These results indicate that the EL-box is involved in the stimulation of Dbp promoter activity by BMAL1/CLOCK or BMAL1/NPAS2. 3.6. Effects of negative factors on the enhancer activity of the EL-box
Fig. 5. Mutational analysis of EL-box elements. One or two nucleotides in the Ank EL box sequence were replaced and subjected to luciferase reporter assays. A series of report constructs was cotransfected with a control vector (open bars) or expression vectors for BMAL1 and CLOCK (closed bars) into NIH3T3 cells and luciferase activities were determined. *, P b 0.05; **, P b 0.01; ***, P b 0.001; ns, not significant. Fold induction rates by CLOCK/BMAL1 are shown in Supplemental Fig. 4.
Next, we examined the effects of negative factors such as PER, CRY, and DEC on BMAL1/CLOCK-induced transcription from the ELbox-containing promoter. Co-transfection of CRY1, DEC1, or DEC2 significantly reduced luciferase activities of both Ank EL-box- and Dec1 Ebox-containing constructs induced by BMAL1/CLOCK (Fig. 7A). However, the suppressive activity of DEC1 and DEC2 was less on the Ank EL-box than on the Dec1 E-box. Since the EL-box consensus sequence contains an N-box sequence (CACNAG) to which HES family transcription factors bind (Sasai et al., 1992), we examined the suppressive activity of HES1
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Fig. 6. Both EL-box and NC E-box elements in the Dbp promoter are responsible for the induction by BMAL1/CLOCK or BMAL1/NPAS2. (A) Various lengths of Dbp promoter constructs were cotransfected with expression vectors for BMAL1 and CLOCK or NPAS2 into NIH3T3 cells and luciferase activities were measured. (B) Effect of a EL-box mutation in the Dbp promoter was analyzed. *, P b 0.05; **, P b 0.01; ***, P b 0.001.
and HES2: HES1 significantly decreased EL-box activity in the presence of BMAL1/CLOCK, but did not decrease E-box activity. For further analysis of the difference of suppressive activities of these negative factors on E-box and EL-box elements, we examined the effects of increasing doses of CRY1, DEC1, DEC2, HES1 and HES2 and calculated IC50. IC50 of CRY1 on the Ank EL-box reporter was similar to that on the Dec1 E-box reporter, whereas those of DEC1 and DEC2 were higher on the EL-box than on the E-box (Fig. 7B). The results also showed that the effects of DEC1 and DEC2 on the EL-box were much weaker than those on the E-box, whereas the effect of CRY1 on the EL-box was similar to that on the E-box (Supplemental Fig. 3). In contrast, IC50 of HES1 was lower on the EL-box reporter than on the E-box. These results indicated that HES1 may serve as a negative regulator like CRY and DEC. 4. Discussion In the present study, we identified several sequence motifs that are correlated with circadian phases of rhythmic genes in the
cartilage. Although it is not known whether these genes are expressed in a circadian manner in other tissues, some of these genes, such as Lox, Mmp14, Tgfa, Ank, Gilz, Slc20a1, and Mgea5, are actually involved in the morphology and/or function of chondrocytes. On these motifs the EL-box showed responsiveness to BMAL1/ CLOCK or BMAL1/NPAS2, which controls the clock system ubiquitously. Although its transcriptional activity was similar to those of E-box and E′-box, the suppressive effects of DEC1 and DEC2 were less potent on the EL-box than on the E-box. Furthermore, the induction of the EL-box-containing promoter by BMAL1/CLOCK or BMAL1/ NPAS2 was suppressed by HES1, whereas HES1 does not show any suppressive effect on the E-box element. DEC1 and DEC2 are transcription factors that bind to E-box elements and suppress transcription from their target genes (Hamaguchi et al., 2004; Honma et al., 2002; Kawamoto et al., 2004). In addition to strong binding affinity for the sequence CACGTG (E-box), DEC1 and DEC2 have weaker affinity for the sequences CACGTT (E′-box) and CACGCG (Fujimoto et al., 2007; Nakashima et al., 2008). DEC1 and DEC2 may have weaker affinity for EL-boxes than for the E-box. The difference of binding affinity
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Fig. 7. HES1 suppresses EL-box activity induced by BMAL1/CLOCK. (A) Effect of negative factors such as CRY1, DEC1, DEC2, HES1 and HES2 on Ank EL-box and Dec1 E-box elements was examined. An expression vector (20 ng) of one of these factors was cotransfected with expression vectors (20 ng each) for BMAL1 and CLOCK together with a reporter construct (50 pg) for the Ank EL-box or Dec1 E-box into NIH3T3 cells and luciferase activity was measured after 48 h of culture. Some of factors significantly decreased the activity induced by BMAL1/CLOCK. ***, P b 0.001. (B) Fifty percent inhibitory concentration (IC50) of negative factors for BMAL1/CLOCK-induced luciferase activity was determined. Various amounts (0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 ng) of expression vectors were used to examine the dose-dependency. Total amount of plasmid DNA for transfection was adjusted by an empty vector.
for EL-box and E-box sequences accounts for weaker suppressive activities of DEC1 and DEC2 to EL-box elements observed in this study. HES1, which has sequence homology to DEC1 and DEC2 (Fujimoto et al., 2001; Shen et al., 1997), is a transcription factor that binds to N-boxes and suppresses transcription of target genes (Sasai et al., 1992). As shown in the present study, the EL-box sequence contains the N-box consensus sequence (CACNAG), and HES1 had a suppressive effect on BMAL1/CLOCK-induced expression of the EL-box-containing promoter. The sequence difference between the EL-box and E-box resulted in distinct responsiveness to HES1 as observed in this study. Although Hes1 expression in cultured cells shows oscillation with 2-h periodicity after serum treatment (Hirata et al., 2002), it did not show significant circadian rhythmicity in rat rib cartilage (data not shown). However, the cross-talk between HES and clock systems may have physiological roles through EL-boxes in some situations.
In our in silico analysis, E-box and E′-box elements were not identified as a candidate sequences for circadian regulation. The EL-box sequence (GGCACGAGGC) is longer than an E-box or E′-box sequence, which consists of only 6 nucleotides. A longer consensus sequence may have provided an advantage for in silico analysis because identification of sequence motifs in clock-related genes depends on the probability of existence in relation to circadian phases. In addition, some E/E′-box elements do not seem to function in circadian regulation, since nonclock transcription factors such as c-MYC also transactivate their target genes via E-box elements. Recent studies demonstrated that double-E-box-like elements with a 6 or 7-bp spacer located in various clock genes have strong ability to produce BMAL1/CLOCK-induced expression (Nakahata et al., 2008). Among eleven EL-box-containing genes, only the Tef promoter contains the CACGTG E-box immediately upstream of an
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EL-box element. These findings suggest that the mechanism involved in EL-box-mediated activation may differ from that for the direct repeat of E-box-like elements. On the other hand, some of the EL-box-containing genes were shown to contain other clock elements such as E-box, E', and NC E-box elements in their regulatory regions. For example, an EL-box in the Dbp promoter worked with an NC E-box synergistically in luciferase reporter assays. In addition to these elements, the Dbp gene also contains two functional E-boxes in its intron region (Ripperger et al., 2000; Ueda et al., 2005). Thus, multiple elements seem to contribute to the unique regulation of clock-related genes. Although we could not determine whether all of EL-boxes identified in this study are functional, our results suggest that, in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes. Acknowledgments This work was supported by grants-in-aid for science from the Ministry of Education, Culture, Sport, Science and Technology of Japan. We thank Maki Ukai-Tadenuma and Hiroki R. Ueda for their technical support and critical reading of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.08.022. References Fujimoto, K., et al., 2001. Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins. Biochem. Biophys. Res. Commun. 280, 164–171. Fujimoto, K., et al., 2007. Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. Int. J. Mol. Med. 19, 925–932. Gekakis, N., et al., 1998. Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569. Gotoh, O., 1999. Multiple sequence alignment: algorithms and applications. Adv. Biophys. 36, 159–206. Hamaguchi, H., et al., 2004. Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system. Biochem. J. 382, 43–50. Hastings, M.H., Reddy, A.B., Maywood, E.S., 2003. A clockwork web: circadian timing in brain and periphery, in health and disease. Nat. Rev. Neurosci. 4, 649–661. Hirata, H., et al., 2002. Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 298, 840–843.
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Honma, S., et al., 2002. Dec1 and Dec2 are regulators of the mammalian molecular clock. Nature 419, 841–844. Kawamoto, T., et al., 2004. A novel autofeedback loop of Dec1 transcription involved in circadian rhythm regulation. Biochem. Biophys. Res. Commun. 313, 117–124. Kiyohara, Y.B., Nishii, K., Ukai-Tadenuma, M., Ueda, H.R., Uchiyama, Y., Yagita, K., 2008. Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector-based strategy. Nucleic Acids Res. 36, e23. Mitsui, S., Yamaguchi, S., Matsuo, T., Ishida, Y., Okamura, H., 2001. Antagonistic role of E4BP4 and PAR proteins in the circadian oscillatory mechanism. Genes Dev. 15, 995–1006. Nakahata, Y., et al., 2008. A direct repeat of E-box-like elements is required for cellautonomous circadian rhythm of clock genes. BMC Mol. Biol. 9, 1. Nakashima, A., et al., 2008. DEC1 modulates the circadian phase of clock gene expression. Mol. Cell. Biol. 28, 4080–4092. Pavesi, G., Mereghetti, P., Mauri, G., Pesole, G., 2004. Weeder Web: discovery of transcription factor binding sites in a set of sequences from co-regulated genes. Nucleic Acids Res. 32, W199–W203. Pittendrigh, C.S., 1993. Temporal organization: reflections of a Darwinian clockwatcher. Annu. Rev. Physiol. 55, 16–54. Preitner, N., et al., 2002. The orphan nuclear receptor REV-ERBalpha controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110, 251–260. Reick, M., Garcia, J.A., Dudley, C., McKnight, S.L., 2001. NPAS2: an analog of clock operative in the mammalian forebrain. Science 293, 506–509. Reppert, S.M., Weaver, D.R., 2002. Coordination of circadian timing in mammals. Nature 418, 935–941. Ripperger, J.A., Shearman, L.P., Reppert, S.M., Schibler, U., 2000. CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev. 14, 679–689. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., Nakanishi, S., 1992. Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and enhancer of split. Genes Dev. 6, 2620–2634. Shen, M., et al., 1997. Molecular characterization of the novel basic helix-loop-helix protein DEC1 expressed in differentiated human embryo chondrocytes. Biochem. Biophys. Res. Commun. 236, 294–298. Simmons, D.J., 1966. Periodicity of S35 uptake in rat femurs. Experientia 20, 137–138. Simmons, D.J., Arsenis, C., Whitson, S.W., Kahn, S.E., Boskey, A.L., Gollub, N., 1983. Mineralization of rat epiphyseal cartilage: a circadian rhythm. Miner. Electrolyte. Metab. 9, 28–37. Suzuki, T., Fujisawa, J.I., Toita, M., Yoshida, M., 1993. The trans-activator tax of human T-cell leukemia virus type 1 (HTLV-1) interacts with cAMP-responsive element (CRE) binding and CRE modulator proteins that bind to the 21-base-pair enhancer of HTLV-1. Proc. Natl. Acad. Sci. U. S. A. 90, 610–614. Ueda, H.R., Matsumoto, A., Kawamura, M., Iino, M., Tanimura, T., Hashimoto, S., 2002. Genome-wide transcriptional orchestration of circadian rhythms in Drosophila. J. Biol. Chem. 277, 14048–14052. Ueda, H.R., et al., 2005. System-level identification of transcriptional circuits underlying mammalian circadian clocks. Nat. Genet. 37, 187–192. Yoo, S.H., et al., 2005. A noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo. Proc. Natl. Acad. Sci. U.S.A. 102, 2608–2613. Zhao, F., Xuan, Z., Liu, L., Zhang, M.Q., 2005. TRED: a transcriptional regulatory element database and a platform for in silico gene regulation studies. Nucleic Acids Res. 33, D103–D107. Zvonic, S., et al., 2007. Circadian oscillation of gene expression in murine calvarial bone. J. Bone Miner. Res. 22, 357–365.
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Identification of a new clock-related element EL-box involved in circadian regulation by BMAL1/CLOCK and HES1 Taichi Ueshima a, Takeshi Kawamoto a,⁎, Kiyomasa K. Honda a, Mitsuhide Noshiro a, Katsumi Fujimoto a, Sanae Nakao b, Natsuhiro Ichinose b, Seiichi Hashimoto c, Osamu Gotoh b, Yukio Kato a, d,⁎⁎ a
Department of Dental and Medical Biochemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734‐8553, Japan Department of Intelligence Science and Technology, Graduate School of Informatics, Kyoto University, Sakyo-ku, Kyoto 606‐8501, Japan Molecular Medicine Laboratories, Institute for Drug Discovery Research, Astellas Pharma Inc., Tsukuba, Ibaraki 305‐8585, Japan d Japan Science and Technology Agency, JST Innovation Plaza Hiroshima, Higashi-Hiroshima 739‐0046, Japan b c
a r t i c l e
i n f o
Article history: Accepted 16 August 2012 Available online 29 August 2012 Keywords: Circadian rhythms Transcriptional regulation Cartilage Sequence elements Clock Transcription factor
a b s t r a c t Several cis-acting elements play critical roles in maintaining circadian expression of clock and clock-controlled genes. Using in silico analysis, we identified 10 sequence motifs that are correlated with the circadian phases of gene expression in the cartilage. One of these motifs, an E-box-like clock-related element (EL-box; GGCACGAGGC), can mediate BMAL1/CLOCK-induced transcription, which is typically regulated through an E-box or E′-box. Expression of EL-box-containing genes, including Ank, Dbp, and Nr1d1 (Rev-erbα), was induced by BMAL1/CLOCK or BMAL1/NPAS2. Compared with the E-box, the EL-box elements had distinct responsiveness to DEC1, DEC2, and HES1: suppressive actions of DEC1 and DEC2 on the EL-box were less potent than those on the E-box. HES1, which is known to bind to the N-box (CACNAG), suppressed enhancer activity of the EL-box, but not the E-box. In the Dbp promoter, an EL-box worked cooperatively with a noncanonical (NC) E-box to mediate BMAL1/CLOCK actions. These findings suggest that in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Circadian rhythms are one of the most critical biorhythms that are conserved among various species (Pittendrigh, 1993). In mammals, the central pacemaker in the suprachiasmatic nucleus (SCN) regulates diurnal rhythms of various physiological functions such as behavior, feeding, blood pressure, and hormonal secretion, whereas peripheral clocks synchronize various cellular activities, including metabolism and cell cycles, in a tissue-specific manner (Reppert and Weaver, 2002). About 5–9% of the transcriptomes in the liver, heart, and SCN are under circadian control (Hastings et al., 2003). Rhythmicity of gene expression in the SCN and peripheral tissues is thought to be tightly regulated by the molecular clock system, which consists of transcription factors and their modulators. Three clock elements, E/E′-box, D-box, and RORE, are pivotal in circadian expression of clock and clockcontrolled genes (Gekakis et al., 1998; Mitsui et al., 2001; Preitner et al., Abbreviations: SCN, suprachiasmatic nucleus; RORE, ROR-responsive element; ANOVA, analysis of variance; LD, light–dark; DD, constant darkness; NC E-box, noncanonical E-box; EL, E-box-like element; TSS, transcriptional start site; TRED, transcriptional regulatory database; ZT, Zeitgeber time; IC50, 50% inhibitory concentration; DNAP, DNA affinity precipitation; ChIP, chromatin immunoprecipitation. ⁎ Corresponding author. ⁎⁎ Correspondence to: Y. Kato, Japan Science and Technology Agency, JST Innovation Plaza Hiroshima, Higashi-Hiroshima 739‐0046, Japan. E-mail addresses: [email protected] (T. Kawamoto), [email protected] (Y. Kato). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.08.022
2002; Ueda et al., 2005; Yoo et al., 2005). Transcription factors that bind to these elements have the ability to induce or maintain circadian expression of their target genes. These promoter elements are also involved in determination of circadian phase. In the clock system, the heterodimer of BMAL1 and CLOCK plays a major role by binding to CACGTG E-box and CATGTT E′-box sequences and inducing circadian oscillation of its target genes (Gekakis et al., 1998; Ueda et al., 2005; Yoo et al., 2005). NPAS2 also forms a heterodimer with BMAL1 and activates the mRNA expression of E/E′-box-containing genes (Reick et al., 2001; Ueda et al., 2005). Recently, another noncanonical (NC) E-box (CATGTG) was found to be involved in circadian regulation (Kiyohara et al., 2008). Accordingly, various sequences similar to E-boxes may be responsible for circadian regulation via interaction with BMAL1/CLOCK or BMAL1/NPAS2. In addition, negative regulators such as PER, CRY, and DEC are crucial for producing circadian rhythmicity of E/E'-box-containing clock genes. One of negative regulators, DEC1, can directly bind to the same E/E′-boxes as BMAL1/CLOCK binds, and its binding affinity for E-box is much stronger than that for E'-box (Nakashima et al., 2008). Overexpression of DEC1 causes phase shifts of expression profiles of E-box-containing clock genes, but not those of E′-box-containing genes. Although these clock genes and clock elements are responsible for both central and peripheral autonomous oscillation, it is not known whether all kinds of circadian rhythms in various tissues can be accounted for by them. In addition, less than 10% of oscillated genes
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are common to any two tissues examined (Hastings et al., 2003). Zvonic et al. (2007) also reported that more than 26% of the calvarial transcriptome exhibits circadian rhythms, but only 3% of them are shared among bone, brown and white adipose tissues, and liver. These findings suggest the existence of another mechanism by which the circadian gene expression is regulated in a tissue-specific manner. In the previous studies, Simmons (1966) and Simmons et al. (1983) reported that incorporation of [ 35S]sulfate into chondroitin sulfate in rat growth plate cartilage was high in daytime and low at night, and alkaline phosphatase activity was high at night, suggesting the importance of circadian rhythmicity in growth and development of bones. We recently identified 277 genes that are expressed in growth plate cartilage in a circadian oscillated manner (Honda et al., submitted for publication). Most of these oscillating genes have not been shown to be expressed circadian-dependently in other tissues. In the present study, we attempted to identify new clock elements that produce circadian rhythms of these genes in the cartilage using in silico analysis. Here, we show 10 sequence motifs that may be responsible for circadian regulation of gene expression in the cartilage in a phase-dependent manner. Among these motifs, an E-box-like element (EL-box) showed similar properties to E-boxes, but had different responsiveness to DEC1 and HES1. These motifs may provide new insights into the circadian regulation of gene expression. 2. Materials and methods 2.1. Animals, RNA extraction and DNA microarray analysis Six-week-old male Sprague–Dawley rats (Crea Japan, Tokyo) were housed under a 12:12 h light–dark (LD) cycle for 14 days. Following this, rats of each group were housed under LD conditions or constant darkness (DD), and three of the rats were sacrificed every 4 h (n = 3) for 48 h. Total RNA was extracted from rib growth plates and resting cartilage. Biotinylated cRNA prepared from the RNA was subjected to DNA microarray analysis using GeneChip Rat Expression Array 230A/ B (Affimetrix, Santa Clara, CA). Details of the analysis will be described elsewhere (Honda, KK et al., submitted). Briefly, analysis was performed as described previously (Ueda et al., 2002). The time course for the expression profile obtained with each probe set was depicted, and any similarity between the profiles for the first and last 24 h was examined. In addition, any similarity between these profiles and that of a standard cosine curve, and between the profiles for 48 h under LD and DD conditions, was examined. Using these criteria, we selected candidates of oscillating genes. The rhythms of gene expression in DNA microarray analysis were evaluated by one-way ANOVA using the expression levels (n = 4) at ZT/CT 2, 6, 10, 14, 18, and 22, which were obtained regardless of whether they were in the first or second 24 h, and regardless of LD or DD. All procedures were performed in compliance with standard principles and guidelines for the care and use of laboratory animals, Hiroshima University Graduate School of Biomedical Sciences. 2.2. Isolation of clock elements Analyzing the microarray data, we identified 254 genes that showed statistically significant (Pb 0.05) circadian expression (Honda et al., submitted). The promoter of a gene is defined as the region 1000 bp upstream from the transcriptional start site (TSS) to 200 bp downstream from the TSS. We selected 54 genes for further analysis, because half of identified genes were unknown genes at the time we started the analysis and some of promoter regions were not conserved among mice, rats, and humans. We consulted transcriptional regulatory database (TRED) (Zhao et al., 2005) to retrieve reliable TSSs, and chose only the promoters categorized into levels 1–3 in TRED (Supplemental Table 1).
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To reduce noisy outcomes of motif finding programs, we first obtained a multiple sequence alignment of the promoter regions that were orthologous among rat, mouse and human genes. For alignment, we progressively applied Aln (Gotoh, 1999) in the semi-global mode. We then scanned the alignment columns with a window of a fixed size of five. If four or all of the five positions in a window are completely conserved among the three species, we regarded the sites within the window as “informative” and other sites as “non-informative”. We assigned ʻN ’ to a non-informative site, the most frequent nucleotide to a completely or partially conserved site, and the nucleotide of the rat genome to a completely divergent site. We finally obtained 54 promoter sequences that fulfilled all the criteria mentioned above. We then categorized the genes with these promoter sequences into six groups according to the peak times {Zeitgeber time (ZT) 0–4, 4–8, 8–12. 12–16, 16–20, 20–24} and then individually applied a motif finding program Weeder (Pavesi et al., 2004) to each group of the masked promoter sequences. We used the “large” and “zoop” options of Weeder for the analysis, which can detect motifs of sizes of 6, 8, 10, or 12. Then, we used Weeder's Motif Locator tool to search for instances of individual motifs thus found in all the 54 promoter sequences allowing for up to two mismatches compared with the consensus sequence. To test whether a motif thus found is correlated with a specific phase of circadian expression profile, we conducted a χ2 or Fisher's exact test with a 2 ×2 contingency table. Each element of the table represents the number of promoters conditioned by the presence or absence of the relevant motif, and the membership of the corresponding gene in the specific category of expression phase. The candidate motifs were sorted by the P-values of this test, and most significantly correlated motifs were selected. 2.3. Plasmid constructions Promoter regions of mouse Ank (−1107 to +293), Dbp (−1289 to +196) and Nr1d1 (−780 to +470) were amplified by PCR from mouse (C57BL/6J) genomic DNA and subcloned into pGL3-Basic (Promega). Sequences of primers used in this study are shown in Supplemental Table 2. Parts of Dbp promoter regions (−734 to +196 and −394 to +196) were also amplified and subcloned into pGL3-Basic. Mouse Bmal1, Clock, Npas2, Hes1, and Hes2 cDNAs were obtained by RT-PCR from mouse liver and subcloned into pcDNA4-V5-HisA (Invitrogen) or p3xFLAG-26 (Sigma). Oligonucleotides of three tandem sequences containing mouse Ank, Dbp, or Nr1d1 EL-box with 4-bp of flanking sequences (Supplemental Table 3) were subcloned into pGL3-TK. To construct a series of mutants of Ank EL-box, oligonucleotides of three tandem sequences containing mutated Ank EL-boxes with 4-bp of flanking sequence (Supplemental Table 3) were subcloned into pGL3-TK. Expression vectors for Bmal1 and Dec1 have been described previously (Kawamoto et al., 2004). Cry1 expression vector was generously supplied by M. Ikeda, Saitama Medical School. To prepare probes for DNAP assay, phosphorylated oligonucleotides of three tandem sequences containing human Dec1 E-box (CACGTG) with 6-bp of flanking sequence or mouse Ank or Dbp EL-box with 4-bp of flanking sequence with sticky ends were self-ligated using ligation high (Toyobo) and subjected to 8% acrylamide gel electrophoresis. DNA elements around 300 bp long were extracted from the gel and subcloned into pEGFP-N1 (Clontech) and resulting plasmids were named pEGFPDec1-E-boxX10, pEGFP-Ank-EL-boxX8 and pEGFP-Dbp-E-boxX7. All constructed described above were validated by nucleotide sequencing. 2.4. Transient transfection and luciferase reporter assay NIH3T3 cells were seeded at 1 × 10 4 cells per well in a 96-well plate 24 h before transfection. Luciferase reporter plasmid (4 ng per well) and 0.05 ng of pRL-SV40 (Promega), as an internal standard, were cotransfected with mouse Bmal1, Flag-Clock, and Flag-Npas2 expression vectors (each 60 ng per well) using Lipofectamine 2000. The total concentration of DNA was adjusted to 124.05 ng per well with
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an empty vector (pcDNA4-V5-HisA). The cells were incubated for 48 h and subjected to luciferase reporter assays using Dual-Luciferase Reporter Assay System (Promega). Luciferase activities were normalized by internal control activities. The values represent the mean±SEM for three wells. Statistical significance was analyzed by Student t test (Fig. 5) or one-way analysis of variance (ANOVA) followed by Turkey's multiple comparison test (Figs. 3, 6, and 7). Various amounts of expression vectors for Cry1, Dec1, Dec2, Hes1, and Hes2 were added to determine the dose-dependent inhibition of the BMAL1/CLOCK activity. The amounts of each expression vector that resulted in the half-maximal activity were determined as 50% inhibitory concentration (IC50) by manual plotting.
time range of circadian expression and the existence of short sequence motifs in conserved regulatory regions in the promoters among humans, mice and rats. From this analysis, we found 10 sequence motifs commonly present in a group of genes, the circadian phases of which were significantly associated with the same peak
2.5. DNA affinity precipitation (DNAP) assay To examine the direct binding of BMAL1/CLOCK or BMAL1/NPAS2 to EL-boxes, DNAP assay was performed as described previously (Suzuki et al., 1993). Expression vectors (9 μg each) for BMAL1 and FLAG-tagged CLOCK, BMAL1 and FLAG-tagged NPAS2, or the same amounts of an empty vector were transfected into COS7 cells seeded in 10 cm dishes, and nuclear extracts were prepared from the transfected cells using Nuclear Extract Kit (Active motif) and dialyzed against DNAP buffer, pH7.9 (20 mM HEPES–KOH, 80 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 0.5 mM, DTT, 10% glycerol, 0.1% tritonX-100 and 0.1 mM PMSF). For preparation of biotinylated probes, multicopies of Dec1 E-box, Ank EL-box and Dbp EL-box were amplified by PCR from pEGFP-Dec1-E-boxX10, pEGFPAnk-EL-boxX8 and pEGFP-Dbp-E-boxX7, respectively, using biotinylated primers. As a negative control, a part of the luc2P gene (273 bp) was also amplified from pGL4.11 (Promega). The biotinylated DNA probes (1 μg) were incubated with the dialyzed nuculear extracts (100 μg) in 500 μl of DNAP buffer containing poly(dI–dC) (15 μg) at 4 °C for 2 h. Then, streptavidin beads (Dynabeads M-280, Dynal) were added to the mixture and mixed by rotation for 30 min. The Dynabeads were collected with a magnet and washed 5 times with DNAP buffer. The trapped proteins were analyzed by SDS-PAGE followed by immunoblotting with anti-FLAG antibodies (Sigma). The experiments were repeated 3 times and similar results were obtained. 2.6. Chromatin immunoprecipitation (ChIP) assay ChIP assays for Ank and Dbp EL-boxes were performed using Chromatin Immunoprecipitation Assay Kit (Upstate) according to the manufacturer's instructions. Forty-eight hours before the assays, expression plasmids (12 μg each) for BMAL1 and FLAG-tagged CLOCK or FLAG-tagged NPAS2 were transfected into ATDC5 cells. Extracts prepared from the cells were subjected to precipitation with anti-FLAG antibodies or control IgG. The precipitated DNA samples were subjected to PCR using gene specific primers (Supplemental Table 2). The PCR was carried out under the following condition: denaturation at 98 °C for 10 s, annealing at 60 °C for 15 s, and extension at 68 °C for 60 s using PrimeSTAR GXL DNA Polymerase (Takara). After 30 cycles of PCR, obtained products were analyzed by agarose gel electrophoresis. The experiments were repeated 3 times and similar results were obtained. 3. Results 3.1. Sequence motifs correlated with the circadian phases of rhythmic gene expression in growth plate cartilage Using microarray analysis we identified various genes, the expression of which showed circadian rhythmicity in the growth plate of rat rib (Honda et al., submitted for publication). To determine promoter elements that govern circadian expression of these rhythmic genes, we subjected 54 gene promoters, which had been well characterized among three species (up to 1 kb from the transcription initiation site; Supplemental Table 1), into correlation analysis between the peak
Fig. 1. Sequence motifs correlated with the circadian phases of rhythmic gene expression in growth plate cartilage. Sequence motifs were extracted from each promoter group with a specific range of peak time, and then their locations in all the 54 promoter sequences were searched for by a motif locator. The fourth column lists the genes that harbor the relevant motif and the fifth column indicates their peak time. Ten sequence motifs showed significant correlation with the peak times as evaluated by t-test or Fisher's exact test. Some genes listed are out of the peak time range, even though all genes carrying the motifs are listed here.
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time classification (Fig. 1). In this study, we focused on the EL-box that had a GGCACGAGGC consensus sequence (Fig. 1). 3.2. A new clock element (EL-box) responsible for induction by BMAL1/ CLOCK The genes containing the EL-box showed clear circadian rhythms (Supplemental Fig. 1). The existence of the EL-box was significantly correlated with the peak times of ZT 8–12 (Fig. 2; P b 0.001): Nr1d1, Slc20a1, Angptl2, Emp3, and Dbp showed the peak times between ZT 8 and ZT 12. In addition, Tef, Klf9, Ank, and Klf15 had similar peak times (ZT 12.0, ZT 12.4, ZT 12.8, and ZT 13.2). Since the EL-boxes contained a sequence, CACGAG, similar to the E-box (CACGTG) element, we hypothesized that EL-boxes may mediate circadian regulation through interaction with BMAL1/CLOCK or BMAL1/ NPAS2. To test this hypothesis, we examined whether EL-box elements have enhancer activities responsive to these transcription factors. ELboxes of Ank, Dbp, and Nr1d1 were subcloned into a luciferase reporter plasmid and co-transfected into NIH/3T3 cells with expression vectors for BMAL1 and CLOCK or BMAL1 and NPAS2. The results demonstrated that BMAL1/CLOCK or BMAL1/NPAS2 markedly increased expression levels of reporter genes carrying EL-box elements (Fig. 3A), whereas BMAL1 or CLOCK alone had no activity on the E- or EL-box-mediated transcription (Supplemental Fig. 2). The native promoters of these genes also showed similar responsiveness to BMAL1/CLOCK or BMAL1/ NPAS2 (Fig. 3B).
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NPAS2 were co-expressed in COS7 cells. Nuclear extracts were prepared from these cells and incubated with biotinylated DNA probes of Ank EL-box, Dbp EL-box and Dec1 E-box elements, and protein-DNA complexes were precipitated with streptavidin beads. As shown in Fig. 4A, binding of BMAL1/CLOCK and BMAL1/NPAS2 to oligonucleotides containing EL-boxes and Dec1 E-box was observed, whereas their binding to a control luc2P gene fragment was not detected. To confirm that BMAL1/CLOCK or BMAL1/NPAS2 actually binds to EL-box-containing promoters in living cells, ChIP assay was performed. Both BMAL1/CLOCK and BMAL1/NPAS2 heterodimers bound to the Dbp and Ank promoters (Fig. 4B). These findings in DNAP and ChIP assays, taken together, revealed that BMAL1/CLOCK and BMAL1/NPAS2 can bind to EL-boxes in oscillating genes. 3.4. Mutational analysis of EL-box elements In addition to E-box, E′-box, and NC E-box elements, the EL-box element may provide a new option for BMAL1/CLOCK action. For further characterization of the EL-box ability, we examined which nucleotides in Ank EL-box are essential for induction by BMAL1 and CLOCK. For this purpose, we generated a series of mutant constructs and performed luciferase reporter assays using these constructs together with expression vectors for BMAL1and CLOCK. All one or two point mutations,
3.3. Binding of BMAL1/CLOCK and BMAL1/NPAS2 to EL-box elements Since the BMAL1/CLOCK heterodimer and BMAL1/NPAS2 heterodimer enhanced the expression of EL-box-containing reporter genes, we performed DNAP (DNA affinity precipitation) assay to show their binding to EL-box elements. BMAL1 and FLAG-tagged CLOCK or FLAG-tagged
Fig. 2. Sequence comparison of newly identified clock elements (designated EL-box elements). Identified 54 genes, expression of which showed circadian rhythmicity, were categorized into six groups according to the peak times (ZT 0–4, 4–8, 8–12, 12–16, 16–20, and 20–24). EL-box elements exist in the promoter regions of 11 genes out of 54 oscillated genes and expression levels of 5 genes were peaked between ZT 8 and ZT 12. The DNA sequence logo describing a graphical representation of the EL-box consensus was generated using WebLogo (http://weblogo.berkeley.edu/).
Fig. 3. EL-box elements are able to mediate the induction by BMAL1/CLOCK or BMAL1/ NPAS2. (A) Activities of Ank, Dbp, and Nr1d1 EL-boxes were examined by luciferase assay. Reporter plasmids carrying three copies of EL-box connected to the TK promoter were cotransfected with expression vectors for BMAL1 and CLOCK or NPAS2 into NIH3T3 cells, and luciferase activities were measured. **, Pb 0.01; ***, Pb 0.001. (B) Promoter constructs of mouse Ank (−1107 to +293), Dbp (−1289 to +196), and Nr1d1 (−780 to +470) were also used for reporters to examine the induction by BMAL1/CLOCK or BMAL1/NPAS2.
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Fig. 4. BMAL1/CLOCK or BMAL1/NPAS2 heterodimers bind to EL-box elements. (A) DNA affinity precipitation (DNAP) assay. Expression plasmids for BMAL1 and FLAG-tagged CLOCK or FLAG-tagged NPAS2 were transfected into COS7 cells. Nuclear extracts prepared from the transfected cells were incubated with biotinylated probes of Dec1 E-box, Ank EL-box and Dbp EL-box. As a negative control, the luc2P gene was used. Proteins bound to the probes were precipitated with streptavidin beads and analyzed by SDS-PAGE followed by immunoblotting using anti-FLAG antibodies. (B) Chromatin immunoprecipitation (ChIP) assay. ATDC5 cells were transfected with expression vectors for BMAL1 and FLAG-tagged CLOCK or FLAG-tagged NPAS2. DNA samples precipitated from the cells with anti-FLAG antibodies or control IgG were subjected to PCR using gene specific primers for Ank and Dbp. The amplified regions by PCR were schematically shown in the bottom of the figure.
except for the substitution of CG in the first two nucleotides to TT (M1), caused significant reduction in the BMAL1/CLOCK-induced activation of reporter genes (Fig. 5 and Supplemental Fig. 4). However, the effect of M5 mutation was modest as compared with almost complete abolishment with M2, M6, M7, and M8 mutations. In addition, M5 mutation had lesser effect than M3 and M4 mutations. M5 mutant still showed more than 3-fold induction by BMAL1/CLOCK, which is only 25% less than about 4-fold induction in the wild-type construct. Thus, the 6-bp sequence (CACGAG) from the EL-box consensus, which is similar to
the E-box consensus, is critical, although its surrounding sequences have some activities. 3.5. Role of EL-box element in the Dbp promoter The transcriptional activity of the Dbp promoter has been reported to be inducible by the BMAL1/CLOCK heterodimer, which binds to a NC E-box element (CATGTG) in the promoter (Kiyohara et al., 2008). In this study, we examined whether the EL-box in the Dbp promoter actually responds to BMAL1/CLOCK. As shown in Fig. 6A, luciferase activities of the Dbp promoter constructs were enhanced by BMAL1/CLOCK or BMAL1/ NPAS2; the activity was markedly reduced by the deletion of the −734 to −394 region, which contained the NC E-box. However, significant induction of the promoter activity by BMAL1/CLOCK or BMAL1/NPAS2 was still observed even with a minimal promoter construct with the EL-box without the NC E-box. To further confirm the involvement of the Dbp EL-box in BMAL1/CLOCK or BMAL1/NPAS2 induction, we made another Dbp promoter construct that contained a mutated EL-box sequence (Fig. 6B). This substitution markedly reduced BMAL1/CLOCK- or BMAL1/ NPAS2-mediated transcription. These results indicate that the EL-box is involved in the stimulation of Dbp promoter activity by BMAL1/CLOCK or BMAL1/NPAS2. 3.6. Effects of negative factors on the enhancer activity of the EL-box
Fig. 5. Mutational analysis of EL-box elements. One or two nucleotides in the Ank EL box sequence were replaced and subjected to luciferase reporter assays. A series of report constructs was cotransfected with a control vector (open bars) or expression vectors for BMAL1 and CLOCK (closed bars) into NIH3T3 cells and luciferase activities were determined. *, P b 0.05; **, P b 0.01; ***, P b 0.001; ns, not significant. Fold induction rates by CLOCK/BMAL1 are shown in Supplemental Fig. 4.
Next, we examined the effects of negative factors such as PER, CRY, and DEC on BMAL1/CLOCK-induced transcription from the ELbox-containing promoter. Co-transfection of CRY1, DEC1, or DEC2 significantly reduced luciferase activities of both Ank EL-box- and Dec1 Ebox-containing constructs induced by BMAL1/CLOCK (Fig. 7A). However, the suppressive activity of DEC1 and DEC2 was less on the Ank EL-box than on the Dec1 E-box. Since the EL-box consensus sequence contains an N-box sequence (CACNAG) to which HES family transcription factors bind (Sasai et al., 1992), we examined the suppressive activity of HES1
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Fig. 6. Both EL-box and NC E-box elements in the Dbp promoter are responsible for the induction by BMAL1/CLOCK or BMAL1/NPAS2. (A) Various lengths of Dbp promoter constructs were cotransfected with expression vectors for BMAL1 and CLOCK or NPAS2 into NIH3T3 cells and luciferase activities were measured. (B) Effect of a EL-box mutation in the Dbp promoter was analyzed. *, P b 0.05; **, P b 0.01; ***, P b 0.001.
and HES2: HES1 significantly decreased EL-box activity in the presence of BMAL1/CLOCK, but did not decrease E-box activity. For further analysis of the difference of suppressive activities of these negative factors on E-box and EL-box elements, we examined the effects of increasing doses of CRY1, DEC1, DEC2, HES1 and HES2 and calculated IC50. IC50 of CRY1 on the Ank EL-box reporter was similar to that on the Dec1 E-box reporter, whereas those of DEC1 and DEC2 were higher on the EL-box than on the E-box (Fig. 7B). The results also showed that the effects of DEC1 and DEC2 on the EL-box were much weaker than those on the E-box, whereas the effect of CRY1 on the EL-box was similar to that on the E-box (Supplemental Fig. 3). In contrast, IC50 of HES1 was lower on the EL-box reporter than on the E-box. These results indicated that HES1 may serve as a negative regulator like CRY and DEC. 4. Discussion In the present study, we identified several sequence motifs that are correlated with circadian phases of rhythmic genes in the
cartilage. Although it is not known whether these genes are expressed in a circadian manner in other tissues, some of these genes, such as Lox, Mmp14, Tgfa, Ank, Gilz, Slc20a1, and Mgea5, are actually involved in the morphology and/or function of chondrocytes. On these motifs the EL-box showed responsiveness to BMAL1/ CLOCK or BMAL1/NPAS2, which controls the clock system ubiquitously. Although its transcriptional activity was similar to those of E-box and E′-box, the suppressive effects of DEC1 and DEC2 were less potent on the EL-box than on the E-box. Furthermore, the induction of the EL-box-containing promoter by BMAL1/CLOCK or BMAL1/ NPAS2 was suppressed by HES1, whereas HES1 does not show any suppressive effect on the E-box element. DEC1 and DEC2 are transcription factors that bind to E-box elements and suppress transcription from their target genes (Hamaguchi et al., 2004; Honma et al., 2002; Kawamoto et al., 2004). In addition to strong binding affinity for the sequence CACGTG (E-box), DEC1 and DEC2 have weaker affinity for the sequences CACGTT (E′-box) and CACGCG (Fujimoto et al., 2007; Nakashima et al., 2008). DEC1 and DEC2 may have weaker affinity for EL-boxes than for the E-box. The difference of binding affinity
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Fig. 7. HES1 suppresses EL-box activity induced by BMAL1/CLOCK. (A) Effect of negative factors such as CRY1, DEC1, DEC2, HES1 and HES2 on Ank EL-box and Dec1 E-box elements was examined. An expression vector (20 ng) of one of these factors was cotransfected with expression vectors (20 ng each) for BMAL1 and CLOCK together with a reporter construct (50 pg) for the Ank EL-box or Dec1 E-box into NIH3T3 cells and luciferase activity was measured after 48 h of culture. Some of factors significantly decreased the activity induced by BMAL1/CLOCK. ***, P b 0.001. (B) Fifty percent inhibitory concentration (IC50) of negative factors for BMAL1/CLOCK-induced luciferase activity was determined. Various amounts (0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 ng) of expression vectors were used to examine the dose-dependency. Total amount of plasmid DNA for transfection was adjusted by an empty vector.
for EL-box and E-box sequences accounts for weaker suppressive activities of DEC1 and DEC2 to EL-box elements observed in this study. HES1, which has sequence homology to DEC1 and DEC2 (Fujimoto et al., 2001; Shen et al., 1997), is a transcription factor that binds to N-boxes and suppresses transcription of target genes (Sasai et al., 1992). As shown in the present study, the EL-box sequence contains the N-box consensus sequence (CACNAG), and HES1 had a suppressive effect on BMAL1/CLOCK-induced expression of the EL-box-containing promoter. The sequence difference between the EL-box and E-box resulted in distinct responsiveness to HES1 as observed in this study. Although Hes1 expression in cultured cells shows oscillation with 2-h periodicity after serum treatment (Hirata et al., 2002), it did not show significant circadian rhythmicity in rat rib cartilage (data not shown). However, the cross-talk between HES and clock systems may have physiological roles through EL-boxes in some situations.
In our in silico analysis, E-box and E′-box elements were not identified as a candidate sequences for circadian regulation. The EL-box sequence (GGCACGAGGC) is longer than an E-box or E′-box sequence, which consists of only 6 nucleotides. A longer consensus sequence may have provided an advantage for in silico analysis because identification of sequence motifs in clock-related genes depends on the probability of existence in relation to circadian phases. In addition, some E/E′-box elements do not seem to function in circadian regulation, since nonclock transcription factors such as c-MYC also transactivate their target genes via E-box elements. Recent studies demonstrated that double-E-box-like elements with a 6 or 7-bp spacer located in various clock genes have strong ability to produce BMAL1/CLOCK-induced expression (Nakahata et al., 2008). Among eleven EL-box-containing genes, only the Tef promoter contains the CACGTG E-box immediately upstream of an
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EL-box element. These findings suggest that the mechanism involved in EL-box-mediated activation may differ from that for the direct repeat of E-box-like elements. On the other hand, some of the EL-box-containing genes were shown to contain other clock elements such as E-box, E', and NC E-box elements in their regulatory regions. For example, an EL-box in the Dbp promoter worked with an NC E-box synergistically in luciferase reporter assays. In addition to these elements, the Dbp gene also contains two functional E-boxes in its intron region (Ripperger et al., 2000; Ueda et al., 2005). Thus, multiple elements seem to contribute to the unique regulation of clock-related genes. Although we could not determine whether all of EL-boxes identified in this study are functional, our results suggest that, in addition to known clock elements, the EL-box element may contribute to circadian regulation of clock and clock-controlled genes. Acknowledgments This work was supported by grants-in-aid for science from the Ministry of Education, Culture, Sport, Science and Technology of Japan. We thank Maki Ukai-Tadenuma and Hiroki R. Ueda for their technical support and critical reading of the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.gene.2012.08.022. References Fujimoto, K., et al., 2001. Molecular cloning and characterization of DEC2, a new member of basic helix-loop-helix proteins. Biochem. Biophys. Res. Commun. 280, 164–171. Fujimoto, K., et al., 2007. Transcriptional repression by the basic helix-loop-helix protein Dec2: multiple mechanisms through E-box elements. Int. J. Mol. Med. 19, 925–932. Gekakis, N., et al., 1998. Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569. Gotoh, O., 1999. Multiple sequence alignment: algorithms and applications. Adv. Biophys. 36, 159–206. Hamaguchi, H., et al., 2004. Expression of the gene for Dec2, a basic helix-loop-helix transcription factor, is regulated by a molecular clock system. Biochem. J. 382, 43–50. Hastings, M.H., Reddy, A.B., Maywood, E.S., 2003. A clockwork web: circadian timing in brain and periphery, in health and disease. Nat. Rev. Neurosci. 4, 649–661. Hirata, H., et al., 2002. Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 298, 840–843.
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