{
 "key": "SMAD",
 "name": "SMAD",
 "class": "SMAD",
 "aka": [
  "SBE",
  "Smad-binding element",
  "CAGA box",
  "CAGAC motif",
  "5GC motif",
  "GC-SBE",
  "BMP-responsive element (BRE)"
 ],
 "definition": "SBE GTCT/AGAC and GC-rich SMAD motifs. SMAD1-5, SMAD9.",
 "consensus": "GTCT / AGAC (palindrome GTCTAGAC); also the 5-bp GC motif GGC(GC/CG)",
 "dataConsensus": "CtAGACa",
 "motifCount": 20,
 "bySource": {
  "JASPAR": 4,
  "HOCOMOCO": 8,
  "CIS-BP": 4,
  "Vierstra": 4
 },
 "symbols": [
  "SMAD",
  "SMAD2",
  "SMAD3",
  "SMAD4",
  "SMAD5",
  "SMAD9"
 ],
 "representative": "M05889_2.00",
 "cohesion": 0.76,
 "short": "SMAD proteins are the intracellular effectors of TGF-beta family signaling. Their MH1 domain inserts a beta-hairpin into the major groove and reads just four bases, GTCT or its reverse complement AGAC, often written as the CAGAC box or as the 8-bp palindrome GTCTAGAC. SMAD3, SMAD4 and the BMP-activated SMAD1/5/9 also bind a separate 5-bp GC-rich element, GGCGC or GGCCG. Both elements are far too short to be informative alone, and genome-wide SMAD occupancy is set mainly by whichever lineage-determining factor is present, so most SMAD ChIP peaks are better explained by a partner's motif than by an SBE. This is a family where a motif hit needs corroboration more than almost any other.",
 "long": "## The factors\n\nEight human SMADs fall into three groups. The receptor-regulated R-SMADs are SMAD2 and SMAD3\n(phosphorylated by TGF-beta and activin receptors) and SMAD1, SMAD5 and SMAD9/SMAD8\n(phosphorylated by BMP receptors). SMAD4 is the shared co-SMAD. SMAD6 and SMAD7 are inhibitory\nand are not sequence-specific activators. DNA contact is made by the N-terminal MH1 domain, a\ncompact domain with a bound zinc ion whose 11-residue beta-hairpin inserts into the major groove [PMID:9741623];\nthe C-terminal MH2 domain mediates trimerization, so the functional unit on DNA is normally a\ntrimer of two R-SMADs plus SMAD4, giving two or three DNA-contacting hairpins [PMID:29234012]. One paralogue is a\nspecial case: wild-type SMAD2 carries a 30-residue insert encoded by exon 3 immediately before the\nhairpin that blocks direct DNA binding [PMID:9873005]. Full-length SMAD2 reaches DNA through partners, most\nclearly the forkhead protein FOXH1/FAST1 [PMID:9702197,9873005]; the naturally occurring SMAD2 delta-exon-3 splice form\ndoes bind DNA, like SMAD3 [PMID:9873005].\n\n## The motif\n\nThe classical Smad-binding element is 4 bp, GTCT, read on the other strand as AGAC and usually\nquoted with a flanking base as CAGAC (\"CAGA box\") or CAGACA. Zawel and colleagues showed that\nSMAD3 and SMAD4 both recognize an identical 8-bp palindrome, GTCTAGAC [PMID:9660945], and the Smad3 MH1 crystal\nstructure explained why: the hairpin reads GTCT and two hairpins from a trimer can occupy the two\nhalves of the palindrome [PMID:9741623]. Real promoters usually contain tandem CAGA repeats rather than a perfect\npalindrome; the TGF-beta response element of the PAI-1/SERPINE1 promoter is the canonical example [PMID:9606191].\nSeparately, SMADs bind a 5-bp GC-rich element with consensus GGC(GC) or GGC(CG). This was first\ndescribed as a BMP-responsive element for SMAD1/5 [PMID:10679014], but structural work showed that SMAD3 and SMAD4\nbind the same 5GC motifs, and that these motifs occur in clusters in SMAD-bound regions\ngenome-wide [PMID:29234012]. So the family has two chemically distinct consensus sequences, not one; a database\nentry showing GTCTAGAC and one showing a GC-rich string can both be correct. The data for this\nfamily reflects that split, and its cohesion (0.76) is the lowest in this batch. The CAGAC-type\nand 5GC-type models do not interconvert, and paralogues are not separable from sequence within\neither type. The CIS-BP SMAD9 entry is a GC-rich model transferred from a Drosophila protein at\n91% MH1 identity, not a human measurement.\n\n## Where it acts\n\nSMAD sites are distributed across promoters and enhancers with no CpG-island preference for the\nCAGAC form, while 5GC clusters are by definition GC-rich and promoter-biased. What matters more is\nthat SMAD occupancy is almost entirely conditional. Smad3 ChIP-seq in three different cell types\nrecovers three largely non-overlapping site sets, each co-occupied by that cell type's\nlineage-determining factor: OCT4 in embryonic stem cells, MYOD1 in myotubes, PU.1 in pro-B cells [PMID:22036565].\nIn other systems the recurring partners are FOXH1 and other forkhead proteins, RUNX1/2, AP-1\n(JUN/FOS), TEAD, and the repressors TGIF1, SKI and SnoN. Named composite elements include the\nactivin-responsive element of the Mix.2 promoter (a forkhead site next to a SMAD site) [PMID:9702197,9873005] and the\nBMP-responsive elements of the Id1 and Smad7 promoters. In practice a SMAD peak set looks like the\npartner's peak set with a weak SBE enrichment layered on top.\n\n## Biology and disease\n\nTGF-beta/BMP signaling through SMADs controls epithelial growth arrest, extracellular matrix\nproduction, epithelial-mesenchymal transition, regulatory T cell induction, bone and cartilage\nformation, and left-right and dorsoventral patterning. Smad3-null mice die within months from\nimpaired mucosal immunity with defective T cell responses to TGF-beta [PMID:10064594]. SMAD4 was identified as\nDPC4, a candidate tumor suppressor at 18q21.1 that was homozygously deleted in 25 of 84 pancreatic\ncarcinomas (about 30%), with potentially inactivating mutations in 6 of 27 of the others [PMID:8553070]; germline SMAD4 mutations cause juvenile polyposis and, with an overlapping\nphenotype, hereditary hemorrhagic telangiectasia [PMID:9582123,15031030]. SMAD4 and SMAD2/SMAD3 are also recurrently\nmutated in colorectal cancer, and loss of SMAD signaling converts TGF-beta from a growth\nsuppressor to a driver of invasion and immune evasion [PMID:37714133]. SMAD9 mutations cause a form of pulmonary\narterial hypertension, consistent with the BMP9/BMPR2 axis [PMID:19211612].\n\n## Reading a hit\n\nTreat an isolated SBE match as essentially no evidence. GTCT or AGAC occurs about once every 128\nbases on either strand, so a 4-bp core appears tens of millions of times in the human genome; even\nthe 8-bp palindrome GTCTAGAC is expected tens of thousands of times by chance. A SMAD hit becomes\nmeaningful only in one of three forms: several CAGA repeats within a short window, a cluster of\n5GC motifs, or an SBE within a few base pairs of a validated partner site (a forkhead, RUNX,\nAP-1 or TEAD motif). The second problem is that ChIP-derived SMAD motifs in these collections are\ncontaminated by indirect binding almost by construction, since SMAD complexes are recruited by\nlineage factors. Every HOCOMOCO SMAD2 model here rests on ChIP-seq only (evidence code P), and\nwild-type SMAD2 cannot bind DNA directly [PMID:9873005], so those matrices describe the composition of SMAD2\npeaks rather than SMAD2's own specificity. Finally, do not use a hit to name a paralogue. CAGAC\ndoes not mean TGF-beta rather than BMP, because SMAD1/5 bind CAGAC-type sites too and SMAD3/4 bind\nGC-type sites; the original \"TGF-beta uses CAGAC, BMP uses GC\" division does not survive the\nstructural and genome-wide data [PMID:29234012,33510867].",
 "caveats": "The lowest-confidence family in this batch. A 4-bp SBE is not a finding; even the 8-bp palindrome is common by chance. The family has two unrelated consensus sequences (CAGAC-type and 5GC-type), so conflicting database entries may both be right, and paralogues are not separable within either. Most SMAD ChIP peaks are explained by a partner's motif, not by an SBE, and all HOCOMOCO SMAD2 models here are ChIP-only while full-length SMAD2 cannot bind DNA directly. The CIS-BP SMAD9 model is transferred from Drosophila. Use SMAD hits only as supporting evidence alongside a partner site or experimental occupancy.",
 "references": [
  {
   "cite": "Shi Y, Wang YF, Jayaraman L, et al. Crystal structure of a Smad MH1 domain bound to DNA: insights on DNA binding in TGF-beta signaling. Cell 1998;94:585-94.",
   "pmid": "9741623",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9741623/"
  },
  {
   "cite": "Zawel L, Dai JL, Buckhaults P, et al. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1998;1:611-7.",
   "pmid": "9660945",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9660945/"
  },
  {
   "cite": "Dennler S, Itoh S, Vivien D, et al. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 1998;17:3091-100.",
   "pmid": "9606191",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9606191/"
  },
  {
   "cite": "Martin-Malpartida P, Batet M, Kaczmarska Z, et al. Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors. Nat Commun 2017;8:2070.",
   "pmid": "29234012",
   "url": "https://pubmed.ncbi.nlm.nih.gov/29234012/"
  },
  {
   "cite": "Kusanagi K, Inoue H, Ishidou Y, et al. Characterization of a bone morphogenetic protein-responsive Smad-binding element. Mol Biol Cell 2000;11:555-65.",
   "pmid": "10679014",
   "url": "https://pubmed.ncbi.nlm.nih.gov/10679014/"
  },
  {
   "cite": "Ruiz L, Kaczmarska Z, Gomes T, et al. Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes. Comput Struct Biotechnol J 2021;19:632-646.",
   "pmid": "33510867",
   "url": "https://pubmed.ncbi.nlm.nih.gov/33510867/"
  },
  {
   "cite": "Mullen AC, Orlando DA, Newman JJ, et al. Master transcription factors determine cell-type-specific responses to TGF-β signaling. Cell 2011;147:565-76.",
   "pmid": "22036565",
   "url": "https://pubmed.ncbi.nlm.nih.gov/22036565/"
  },
  {
   "cite": "Labbé E, Silvestri C, Hoodless PA, et al. Smad2 and Smad3 positively and negatively regulate TGF beta-dependent transcription through the forkhead DNA-binding protein FAST2. Mol Cell 1998;2:109-20.",
   "pmid": "9702197",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9702197/"
  },
  {
   "cite": "Yagi K, Goto D, Hamamoto T, et al. Alternatively spliced variant of Smad2 lacking exon 3. Comparison with wild-type Smad2 and Smad3. J Biol Chem 1999;274:703-9.",
   "pmid": "9873005",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9873005/"
  },
  {
   "cite": "Yang X, Letterio JJ, Lechleider RJ, et al. Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta. EMBO J 1999;18:1280-91.",
   "pmid": "10064594",
   "url": "https://pubmed.ncbi.nlm.nih.gov/10064594/"
  },
  {
   "cite": "Hahn SA, Schutte M, Hoque AT, et al. DPC4, a candidate tumor suppressor gene at human chromosome 18q21.1. Science 1996;271:350-3.",
   "pmid": "8553070",
   "url": "https://pubmed.ncbi.nlm.nih.gov/8553070/"
  },
  {
   "cite": "Massagué J, Sheppard D. TGF-β signaling in health and disease. Cell 2023;186:4007-4037.",
   "pmid": "37714133",
   "url": "https://pubmed.ncbi.nlm.nih.gov/37714133/"
  },
  {
   "cite": "Howe JR, Roth S, Ringold JC, et al. Mutations in the SMAD4/DPC4 gene in juvenile polyposis. Science 1998;280:1086-8.",
   "pmid": "9582123",
   "url": "https://pubmed.ncbi.nlm.nih.gov/9582123/"
  },
  {
   "cite": "Gallione CJ, Repetto GM, Legius E, et al. A combined syndrome of juvenile polyposis and hereditary haemorrhagic telangiectasia associated with mutations in MADH4 (SMAD4). Lancet 2004;363:852-9.",
   "pmid": "15031030",
   "url": "https://pubmed.ncbi.nlm.nih.gov/15031030/"
  },
  {
   "cite": "Shintani M, Yagi H, Nakayama T, et al. A new nonsense mutation of SMAD8 associated with pulmonary arterial hypertension. J Med Genet 2009;46:331-7.",
   "pmid": "19211612",
   "url": "https://pubmed.ncbi.nlm.nih.gov/19211612/"
  }
 ],
 "timeline": [
  {
   "year": 1998,
   "finding": "SMAD3 and SMAD4 each recognize the same 8-bp palindrome, GTCTAGAC, and tandem copies confer TGF-beta responsiveness that is lost in SMAD4-deleted cells, establishing SMADs as sequence-specific DNA-binding factors.",
   "methods": "Binding-site selection from random oligonucleotides, reporter assays, SMAD4-null cells",
   "confidence": "medium",
   "pmids": [
    "9660945"
   ]
  },
  {
   "year": 1998,
   "finding": "Three CAGA boxes in the PAI-1/SERPINE1 promoter are bound directly by SMAD3 and SMAD4, are required for TGF-beta induction, and respond to TGF-beta and activin but not BMP; recombinant SMAD1 and SMAD2 do not bind them.",
   "methods": "Promoter mutagenesis, EMSA with recombinant proteins and nuclear extracts, reporter assays",
   "confidence": "medium",
   "pmids": [
    "9606191"
   ],
   "open": "What, if anything, the BMP-activated SMADs bind directly."
  },
  {
   "year": 1998,
   "finding": "The 2.8 A structure of the SMAD3 MH1 domain on DNA shows that base-specific recognition is made entirely by a conserved 11-residue beta-hairpin in the major groove.",
   "methods": "X-ray crystallography",
   "confidence": "high",
   "pmids": [
    "9741623"
   ]
  },
  {
   "year": 1999,
   "finding": "Wild-type SMAD2 does not bind DNA directly because of the exon 3 insert before the hairpin; it reaches target promoters with SMAD4 through the forkhead partner FAST/FOXH1, whereas the delta-exon-3 isoform and SMAD3 bind DNA themselves.",
   "methods": "EMSA, reporter assays, splice-variant comparison",
   "confidence": "medium",
   "pmids": [
    "9702197",
    "9873005"
   ]
  },
  {
   "year": 2000,
   "finding": "BMP-activated SMAD1 with SMAD4 binds the GC-rich GCCGnCGC element first defined for Drosophila Mad, weakly and only with multiple repeats, and GCCG reporters respond to BMP but not TGF-beta, supporting a split between GC-type BMP sites and CAGA-type TGF-beta sites.",
   "methods": "EMSA, multimerized reporter assays, mutagenesis",
   "confidence": "medium",
   "pmids": [
    "10679014"
   ],
   "open": "Whether the GC versus CAGA division reflected intrinsic specificity or the reporter contexts tested."
  },
  {
   "year": 2011,
   "finding": "SMAD3 ChIP-seq in embryonic stem cells, myotubes and pro-B cells gives largely distinct site sets, each co-occupied with and dependent on that cell's lineage-determining factor (OCT4, MYOD1, PU.1); inducing MYOD1 redirects SMAD3.",
   "methods": "ChIP-seq, knockdown and ectopic expression",
   "confidence": "medium",
   "pmids": [
    "22036565"
   ]
  },
  {
   "year": 2017,
   "finding": "Structures of SMAD3 and SMAD4 on goosecoid promoter DNA define the 5-bp GC motif GGC(GC)|(CG) as a site for TGF-beta-activated, BMP-activated and common SMADs alike, and 5GC clusters are frequent in SMAD-bound regions genome-wide. This overturns the view that CAGAC is the TGF-beta site and GC-rich elements the BMP site.",
   "methods": "X-ray crystallography, binding assays, ChIP-seq motif analysis",
   "confidence": "high",
   "pmids": [
    "29234012"
   ]
  },
  {
   "year": 2021,
   "finding": "SMAD5 and SMAD8 MH1 structures show all MH1 domains bind 5GC motifs similarly, but SMAD2/3/4 MH1 domains bind as monomers while SMAD1/5/8 form helix-swapped dimers, and site clustering and spacing in ChIP-seq peaks differ between BMP- and TGF-beta-responsive genes.",
   "methods": "X-ray crystallography, NMR, chimeric domains, ChIP-seq motif analysis",
   "confidence": "medium",
   "pmids": [
    "33510867"
   ],
   "open": "Whether monomer-versus-dimer propensity actually drives pathway-specific target selection in cells."
  }
 ],
 "openQuestions": [
  "How TGF-beta and BMP SMADs reach different targets is unresolved now that both bind CAGAC-type and 5GC sites: one proposal is that MH1 monomer (SMAD2/3/4) versus helix-swapped dimer (SMAD1/5/8) propensity sets site spacing and clustering, the alternative is that partner factors such as FOXH1 or lineage-determining factors choose the sites and the SMAD motif contributes little.",
  "The ChIP-derived HOCOMOCO SMAD2, SMAD3 and SMAD4 models (for example tGTCTGdCACc and AGGTGncAGACA) do not match the in vitro palindrome GTCTAGAC, and it is not settled whether they describe composite SMAD-partner sites or simply peak composition shaped by recruiting factors."
 ],
 "members": [
  {
   "id": "SMAD3 (MA0795.1)",
   "source": "JASPAR",
   "consensus": "cGTCTAGACA",
   "bits": 15.6
  },
  {
   "id": "M03482_2.00",
   "source": "CIS-BP",
   "consensus": "cGTCTAGACA",
   "bits": 15.6,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "SMAD3.H14CORE.0.S.B",
   "source": "HOCOMOCO",
   "consensus": "GTCTAGACA",
   "bits": 13.9,
   "prov": {
    "evidence": "S",
    "grade": "B"
   }
  },
  {
   "id": "SMAD5 (MA1557.1)",
   "source": "JASPAR",
   "consensus": "GTCTAGACA",
   "bits": 13.5
  },
  {
   "id": "M05743_2.00",
   "source": "CIS-BP",
   "consensus": "GTCTAGACA",
   "bits": 13.5,
   "prov": {
    "inferred": false
   }
  },
  {
   "id": "AC0323:SMAD:SMAD",
   "source": "Vierstra",
   "consensus": "GTCTAGAC",
   "bits": 13.3
  },
  {
   "id": "M05889_2.00",
   "source": "CIS-BP",
   "consensus": "CtAGACa",
   "bits": 11.7,
   "prov": {
    "species": "Mus musculus",
    "dbdIdentity": 1,
    "inferred": false
   }
  },
  {
   "id": "Smad4 (MA1153.2)",
   "source": "JASPAR",
   "consensus": "tGTCTaG",
   "bits": 11.5
  },
  {
   "id": "SMAD4.H14CORE.1.P.B",
   "source": "HOCOMOCO",
   "consensus": "tGTCTGdCACc",
   "bits": 11.1,
   "prov": {
    "evidence": "P",
    "grade": "B"
   }
  },
  {
   "id": "SMAD2 (MA1964.2)",
   "source": "JASPAR",
   "consensus": "CCAGAC",
   "bits": 10.6
  },
  {
   "id": "AC0597:SMAD:SMAD",
   "source": "Vierstra",
   "consensus": "gGTGhcAGACa",
   "bits": 10.6
  },
  {
   "id": "SMAD2.H14CORE.1.P.B",
   "source": "HOCOMOCO",
   "consensus": "CTGTCTG",
   "bits": 10.4,
   "prov": {
    "evidence": "P",
    "grade": "B"
   }
  },
  {
   "id": "SMAD3.H14CORE.1.P.B",
   "source": "HOCOMOCO",
   "consensus": "CCAGACAg",
   "bits": 10.4,
   "prov": {
    "evidence": "P",
    "grade": "B"
   }
  },
  {
   "id": "SMAD2.H14CORE.0.P.B",
   "source": "HOCOMOCO",
   "consensus": "tGTCTGdCACc",
   "bits": 10.1,
   "prov": {
    "evidence": "P",
    "grade": "B"
   }
  },
  {
   "id": "SMAD3.H14CORE.2.P.C",
   "source": "HOCOMOCO",
   "consensus": "AGGTGncAGACA",
   "bits": 9.9,
   "prov": {
    "evidence": "P",
    "grade": "C"
   }
  },
  {
   "id": "AC0371:SMAD:['SMAD/NF-1_DNA-binding_domain_factors']",
   "source": "Vierstra",
   "consensus": "GTCTGG",
   "bits": 9.9
  },
  {
   "id": "SMAD5.H14CORE.0.M.C",
   "source": "HOCOMOCO",
   "consensus": "CGTCT",
   "bits": 9.6,
   "prov": {
    "evidence": "M",
    "grade": "C"
   }
  },
  {
   "id": "SMAD4.H14CORE.0.P.B",
   "source": "HOCOMOCO",
   "consensus": "tGTCTGg",
   "bits": 8.6,
   "prov": {
    "evidence": "P",
    "grade": "B"
   }
  },
  {
   "id": "M06446_2.00",
   "source": "CIS-BP",
   "consensus": "GCGhCGcCmG",
   "bits": 8.6,
   "prov": {
    "species": "Drosophila melanogaster",
    "dbdIdentity": 0.91,
    "inferred": true
   }
  },
  {
   "id": "AC0598:SMAD:SMAD",
   "source": "Vierstra",
   "consensus": "CTghCacCt",
   "bits": 7
  }
 ],
 "url": "https://motif.zhoulab.io/families/smad/",
 "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/"
}