Genome-Wide Profiling of the Core Clock Protein BMAL1 Targets

Genome-Wide Proﬁling of the Core Clock Protein BMAL1 Targets Reveals a Strict Relationship with Metabolismᰔ Fumiyuki Hatanaka,1,2,3 Chiaki Matsubara,1 Jihwan Myung,1,3,4 Takashi Yoritaka,3 Naoko Kamimura,5 Shuichi Tsutsumi,5 Akinori Kanai,6 Yutaka Suzuki,6 Paolo Sassone-Corsi,7 Hiroyuki Aburatani,5 Sumio Sugano,6 and Toru Takumi1,2,3,8* Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan1;GraduateSchoolofMedicine,KyotoUniversity,Sakyo,Kyoto606-8501, Downloaded from Japan2;GraduateSchoolofBiomedicalSciences,HiroshimaUniversity,Minami,Hiroshima734-8553,Japan3; Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8501, Japan4; Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Meguro, Tokyo 153-8904, Japan5;DepartmentofMedicalGenomeSciences,GraduateSchoolofFrontierSciences,Universityof Tokyo, Minato, Tokyo 108-8639, Japan6; Department of Pharmacology, School of Medicine, University of California, Irvine, California 926977; and JST, CREST, 102-0075 Tokyo, Japan8

Received 8 July 2010/Returned for modiﬁcation 4 August 2010/Accepted 14 September 2010 http://mcb.asm.org/

Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. Since a signiﬁcant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. We used multiple high-throughput approaches, including chromatin immunoprecipitation-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genome-wide proﬁles of BMAL1 target genes. We reveal that, in addition to E-boxes, the CCAATG element contributes to elicit robust circadian expression. BMAL1

occupancy is found in more than 150 sites, including all known clock genes. Importantly, a signiﬁcant on August 6, 2012 by HIROSHIMA UNIVERSITY proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions.

Many physiological phenomena in almost all of organisms (30). These notions indicate that global mechanisms of gene are regulated in a circadian manner. This is possible through expression, specifically chromatin remodeling (23), must oper- the function of an intrinsic biological clock or pacemaker. The ate to accommodate these genome-wide oscillations. circadian clock operates independently of external cues, but it The transcriptional-translational feedback loops that consti- has remarkable plasticity and so can adapt to changing envi- tute the core circadian oscillator have been dissected. At the ronmental conditions. Dysfunctions of the circadian clock are core of the circadian machinery lies the BMAL1 protein, a associated with a wide variety of disorders in humans, includ- basic helix-loop-helix (bHLH) type transcription factor that ing insomnia, depression, cardiovascular disorders, and cancer forms hetero-complexes with either CLOCK or NPAS2, two (13, 35, 40). The central circadian pacemaker in mammals is other bHLH nuclear activators with different tissue distribu- located within the suprachiasmatic nucleus (SCN) in the ante- tions (10, 50). These dimers activate the transcription of clock- rior hypothalamus (13). controlled genes (CCGs) and of genes encoding other ele- A critical advance in the field has been the discovery of ments of the clock machinery, specifically the Per and Cry circadian clocks present in peripheral tissues and in cultured genes. Once synthesized, PERs and CRYs inhibit CLOCK/ cells (13). The SCN appears to function by orchestrating pe- BMAL1 activation potential, resulting in the downregulation ripheral clocks (37), likely by using a specific group of humoral of their own transcription (31). The circadian transcription of signals as synchronizing elements, including glucocorticoids the Bmal1 gene is itself regulated by nuclear receptors posi- and retinoic acid (12). This complex network relies on a highly tively by RORs and negatively by REV-ERBs (2, 29, 36). controlled system of gene expression, in which interlocked Importantly, Bmal1-deficent mice exhibit arrhythmic locomo- transcriptional-translational feedback loops operate (20, 31, 50). Also, microarray studies have revealed that ca. 10 to 15% tor activity in constant darkness, a unique case as single-gene of all transcripts in different tissues display circadian oscillation ablation among all clock genes (5), indicating that BMAL1 is (3, 9, 26, 38). More recent data suggest that the expression of indeed indispensable to generate circadian gene expression. as much as 50% of all genes oscillates in a circadian manner DNA microarray analyses in both SCN and peripheral tissues (26, 38, 46) and further bioinformatic analyses determined that circadian transcription may be elicited through three pro- * Corresponding author. Mailing address: Laboratory of Integrative moter elements (E-box, RORE, and DBPE) (47, 52). Among Bioscience, Graduate School of Biomedical Sciences, Hiroshima Univer- these, the E-box appears to play a major role as it is highly sity, 1-2-3 Kasumi, Minami, Hiroshima 734-8553, Japan. Phone: 81-82- 257-5115. Fax: 81-82-257-5119. E-mail: [email protected]. abundant in the mammalian genome and responsible in driving ᰔ Published ahead of print on 11 October 2010 . the expression of most CCGs (47). However, with the excep-

5636 VOL. 30, 2010 PROFILES OF CORE CLOCK PROTEIN BMAL1 TARGETS 5637 tion of a limited number of individually studied genes (11, 24), The DNA was recovered by phenol-chloroform-isoamyl alcohol (25:24:1) extrac- the extent of BMAL1-mediated control of circadian transcription and ethanol precipitation. Sequencing. Bmal1-bound DNA was purified by SDS-PAGE to obtain 150- to tion at a genome-wide level remains unknown. 250-bp fragments and sequenced on an Illumina GA sequencer. About 15,000 to We describe here results obtained by a systematic analysis 20,000 clusters were generated per “tile,” and 26 cycles of the sequencing reac- using a chromatin immunoprecipitation (ChIP)-based technol- tions were performed according to the manufacturer’s instructions. ogy and a genome-wide microarray. We have applied ChIP- Tiling array. ChIP and control samples were amplified by two cycles of in vitro on-chip (ChIP-chip) (17) and ChIP coupled with ultra-high- transcription and hybridized on separate Affymetrix human promoter 1.0 oligo- nucleotide tiling arrays as described previously (49). Enrichment values (ChIP/ throughput DNA sequencing (ChIP-seq) methods (4, 7, 15, 33) control) were calculated with the MAT algorithm as described previously to reveal BMAL1 target genes at a whole genome level. All (16, 49). known CCGs driven by E-boxes are present in our systematic Data processing. The obtained sequences were mapped onto mouse genomic ChIP results and the profiling of BMAL1 targets reveals strict sequences (mm9 as of UCSC Genome Browser [http://genome.ucsc.edu/]) using

the sequence alignment program Eland. Unmapped or redundantly mapped Downloaded from relationship with metabolism. Our analysis has revealed unex- sequences were removed from the data set. For uniquely mapped sequences, pected features of circadian gene control and constitutes a relative positions to RefSeq genes were calculated based on the respective valuable resource to study the link between clock control and genomic coordinates. Genomic coordinates of exons and other information of cellular metabolism. the RefSeq transcripts are as described in mm9 as of UCSC Genome Browser. GO (as of 14 June 2007) and KEGG (release 42) terms were associated with RefSeq genes by using loc2go (as of 14 June 2007) using NCBI Entrez Gene database (http://www.ncbi.nlm.nih.gov/sites/entrez?dbϭgene). Details and ratio- MATERIALS AND METHODS nalization of the procedure were as described previously (43).

Cell culture. NIH 3T3 and WI38 cells were maintained at 37°C and 5% CO2 Motif analysis. MEME (for Multiple Em for Motif Elicitation) with default in Dulbecco modified Eagle medium (Nacalai Tesque, Kyoto, Japan) supple- parameters was used to identify statistically overrepresented consensus motifs http://mcb.asm.org/ mented with 10% newborn bovine serum (NBS; ICN Biomedicals, Inc.) and within the inferred binding sites. antibiotics. Quantitative real-time RT-PCR. Each quantitative real-time reverse transcrip- Animals. Male BALB/c mice purchased 5 weeks postpartum from Japan SLC tion-PCR (RT-PCR) was performed using the ABI Prism 7900HT sequence (Hamamatsu, Japan) were exposed to 2 weeks of 12:12 light-dark (LD) cycles detection system as described previously (25), The PCR primers were designed and then kept in complete darkness as a continuation of the dark phase of the with Primer Express software (Applied Biosystems), and the sequences of the last LD cycle. Liver mRNA levels were examined in the third dark-dark (DD) primers are shown in Table S5 at the URL in “Antibody,” above. The reaction cycle. Adult Bmal1Ϫ/Ϫ (5) and wild-type mice were kept in a LD cycle and killed was first incubated at 50°C for 2 min and then at 95°C for 10 min, followed by 40 at Zeitgeber time 0 (ZT0) and ZT12. Tissues were immediately frozen in liquid cycles at 95°C for 15 s and 60°C for 1 min. nitrogen and stored at Ϫ80°C until processed for RNA. All protocols of exper- Rhythmicity and acrophase estimation. We use similarity between data and iments using animals in the present study were approved by the OBI (Osaka the best-fitting cosine as a measure of rhythmicity. This is in line with the popular on August 6, 2012 by HIROSHIMA UNIVERSITY Bioscience Institute) Animal Research Committee. method for testing rhythmicity in microarray data, where a cosine fit is selected Antibody. Purified glutathione S-transferase (GST)-mBMAL1 N-terminal that gives maximal correlation with data (51). We find the cosine fits first and use (amino acids 1 to 100) antigen (41) was produced to immunize rabbits. After Pearson correlation coefficient to quantify the closeness to the ideal oscillation, removing possible GST-recognizing antibody by using HiTrap-NHS conjugated which we call “rhythmicity.” Rhythmicity is directly related to robustness of with GST, the antiserum was subjected to affinity purification using HiTrap-NHS circadian gene cycling. Each set, not average, of PCR data is fit for a cosine curve conjugated with the antigen. The anti-mBMAL1 antibody specifically recognizes using nonlinear linear squares as the following equation, where bi represents the its target protein in immunochemical analysis (see Fig. S10 [all supplemental acrophase: si (t) ϭ ai cos [2␲ (t Ϫ bi)/24] ϩ ci. material may be found at this URL at http://home.hiroshima-u.ac.jp/anatomy2/]). The circadian rhythmicity is assumed for all samples, and the period is fixed to Normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) was used as a control. 24 h (51). A higher correlation means a higher rhythmicity, which reaches a ChIP. NIH 3T3 cells were cultured in 10-cm plates to 1.5 ϫ 107 cells, and 10 maximum at 1. Higher sample variance implies less robustness of oscillation and plates were used per immunoprecipitation. WI38 cells were cultured in 10-cm poorer rhythmicity. Statistical significance of the rhythmicity is tested against plates to 1.5 ϫ 106 cells, and five plates were used per immunoprecipitation. Cells random surrogates of the same sample size and dynamic range as PCR data. P were fixed with 1% formaldehyde for 15 min at room temperature with swirling. values from one-sided t test estimate separation between the data and the Glycine was added to a final concentration of 0.125 M, and the incubation was surrogate. A nonoverlapping linear trend exists between rhythmicity and the P continued for an additional 15 min. After washing twice with ice-cold phosphate- value in rhythmicity interval between 0.7 and 1.0, which corresponds to P value buffered saline, the cells were harvested by scraping, pelleted, and resuspended below 0.2 (see Fig. S6 at the URL in “Antibody,” above). This implies the in 500 ␮l of ice-cold cell lysis buffer (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% rhythmicity above 0.7 is above the chance level. NP-40, and protease inhibitors). After incubation at 4°C for 15 min, samples IV-ROMS. NIH 3T3 cells at 105 plated in Opti-MEM (Gibco) supplemented were centrifuged at 3,000 rpm at 4°C for 5 min, and the precipitations were with 10% NBS in 35-mm dishes were transfected with the desired plasmids by resuspended in 1 ml of nuclei lysis buffer (50 mM Tris-HCl [pH 8.0], 10 mM using Lipofectamine and Plus reagent (Invitrogen). At 24 h after transfection, EDTA, 1% sodium dodecyl sulfate [SDS], and protease inhibitors). After incu- the medium was exchanged for 100 nM dexamethasone containing medium, and bation on ice for 20 min, the samples were sonicated 10 times for 10 s each time 2 h later this medium was replaced with Opti-MEM supplemented with 1% NBS at intervals of 50 s with a Microson (Misonix, Inc.). Samples were centrifuged at and 0.1 mM luciferin–10 mM HEPES (pH 7.2). Bioluminescence was measured 15,000 rpm at 4°C for 10 min. After removal of a control aliquot (whole-cell by using in vitro real-time oscillation monitoring system (IV-ROMS; Hamamatsu extract), supernatants were diluted 10-fold in ChIP dilution buffer (50 mM Photonics) as described previously (2, 22, 52). Tris-HCl [pH 8.0], 167 mM NaCl, 1.1% Triton X-100, 0.11% sodium deoxy- Luciferase assay. NIH 3T3 cells were cultured and transfected with the desired cholate, protease inhibitor). Nonspecific background was removed by incubating plasmids by using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 h samples with a salmon sperm DNA/protein A-agarose slurry at 4°C for 2 h with after transfection, and cell lysates were prepared and then used in the dual rotation. The samples were centrifuged at 2,000 rpm at 4°C for 2 min, and a 0.1 luciferase assay system (Promega). volume of the recovered supernatants was stored as an input sample, whereas the Microarray analysis. Applied Biosystems mouse genome survey arrays were rest was incubated overnight with 3 ␮g of indicated antibodies at 4°C with used to analyze the transcriptional profiles of liver samples as described previ- rotation. The immunocomplexes were collected with 70 ␮l of salmon sperm ously (48). Digoxigenin-UTP-labeled cRNA was generated and linearly ampli- DNA/protein A-agarose at 4°C for 3 h with rotation. The beads were sequentially fied from 2 ␮g of total RNA using Applied Biosystems Chemiluminescent RT- washed with the following buffers: radioimmunoprecipitation assay (RIPA) buff- IVT Labeling Kit V2.0 according to the manufacturer’s protocol. Portions (10 er–150 mM NaCl, RIPA buffer–500 mM NaCl, and LiCl wash solution. Finally, ␮g) of labeled cRNA were hybridized to each pre-hybridized microarray in a the beads were washed twice with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. 1.5-ml volume at 55°C for 16 h. Array hybridization and chemiluminescence The immunocomplexes were then eluted by the addition of 200 ␮l of ChIP direct detection were performed with a Applied Biosystems chemiluminescence detec- elution buffer (10 mM Tris-HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA, 0.5% tion kit according to the manufacturer’s protocol. Images were collected for each SDS) and rotated for 15 min at room temperature and incubated for 4 h at 65°C. microarray using a 1700 analyzer. Images were auto-gridded, and the chemilu- 5638 HATANAKA ET AL. MOL.CELL.BIOL. minescent signals were quantified, corrected for background and spot, and spa- In the ChIP-chip analysis with the human lung fibroblast tially normalized. cells, high signals were detected in the Per1 promoter region Site-directed mutagenesis. Site-directed mutagenesis was performed in a single step by PCR to modify the specific hDbp CCAATG element or CATGTG to (87.65-fold change versus IgG) and in the intron region of AGTCAT or CATTGG, respectively. Mutated DNA was identified by DpnI Rev-erb␣ (10.3-fold change versus IgG) on human chromo- selection for hemimethylated DNA, and the sequences were checked. The oli- some 17. ChIP-seq analysis with mouse embryonic fibroblasts gonucleotide primers used were 5Ј-CGG ACC CAG AGG CCC TAC TGA CTG revealed the positions of BMAL1 occupancy at higher resolu- mut TGC GTC TCA AGG (631 ), 5Ј-CCC CCA GTA CCG CCT CCT ACT GAG tion. Two clusters were detected upstream of Per1 and three CAA ATG TAG GTC AGT G (738 mut), 5Ј-CCC CTC CCG CCT GCC TTA CTG ACC CAA ACT GGG (784 mut), and 5Ј-GCA CGA GCA GAG CCA TTG clusters were detected on the Rev-erb␣ locus on mouse chro- GCT TCC CCC TCC C (E-box-like, CATTGG). mosome 11 (see Fig. S3 and S4 at the URL given in “Anti- body” in Materials and Methods). By focusing further on the 5 kb upstream of the Per1 gene TSS, two E-box and two

RESULTS Downloaded from CCAATG elements were located with high tag numbers (see ChIP-chip and ChIP-seq identify BMAL1 targets. For ChIP Fig. S3 and S4 at the URL given in “Antibody” in Materials experiments, we generated speciﬁc antibody against BMAL1. and Methods). The precise locations of ChIP-seq tags for other To identify the BMAL1-binding sites at a genome-wide level, 6 genes (Per2, Cry1, Cry2, Dbp, Tef, and Gm129) are also we performed a ChIP-chip analysis. We used WI38 human represented in Fig. S1 to S4 and Table S3 at the URL given in ﬁbroblasts from which we immunoprecipitated BMAL1 (or “Antibody” in Materials and Methods. It is notable that these IgG as control) to use it for hybridization on human promoter elements are highly conserved in the genomes of vertebrates, tiling arrays. The number of sites with the ratio of BMAL1/ underscoring the importance of these regions during evolution.

IgG, Ͼ5.0 and Ͼ3.0 were 32 and 183, respectively (Ͼ5.0 in BMAL1 target genes display circadian expression. To de- http://mcb.asm.org/ Table S1 at the URL given in “Antibody” in Materials and termine whether BMAL1 target genes are expressed in a cir- Methods; Ͼ3.0 with ChIP-seq signals in Table S2 at the URL cadian manner, we performed quantitative RT-PCR using given in “Antibody” in Materials and Methods). As predicted, mouse liver tissues sampled every 4 h. All eight overlapped the clock genes—Per1, Per2, Cry1, and Cry2—were identified genes (categorized as ChIP-seq Ͼ consecutive 10.0 and ChIP- among the genes with Ͼ5.0 ratio (Table 1). To further identify chip Ͼ 5.0) displayed robust circadian oscillation with high and confirm the BMAL1-DNA interaction at high resolution, amplitudes in the liver (Fig. 2). In the other category (ChIP- we performed ChIP-seq using NIH 3T3 mouse fibroblasts. We seq Ͼ consecutive 10.0 and ChIP-chip Ͻ 5.0, ChIP-seq Ͼ obtained 2,990 tags from BMAL1-bound DNA compared to nonconsecutive 10.0 and ChIP-chip Ͼ 2.0), several genes such on August 6, 2012 by HIROSHIMA UNIVERSITY 1,248 tags in the control whole-cell lysate. We then collected as Rev-erb␤ (Nr1d2), Hlf, Dec2 (Bhlhe41), Per3, Thra, and Klf11 172 ChIP-seq signals with more than 10 consecutive tags (1 tag exhibited clear circadian expression. In the lower category consists of 100 bp; consecutive tags were defined as tags situ- (ChIP-seq Ͻ nonconsecutive 10.0 and ChIP-chip Ͼ 3.0, ChIP- ated directly adjacent to each other). Most of BMAL1-binding chip Ͼ 10.0 without ChIP-seq signals), some genes such as sites (44%) were located in promoter regions (Fig. 1A), and Asna1, Coq4, Hus1, Mrpl45, Vps33a, Vps11, and Bloc1s3 dis- almost all of these sites were close to the TSS (transcriptional played weak circadian expression and very small amplitude in start site) (Fig. 1B). We uncovered 32 BMAL1-binding sites on oscillation. The analysis by cosine fitting confirmed theses fea- promoters with a Ͼ5.0-fold change from ChIP-chip analysis tures of cyclic expression (see Fig. S5 to S7 at the URL given (see Table S1 at the URL given in “Antibody” in Materials and in “Antibody” in Materials and Methods). Methods), whereas 20 sites with more than 10.0 consecutive The CCAATG element contributes to robust circadian ex- tags from ChIP-seq (Table 1). Eight overlapped sites were pression. Since the CCAATG element identified in the present found in the promoter regions of the genes Per1, Per2, Cry1, study has not been examined as a clock element to date, we Cry2, Rev-erb␣ (Nr1d1), Dbp, and Tef, as well as in the pro- sought to determine whether the CCAATG element located in moter of an as-yet-unidentified gene, Gm129 (Fig. 1C, Table the predicted BMAL1-binding sites is indeed functional. To do 1). Substantial tags were accumulated in each site where so, we generated mutations within the CCAATG elements to BMAL1 binds (see Fig. S1 to S4 at the URL given in “Anti- AGTCAT in a hDbp:luciferase reporter construct (hDbp/ body” in Materials and Methods). Motif analysis by MEME pGL3B, Fig. 3A). In the hDbp promoter region (Ϫ894 to Ϫ1 (using motif’s length of 5 to 10 bp) on identified BMAL1- from the translational start site), one E-box-like and three binding sites from ChIP-seq unexpectedly revealed two con- CATTGG elements are located with adequate tag numbers sensus DNA-binding motifs (Fig. 1D). The first consensus from ChIP-seq (see Table S3 at the URL given in “Antibody” motif (Motif1) was CACGTG (E-value ϭ 2.9eϪ14), which pre- in Materials and Methods). NIH 3T3 cells were transfected dictably corresponded to the E-box. The second motif (Motif2) with the hDbp/pGL3B reporter or its mutated forms and then was CCAATG (E-value ϭ 5.5eϪ1), a motif highly similar to the stimulated with 100 nM dexamethasone. Light emission was so-called CCAAT-box, a well-characterized promoter element measured in the presence of luciferin after dexamethasone (21). In eight common BMAL1-binding sites from both ChIP- treatment. Although mutations at the bp Ϫ784 or bp Ϫ738 seq and ChIP-chip analyses, most of the two consensus motifs (Ϫ784mut or Ϫ738mut) site had little effect on the amplitude of (E-box or E-box-like and CCAATG elements) were located circadian oscillation, mutation at bp Ϫ631 (Ϫ631mut) caused a upstream to the TSS (Fig. 1E; also see Table S3 at the URL loss of robust circadian oscillation and a drastic decrease in the given in “Antibody” in Materials and Methods). This result is amplitude compared to hDbp (wt) (Fig. 3B). Furthermore, consistent with studies (24, 47) indicating that the CLOCK/ disruption of circadian oscillation was observed by using a BMAL1 complex positively regulates the activity of E-box con- promoter construct in which the E-box-like sequence was re- taining promoters. placed by the CATTGG element. These results were consis- VOL. 30, 2010 PROFILES OF CORE CLOCK PROTEIN BMAL1 TARGETS 5639

TABLE 1. Result of ChIP-seq and ChIP-chip analysesa

Analysis details (ChIP-chip fold Chromosome Position ChIP-chip fold b Distribution Tag no. change) and gene (mouse) Start End change ChIP-seq, tag Ͼ 10.0, c (Ͼ5.0) Per2 1 93355701 93355800 Exon 1, intron 1 10 20.49 93355801 93355900 Exon 1 52 93355901 93356000 Promoter 34 93356001 93356100 Promoter 50 Cry2 2 92264001 92264100 Intron 3 10 5.81 92274301 92274400 Promoter 28 92274401 92274500 Promoter 13

Gm129 3 95686101 95686200 Promoter exon 1 19 31.91 Downloaded from 95686201 95686300 Promoter 17 Dbp 7 52960101 52960200 Promoter 20 9.07 52960201 52960300 Promoter 7 52960301 52960400 Promoter 5 52960401 52960500 Promoter 10 52961401 52961500 Intron 1 11 52963001 52963100 Intron 2 11 52963101 52963200 Intron 2 15 Cry1 10 84647801 84647900 Promoter 19 8.75

84647901 84648000 Promoter 5 http://mcb.asm.org/ 84648001 84648100 Promoter 16 84648101 84648200 Promoter 27 Per1 11 68908301 68908400 Intergenic 52 87.65 68908401 68908500 Intergenic 24 68908501 68908600 Intergenic 15 68908601 68908700 Intergenic 22 68908701 68908800 Intergenic 8 68912201 68912300 Promoter 12 68912301 68912400 Promoter 10 68912401 68912500 Exon 1 10 Rev-erb␣ 11 98635101 98635200 Intron 1 81 10.3 on August 6, 2012 by HIROSHIMA UNIVERSITY 98635201 98635300 Intron 1 18 98635301 98635400 Intron 1 5 98635401 98635500 Intron 1 32 98636501 98636600 Promoter exon 1 12 NDc 98636601 98636700 Promoter 27 98644601 98644700 Intergenic 17 98644701 98644800 Intergenic 50 98644801 98644900 Intergenic 19 Tef 15 81641301 81641400 Intron 1 64 5.02 81641401 81641500 Intron 1 69 81641501 81641600 Intron 1 10 81641601 81641700 Intron 1 24

ChIP-seq, tag Ͼ 10.0, c (Ͻ5.0) Igsf8 1 174240201 174240300 Intergenic 13 2.66 174240301 174240400 Intergenic 15 E530001K10Rik Mir670 2 94113401 94113500 Promoter 14 ND 94113501 94113600 Promoter 17 2310035K24Rik 2 131030401 131030500 Intergenic 13 ND 131030501 131030600 Intergenic 26 131030601 131030700 Intergenic 8 6 91549101 91549200 Intergenic 49 ND 91549201 91549300 Intergenic 15 Dec2 6 145804301 145804400 Intergenic 44 3.57 145804401 145804500 Intergenic 32 Crispld2 8 122570301 122570400 Intron 1 11 ND 122570401 122570500 Intron 1 10 Nptn 9 58429801 58429900 Promoter 11 ND 58429901 58430000 Promoter 26 Hlf 11 90279301 90279400 Intergenic 7 ND 90279401 90279500 Intergenic 26 90279501 90279600 Intergenic 22 Rsad1 11 94410201 94410300 Intron 1 12 ND 94410301 94410400 Exon 1 26 Rev-erb␤ 14 19071001 19071100 Intron 1 22 ND 19071101 19071200 Intron 1 43 Continued on following page 5640 HATANAKA ET AL. MOL.CELL.BIOL.

TABLE 1—Continued

Analysis details (ChIP-chip fold Chromosome Position ChIP-chip fold b Distribution Tag no. change) and gene (mouse) Start End change 19071201 19071300 Intron 1 23 Ighmbp2 19 3282901 3283000 Promoter 10 ND Mrpl21 3283001 3283100 Promoter exon 1 11 X 137054801 137054900 Intergenic 10 ND 137054901 137055000 Intergenic 13

ChIP-seq, tag Ͼ 10.0, nc (Ͼ2.0) Cops7b 1 88483601 88483700 Promoter 15 2.42

88483701 88483800 Exon 1 6 Downloaded from Per3 4 150418701 150418800 Promoter exon 1 29 2.65 150418801 150418900 Promoter 7 150418901 150419000 Promoter 5 C030048B08Rik Pex1 5 3596001 3596100 Exon 1, promoter 10 5.04 3596101 3596200 Exon 1 7 Krr1 10 111409701 111409800 Promoter 12 3.88 Pex13 11 23566301 23566400 Promoter 11 3.15 Pus10 Exon 2 Thra 11 98608901 98609000 Intron 1 13 3.16

Klf11 12 25336001 25336100 Promoter 11 5.16 http://mcb.asm.org/ Ccdc85b 19 5457501 5457600 Promoter exon 1 11 2.53

a Eight genes are overlapped from ChIP-seq with high tags (more than 10 tags and consecutive) and ChIP-chip with a Ͼ5.0-fold change. Eleven genes and 1 micro-RNA are extracted from ChIP-seq with more than 10 tags (consecutive) and ChIP-chip with a Ͻ5.0-fold change. Ten genes are extracted from ChIP-seq with more than 10 tags (nonconsecutive) and ChIP-chip with a Ͼ2.0-fold change. The 2-kb region upstream of transcription start site is defined as the promoter. The chromosome, start, end, distribution, and tag number columns indicate ChIP-seq data from mouse fibroblasts. The ChIP-chip fold change shows ChIP-chip data from human fibroblasts. b c, consecutive; nc, nonconsecutive. The ChIP-chip fold change for each type of analysis is indicated in parentheses. c ND, not determined. on August 6, 2012 by HIROSHIMA UNIVERSITY tent with those of additional luciferase (luc) assays (Fig. 3C). vitro real-time luminescence monitoring. Gm129/pGL3B ex- hDbp-784mut,hDbp-738mut, and hDbp-631mut expressing hibited a robust circadian oscillation that was antiphasic to BMAL1 and CLOCK displayed a lower luciferase activity com- hBmal1/pGL3B (Fig. 4B). mRNA levels of Gm129 in the liver pared to Ϫ894 hDbpwt (P Ͻ 0.005, Fig. 3C, black bar [Student also showed a circadian oscillation with an amplitude as high as t test]). A deletion construct with Ϫ784 to Ϫ1 of hDbp/pGL3B Per2 (Fig. 4C). Gm129 and Bmal1 are almost antiphasic in (Ϫ784 hDbpwt), which did not include E-box-like sequence, expression, with acrophases separated by 10 h (see Fig. S5B at also exhibited a reduced luciferase activity. Interestingly, a the URL given in “Antibody” in Materials and Methods). In promoter construct with mutation from the E-box-like se- the SCN, the mammalian circadian central pacemaker, Gm129 quence to the CATTGG element (hDbpE-C) displayed a high expression displayed a circadian oscillation as robust as Bmal1 luciferase activity. Ectopic coexpression of BMAL1 and (Fig. 4D). These results suggest that Gm129 is a novel CCG. CLOCK stimulated transcription from the basal reporter, Two groups of CCGs under BMAL1 control. We then stud- whereas they had no effect on the Ϫ784 hDbpwt and hDbpE-C ied the expression profiles for representative liver genes at ZT0 mutant reporters. In addition, CRY2 inhibited the BMAL1/ and ZT12, the trough and peak of Bmal1 expression, respec- CLOCK-mediated transactivation in all of the CCAATG- tively (46), comparing wild-type and Bmal1Ϫ/Ϫ mice (Fig. 5). based reporters, indicating that CRY-mediated negative regu- While Bmal1 was highly expressed at ZT0 and at almost un- lation is through a pathway independent of the E-box-like detectable levels at ZT12 in wild-type mice, all BMAL1 target sequence on promoters (see Fig. S8 at the URL given in genes except Cry1 were expressed in an anti-phase manner “Antibody” in Materials and Methods). These results suggest (Fig. 5, solid line). In contrast, the expression of Dbp, Rev-erb␣, that the CCAATG element contributes to activate hDbp tran- Rev-erb␤, Gm129, and Dec2 was drastically decreased at both scription and that the E-box-like sequence is critical for the ZT0 and ZT12 in Bmal1Ϫ/Ϫ mice (Fig. 5, dashed line). Impor- regulation of circadian oscillation. tantly, this loss of expression was not observed for Per1, Per2, Gm129 is a novel CCG. Our ChIP-seq and ChIP-chip anal- Cry1, Cry2, Tef, and Hlf. Thus, at least two distinct groups of yses pointed at Gm129 as a novel candidate for a BMAL1- CCGs appear to be controlled differentially by BMAL1. The regulated gene. The promoter region of Gm129 includes three first group (including Dbp, etc.,) strictly requires BMAL1 to E-box elements, one E-box-like element, and one CCAATG display circadian oscillation, since expression is virtually abol- element with mild tag numbers of ChIP-seq (Fig. 4A). We ished in Bmal1Ϫ/Ϫ mice. The second group (including Per1, hypothesized that the circadian expression of Gm129 is linked etc.) of genes, although circadian, undergoes a rhythmic reg- to activation elicited by BMAL1. To test this hypothesis, we ulation that is BMAL1 independent. To gain further insights subcloned a DNA fragment corresponding to the region from into the molecular regulation by BMAL1, we performed com- bp Ϫ2033 to Ϫ1 upstream of the ATG and generated a lucif- parative whole-genome microarrays at ZT0 and ZT12 in the erase reporter construct (Gm129/pGL3B) to perform an in liver of wild-type and Bmal1Ϫ/Ϫ mice. We identified 23 genes VOL. 30, 2010 PROFILES OF CORE CLOCK PROTEIN BMAL1 TARGETS 5641 Downloaded from http://mcb.asm.org/

FIG. 1. High-resolution genome-wide mapping of the BMAL1 target with ChIP-seq and ChIP-chip. (A) Distribution of binding sites across the genome. The 2-kb region upstream of transcription start site (TSS) is defined as the promoter. (B) Distribution of tags from ChIP-seq relative to the TSS. (C) BMAL1 binds 20 sites from ChIP-seq in NIH 3T3 cells (tag numbers were more than consecutive 10.0) and 32 sites from ChIP-chip on August 6, 2012 by HIROSHIMA UNIVERSITY in WI38 cells (fold change is more than 5.0). Eight sites are overlapped in ChIP-seq and ChIP-chip. (D) Logo of the BMAL1 binding element that is identified as 20 sites from ChIP-seq. Motif 1: width, 10; sites, 50; log likelihood, 407; E-value, 2.9eϪ14. Motif 2: width, 9; sites, 16; log likelihood, 158; E-value, 5.5eϪ1. (E) Distribution of tags from eight genes and locations of E-box, E-box-like, and CCAATG elements. that fluctuated in wild-type mice and displayed reduced expres- DISCUSSION sion levels in Bmal1Ϫ/Ϫ mice (Table 2). Because 20 genes, besides Per3, Rev-erb␤, and Dbp, were not found in our ChIP- Circadian regulation is exerted mostly at transcriptional seq and ChIP-chip analyses, we conclude that these genes are level and is critical for a wide range of physiological and met- located downstream of BMAL1 but are not directly regulated abolic functions. Deciphering how the circadian clock machin- by BMAL1. ery operates at the genome-wide level is thereby a critical step BMAL1 target genes are related to metabolism. To establish toward the understanding of cellular physiology. Our system- a systematic classification of the BMAL1 target genes identi- atic analyses by ChIP-chip and ChIP-seq uncovered hundreds fied by the ChIP-chip and ChIP-seq approaches, we analyzed of targeted candidates for the core clock transcription factor gene ontology by DAVID Bioinformatics Database for associ- BMAL1. Importantly, all known CCGs driven by E-boxes were ations with particular gene ontology terms (Fig. 6; see Table S4 present in our systematic ChIP results, validating our experi- at the URL given in “Antibody” in Materials and Methods). mental approach. The outcome of this analysis revealed that BMAL1 target In order to obtain highly reliable data, we used the combi- genes are related to rhythmic processes, metabolic pathways nation of two methods to reveal binding sites for BMAL1. We and transcription as a biological process, DNA binding or identified eight common BMAL1-binding sites that contain transcription as a molecular function, and nucleus as a cellular both E-box and CATTGG motifs. Luciferase reporter experi- component. Furthermore, the expressions of key genes related ments demonstrated that E-box and/or E-box-like sequences to glucose metabolism such as Glut2, Por, Pck1 and Gys2 were are essential to drive circadian gene expression and that the greatly diminished in the livers of Bmal1Ϫ/Ϫ mice compared to CATTGG element regulates transcriptional activity for the wild-type mice. Also, genes relevant to drug and cholesterol hDbp promoter. It is recognized that the ChIP-chip approach metabolism (Cyp2a4, Cyp2a5, Cyp4a14, Cyp7a1, and Cyp2c55) has some limitations, such as the construction of specific were differently expressed in Bmal1Ϫ/Ϫ mice compared to the probes and pseudo-signals of hybridization (14), whereas wild-type littermates (see Fig. S9 at the URL given in “Anti- ChIP-seq provides markedly precise locations in a genome- body” in Materials and Methods), although we could not find wide analysis. Our results confirm this view since we revealed these genes in ChIP-chip and ChIP-seq signals. It is therefore precise locations of the BMAL1-binding sites in the ChIP-seq possible that these genes related to metabolism are down- analysis. Correlation between ChIP signals and rhythmic ex- stream targets of BMAL1. pression also supports this view (see Fig. S7 at the URL given Downloaded from http://mcb.asm.org/ on August 6, 2012 by HIROSHIMA UNIVERSITY

FIG. 2. Temporal mRNA expression of BMAL1-regulating genes. The temporal mRNA expression of selected genes in mouse liver was measured by using quantitative RT-PCR. The abscissa indicates the circadian time (CT), and ordinate indicates the mRNA amounts. The relative level of each mRNA is normalized to the corresponding G3-PDH RNA level. The maximum RNA amount is set to 100. The data are presented as means Ϯ the standard errors (SE) of triplicate samples.

5642 VOL. 30, 2010 PROFILES OF CORE CLOCK PROTEIN BMAL1 TARGETS 5643 Downloaded from http://mcb.asm.org/ on August 6, 2012 by HIROSHIMA UNIVERSITY

FIG. 3. E-box-like and CATTGG elements on the hDbp promoter required for circadian oscillation and transactivation. (A) Schematic representation of E-box-like and CATTGG elements on hDbp promoter. ϩ1 corresponds to the translational start site. TSS, transcriptional start site. (B) Ϫ784mut and Ϫ738mut hDbp:Luc exhibit a low-amplitude oscillation, and Ϫ631mut exhibits a dampened oscillation of very low amplitude compared to the wild type (wt). Mutated construct that is converted from E-box-like to CATTGG disrupted circadian activity. Black and gray lines indicate the bioluminescence of mutants and the wild type, respectively. Each shadow indicates the standard error of five or six samples. The abscissa indicates the day, and the ordinate indicates the relative luciferase intensity. (C) The effects of overexpression of BMAL1 and/or CLOCK and CRY2 protein on the transactivation of each hDbp construct are evaluated by using a luciferase assay. The basal transcriptional activity of hDbp promoter wild type (WT) (bp Ϫ894 to Ϫ1 from the translational start site)/pGL3B was set to 1. The data represent the means Ϯ the SE of eight samples (*, P Ͻ 0.001; **, P Ͻ 0.0001; Student t test). in “Antibody” in Materials and Methods). Specifically for Per1, quences of these genes, besides the proximal promoter re- Dbp, Rev-erb␣, and Tef,thesesiteswerefoundnotonlyinthe gions, may be involved in driving robust circadian oscilla- proximal promoter region but also within intergenic regions tion. This is not necessarily the case for all CCGs. In the and introns. This result suggests that other genomic se- case of Per2,theproximalregionissolelysufficientinac- 5644 HATANAKA ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/

FIG. 4. Gm129 shows circadian expression. (A) BMAL1 binding promoter of Gm129 from ChIP-seq in UCSC genome browser view. (B) Gm129 and Bmal1 promoter-luciferase reporter activities in NIH 3T3 cells. (C) Temporal mRNA expression of Bmal1, Per2, and Gm129 in mouse liver by quantitative RT-PCR. The abscissa indicates the time circadian time (CT), and ordinate indicates the mRNA amounts. The relative on August 6, 2012 by HIROSHIMA UNIVERSITY level of each mRNA is normalized to the corresponding G3-PDH RNA levels. The maximum RNA amount is set to 100. The data are presented as the means Ϯ the SE of triplicate samples. Gm129 shows circadian expression. The expression of Gm129 and Bmal1 is almost anti-phasic with acrophases separated by 10 h. The temporal proﬁles of Gm129 and Bmal1 expression are distinct [gene ϫ time ϭ F(5, 24) ϭ 60.421, P Ͻ 0.0001, analysis of variance]. (D) Temporal mRNA expression of Bmal1 and Gm129 in mouse suprachiasmatic nuclei as determined by quantitative RT-PCR. The abscissa indicates the Zeitgeber time (ZT), and the ordinate indicates the mRNA amounts. The relative levels of each mRNA are normalized to the corresponding G3-PDH RNA levels. The maximum RNA amount is set to 100. The data are presented as means.

cordance with previous studies (1, 24, 55). In addition, ro- BMAL1-regulated genes with rhythmicities of Ͼ0.7 (see Fig. bustness of oscillation directly correlates with the strength S5 and S6 at the URL given in “Antibody” in Materials and of BMAL1 binding, suggesting that oscillation is generated Methods) possess E-box and E-box-like sequences in their by occupancy of BMAL1 (see Fig. S5 to S7 at the URL given promoters. Further studies will provide valuable information in “Antibody” in Materials and Methods). Interestingly, the on the physiological roles of these clock-controlled genes. detected tags in ChIP-seq for BMAL1 were located at the Many genes that normally show oscillatory expression lost their same region from ChIP-seq of RNA polymerase II and rhythmicity in the liver of Bmal1Ϫ/Ϫ mice. Importantly, our ChIP H3K4me3, a histone modification generally associated with analysis revealed two distinct classes of BMAL1-regulated genes. transcriptional activation (4). These results suggest that Genes in the first group, which include Dbp,showatotallossof transcriptional factor BMAL1, RNA polymerase II, and a Ϫ/Ϫ cyclic transcription in Bmal1 mice. Genes in the second group histone methyltransferase responsible for H3K4me3 are re- still have substantial and rhythmic expression in Bmal1Ϫ/Ϫ mice. cruited in a coordinate manner, confirming the notion that We concluded that cyclic expression of the genes in the second circadian transcription and chromatin modifications are in- group defines a set of CCGs in the liver which does not solely timately linked (8, 23, 32). Our analysis identified Gm129, a BMAL1 target whose func- utilize the canonical CLOCK/BMAL1 core clock complex. These tion is as yet unknown and absent in the database. Expression observations may be related to previous studies implicating cyclic of Gm129 in the SCN and liver displays a robust circadian AMP-response element binding protein (CREB) in the coregu- rhythm with a profile similar to Per2 and anti-phasic to Bmal1. lation of CCGs. Indeed, CREB has been reported to activate the Our preliminary data show that coexpression of Gm129 results same group of genes that we identify not to be transcriptionally Ϫ/Ϫ in inhibition of E-box driven transcription. Thus, the protein silenced in the liver of Bmal1 mice (56). Furthermore, a sim- encoded by the Gm129 might operate in a manner similar to ilar regulation is seen in the SCN with a light pulse and in the CRY and could thereby participate in the circadian loop. retina of Bmal1Ϫ/Ϫ mice (28, 39). Therefore, core circadian genes Gm129-null mice will help understanding what role of Gm129 such as Per1, Per2, Cry1,andCry2 appear to be regulated by a may play in circadian and physiological functions. Other combination of BMAL1 with other transcription factors for VOL. 30, 2010 PROFILES OF CORE CLOCK PROTEIN BMAL1 TARGETS 5645

gene ontology support this view. Previous transcriptome analysis has revealed global changes in metabolic pathways in a circadian manner (26). Furthermore, mice deficient in either Bmal1 or Clock gene display abnormal metabolic activities (34, 45). Our study uncovered additional molecular couplings between circadian and metabolic transcription networks. More- over, our genome-wide profile can contribute to the discovery of new key genes in metabolic pathways coupled with BMAL1 in a manner similar to what reported for REV-ERB␣ (19, 54). Genes whose expression was downregulated in Bmal1Ϫ/Ϫ mice but were not found in the ChIP analyses could be candidates to Downloaded from be downstream from BMAL1. However, further studies are needed in order to provide a comprehensive interpretation of the data. In the liver of Bmal1Ϫ/Ϫ mice, many genes relevant to glucose and drug metabolism seem to be differently expressed compared to wild-type tissues (see Fig. S9A at the URL given in “Antibody” in Materials and Methods). It has been reported that a large proportion of nuclear receptors (NRs) display circadian expression in key metabolic tissues (53). Importantly, http://mcb.asm.org/ other than Rev-erbs, no other NRs appear to be expressed differently in the livers of Bmal1Ϫ/Ϫ mice (see Fig. S9B at the Bmal1Ϫ/Ϫ FIG. 5. Expression patterns of BMAL1-regulating genes in URL given in “Antibody” in Materials and Methods). While mouse. Quantitative RT-PCR analysis of selected genes whose expression is dependent on Bmal1 was performed. Solid lines with circles and dotted this could appear surprising, considering the drastic changes in lines with triangles represent wild-type and Bmal1Ϫ/Ϫ,respectively.The the expression of metabolic genes in the Bmal1Ϫ/Ϫ livers, one relative levels of each mRNA are normalized to the corresponding G3- interpretation may be that NR expression is prominently en- PDH RNA level. The minimum RNA amount is set to 1. The data are Ϫ/Ϫ presented as the means Ϯ the SE of three samples. trained by feeding (6) or that Bmal1 mice show robust food anticipatory activity (27). Altogether, our findings indicate that the hierarchical transcriptional system based on the circadian on August 6, 2012 by HIROSHIMA UNIVERSITY which many binding elements exist in their promoter regions (18, clock coordinates physiological functions in metabolism. In 24, 42). this scenario, the specific contribution of the core clock com- Recent studies have revealed intimate links between clock ponent BMAL1 appears to be central, although a number of control and cellular metabolism (10, 12, 44, 50). Our results of genes commonly thought to be under its control may utilize

TABLE 2. Results of competitive microarray analysis of Bmal1-regulated genes in the livers of wild-type and Bmal1Ϫ/Ϫ micea

Wild-type mice Bmal1Ϫ/Ϫ mice Gene Description ZT0 ZT12 ZT0 ZT12 Mfsd2 Major facilitator superfamily domain containing 2 11.07 101.98 16.81 18.49 Mafb v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (avian) 39.76 142.58 36.71 32.20 Maff v-maf musculoaponeurotic fibrosarcoma oncogene family, protein F (avian) 2.42 13.94 2.59 2.35 Brunol5 Bruno-like 5, RNA-binding protein (Drosophila) 0.46 2.21 0.65 1.00 Gpr64 G-protein-coupled receptor 64 1.15 3.27 0.26 0.62 Gdap10 Ganglioside-induced differentiation-associated-protein 10 11.88 31.82 15.75 13.28 Slc2a5 Solute carrier family 2 (facilitated glucose transporter), member 5 5.58 17.69 5.09 2.83 Per3 Period homolog 3 (Drosophila) 1.29 22.34 6.81 4.00 4631416L12Rik Riken cDNA 4631416L12 gene 35.00 113.73 45.38 39.85 3010003L21Rik Riken cDNA 3010003L21 gene 1.67 5.72 2.11 1.85 Rev-erb␤ Nuclear receptor subfamily 1, group D, member 2 1.74 10.86 1.62 1.18 Nrg4 Neuregulin 4 2.55 9.18 1.21 0.74 Ibrdc3 IBR domain containing 3 3.34 8.98 2.27 3.76 Dbp D-site albumin promoter binding protein 4.25 249.19 12.37 7.80 Gys2 Glycogen synthase 2 266.31 851.86 206.32 275.26 Cxcl1 Chemokine (C-X-C motif) ligand 1 8.89 21.82 7.20 4.28 Homer2 Homer homolog 2 (Drosophila) 49.03 99.04 36.85 40.74 Klf13 Kruppel-like factor 13 2.42 20.41 1.56 9.56 Cabyr Calcium-binding tyrosine-(Y)-phosphorylation regulated (fibrousheathin 2) 2.33 20.03 4.11 8.88 Spry2 Sprouty homolog 2 (Drosophila) 1.53 3.19 1.41 1.35 6430710M23Rik Riken cDNA 6430710M23 gene 2.28 5.06 1.64 2.29 Coq10b Coenzyme Q10 homolog B (S. cerevisiae) 5.66 14.73 5.96 6.08 Ccrn4l CCR4 carbon catabolite repression 4-like (S. cerevisiae) 9.83 33.54 8.00 6.50

a These genes were altered by at least 2.0-fold compared to ZT0 to ZT12 in the livers of wild-type mice and are decreased by at least 2.0-fold at both ZT0 and ZT12 of Bmal1Ϫ/Ϫ mice compared to the ZT12 of the wild type. The values indicate signal intensities after global normalization from microarrays. 5646 HATANAKA ET AL. MOL.CELL.BIOL. Downloaded from http://mcb.asm.org/ on August 6, 2012 by HIROSHIMA UNIVERSITY

FIG. 6. Gene ontology terms from BMAL1-regulating genes. Fifty-three genes identiﬁed in ChIP-seq and ChIP-chip results were analyzed by using the DAVID Bioinformatics Database (http://david.abcc.ncifcrf.gov/) for associations with particular Gene Ontology terms. The result reveals an enrichment of genes related to rhythmic process, metabolic process and transcription. BP (blue), MF (pink), and CC (yellow) indicate the biological process, molecular function, and cellular component, respectively. alternative pathways of regulation. Future genome-wide stud- contribution at the initial stages of this project, T. Tamaru and Y. Naka- ies with combinations of antibodies for CLOCK, NPAS2, hata for their providing information on the BMAL1 antibody, T. Akagi for WI38 cells, and C. Bradﬁeld for Bmal1 (Mop3) knockout mice. REV-ERB, and other regulators will provide additional insight This study was supported in part by the MEXT, the Sumitomo into the whole transcriptional network of clock genes and their Foundation, the Takeda Foundation, Sony Corporation, and Nippon diverse functions in vivo. Boehringer Ingelheim Co., Ltd. REFERENCES ACKNOWLEDGMENTS 1. Akashi, M., T. Ichise, T. Mamine, and T. Takumi. 2006. Molecular mechanism of cell-autonomous circadian gene expression of Period2, a crucial We thank the technicians of the Takumi laboratory for their valuable regulator of the mammalian circadian clock. Mol. Biol. Cell 17:555–565. assistance. We also acknowledge T. Ishibashi and H. Kimura for their 2. Akashi, M., and T. Takumi. 2005. The orphan nuclear receptor ROR␣ VOL. 30, 2010 PROFILES OF CORE CLOCK PROTEIN BMAL1 TARGETS 5647

regulates circadian transcription of the mammalian core-clock Bmal1. Nat. T. Takumi. 2009. Abnormal behavior in a chromosome-engineered mouse Struct. Mol. Biol. 12:441–448. model for human 15q11-13 duplication seen in autism. Cell 137:1235–1246. 3. Akhtar, R. A., A. B. Reddy, E. S. Maywood, J. D. Clayton, V. M. King, A. G. 26. Panda, S., M. P. Antoch, B. H. Miller, A. I. Su, A. B. Schook, M. Straume, Smith, T. W. Gant, M. H. Hastings, and C. P. Kyriacou. 2002. Circadian P. G. Schultz, S. A. Kay, J. S. Takahashi, and J. B. Hogenesch. 2002. cycling of the mouse liver transcriptome, as revealed by cDNA microarray, is Coordinated transcription of key pathways in the mouse by the circadian driven by the suprachiasmatic nucleus. Curr. Biol. 12:540–550. clock. Cell 109:307–320. 4. Barski, A., S. Cuddapah, K. Cui, T. Y. Roh, D. E. Schones, Z. Wang, G. Wei, 27. Pendergast, J. S., W. Nakamura, R. C. Friday, F. Hatanaka, T. Takumi, and I. Chepelev, and K. Zhao. 2007. High-resolution profiling of histone methyl- S. Yamazaki. 2009. Robust food anticipatory activity in BMAL1-deficient ations in the human genome. Cell 129:823–837. mice. PLoS One 4:e4860. 5. Bunger, M. K., L. D. Wilsbacher, S. M. Moran, C. Clendenin, L. A. Radcliffe, 28. Porterfield, V. M., H. Piontkivska, and E. M. Mintz. 2007. Identification of J. B. Hogenesch, M. C. Simon, J. S. Takahashi, and C. A. Bradfield. 2000. novel light-induced genes in the suprachiasmatic nucleus. BMC Neurosci. Mop3 is an essential component of the master circadian pacemaker in 8:98. mammals. Cell 103:1009–1017. 29. Preitner, N., F. Damiola, L. Lopez-Molina, J. Zakany, D. Duboule, U. Al- 6. Canaple, L., J. Rambaud, O. Dkhissi-Benyahya, B. Rayet, N. S. Tan, L. brecht, and U. Schibler. 2002. The orphan nuclear receptor REV-ERBalpha Michalik, F. Delaunay, W. Wahli, and V. Laudet. 2006. Reciprocal regula- controls circadian transcription within the positive limb of the mammalian tion of brain and muscle Arnt-like protein 1 and peroxisome proliferator- circadian oscillator. Cell 110:251–260. Downloaded from activated receptor alpha defines a novel positive feedback loop in the rodent 30. Ptitsyn, A. A., S. Zvonic, S. A. Conrad, L. K. Scott, R. L. Mynatt, and J. M. liver circadian clock. Mol. Endocrinol. 20:1715–1727. Gimble. 2006. Circadian clocks are resounding in peripheral tissues. PLoS 7. Chen, X., H. Xu, P. Yuan, F. Fang, M. Huss, V. B. Vega, E. Wong, Y. L. Orlov, Comput. Biol. 2:e16. W. Zhang, J. Jiang, Y. H. Loh, H. C. Yeo, Z. X. Yeo, V. Narang, K. R. 31. Reppert, S. M., and D. R. Weaver. 2002. Coordination of circadian timing in Govindarajan, B. Leong, A. Shahab, Y. Ruan, G. Bourque, W. K. Sung, N. D. mammals. Nature 418:935–941. Clarke, C. L. Wei, and H. H. Ng. 2008. Integration of external signaling 32. Ripperger, J. A., and U. Schibler. 2006. Rhythmic CLOCK-BMAL1 binding pathways with the core transcriptional network in embryonic stem cells. Cell to multiple E-box motifs drives circadian Dbp transcription and chromatin 133:1106–1117. transitions. Nat. Genet. 38:369–374. 8. Doi, M., J. Hirayama, and P. Sassone-Corsi. 2006. Circadian regulator 33. Robertson, G., M. Hirst, M. Bainbridge, M. Bilenky, Y. Zhao, T. Zeng, G. CLOCK is a histone acetyltransferase. Cell 125:497–508. Euskirchen, B. Bernier, R. Varhol, A. Delaney, N. Thiessen, O. L. Griffith, A.

9. Duffield, G. E., J. D. Best, B. H. Meurers, A. Bittner, J. J. Loros, and J. C. He, M. Marra, M. Snyder, and S. Jones. 2007. Genome-wide profiles of http://mcb.asm.org/ Dunlap. 2002. Circadian programs of transcriptional activation, signaling, STAT1 DNA association using chromatin immunoprecipitation and mas- and protein turnover revealed by microarray analysis of mammalian cells. sively parallel sequencing. Nat. Methods 4:651–657. Curr. Biol. 12:551–557. 34. Rudic, R. D., P. McNamara, A. M. Curtis, R. C. Boston, S. Panda, J. B. 10. Eckel-Mahan, K., and P. Sassone-Corsi. 2009. Metabolism control by the Hogenesch, and G. A. Fitzgerald. 2004. BMAL1 and CLOCK, two essential circadian clock and vice versa. Nat. Struct. Mol. Biol. 16:462–467. components of the circadian clock, are involved in glucose homeostasis. 11. Gekakis, N., D. Staknis, H. B. Nguyen, F. C. Davis, L. D. Wilsbacher, D. P. PLoS Biol. 2:e377. King, J. S. Takahashi, and C. J. Weitz. 1998. Role of the CLOCK protein in 35. Sahar, S., and P. Sassone-Corsi. 2009. Metabolism and cancer: the circadian the mammalian circadian mechanism. Science 280:1564–1569. clock connection. Nat. Rev. Cancer 9:886–896. 12. Green, C. B., J. S. Takahashi, and J. Bass. 2008. The meter of metabolism. 36. Sato, T. K., S. Panda, L. J. Miraglia, T. M. Reyes, R. D. Rudic, P. Cell 134:728–742. McNamara, K. A. Naik, G. A. FitzGerald, S. A. Kay, and J. B. Hogenesch. 13. Hastings, M. H., A. B. Reddy, and E. S. Maywood. 2003. A clockwork web: 2004. A functional genomics strategy reveals Rora as a component of the circadian timing in brain and periphery, in health and disease. Nat. Rev. mammalian circadian clock. Neuron 43:527–537. on August 6, 2012 by HIROSHIMA UNIVERSITY Neurosci. 4:649–661. 37. Schibler, U., and P. Sassone-Corsi. 2002. A web of circadian pacemakers. 14. Johnson, D. S., W. Li, D. B. Gordon, A. Bhattacharjee, B. Curry, J. Ghosh, Cell 111:919–922. L. Brizuela, J. S. Carroll, M. Brown, P. Flicek, C. M. Koch, I. Dunham, M. 38. Storch, K. F., O. Lipan, I. Leykin, N. Viswanathan, F. C. Davis, W. H. Wong, Bieda, X. Xu, P. J. Farnham, P. Kapranov, D. A. Nix, T. R. Gingeras, X. and C. J. Weitz. 2002. Extensive and divergent circadian gene expression in Zhang, H. Holster, N. Jiang, R. D. Green, J. S. Song, S. A. McCuine, E. liver and heart. Nature 417:78–83. Anton, L. Nguyen, N. D. Trinklein, Z. Ye, K. Ching, D. Hawkins, B. Ren, 39. Storch, K. F., C. Paz, J. Signorovitch, E. Raviola, B. Pawlyk, T. Li, and C. J. P. C. Scacheri, J. Rozowsky, A. Karpikov, G. Euskirchen, S. Weissman, M. Weitz. 2007. Intrinsic circadian clock of the mammalian retina: importance Gerstein, M. Snyder, A. Yang, Z. Moqtaderi, H. Hirsch, H. P. Shulha, Y. Fu, for retinal processing of visual information. Cell 130:730–741. Z. Weng, K. Struhl, R. M. Myers, J. D. Lieb, and X. S. Liu. 2008. Systematic 40. Takahashi, J. S., H. K. Hong, C. H. Ko, and E. L. McDearmon. 2008. The evaluation of variability in ChIP-chip experiments using predefined DNA genetics of mammalian circadian order and disorder: implications for phys- targets. Genome Res. 18:393–403. iology and disease. Nat. Rev. Genet. 9:764–775. 15. Johnson, D. S., A. Mortazavi, R. M. Myers, and B. Wold. 2007. Genome- 41. Tamaru, T., Y. Isojima, G. T. van der Horst, K. Takei, K. Nagai, and K. wide mapping of in vivo protein-DNA interactions. Science 316:1497–1502. Takamatsu. 2003. Nucleocytoplasmic shuttling and phosphorylation of 16. Johnson, W. E., W. Li, C. A. Meyer, R. Gottardo, J. S. Carroll, M. Brown, BMAL1 are regulated by circadian clock in cultured fibroblasts. Genes Cells and X. S. Liu. 2006. Model-based analysis of tiling-arrays for ChIP-chip. 8:973–983. Proc. Natl. Acad. Sci. U. S. A. 103:12457–12462. 42. Travnickova-Bendova, Z., N. Cermakian, S. M. Reppert, and P. Sassone- 17. Kim, T. H., and B. Ren. 2006. Genome-wide analysis of protein-DNA inter- Corsi. 2002. Bimodal regulation of mPeriod promoters by CREB-dependent actions. Annu. Rev. Genomics Hum. Genet. 7:81–102. signaling and CLOCK/BMAL1 activity. Proc. Natl. Acad. Sci. U. S. A. 18. Kumaki, Y., M. Ukai-Tadenuma, K. D. Uno, J. Nishio, K. H. Masumoto, M. 99:7728–7733. Nagano, T. Komori, Y. Shigeyoshi, J. B. Hogenesch, and H. R. Ueda. 2008. 43. Tsuchihara, K., Y. Suzuki, H. Wakaguri, T. Irie, K. Tanimoto, S. Hashimoto, Analysis and synthesis of high-amplitude Cis-elements in the mammalian K. Matsushima, J. Mizushima-Sugano, R. Yamashita, K. Nakai, D. Bentley, circadian clock. Proc. Natl. Acad. Sci. U. S. A. 105:14946–14951. H. Esumi, and S. Sugano. 2009. Massive transcriptional start site analysis of 19. Le Martelot, G., T. Claudel, D. Gatfield, O. Schaad, B. Kornmann, G. L. human genes in hypoxia cells. Nucleic Acids Res. 37:2249–2263. Sasso, A. Moschetta, and U. Schibler. 2009. REV-ERBalpha participates in 44. Tu, B. P., and S. L. McKnight. 2006. Metabolic cycles as an underlying basis circadian SREBP signaling and bile acid homeostasis. PLoS Biol. of biological oscillations. Nat. Rev. Mol. Cell. Biol. 7:696–701. 7:e1000181. 45. Turek, F. W., C. Joshu, A. Kohsaka, E. Lin, G. Ivanova, E. McDearmon, A. 20. Liu, A. C., W. G. Lewis, and S. A. Kay. 2007. Mammalian circadian signaling Laposky, S. Losee-Olson, A. Easton, D. R. Jensen, R. H. Eckel, J. S. Taka- networks and therapeutic targets. Nat. Chem. Biol. 3:630–639. hashi, and J. Bass. 2005. Obesity and metabolic syndrome in circadian Clock 21. Mitchell, P. J., and R. Tjian. 1989. Transcriptional regulation in mammalian mutant mice. Science 308:1043–1045. cells by sequence-specific DNA binding proteins. Science 245:371–378. 46. Ueda, H. R., W. Chen, A. Adachi, H. Wakamatsu, S. Hayashi, T. Takasugi, 22. Nakahata, Y., M. Akashi, D. Trcka, A. Yasuda, and T. Takumi. 2006. The in M. Nagano, K. Nakahama, Y. Suzuki, S. Sugano, M. Iino, Y. Shigeyoshi, and vitro real-time oscillation monitoring system identifies potential entrainment S. Hashimoto. 2002. A transcription factor response element for gene ex- factors for circadian clocks. BMC Mol. Biol. 7:5. pression during circadian night. Nature 418:534–539. 23. Nakahata, Y., B. Grimaldi, S. Sahar, J. Hirayama, and P. Sassone-Corsi. 47. Ueda, H. R., S. Hayashi, W. Chen, M. Sano, M. Machida, Y. Shigeyoshi, M. 2007. Signaling to the circadian clock: plasticity by chromatin remodeling. Iino, and S. Hashimoto. 2005. System-level identification of transcriptional Curr. Opin. Cell Biol. 19:230–237. circuits underlying mammalian circadian clocks. Nat. Genet. 37:187–192. 24. Nakahata, Y., M. Yoshida, A. Takano, H. Soma, T. Yamamoto, A. Yasuda, T. 48. Watanabe, Y., K. Inoue, A. Okuyama-Yamamoto, N. Nakai, J. Nakatani, K. Nakatsu, and T. Takumi. 2008. A direct repeat of E-box-like elements is Nibu, N. Sato, Y. Iiboshi, K. Yusa, G. Kondoh, J. Takeda, T. Terashima, and T. required for cell-autonomous circadian rhythm of clock genes. BMC Mol. Takumi. 2009. Fezf1 is required for penetration of the basal lamina by olfactory Biol. 9:1. axons to promote olfactory development. J. Comp. Neurol. 515:565–584. 25. Nakatani, J., K. Tamada, F. Hatanaka, S. Ise, H. Ohta, K. Inoue, S. To- 49. Wendt, K. S., K. Yoshida, T. Itoh, M. Bando, B. Koch, E. Schirghuber, S. monaga, Y. Watanabe, Y. J. Chung, R. Banerjee, K. Iwamoto, T. Kato, M. Tsutsumi, G. Nagae, K. Ishihara, T. Mishiro, K. Yahata, F. Imamoto, H. Okazawa, K. Yamauchi, K. Tanda, K. Takao, T. Miyakawa, A. Bradley, and Aburatani, M. Nakao, N. Imamoto, K. Maeshima, K. Shirahige, and J. M. 5648 HATANAKA ET AL. MOL.CELL.BIOL.

Peters. 2008. Cohesin mediates transcriptional insulation by CCCTC-binding Waitt, D. J. Parks, K. H. Pearce, G. B. Wisely, and M. A. Lazar. 2007. factor. Nature 451:796–801. Rev-erb␣, a heme sensor that coordinates metabolic and circadian pathways. 50. Wijnen, H., and M. W. Young. 2006. Interplay of circadian clocks and Science 318:1786–1789. metabolic rhythms. Annu. Rev. Genet. 40:409–448. 55. Yoo, S. H., C. H. Ko, P. L. Lowrey, E. D. Buhr, E. J. Song, S. Chang, O. J. 51. Yamada, R., and H. R. Ueda. 2007. Microarrays: statistical methods for Yoo, S. Yamazaki, C. Lee, and J. S. Takahashi. 2005. A noncanonical E-box circadian rhythms. Methods Mol. Biol. 362:245–264. enhancer drives mouse Period2 circadian oscillations in vivo. Proc. Natl. 52. Yamamoto, T., Y. Nakahata, H. Soma, M. Akashi, T. Mamine, and T. Acad. Sci. U. S. A. 102:2608–2613. Takumi. 2004. Transcriptional oscillation of canonical clock genes in mouse 56. Zhang, X., D. T. Odom, S. H. Koo, M. D. Conkright, G. Canettieri, J. Best, peripheral tissues. BMC Mol. Biol. 5:18. H. Chen, R. Jenner, E. Herbolsheimer, E. Jacobsen, S. Kadam, J. R. Ecker, 53. Yang, X., M. Downes, R. T. Yu, A. L. Bookout, W. He, M. Straume, D. J. B. Emerson, J. B. Hogenesch, T. Unterman, R. A. Young, and M. Montminy. Mangelsdorf, and R. M. Evans. 2006. Nuclear receptor expression links the 2005. Genome-wide analysis of cAMP-response element binding protein circadian clock to metabolism. Cell 126:801–810. occupancy, phosphorylation, and target gene activation in human tissues. 54. Yin, L., N. Wu, J. C. Curtin, M. Qatanani, N. R. Szwergold, R. A. Reid, G. M. Proc. Natl. Acad. Sci. U. S. A. 102:4459–4464. Downloaded from http://mcb.asm.org/ on August 6, 2012 by HIROSHIMA UNIVERSITY