{
 "key": "CLOCK_BMAL",
 "name": "CLOCK / BMAL1",
 "class": "bHLH-PAS",
 "aka": [
  "Circadian E-box",
  "CACGTG E-box",
  "Tandem E-box (E1-E2)",
  "E'-box (CACGTT)",
  "BMAL1/ARNTL (MOP3)",
  "NPAS2 (MOP4)",
  "bHLH-PAS factors"
 ],
 "definition": "Tandem E-boxes CACGTG of the circadian oscillator. CLOCK, ARNTL/BMAL1, NPAS2.",
 "consensus": "CACGTG",
 "dataConsensus": "cCACGTGc",
 "motifCount": 12,
 "bySource": {
  "JASPAR": 3,
  "HOCOMOCO": 6,
  "CIS-BP": 3
 },
 "symbols": [
  "ARNTL",
  "BMAL1",
  "CLOCK",
  "NPAS2"
 ],
 "representative": "M04129_2.00",
 "cohesion": 0.86,
 "short": "CLOCK, BMAL1 (gene symbol ARNTL) and NPAS2 are basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) transcription factors that form the activating limb of the mammalian circadian clock. BMAL1 pairs with CLOCK or with its paralogue NPAS2 and binds the palindromic E-box CACGTG together with a small set of E-box-like variants. The complex reads seven base pairs rather than six, because a flanking A-T pair next to the core contributes to affinity. Strong circadian promoters usually carry two E-boxes in tandem six to seven base pairs apart, and it is the pair, not a single hexamer, that produces rhythmic transcription. Occupancy oscillates once a day and drives Per, Cry, Rev-erb and thousands of output genes. Because CACGTG is also the core read by MYC/MAX, USF and SREBP, sequence alone cannot assign an E-box to the clock.",
 "long": "## The factors\n\nCLOCK, BMAL1 (*ARNTL*, formerly MOP3) and NPAS2 (MOP4) are bHLH-PAS proteins: an N-terminal basic helix-loop-helix domain followed by two PAS domains (PAS-A, PAS-B) and a PAC extension. The bHLH contacts DNA; the PAS domains set dimerization specificity and provide cofactor and small-molecule surfaces. Neither subunit binds DNA productively alone. BMAL1 is the obligate partner, heterodimerizing with CLOCK or with NPAS2 [PMID:9576906]; BMAL2 (*ARNTL2*) is a lower-abundance paralogue. The crystal structure of the mouse CLOCK:BMAL1 bHLH-PAS region shows an asymmetric, interdigitated assembly with three separate interfaces, so the subunits wrap around one another rather than pairing symmetrically (Huang et al. 2012) [PMID:22653727]. CLOCK and BMAL1 are broadly expressed; NPAS2 is enriched in forebrain and some peripheral tissues and can substitute for CLOCK, which is why *Clock*-null mice keep behavioral rhythms (DeBruyne et al. 2006) [PMID:16675400] while *Bmal1*-null mice are arrhythmic in constant darkness (Bunger et al. 2000) [PMID:11163178]. This family holds 12 motifs from JASPAR, HOCOMOCO and CIS-BP labeled ARNTL/BMAL1, CLOCK or NPAS2, with none flagged as probably mislabeled.\n\n## The motif\n\nThe core is the palindromic E-box CACGTG. Each basic helix enters the major groove and reads one CAC half-site; because the hexamer is its own reverse complement, the two subunits occupy equivalent halves. A structure of the human CLOCK:BMAL1 bHLH domains on a canonical E-box resolved the hydrogen-bond network and showed BMAL1 Ile80 contacting the base pair immediately 3' of the core, so the recognized unit is 7 bp, CACGTGA, with that flanking A-T pair indispensable; the same study measured single substitutions and defined two high-affinity non-canonical boxes, AACGTGA and CATGTGA (Wang et al. 2013) [PMID:23229515]. This resolves an apparent split in the databases. The methyl-HT-SELEX models CLOCK.H14CORE.0.M.C and NPAS2.H14CORE.1.M.C recover ACACATGT and ACATGTGT, the CATGTG variant, while the grade-A models BMAL1.H14CORE.0.PSM.A and CLOCK.H14CORE.1.PS.A recover CACGTG with a flanking base. The pooled consensus computed from the members is cCACGTGc; it does not contradict the literature 7-mer, because averaging a strand-ambiguous palindrome symmetrizes the flanks. The other commonly cited variant is the E'-box CACGTT. Functionally rhythmic promoters generally carry tandem E-boxes 6-7 bp apart, and the pair rather than either hexamer confers cell-autonomous rhythmicity (Nakahata et al. 2008) [PMID:18177499]; the most information-rich model here, NPAS2.H14CORE.2.S.B, is itself a tandem (CACGTGbnndaCACGTGt, about 21 bits). No member of the family can be told from another by sequence.\n\n## Where it acts\n\nBinding is promoter-biased. ChIP-seq in mouse liver puts most confident sites within a few hundred base pairs of transcription start sites, often in CpG-island promoters, with roughly one to three thousand sites per time point (Rey et al. 2011; Koike et al. 2012) [PMID:21364973,22936566]. Occupancy itself cycles, peaking once per day ahead of the RNA polymerase II and H3K4me3/H3K9ac rhythms it drives (Koike et al. 2012) [PMID:22936566]. Core targets are *Per1*, *Per2*, *Cry1*, *Cry2*, *Nr1d1*/*Nr1d2* and *Dbp*; the wider output program layers E-boxes with D-boxes (DBP/HLF/TEF) and ROREs (ROR/REV-ERB) so that one clock input can generate many output phases (Takahashi 2017) [PMID:27990019]. A specific hexamer is expected about 7 x 10^5 times in a 3-Gb genome under uniform base composition; CpG depletion lowers the real count, but matches still outnumber occupied sites by orders of magnitude.\n\n## Biology and disease\n\nCLOCK:BMAL1 was identified as the activator of *Per1* through E-boxes, with the dominant-negative *Clock* mutation lengthening period (Gekakis et al. 1998) [PMID:9616112]. *Bmal1* loss abolishes free-running rhythms (Bunger et al. 2000) [PMID:11163178] and produces early-onset aging phenotypes including cataracts, sarcopenia and reduced lifespan (Kondratov et al. 2006) [PMID:16847346]. Pancreatic-islet disruption of *Clock* or *Bmal1* causes hypoinsulinemia and diabetes (Marcheva et al. 2010) [PMID:20562852]. In humans the clock is implicated in sleep-phase disorders, metabolic syndrome and shift-work-associated disease, though the strongest Mendelian sleep-timing mutations lie in *PER2* and *CSNK1D* rather than in CLOCK or BMAL1 (Takahashi 2017) [PMID:27990019].\n\n## Reading a hit\n\nA CACGTG match by itself says almost nothing about which bHLH protein bound it: MYC/MAX, MXI1, USF1/2, SREBF1/2, TFE3/MITF, ARNT homodimers and HIF:ARNT all read the same or an overlapping core, and several CLOCK/BMAL models in this family have a closer neighbor in another family than within their own. Features that shift the interpretation toward the clock are a tandem pair 6-7 bp apart, an A or T immediately 3' of the core, a promoter-proximal CpG-island location, and rhythmic accessibility or expression of the nearby gene. The hit cannot separate CLOCK from NPAS2, BMAL1 from BMAL2, or either from an ARNT homodimer. Evidence codes matter: the grade-A BMAL1 and CLOCK models carry in vitro support (HT-SELEX and methyl-HT-SELEX), whereas ChIP-only models can reflect tethering to partner factors, and CLOCK/BMAL1 ChIP peaks include many regions with no recognizable E-box.",
 "caveats": "Treat a hit as \"an E-box\" first and a clock site only with extra evidence. The core CACGTG is shared with the MYC/MAX, USF, SREBP and MiT bHLH groups and with HIF:ARNT, and in this family several models sit closer to motifs outside the family than inside it. Nothing in the sequence distinguishes CLOCK from NPAS2 or BMAL1 from BMAL2. The non-canonical CATGTG-type models come from methyl-HT-SELEX at grade C and are lower confidence than the grade-A CACGTG models. ChIP-derived versions include indirect binding, so peak-derived motifs are not proof that the tagged subunit touched the DNA. Tandem spacing and rhythmic behavior of the locus are more informative than the match score.",
 "references": [
  {
   "cite": "Gekakis N, Staknis D, Nguyen HB, et al. Role of the CLOCK protein in the mammalian circadian mechanism. Science 1998;280:1564-9.",
   "pmid": "9616112",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9616112/"
  },
  {
   "cite": "Hogenesch JB, Gu YZ, Jain S, et al. The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci U S A 1998;95:5474-9.",
   "pmid": "9576906",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9576906/"
  },
  {
   "cite": "Bunger MK, Wilsbacher LD, Moran SM, et al. Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 2000;103:1009-17.",
   "pmid": "11163178",
   "url": "https://pubmed.ncbi.nlm.nih.gov/11163178/"
  },
  {
   "cite": "Huang N, Chelliah Y, Shan Y, et al. Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex. Science 2012;337:189-94.",
   "pmid": "22653727",
   "url": "https://pubmed.ncbi.nlm.nih.gov/22653727/"
  },
  {
   "cite": "Wang Z, Wu Y, Li L, et al. Intermolecular recognition revealed by the complex structure of human CLOCK-BMAL1 basic helix-loop-helix domains with E-box DNA. Cell Res 2013;23:213-24.",
   "pmid": "23229515",
   "url": "https://pubmed.ncbi.nlm.nih.gov/23229515/"
  },
  {
   "cite": "Nakahata Y, Yoshida M, Takano A, et al. A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes. BMC Mol Biol 2008;9:1.",
   "pmid": "18177499",
   "url": "https://pubmed.ncbi.nlm.nih.gov/18177499/"
  },
  {
   "cite": "Rey G, Cesbron F, Rougemont J, et al. Genome-wide and phase-specific DNA-binding rhythms of BMAL1 control circadian output functions in mouse liver. PLoS Biol 2011;9:e1000595.",
   "pmid": "21364973",
   "url": "https://pubmed.ncbi.nlm.nih.gov/21364973/"
  },
  {
   "cite": "Koike N, Yoo SH, Huang HC, et al. Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 2012;338:349-54.",
   "pmid": "22936566",
   "url": "https://pubmed.ncbi.nlm.nih.gov/22936566/"
  },
  {
   "cite": "Debruyne JP, Noton E, Lambert CM, et al. A clock shock: mouse CLOCK is not required for circadian oscillator function. Neuron 2006;50:465-77.",
   "pmid": "16675400",
   "url": "https://pubmed.ncbi.nlm.nih.gov/16675400/"
  },
  {
   "cite": "Kondratov RV, Kondratova AA, Gorbacheva VY, et al. Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock. Genes Dev 2006;20:1868-73.",
   "pmid": "16847346",
   "url": "https://pubmed.ncbi.nlm.nih.gov/16847346/"
  },
  {
   "cite": "Marcheva B, Ramsey KM, Buhr ED, et al. Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes. Nature 2010;466:627-31.",
   "pmid": "20562852",
   "url": "https://pubmed.ncbi.nlm.nih.gov/20562852/"
  },
  {
   "cite": "Takahashi JS. Transcriptional architecture of the mammalian circadian clock. Nat Rev Genet 2017;18:164-179.",
   "pmid": "27990019",
   "url": "https://pubmed.ncbi.nlm.nih.gov/27990019/"
  }
 ],
 "timeline": [
  {
   "year": 1998,
   "finding": "CLOCK:BMAL1 heterodimers were shown to activate the mouse Per1 promoter through its E-boxes, and the dominant-negative Clock mutant protein to block that activation; this tied the behavioral period mutation to E-box-driven transcription.",
   "methods": "Transfection reporter assays, gel-shift, yeast two-hybrid",
   "confidence": "medium",
   "pmids": [
    "9616112"
   ],
   "open": "Which E-box variants beyond CACGTG the complex could use, and how occupancy was timed in vivo, were not addressed."
  },
  {
   "year": 1998,
   "finding": "MOP3, later renamed BMAL1/ARNTL, was identified as the bHLH-PAS subunit that dimerizes with both circadian (CLOCK, NPAS2) and hypoxia (HIF-1alpha) partners, placing the clock E-box inside the wider ARNT-like beta-subunit class.",
   "methods": "Two-hybrid screening, co-immunoprecipitation, reporter assays",
   "confidence": "medium",
   "pmids": [
    "9576906"
   ],
   "open": "Whether the shared beta subunit left any sequence feature that separates clock sites from hypoxia sites."
  },
  {
   "year": 2000,
   "finding": "Mice lacking Bmal1 lose free-running behavioral rhythms in constant darkness, establishing BMAL1 as a non-redundant component of the activating limb.",
   "methods": "Mouse germline knockout, wheel-running behavior",
   "confidence": "high",
   "pmids": [
    "11163178"
   ]
  },
  {
   "year": 2006,
   "finding": "Clock-null mice were found to keep circadian behavior, reversing the assumption that CLOCK is indispensable; NPAS2 substitutes for it in the suprachiasmatic nucleus, so the partner of BMAL1 is interchangeable in a way the beta subunit is not.",
   "methods": "Mouse germline knockout, behavior, SCN slice bioluminescence",
   "confidence": "high",
   "pmids": [
    "16675400",
    "11163178"
   ],
   "open": "Whether CLOCK and NPAS2 occupy the same sites genome-wide, or only overlap at core clock genes."
  },
  {
   "year": 2008,
   "finding": "Cell-autonomous rhythmicity was shown to require a direct repeat of two E-box-like elements spaced six to seven base pairs apart, not a single hexamer, making spacing part of the functional definition of a clock site.",
   "methods": "Reporter assays with spacing variants in cultured cells",
   "confidence": "medium",
   "pmids": [
    "18177499"
   ],
   "open": "Whether tandem spacing is required at endogenous loci genome-wide or mainly at the core clock promoters tested."
  },
  {
   "year": 2012,
   "finding": "The crystal structure of the mouse CLOCK:BMAL1 bHLH-PAS region showed an asymmetric, interdigitated heterodimer held by three separate interfaces rather than a symmetric pairing.",
   "methods": "X-ray crystallography",
   "confidence": "high",
   "pmids": [
    "22653727"
   ],
   "open": "The construct did not include DNA, so base-specific contacts remained unresolved."
  },
  {
   "year": 2012,
   "finding": "Genome-wide ChIP-seq across the circadian cycle in mouse liver put most confident CLOCK:BMAL1 sites near transcription start sites, at roughly one to three thousand per time point, with occupancy itself cycling ahead of the RNA polymerase II and histone-modification rhythms it drives.",
   "methods": "ChIP-seq time course, nascent-RNA and chromatin profiling",
   "confidence": "high",
   "pmids": [
    "22936566",
    "21364973"
   ],
   "open": "Why only a small fraction of genomic CACGTG matches is ever occupied."
  },
  {
   "year": 2013,
   "finding": "A structure of the human CLOCK:BMAL1 bHLH domains on an E-box, with accompanying affinity measurements, showed that BMAL1 Ile80 contacts the base pair immediately 3' of the core, making the recognized unit 7 bp (CACGTGA), and defined two high-affinity non-canonical boxes, AACGTGA and CATGTGA.",
   "methods": "X-ray crystallography with substitution series",
   "confidence": "high",
   "pmids": [
    "23229515"
   ],
   "open": "Whether the non-canonical boxes are used at endogenous rhythmic promoters."
  }
 ],
 "openQuestions": [
  "Whether the non-canonical boxes AACGTGA and CATGTGA are used at endogenous clock sites is unsettled: they bind with high affinity in vitro and the methyl-HT-SELEX models in this family recover the CATGTG form, but the grade-A models and ChIP-derived sites are dominated by CACGTG.",
  "It is not resolved what selects the one to three thousand occupied sites from the roughly 7 x 10^5 CACGTG matches in the genome; tandem spacing, pre-existing chromatin accessibility and partner factors have each been proposed, and no model predicts occupancy from sequence alone.",
  "Whether CLOCK and NPAS2 regulate distinguishable target sets is open: they cannot be separated by sequence and NPAS2 can sustain behavioral rhythms without CLOCK, yet their tissue distributions differ.",
  "A substantial share of CLOCK:BMAL1 ChIP peaks contains no recognizable E-box, and whether these reflect tethering through partner proteins or binding to degenerate sites below the detection threshold of current models has not been settled."
 ],
 "members": [
  {
   "id": "NPAS2.H14CORE.2.S.B",
   "source": "HOCOMOCO",
   "consensus": "CACGTGbnndaCACGTGt",
   "bits": 21.1,
   "prov": {
    "evidence": "S",
    "grade": "B"
   }
  },
  {
   "id": "CLOCK.H14CORE.1.PS.A",
   "source": "HOCOMOCO",
   "consensus": "CACGTGTT",
   "bits": 15.2,
   "prov": {
    "evidence": "PS",
    "grade": "A"
   }
  },
  {
   "id": "NPAS2.H14CORE.0.M.B",
   "source": "HOCOMOCO",
   "consensus": "catgtGknaaACATGTGt",
   "bits": 14.5,
   "prov": {
    "evidence": "M",
    "grade": "B"
   }
  },
  {
   "id": "M04158_2.00",
   "source": "CIS-BP",
   "consensus": "aCACGTGt",
   "bits": 13.7,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "CLOCK.H14CORE.0.M.C",
   "source": "HOCOMOCO",
   "consensus": "ACACATGT",
   "bits": 12.4,
   "prov": {
    "evidence": "M",
    "grade": "C"
   }
  },
  {
   "id": "NPAS2.H14CORE.1.M.C",
   "source": "HOCOMOCO",
   "consensus": "ACATGtGt",
   "bits": 12.2,
   "prov": {
    "evidence": "M",
    "grade": "C"
   }
  },
  {
   "id": "Npas2 (MA0626.2)",
   "source": "JASPAR",
   "consensus": "CACGTGt",
   "bits": 11.5
  },
  {
   "id": "BMAL1.H14CORE.0.PSM.A",
   "source": "HOCOMOCO",
   "consensus": "CACGTGa",
   "bits": 11.2,
   "prov": {
    "evidence": "PSM",
    "grade": "A"
   }
  },
  {
   "id": "M04129_2.00",
   "source": "CIS-BP",
   "consensus": "cCACGTGc",
   "bits": 11.2,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "Arntl (MA0603.2)",
   "source": "JASPAR",
   "consensus": "gTCACGTG",
   "bits": 11
  },
  {
   "id": "M04124_2.00",
   "source": "CIS-BP",
   "consensus": "CACGTG",
   "bits": 10.6,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "CLOCK (MA0819.3)",
   "source": "JASPAR",
   "consensus": "ACACGTG",
   "bits": 10.5
  }
 ],
 "url": "https://motif.zhoulab.io/families/clock_bmal/",
 "generated": "2026-10-21",
 "provenance": "Entry text drafted by a language model reading the primary literature; every cited PMID verified against PubMed. Computed fields derive from JASPAR 2024, HOCOMOCO v14, CIS-BP and the Vierstra archetypes. See https://motif.zhoulab.io/families/methods/"
}