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¹ Department of Dental and Medical Biochemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan
² Department of Physiology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
³ Department of Biochemistry, Nihon University School of Medicine, Tokyo 173-8610, Japan

	Abstract

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DEC1 (BHLHB2/Stra13/Sharp2)—a basic helix-loop-helix transcription factor—is known to be involved in various biological phenomena including clock systems and metabolism. In the clock systems, Dec1 expression is dominantly up-regulated by CLOCK : BMAL1 heterodimer, and it exhibits circadian rhythm in the suprachiasmatic nucleus (SCN)—the central circadian pacemaker—and other peripheral tissues. Recent studies have shown that the strong circadian rhythmicity of Dec1 in the SCN was abolished by Clock mutation, whereas that in the liver was affected, but not abolished, by Clock mutation. Moreover, feeding conditions affected hepatic Dec1 expression, which indicates that Dec1 expression is closely linked with the metabolic functions of the liver. Among ligand-activated nuclear receptors examined, LXR and LXRβ with T0901317—agonist for LXR—were found to be potent enhancers for Dec1 promoter activity, and a higher expression level of LXR protein was detected in the liver than in the kidney and heart. T0901317 increased the levels of endogenous Dec1 transcript in hepatoma cells. Chromatin immunoprecipitation assay indicated that LXR bound to the Dec1 promoter, and an LXR-binding site was identified. These observations indicate that hepatic DEC1 mediates the ligand-dependent LXR signal to regulate the expression of genes involved in the hepatic clock system and metabolism.

	Introduction

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DEC1 (BHLHB2/Stra13/Sharp2)—a basic helix-loop-helix (bHLH) transcription factor—is known to be ubiquitously expressed and involved in various biological phenomena including chondrogenesis (Shen et al. 2002), neurogenesis (Rossner et al. 1997), cell growth arrest (Sun & Taneja 2000), hypoxia response and carcinogenesis (Ivanova et al. 2001; Miyazaki et al. 2002), age-induced autoimmunity as a result of impaired T-lymphocyte activation (Sun et al. 2001), and the clock system (Honma et al. 2002; Kawamoto et al. 2004; Nakashima et al. 2008).

Our recent studies have shown that Dec1 and a structurally related gene, Dec2 (BHLHB3/Sharp1), exhibit robust circadian rhythm in various organs including the suprachiasmatic nucleus (SCN)—the master circadian pacemaker in mammals—and that their proteins down-regulate various clock-controlled genes, including Per1, Dec1 and Dec2 by binding to CACGTG E-box, which is also responsible for CLOCK : BMAL1 binding. Dec1 and Dec2 genes themselves are thus regulated by the positive regulators—CLOCK, NPAS2, BMAL1 and BMAL2—and the negative regulators—PER1, PER2, CRY1, CRY2, DEC1 and DEC2—through E-box elements (Honma et al. 2002; Hamaguchi et al. 2004; Kawamoto et al. 2004; Nakashima et al. 2008). These lines of evidence established that DEC1 and DEC2 are components in the molecular clock system.

On the other hand, we found that while hepatic circadian expression profiles of Dec1 in homozygous Clock mutant mice (Ck/Ck)—mutant mice expressing dominant negative CLOCK protein (Gekakis et al. 1998)—were influenced, Dec1 mRNA expression remained at similar levels, indicating that hepatic Dec1 is up-regulated by some other factor(s) in addition to CLOCK : BMAL1. In contrast, the expressions of Dec1 in the SCN and heart of Clock mutant mice were markedly reduced and became arrhythmic (Butler et al. 2004; Noshiro et al. 2005). In addition, various other stimuli, such as NGF, kainic acid, PDGF, retinoic acid, cAMP, insulin, and TGFβ, rapidly induced the expression of Dec1 mRNA within 1 h as an immediate-early gene (Rossner et al. 1997; Inuzuka et al. 1999; Shen et al. 2001; Zawel et al. 2002; Yamada et al. 2003). More recent study showed that fasting of mice down-regulated Dec1 mRNA expression in peripheral tissues, especially in the liver (Kawamoto et al. 2006). Re-feeding after fasting restored the decreased Dec1 mRNA, but these treatments did not affect the peripheral expression of Dec2 mRNA. Restricted feeding shifted the phase of hepatic expression profile of Dec1 to feeding period. These observations suggest that hepatic Dec1 may have some metabolic functions in the liver.

Members of the nuclear hormone receptor superfamily function as ligand-activated transcription factors to regulate hepatic genes critical in metabolism (Mangelsdorf et al. 1995). A subgroup of these receptors—LXRs, PPARs, FXR, VDR, PXR and CAR—appear to serve as lipid sensors, since they are bound and activated by dietary-derived or endogenous lipids and ultimately regulate various genes to exhibit hepatic functions (Chawla et al. 2001). Consequently, to look for new regulator(s) of Dec1, we tested various nuclear receptors functional in the liver, and found that LXR and LXRβ are potent positive regulators for hepatic Dec1 expression and that DEC1 may participate in mediating the LXR signals to ultimate targets.

	Results

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Multiple regulatory mechanisms exist for hepatic Dec1 expression

First we determined the effects of Clock mutation on hepatic expression of Dec1 and Dec2. In wild-type mice (BALB/c), hepatic expression of Dec1 mRNA showed a circadian rhythm with peaks at ZT 6 (one-way ANOVA, P < 0.05), whereas in Clock mutant mice (Ck/Ck), peaks were delayed by 6 h compared with the peak in wild-type mice, but the pattern maintained a significant rhythmicity (one-way ANOVA, P < 0.05) (Fig. 1A, left). Differences in the patterns between genotypes were significant (two-way ANOVA, P < 0.01) indicating that CLOCK is indeed involved in the regulation of Dec1 but there seem to be some other mechanism(s) (Noshiro et al. 2005). On the other hand, strong circadian rhythm of Dec2 expression with a peak at ZT6 was observed in wild-type mouse liver (one-way ANOVA, P < 0.01), and this was abolished by Clock mutation (two-way ANOVA, P < 0.01) (Fig. 1A, right), indicating that CLOCK : BMAL1 heterodimer dominantly regulates Dec2 expression (Butler et al. 2004; Noshiro et al. 2005).

View larger version (32K): [in this window] [in a new window]   Figure 1  Gene expressions in the liver of Clock mutant (A and B) and starved mice (C and D). Transcript levels of Dec1 were determined by real time RT-PCR for three mice at each time point. Data are shown as mean ± SEM (n = 3). Open and closed bars indicate light and dark phases, respectively. Time series data were analyzed by one-way ANOVA for rhythmicity (*P < 0.05; **P < 0.01; ns, not significant) in the livers of Clock mutant mice (A) and fasted mice (C). Differences between genotypes were tested by two-way ANOVA (*P < 0.05; **P < 0.01; ns, not significant). Area under the curve (AUC) values were calculated for each time series data in order to compare whole day expression levels of the genes in the liver, kidney, heart, lung, and adrenal gland (B). The results are expressed as percentages of AUC values for the respective tissue of wild-type mice. AUC values were calculated for each gene in the liver (D). The results are expressed as percentages of AUC values for the respective gene in control mice.

On the other hand, 24 h-fasting of mice (C57/BL6) significantly repressed Dec1 expression (two-way ANOVA, P < 0.01)(Fig. 1C), and AUC value indicated that the repression of Dec1 was similar to that of hepatic enzymes, including Cyp7a and Hmgcr, involved in cholesterol metabolism (Fig. 1D). However, Dec2 expression was not affected by the fasting. Our previous studies showed that re-feeding after fasting up-regulated Dec1 expression, whereas it did not change Dec2 expression (Kawamoto et al. 2006).

These observations indicate that some factor(s) related to the metabolic pathways regulate the expression of hepatic Dec1.

LXR and LXRβ are potent positive regulators for hepatic Dec1

Several canonical AGGTCA elements and its related elements, binding sites for nuclear receptors, were found in the upstream (–1700 to –2500 nt) of mouse Dec1 promoter (see Fig. 3A), a region highly conserved in several animal species including mouse, rat, human, cow and dog (data not shown). A subgroup of nuclear receptors—LXRs, PPARs, FXR, VDR, PXR and CAR—are activated by dietary-derived or endogenous ligands and ultimately regulate various genes (Chawla et al. 2001). Consequently, we examined the effects of ligand-dependent activation of these nuclear receptors on Dec1 promoter activity. Luciferase reporter carrying 3.9 kb of 5'-flanking region of the mouse Dec1 gene was constructed and we examined the effects of ten ligand-activated nuclear receptors on Dec1 promoter activity in HepG2 cells. RXR as a heterodimer partner and proper agonists were added when they were required. Among these, three nuclear receptors—LXR, LXRβ and CAR—significantly enhanced reporter activity, whereas PXR significantly suppressed Dec1 promoter activity (Fig. 2A). The Dec2 promoter (3.1 kbp) was not induced by addition of LXR/β or other ligand-activated nuclear receptors examined (data not shown), which is in agreement with the absence of AGGTCA response element for nuclear receptors in the promoter region of the Dec2 gene of several animal species (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 3  Structure of mouse Dec1 promoter and detection of LXR-binding site. The structure of mouse Dec1 promoter, positions of AGGTCA half-site and CACGTG E-box, and fragments for luciferase constructs are illustrated (A). Reporter plasmids (20 ng/well) of Dec1 (3.9 kb) in pGL3 and three DNA fragments in pGL3-TK were transfected with LXR-VP16 (50 ng/well), RXR-VP16 (50 ng/well), and pRL-TK (2 ng/well) into HepG2 cells (B). Values were relative luciferase activities to the control level (without activator plasmids) of 3.9-kbp promoter. Significance of differences between respective control and the addition of LXR-VP16 : RXR-VP16 were analyzed by Student's t-test (**, P < 0.01).

View larger version (27K): [in this window] [in a new window]   Figure 2  Effects of nuclear receptors on Dec1 promoter. (A), Expression plasmids (100 ng/well) for each nuclear receptor (NR) were co-transfected with mouse Dec1 (3.9 kbp)-Luc (20 ng/assay) and the internal control plasmid pRL-TK (2 ng/assay) into HepG2 cells. When LXR/β, FXR, VDR, PXR, PPAR, PPAR and RAR were used, ligands T0901317 (T090, 10–7 M), chenodeoxycholic acid (CDCA, 10–5 M), 1,25-dihydroxyvitamin D3 (D3, 10–7 M), lithocholic acid (LC, 10–5 M), WY-14 643 (WY, 10–5 M), troglitazone (Trog, 10–6 M), and retinoic acid (RA, 10–7 M), respectively, dissolved in dimethyl sulfoxide, were added to the culture medium. Rxr with 9-cis retinoic acid (10–7 M) was co-transfected as a heterodimer partner. The luciferase activities were normalized by Renilla luciferase activities of the internal control pRL-TK, and data were plotted as mean values ± SEM in triplicate assay as a ratio to the control assay in the absence of expression plasmid and agonist (none). Significance of differences between respective control (none) and each condition were analyzed by Student's t-test (*P < 0.05; **P < 0.01). All the experiments were repeated at least three times and yielded reproducible results. (B), Mouse Dec1 (3.9 kbp)-Luc was co-transfected with or without expression plasmids for nuclear receptors into Hepa1c1c7 (Hepa1c), NIH3T3, or HEK294 cells. T0901317 (T090, 10–7 M) and 9-cis-retinoic acid (9cis, 10–7 M) were added as indicated. (C), Mouse Dec1 (3.9 kbp)-Luc was co-transfected with or without the expression plasmids into Hepa1c1c7 cultured in the medium containing lipoprotein-deficient serum (Biomedical Tech. Inc.) to minimize endogenous lipophilic ligands. T0901317 (T090, 10–8 to 10–6 M) and 9-cis-retinoic acid (10–7 M) were added. (D), Expression plasmids (100 ng/well) for CLOCK or NPAS2 and BMAL1, and mouse Dec1 (3.9 kbp)-Luc were co-transfected into Hepa1c1c7 cells.

Figure 2B,C show that the enhancement of the Dec1 promoter by LXR : RXR requires the known ligands T0901317 for LXR and 9-cis-retinoic acid for RXR as reported (Schultz et al. 2000; Spencer et al. 2001). In Hepa1c1c7 cells, heterodimer CLOCK : BMAL1 or NPAS2 : BMAL1 enhanced the Dec1 promoter activity (Fig. 2D), which is comparable to the enhanced levels by LXR : RXR in the same cells (Fig. 2B, left).

LXR response element in the Dec1 promoter

To identify the response element(s) for LXR, various promoter fragments of the AGGTCA-rich region were used to construct pGL3-TK luciferase reporter plasmids as shown in Fig. 3A, and we then examined the effects of LXR-VP16 (LXR fused to the active domain of herpesvirus VP16 protein) on them. Dec1 reporter constructs containing DNA fragment (–2603 to –1708) and fragment (–2146 to –1708) were significantly induced by LXR-VP16 : RXR-VP16 as well as 3.9 kbp-promoter of Dec1 (Fig. 3B). When the region of –2146 to –1954 was deleted, the enhancement by LXR-VP16 : RXR-VP16 was abolished, indicating that the response element(s) is located in this region.

In the region of –2146 to –1954, two putative direct repeat 4 (DR4) elements responsible for LXR : RXR heterodimer were found, and these elements were conserved among several animal species including mouse, rat, human, cow and dog (Fig. 4A). To identify the response element for LXR, DNA fragments designated as probes DR4-1 and DR4-2 in Fig. 4B were synthesized and used for EMSA. In the presence of either LXR or LXRβ with heterodimer partner RXR, specific retarded bands were observed only with DR4-2 (lanes 9 and 10) at a position similar to that with mouse Cyp7a LXRE and LXR : RXR (data not shown), but the retarded band was not observed in the absence of the heterodimer partner (lanes 7 and 8), and the mutated element (DR4-2 m) lost binding activity to LXR : RXR (lanes 11 and 12). In contrast, DR4-1 (lanes 1–5) did not show any specific retarded band in the presence of LXR or LXRβ with RXR. DR4-2 is thus identified as the LXR response element for Dec1 gene.

View larger version (47K): [in this window] [in a new window]   Figure 4  LXR and LXRβ proteins with a heterodimer partner RXR bound to the proximal DR4 site of mouse Dec1 promoter. A, Alignment of the region containing DR4-1 and DR4-2 for five animal species was obtained from the UCSC Genome Bioinformatics <http://genome.ucsc.edu/>. The region from –2133 to –2036 containing putative response elements for LXR, and sites of probes (DR4-1 and DR4-2) for EMSA assay are shown (B). Two nucleotides at the middle of each respective half-site were replaced by AA to produce mutated probes (DR4-2 m). EMSA was performed with 32P-labeled double stranded probes (DR4-1, DR4-2, and DR4-2 m), the nuclear receptors, and the agonist T0901317 (10–7 M). The radio-activities of retarded bands were visualized by FLA-3000G fluoro-image analyzer (Fujifilm Co.) (C).

LXR binds to Dec1 gene in vivo

To further confirm the interaction of LXR with Dec1 gene in living cells, ChIP assay was performed using antibodies against LXR and Hepa1c1c7 cells. Two specific primer sets P1 : P2 and P3 : P4 were used to amplify the Dec1 gene around DR4-2 (Fig. 5A): They formed PCR products of expected sizes (302, and 263 bp, respectively) from anti-LXR—immunoprecipitates and the input as shown in Fig. 5B, whereas control IgG did not precipitate the Dec1 promoter DNA. The ChIP data agree with the EMSA results (Fig. 4) and indicate that LXR interacts with the Dec1 gene promoter.

View larger version (44K): [in this window] [in a new window]   Figure 5  ChIP assays of Dec1 gene containing DR4 elements in Hepa1c1c7 cells. One day after addition of agonists T0901317 (10–7 M), cellular genomic DNA was cross-linked with formaldehyde, sonicated, and DNA fragments were precipitated using anti-LXR antibodies (LXR) or control IgG (Cont). The input and the immunoprecipitated DNA fragments were amplified using two primer sets (P1 : P2 and P3 : P4) for mouse Dec1, as shown in A.

Agonist for LXR, T0901317, was added to a confluent culture of HepG2 cells, and Dec1 mRNA levels were examined on the cells at 1, 2, 4 and 24 h after the addition of agonist, since Dec1 transcripts in culture cells were induced immediately after various treatments and then exhibited circadian oscillation every 24 h (Nakashima et al. 2008). Figure 6A shows that even control medium induced Dec1 transcript by more than threefold at 2 h after medium change, although the basal medium was glucose-free DMEM supplemented with lipoprotein-free bovine serum and cholesterol biosynthesis inhibitor (atorvastatin) to minimize endogenous levels of natural LXR ligands in the culture (Mitro et al. 2007). Nevertheless, the addition of T0901317 induced Dec1 transcripts to significantly higher levels than the control medium at 1, 2 and 24 h after the addition. On the contrary, Dec2 transcript levels were not affected by the addition of agonist (Fig. 6B). Two known genes enhanced by ligand-activated LXR—AbcA1 transporter and Srebp1—were examined with the same samples for comparison: Both were induced immediately after the addition as reported (Fig. 6C,D) (Mitro et al. 2007), but control levels slightly decreased. These findings indicate the direct regulation of Dec1 expression by LXR.

View larger version (22K): [in this window] [in a new window]   Figure 6  Agonist-induced Dec1 transcript in HepG2 cells. T0901317 (1 x 10–6 M) or vehicle (DMSO final 0.1%) was added to confluent cultures of HepG2 cells in 6-multiwell plastic plates. Three wells were used for each point. Cells were harvested at 1, 2, 4 and 24 h after the addition of agonist, and total RNA preparations were examined for expression levels of endogenous transcripts of Dec1 (A), Dec2 (B), AbcA1 (C), and Srebp1 (D), using quantitative real-time RT-PCR method. Pair-wise comparisons at each time point were done using Student's t-test (*P < 0.05; **P < 0.01). Data were plotted as mean ± SEM.

High expression of LXR is restricted to the liver, adipose tissue, small intestine, and macrophage, whereas LXRβ is ubiquitously expressed (Zhang & Mangelsdorf 2002). We also observed that the relative mRNA levels of Lxr were higher in the liver than in the kidney and heart (Fig. 7A, left), whereas the relative mRNA level of Lxrβ was higher in the kidney than in the liver and heart (Fig. 7A, right). Expressions of Lxr and Lxrβ were not affected by Clock mutation in any of the three tissues (Fig. 7A) and did not show rhythmicity (data not shown) (Noshiro et al. 2007). The protein level of LXR in the liver was also high, whereas it was low in the kidney and not detectable in the heart (Fig. 7B). Insignificant decrease of the LXR protein level was observed in the liver of Clock mutant mice. The higher expression of Lxrβ in the kidney and heart compared with the liver, apparently does not contribute to Dec1 expression in Clock mutant mice, since overall expressions of Dec1 in the kidney and heart of Clock mutant mice were much reduced (Fig. 1B). Based on the results in Fig. 2B, it is plausible that co-activators required for LXR might be absent or less abundant in the kidney.

View larger version (17K): [in this window] [in a new window]   Figure 7  Lxr and Lxrβ expression in the liver, kidney, and heart of mice. Relative levels of Lxr and Lxrβ mRNA were determined by quantitative real-time RT-PCR (A). Data were plotted as mean ± SEM (n = 4). (B), Western blot of LXR protein in the nuclear extracts of the three tissues. A representative photograph is shown (upper panel) and quantitative values (n = 3) were plotted as mean ± SEM (lower panel). The size of LXR protein (50 kDa) is indicated.

	Discussion

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High expression of LXR is restricted to some tissues including the liver, whereas LXRβ is ubiquitously expressed (Zhang & Mangelsdorf 2002). Higher enhancing activity of LXR for Dec1 promoter than that of LXRβ and higher content of LXR protein in nuclear extract of the liver than the kidney and heart indicate that LXR more likely activates Dec1 promoter to the comparable level activated by CLOCK : BMAL1 heterodimer in the liver. Consequently, in Clock mutant mice, Dec1 expression was remained at relatively high level in the liver although the peak was delayed, whereas it was markedly reduced in the kidney and heart as reported previously (Noshiro et al. 2005).

The natural ligands for LXRs, such as oxysterols, are supplied from diet or endogenously synthesized by cholesterol biosynthesis pathways and hydroxylation of side chain of cholesterol by cytochrome P450 species, including CYP27A, CYP3A4, and CYP46A (Björkhem & Diczfalusy 2002). Availability of dietary ligands for LXR might explain how hepatic Dec1 expression was suppressed by fasting and activated by re-feeding (Kawamoto et al. 2006). DEC1, up-regulated by ligand-activated LXR, may mediate down-regulation by LXR action through E-boxes of target genes involved in the metabolic functions or hepatic clock system as discussed below.

Numerous studies have shown that LXR serves as a transcription factor regulating not only lipogenesis but also carbohydrate metabolism in the liver (Steffensen & Gustafsson 2004; Makishima 2005; Kalaany & Mangelsdorf 2006). The molecular mechanism responsible for LXR-mediated hepatic lipogenesis has been largely attributed to the dramatic increase in expression of the target genes (such as Cyp7a, Srebp1, and AbcA1) as a result of the direct binding of LXR : RXR heterodimer to LXR-responsive element (LXRE) in their promoters when activated in the presence of agonists. On the other hand, expressions of some enzymes involved in gluconeogenesis or lipogenesis—phosphoenol pyruvate carboxykinase (Pepck), glucose-6-phosphatase (G6Pc), and glycerophosphate dehydrogenase 1 (Gpd1)—are down-regulated by the activation of LXR, and LXR response elements (LXRE) have not been identified among their promoters (Steffensen & Gustafsson 2004; Kalaany & Mangelsdorf 2006), which suggests that suppression of these genes by LXR take place in an indirect manner. Since DEC1 is a suppressor for target genes (Honma et al. 2002; Kawamoto et al. 2004), it is plausible that DEC1 mediates the suppressive action of LXR in some genes. It is also noteworthy that CACGTG E-boxes—DEC1 binding sequence—are found in Pepck and Gpd1 genes conserved in mouse, rat, and human genes (data not shown), and that Pepck and G6Pc are known to exhibit strong circadian rhythms (Ishida et al. 2000; Kennaway et al. 2007). Moreover, recent studies have demonstrated that DEC1 down-regulates Pepck expression based on the adenovirus-mediated over-expression of DEC1 in rat hepatocytes and the reporter assay using Pepck promoter and DEC1 (Yamada et al. 2005). DEC1 action on the other possible target genes must be examined in detail in future studies.

In conclusion, hepatic DEC1 is up-regulated by LXR : RXR heterodimer and may mediates the ligand-dependent LXR signal to regulate the expression of genes involved in the hepatic clock system and metabolism.

	Experimental procedures

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Animals and isolation of RNA

Six to eight-week-old male C57/BL6 mice (Crea Japan, Tokyo), Balb/c mice, and Clock mutant mice with a BALB/c background (Noshiro et al. 2005) were housed under a 12 : 12-h light–dark (LD) cycle at constant temperature and given food and water ad libitum before the experiment for at least 2 weeks. All procedures were performed in compliance with standard principles and guidelines for the care and use of laboratory animals in Hiroshima University Graduate School of Biomedical Sciences or Hokkaido University Graduate School of Medicine.

Three to four mice for each time point were decapitated at 4 or 6 h intervals beginning at zeitgeber time 2 (ZT 2, 0800 h) in a normal light–dark cycle. The tissues were quickly dissected, frozen on dry ice, and stored at –80 °C until processing. Total RNA was extracted by Trizol reagent (Invitrogen). All RNA preparations were obtained separately for individual mice and used for the determination of transcript levels of respective genes.

Quantitative real-time RT-PCR analysis

Quantitative real-time RT-PCR analysis was performed using an ABI PRISM 7900 Sequence Detection System instrument and software (Applied Biosystems) as described previously (Gibson et al. 1996). First strand cDNA was synthesized using a ReverTra Ace reverse transcriptase kit (Toyobo Co. Osaka) with total RNA (1 µg) preparations and random primers. Validity of the cDNA preparations was examined by amplification of 18S ribosomal RNA using Ribosomal RNA control kit (Applied Biosystems). The sequences of the primers and TaqMan^TM fluorogenic probes (Applied Biosystems) for Dec1, Dec2, and Dbp used in these analyses were described previously (Noshiro et al. 2005). The sequences of the primers and TaqMan^TM fluorogenic probes for human AbcA1 and Srebp1 were designed according to the ProbeFinder^TM software of Roche Universal Probe Library system (Roche Applied Science): 5'-gcctgctagtggtcatcctg-3', 5'-ccacgctgggatcactgta-3', and #62 fluorescent probe (Roche) for AbcA1 (NM_005502 [GenBank] .2); 5'-gctcctccatcaatgacaaaa-3', 5'-tgcgcaagacagcagattta-3', and #77 fluorescent probe (Roche) for Srebp1 (U00968.1 [GenBank] ).

Western blot analysis

Nuclear extracts were prepared from the liver, kidney, and heart of normal BALB/c mice and Ck/Ck mice (ZT18) using Nuclear Extract Kit (Active Motif, Carlsbad, CA). The equal amounts of the protein samples (20 µg per gel lane) were subjected to Western blot analysis. After SDS-polyacrylamide gel electrophoresis, proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). Rabbit antibodies against human LXR (H-144, Santa Cruz Biotechnology Inc.) were used as primary antibodies (1 : 1000 dilution), and the antibodies were cross-reactive to both human and mouse LXR. Immunoreactive bands were detected using ¹²⁵I-labeled anti-rabbit antibodies (GE Healthcare Bioscience) as second antibodies. Radioactivity of detected areas was quantified using a FLA-3000G fluoro-image analyzer (Fujifilm Co.).

Cell culture and luciferase reporter assay

Human hepatoma HepG2, mouse hepatoma Hepa1c1c7, mouse NIH 3T3, or human HEK293 cells were inoculated at 5 x 10⁴ cells/16 mm diameter well in 24-multiwell plastic plates, and cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) containing 1 mg/mL D-glucose, 10% fetal bovine serum, 50 µg/mL ascorbic acid, 32 U/mL penicillin and 40 µg/mL streptomycin. DNA transfection of the luciferase constructs (20 ng/assay), with internal control plasmid pRL-TK (2 ng/assay), and the expression plasmids to cell cultures was performed using TransIT LT1 (Mirus Corp. Madison, WI) 24 h after cell seeding. When LXR/β, FXR, VDR, PXR, PPAR, PPAR and RAR were used, ligands T0901317 (10^–7 M), chenodeoxycholic acid (10^–5 M), 1, 25-(OH)₂ vitamin D₃ (10^–7 M), lithocholic acid (10^–5 M), WY-14 643 (10^–5 M), troglitazone (10^–6 M) and retinoic acid (10^–7 M), respectively, dissolved in dimethyl sulfoxide, were added to the culture medium (Gottlicher et al. 1992; Willy et al. 1995). Next, 24 h after transfection with the reporter and expression plasmids, these cells were harvested and the cell lysate was analyzed using dual luciferase assay reagents (Promega). Three or four wells were measured for each assay and luciferase activities were normalized to internal control values. At least two independent transfection experiments were performed. DNA fragments containing 3855 bp (–3965 to –111; +1 indicates the translation initiation site), 896 bp (–2603 to –1708), 439 bp (–2146 to –1708), and 246 bp (–1953 to –1708) of mouse Dec1 gene promoter, and 3500 bp (–3500 to +1) of human Dec1 gene promoter were used to construct luciferase reporters as previously described (Kawamoto et al. 2004). DNA fragment of 3.1 kb (–3170 to –83) of mouse Dec2 gene promoter was used to construct luciferase reporter (Hamaguchi et al. 2004). The expression plasmids (pcDNA3.1) for mouse DEC1, DEC2 and rat E4BP4 were prepared as described previously (Noshiro et al. 2004). The expression plasmids (pCMX) for mouse PPAR (NR1C1), rat E4BP4, human VDR (NR1I1), human PXR (NR1I2), human and mouse LXR (NR1H3), human and mouse LXRβ (NR1H2), mouse CAR (NR1I3) and human RXR (NR2B1) were also described previously (Kliewer et al. 1994; Willy et al. 1995; Sueyoshi et al. 1999; Lu et al. 2000). Expression plasmids (pCMX-VP16) for human LXR (LXR-VP16) and RXR (RXR-VP16) fused to activation domain of herpesvirus VP16 protein were previously described (Kaneko et al. 2003). These expression plasmids that fused to VP16 do not require the ligands for activation of nuclear receptors in the reporter assay.

Electrophoretic mobility shift assay (EMSA)

Double-stranded synthetic probes (Fig. 4B) for EMSA were prepared and labeled with -[³²P]dCTP as described previously (Noshiro et al. 2004). The binding reaction mixture contained 20 000 cpm of labeled oligonucleotide probe and the protein factor(s) in 15 µL of 10 mM Tris–HCl (pH 8.0), 50 mM NaCl, 25 mM MgCl₂, 5 mM dithiothreitol, 0.2 µg/µL poly (dI-dC), and 10% glycerol. The mixtures were incubated at room temperature for 10 min and subjected to 5% native polyacrylamide gel electrophoresis and visualized by autoradiography. Electrophoresis was performed at room temperature for 1.5 h at constant 15 W. The protein factors were prepared by the use of TnT Quick coupled in vitro transcription/translation system (Promega) and the expression plasmids.

Chromatin immunoprecipitation (ChIP) assay

Mouse hepatoma Hepa1c1c7 cells and rabbit antibodies against LXR (H-144, Santa Cruz Biotech Inc.) were used. ChIP assay of LXR was performed using ChIP Assay Kit (Upstate) according to the manufacturer's instructions. Recovered DNA samples were subjected to PCR for DEC1 using gene specific primers as follows: P1 (position at –2232), 5'-aagcac ggtagggaaggt ta-3'; P2 (–1950), 5'-gcacaaaggggcagcaccag-3'; P3 (–2146), 5'-gggctagccaggcct tattttagtcat-3'; P4 (–1904), 5'-caagaggtttaggtagtagg-3'. Combinations of P1 : P2 and P3 : P4 formed 302- and 262-bp PCR products, respectively.

Agonist treatment of HepG2

HepG2 cells were inoculated at 1 x 10⁵ cells/35 mm diameter well in 6-multiwell plastic plates, and cultured in the medium described above. One day after inoculation, the medium was changed to glucose-free Dulbecco's modified Eagle's medium (Invitrogen) containing 110 mg/L sodium pyruvate, 10% lipoprotein-deficient serum (Biomedical Tech. Inc.), 32 U/mL penicillin, 40 µg/mL streptomycin, and 1 x 10^–6 M atorvastatin, in the presence or absence of LXR agonist. Cells were harvested at 1, 2, 4 and 24 h after the addition of agonist, and total RNA was extracted by TRIsure reagent (Bioline) and examined for Dec1 expression by quantitative real-time RT-PCR method.

Statistical analysis

Time series data were analyzed by one-way analysis of variance (one-way ANOVA) for rhythm. Differences between two genotypes or two light conditions were tested by two-way ANOVA. Significance of differences between the two groups was analyzed by Student's t-test. Comparisons with P < 0.05 were taken as significant.

	Acknowledgements

 
This work was supported by grants-in-aid for science from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Authors thank the Research Center for Molecular Medicine, Hiroshima University School of Medicine, for the use of their facilities.

	Footnotes

 
Communicated by: Eisuke Nishida

^* Correspondence: noshiro{at}hiroshima-u.ac.jp

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Accepted: 3 October 2008

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