{
 "key": "HES_HEY",
 "name": "HES / HEY (N-box)",
 "class": "bHLH-Orange",
 "aka": [
  "N box",
  "N-box CACNAG",
  "class C site",
  "class B E-box (CACGYG)",
  "hairy/Enhancer-of-split (HES)",
  "HESR/HERP/HRT/CHF/gridlock (HEY)",
  "bHLH-Orange factors"
 ],
 "definition": "Class C bHLH-Orange repressors reading CACGYG (and the N box CACNAG); the CACGTG form is shared with the MYC/USF/MITF/CLOCK E-box families. HES1-7, HEY1/2, HEYL, HELT.",
 "consensus": "CACGYG",
 "dataConsensus": "CACGTGcC",
 "motifCount": 26,
 "bySource": {
  "JASPAR": 7,
  "HOCOMOCO": 7,
  "CIS-BP": 9,
  "Vierstra": 3
 },
 "symbols": [
  "HES",
  "HES1",
  "HES2",
  "HES4",
  "HES5",
  "HES6",
  "HES7",
  "HEY",
  "HEY1",
  "HEY2",
  "HEYL"
 ],
 "representative": "HEY1 (MA0823.1)",
 "cohesion": 0.9,
 "short": "HES1-7, HEY1/2, HEYL and HELT are the vertebrate hairy/Enhancer-of-split relatives: bHLH repressors with an Orange domain that sets partner choice and a C-terminal WRPW or YRPW peptide that recruits TLE/Groucho corepressors. Most are direct Notch targets, so their activity reports Notch signalling. They read a genuinely different site from the class II bHLH activators — a proline in the basic region shifts preference away from CANNTG. Two sites are described: the functionally defined N box CACNAG, and the higher-affinity CACGYG (CACGTG or CACGCG) that the database matrices actually capture. HES1 autorepression through N boxes generates a two-hour expression oscillation; HES7 drives the somite segmentation clock.",
 "long": "## The factors\n\nHES1-HES7, HEY1, HEY2, HEYL and HELT are the vertebrate relatives of *Drosophila* hairy and Enhancer of split, and they are structurally distinct from the class I and class II bHLH proteins that read E-boxes. Three features define them. First, a proline in the basic region, which is why this group is sometimes called the proline-bHLH class and why its sequence preference differs from CANNTG [PMID:12548545]. Second, an Orange domain, a two-helix module immediately C-terminal to the bHLH that restricts which partners a given member will dimerise with. Third, a C-terminal tetrapeptide — WRPW in HES proteins, YRPW in HEY proteins — that recruits TLE/Groucho corepressors [PMID:17586813]. They are repressors, not activators. Most are direct transcriptional targets of Notch signalling through RBPJ/CSL, so their expression reports Notch activity [PMID:17329370]: HES1 broadly, HES5 in neural progenitors, HES7 in presomitic mesoderm, HEY1/HEY2/HEYL in cardiovascular and endothelial tissue. HES2, HES3, HES4 and HES6 are divergent; HES6 does not behave like a canonical repressor [PMID:17329370]. Crucially, these proteins repress by two routes. They bind DNA and recruit corepressors, and they also sequester class II activators: HES1 reduces E47-driven transcription and blocks MyoD- and MASH1-driven differentiation, which requires no HES site at all [PMID:8524259].\n\n## The motif\n\nTwo different sites are attributed to this family, and the distinction matters. The site defined functionally is the N box, CACNAG, the Enhancer-of-split recognition sequence through which HES1 represses transcription [PMID:1340473]. *Drosophila* hairy was then shown to prefer a non-canonical site, CACGCG, and mutating a single CACGCG in the *achaete* promoter abolishes hairy-mediated repression in cells and produces ectopic sensory organs in flies [PMID:7958930,7958929]. Different papers call this site a class C site or a class B E-box; the nomenclature is not consistent, so it is safer to write the sequence than the class name.\n\nWhat the databases contain is neither of these. Twenty-four of the 26 motifs assigned here read CACGTG or CACGYG, usually with GG/CC flanks: GGCACGTGCC. The representative model is JASPAR HEY1 MA0823.1 (CACGTGCC), and almost all the HOCOMOCO models carry in vitro evidence (`S` or `SM`), so this is a real binding preference measured in vitro, not an artefact. Genome-wide, HEY1, HEY2 and HEYL bound preferentially to CACGYG, but a large fraction of efficiently bound promoters contained no E-box at all, implying additional, loosely defined binding modes [PMID:22615585]. The honest summary is that the high-affinity in vitro site is CACGYG, the classical repression site is the lower-affinity N box CACNAG, and the database matrices capture only the former. The atlas's data consensus CACGTGCC therefore disagrees with the ontology definition's N box for a documented reason rather than by error.\n\nCACGTG is a problem sequence. It is the canonical palindromic E-box of MYC/MAX, USF1/2, TFE3/TFEB/MITF, SREBF1/2, BHLHE40/41, ARNT and the CLOCK-BMAL1 heterodimer. The Vierstra archetype `AC0068:HEY/CLOCK:bHLH` states this in its name. Two further Vierstra archetypes here, `AC0263:HES` and `AC0264:HES`, have low-information consensuses (CACACTT, CNCACGT) that resemble nothing else in the family and should not be used as HES models. Two CIS-BP entries, for HES4 and HEYL, are inferred from mouse proteins at 91% DNA-binding-domain identity.\n\n## Where it acts\n\nUnlike the E-box families, HES/HEY sites are promoter-proximal. Chromatin immunoprecipitation of HEY factors in HEK293 cells, cardiomyocytes and mouse hearts found binding clustered in proximal promoter regions, disproportionately at genes encoding transcriptional regulators and developmental control proteins [PMID:22615585]. CACGTG contains a CpG, so these sites are enriched in CpG islands and their occupancy is sensitive to DNA methylation. HES1 binds N boxes in its own promoter, and that negative feedback loop, combined with short protein and mRNA half-lives, generates a roughly two-hour oscillation in HES1 expression [PMID:12399594]. Neighbouring RBPJ/CSL sites are common, because these genes and their targets are Notch-responsive.\n\n## Biology and disease\n\nHES and HEY proteins are the main transcriptional output of Notch signalling. Hes1 and Hes5 maintain neural progenitors and delay neuronal differentiation; removing them causes premature neurogenesis and progenitor depletion [PMID:10205173]. Hes7 drives the segmentation clock in presomitic mesoderm, and its loss disorganises somite formation [PMID:11641270]; in humans, *HES7* mutations cause spondylocostal dysostosis with vertebral and rib malformations [PMID:23897666]. HEY1, HEY2 and HEYL are strikingly redundant, which is why single knockouts are often mild and combined losses phenocopy each other in the heart [PMID:22615585]. HES1 acts downstream of activated NOTCH1 in T-cell acute lymphoblastic leukaemia.\n\n## Reading a hit\n\nA CACGTG hit is one of the least specific results a scan can return: on sequence alone it is compatible with at least eight other families, several of which (MYC/MAX, USF, MITF, CLOCK-BMAL1) are far more abundantly expressed in most cell types than HES or HEY. Treat a CACGTG match as a \"class B E-box\" hit and resolve it with expression and co-factor evidence, not with motif scores. If you are specifically after Notch-effector repression, the functionally validated site is the N box CACNAG, which the database matrices will not find for you; scan for it separately and expect weak scores. The individual HES and HEY paralogues cannot be distinguished. Finally, remember that these proteins can repress without binding DNA, by titrating E-proteins and class II activators, so the absence of a site is not evidence that HES or HEY is not acting on a gene.",
 "caveats": "The site the databases model, CACGTG, is the canonical palindromic E-box shared with MYC/MAX, USF1/2, TFE3/TFEB/MITF, SREBF, BHLHE40/41, ARNT and CLOCK-BMAL1 — one Vierstra archetype here is named HEY/CLOCK for exactly that reason — so a hit is weak evidence for HES or HEY specifically. The classically defined repression site, the N box CACNAG, is lower affinity and is not represented by these matrices, so a scan will miss it. Two low-information Vierstra HES archetypes resemble nothing else in the family. HES4 and HEYL models are inferred from mouse proteins. HES and HEY also repress without binding DNA, by sequestering E-proteins, so absent sites do not exclude their involvement.",
 "references": [
  {
   "cite": "Sasai Y, Kageyama R, Tagawa Y, et al. Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and Enhancer of split. Genes Dev 1992;6:2620-34.",
   "pmid": "1340473",
   "url": "https://pubmed.ncbi.nlm.nih.gov/1340473/"
  },
  {
   "cite": "Ohsako S, Hyer J, Panganiban G, et al. Hairy function as a DNA-binding helix-loop-helix repressor of Drosophila sensory organ formation. Genes Dev 1994;8:2743-55.",
   "pmid": "7958930",
   "url": "https://pubmed.ncbi.nlm.nih.gov/7958930/"
  },
  {
   "cite": "Van Doren M, Bailey AM, Esnayra J, et al. Negative regulation of proneural gene activity: hairy is a direct transcriptional repressor of achaete. Genes Dev 1994;8:2729-42.",
   "pmid": "7958929",
   "url": "https://pubmed.ncbi.nlm.nih.gov/7958929/"
  },
  {
   "cite": "Dawson SR, Turner DL, Weintraub H, et al. Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol Cell Biol 1995;15:6923-31.",
   "pmid": "8524259",
   "url": "https://pubmed.ncbi.nlm.nih.gov/8524259/"
  },
  {
   "cite": "Ohtsuka T, Ishibashi M, Gradwohl G, et al. Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation. EMBO J 1999;18:2196-207.",
   "pmid": "10205173",
   "url": "https://pubmed.ncbi.nlm.nih.gov/10205173/"
  },
  {
   "cite": "Bessho Y, Sakata R, Komatsu S, et al. Dynamic expression and essential functions of Hes7 in somite segmentation. Genes Dev 2001;15:2642-7.",
   "pmid": "11641270",
   "url": "https://pubmed.ncbi.nlm.nih.gov/11641270/"
  },
  {
   "cite": "Hirata H, Yoshiura S, Ohtsuka T, et al. Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 2002;298:840-3.",
   "pmid": "12399594",
   "url": "https://pubmed.ncbi.nlm.nih.gov/12399594/"
  },
  {
   "cite": "Iso T, Kedes L, Hamamori Y. HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 2003;194:237-55.",
   "pmid": "12548545",
   "url": "https://pubmed.ncbi.nlm.nih.gov/12548545/"
  },
  {
   "cite": "Kageyama R, Ohtsuka T, Kobayashi T. The Hes gene family: repressors and oscillators that orchestrate embryogenesis. Development 2007;134:1243-51.",
   "pmid": "17329370",
   "url": "https://pubmed.ncbi.nlm.nih.gov/17329370/"
  },
  {
   "cite": "Fischer A, Gessler M. Delta-Notch--and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids Res 2007;35:4583-96.",
   "pmid": "17586813",
   "url": "https://pubmed.ncbi.nlm.nih.gov/17586813/"
  },
  {
   "cite": "Heisig J, Weber D, Englberger E, et al. Target gene analysis by microarrays and chromatin immunoprecipitation identifies HEY proteins as highly redundant bHLH repressors. PLoS Genet 2012;8:e1002728.",
   "pmid": "22615585",
   "url": "https://pubmed.ncbi.nlm.nih.gov/22615585/"
  },
  {
   "cite": "Sparrow DB, Faqeih EA, Sallout B, et al. Mutation of HES7 in a large extended family with spondylocostal dysostosis and dextrocardia with situs inversus. Am J Med Genet A 2013;161A:2244-9.",
   "pmid": "23897666",
   "url": "https://pubmed.ncbi.nlm.nih.gov/23897666/"
  }
 ],
 "timeline": [
  {
   "year": 1992,
   "finding": "Mammalian HES1 and HES3 are cloned as relatives of Drosophila hairy and Enhancer of split; HES1 represses through the N box CACNAG and separately reduces E-box-driven activation by E47 without binding that site, defining the two repression modes at the outset.",
   "methods": "cDNA cloning, gel shift and reporter assays",
   "confidence": "medium",
   "pmids": [
    "1340473"
   ],
   "open": "The relative importance of DNA-bound repression and activator sequestration in vivo."
  },
  {
   "year": 1994,
   "finding": "Drosophila hairy is shown to be a sequence-specific repressor that prefers the non-canonical site CACGCG rather than the N box, and mutating a single CACGCG in the achaete promoter abolishes repression in cells and produces ectopic sensory organs in flies — direct genetic evidence tying one site to one phenotype.",
   "methods": "binding-site selection, cell reporter assays, transgenic flies",
   "confidence": "high",
   "pmids": [
    "7958930",
    "7958929"
   ],
   "open": "Whether vertebrate HES proteins share the CACGCG preference or use the N box."
  },
  {
   "year": 1995,
   "finding": "Domain swaps place repression specificity outside the bHLH: the bHLH and Orange domains antagonise specific activators such as Scute, while the C-terminal WRPW tetrapeptide represses through corepressor recruitment, so two separable mechanisms operate.",
   "methods": "chimeric proteins assayed in Drosophila sex determination",
   "confidence": "medium",
   "pmids": [
    "8524259"
   ]
  },
  {
   "year": 1999,
   "finding": "Hes1 and Hes5 are established as the Notch effectors that hold neural progenitors undifferentiated, with premature neurogenesis and progenitor depletion on their loss.",
   "methods": "mouse knockouts and progenitor assays",
   "confidence": "medium",
   "pmids": [
    "10205173"
   ]
  },
  {
   "year": 2001,
   "finding": "Dynamic rather than static repression emerges: Hes7 is shown to be required for ordered somite segmentation (2001), and Hes1 is shown to oscillate with a roughly two-hour period driven by negative autoregulation through N boxes in its own promoter plus short mRNA and protein half-lives (2002).",
   "methods": "mouse mutants, serum-synchronised cultures, mathematical modelling",
   "confidence": "medium",
   "pmids": [
    "11641270",
    "12399594"
   ],
   "open": "Whether oscillation depends on the precise affinity of the autoregulatory N boxes."
  },
  {
   "year": 2012,
   "finding": "Genome-wide mapping of HEY1, HEY2 and HEYL finds binding clustered in proximal promoters of transcriptional regulators, a clear preference for class B E-boxes CACGYG rather than the N box, extensive redundancy among the three proteins, and many efficiently bound promoters with no E-box at all.",
   "methods": "ChIP-chip/ChIP-seq and microarray profiling in cells, cardiomyocytes and mouse heart",
   "confidence": "medium",
   "pmids": [
    "22615585"
   ],
   "open": "What the factors recognise at the E-box-free promoters was left undefined."
  },
  {
   "year": 2013,
   "finding": "HES7 mutation is shown to cause spondylocostal dysostosis in a large human family, carrying the segmentation-clock mechanism into human skeletal disease.",
   "methods": "linkage and sequencing in an extended family",
   "confidence": "medium",
   "pmids": [
    "23897666"
   ]
  }
 ],
 "openQuestions": [
  "Which site this family actually uses is unsettled: repression was defined functionally through the N box CACNAG and, in Drosophila, through CACGCG, whereas the in vitro matrices in these databases read CACGTG/CACGYG, and it is unresolved whether the N box is a genuine lower-affinity site used in vivo or an early assignment that the newer binding data supersede.",
  "A large fraction of promoters efficiently bound by HEY proteins contain no E-box, and whether that reflects tethering by other DNA-binding factors, a degenerate site that position weight matrices do not capture, or indirect ChIP signal has not been determined.",
  "How much HES/HEY repression requires DNA binding at all is unquantified: these proteins also repress by sequestering E-proteins and class II activators, a mode that needs no site, and the in vivo balance between the two mechanisms is unknown.",
  "No paralogue-level sequence difference has been demonstrated within the family, and the two low-information Vierstra archetypes grouped here, AC0263:HES (CACACTT) and AC0264:HES (CNCACGT), resemble neither the CACGYG models nor each other, so whether they capture any real HES binding mode is doubtful."
 ],
 "members": [
  {
   "id": "HES1.H14CORE.0.S.C",
   "source": "HOCOMOCO",
   "consensus": "GGCACGTGcCaggCACGtG",
   "bits": 20.2,
   "prov": {
    "evidence": "S",
    "grade": "C"
   }
  },
  {
   "id": "HES7 (MA0822.1)",
   "source": "JASPAR",
   "consensus": "tGGCACGTGCCa",
   "bits": 19.8
  },
  {
   "id": "M02809_2.00",
   "source": "CIS-BP",
   "consensus": "cGGCACGTGCC",
   "bits": 18.5,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "HES5.H14CORE.0.SM.B",
   "source": "HOCOMOCO",
   "consensus": "GGCACGTGtC",
   "bits": 15.8,
   "prov": {
    "evidence": "SM",
    "grade": "B"
   }
  },
  {
   "id": "HEY2.H14CORE.0.S.B",
   "source": "HOCOMOCO",
   "consensus": "GGCACGTGTC",
   "bits": 15.7,
   "prov": {
    "evidence": "S",
    "grade": "B"
   }
  },
  {
   "id": "HES2.H14CORE.0.S.C",
   "source": "HOCOMOCO",
   "consensus": "GGCACGTGc",
   "bits": 15.6,
   "prov": {
    "evidence": "S",
    "grade": "C"
   }
  },
  {
   "id": "HES1.H14CORE.1.S.C",
   "source": "HOCOMOCO",
   "consensus": "GGCACGtG",
   "bits": 14.6,
   "prov": {
    "evidence": "S",
    "grade": "C"
   }
  },
  {
   "id": "HES6 (MA1493.1)",
   "source": "JASPAR",
   "consensus": "GGCACGTGTt",
   "bits": 14.2
  },
  {
   "id": "M04139_2.00",
   "source": "CIS-BP",
   "consensus": "aACACGTGCC",
   "bits": 14.2,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "HEY1.H14CORE.0.S.B",
   "source": "HOCOMOCO",
   "consensus": "gGCACGTG",
   "bits": 14.1,
   "prov": {
    "evidence": "S",
    "grade": "B"
   }
  },
  {
   "id": "HEY1 (MA0823.1)",
   "source": "JASPAR",
   "consensus": "CACGTGcC",
   "bits": 12.3
  },
  {
   "id": "M04060_2.00",
   "source": "CIS-BP",
   "consensus": "gCACGTGCC",
   "bits": 12.3,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "HES2 (MA0616.3)",
   "source": "JASPAR",
   "consensus": "GGCACGTGc",
   "bits": 12
  },
  {
   "id": "HEY2 (MA0649.2)",
   "source": "JASPAR",
   "consensus": "GrCACGTGc",
   "bits": 11.8
  },
  {
   "id": "M02790_2.00",
   "source": "CIS-BP",
   "consensus": "GGCACGTG",
   "bits": 11.5,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "M04191_2.00",
   "source": "CIS-BP",
   "consensus": "CACGTGcC",
   "bits": 11.4,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "M04090_2.00",
   "source": "CIS-BP",
   "consensus": "CACGtGcC",
   "bits": 11,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "HES1 (MA1099.3)",
   "source": "JASPAR",
   "consensus": "GgCaCGTG",
   "bits": 10.5
  },
  {
   "id": "HES5 (MA0821.2)",
   "source": "JASPAR",
   "consensus": "ggCACGTGcc",
   "bits": 10.4
  },
  {
   "id": "HES7.H14CORE.0.S.B",
   "source": "HOCOMOCO",
   "consensus": "gGCACGTG",
   "bits": 10.4,
   "prov": {
    "evidence": "S",
    "grade": "B"
   }
  },
  {
   "id": "M04152_2.00",
   "source": "CIS-BP",
   "consensus": "CACGTG",
   "bits": 9.8,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "M01722_2.00",
   "source": "CIS-BP",
   "consensus": "CACGTGnc",
   "bits": 8.9,
   "prov": {
    "species": "Mus musculus",
    "dbdIdentity": 0.91,
    "inferred": true
   }
  },
  {
   "id": "M01726_2.00",
   "source": "CIS-BP",
   "consensus": "CACGcG",
   "bits": 5.4,
   "prov": {
    "species": "Mus musculus",
    "dbdIdentity": 0.91,
    "inferred": true
   }
  },
  {
   "id": "AC0263:HES:bHLH",
   "source": "Vierstra",
   "consensus": "CACaCtt",
   "bits": 5.4
  },
  {
   "id": "AC0068:HEY/CLOCK:bHLH",
   "source": "Vierstra",
   "consensus": "CACGTG",
   "bits": 5.2
  },
  {
   "id": "AC0264:HES:bHLH",
   "source": "Vierstra",
   "consensus": "cncACgT",
   "bits": 3.5
  }
 ],
 "url": "https://motif.zhoulab.io/families/hes_hey/",
 "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/"
}