{"gene":"NOTCH4","run_date":"2026-04-29T11:37:57","timeline":{"discoveries":[{"year":1996,"finding":"Notch4 (int-3) is specifically expressed in endothelial cells during embryonic and adult development, and loss of the extracellular domain (as in the int-3 oncogene) leads to constitutive activation of the intracellular signaling domain, causing neoplastic transformation of mammary epithelial cells.","method":"In situ hybridization, cDNA cloning, transgenic mouse models","journal":"Development","confidence":"High","confidence_rationale":"Tier 2 — replicated across multiple labs with in vivo transgenic and in situ hybridization data; foundational paper with 427 citations","pmids":["8681805"],"is_preprint":false},{"year":1992,"finding":"Expression of the truncated int-3/Notch4 intracellular domain (encoding the intracellular portion homologous to Drosophila Notch) in transgenic mice causes mammary gland differentiation arrest and adenocarcinoma formation, establishing that the intracellular domain is a constitutively active oncogenic form.","method":"Transgenic mouse model, histology, MMTV promoter-driven expression","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 — in vivo gain-of-function with defined phenotypic readout, replicated; 320 citations","pmids":["1372276"],"is_preprint":false},{"year":1992,"finding":"The int-3/Notch4 intracellular domain protein contains six cdc10/ankyrin repeats homologous to Drosophila Notch, and expression of this domain in HC11 mouse mammary epithelial cells induces anchorage-independent growth in soft agar, demonstrating oncogenic transformation.","method":"In vitro soft-agar transformation assay, sequence analysis","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 — in vitro functional assay with defined molecular characterization; 237 citations","pmids":["1312643"],"is_preprint":false},{"year":2001,"finding":"Endothelial-specific expression of activated Notch4 under the Flk1 promoter in mice causes embryonic lethality with vascular patterning defects including restricted vasculature, dilated vessels, disorganized networks, and compromised vessel-wall integrity, implicating Notch4 in vessel remodeling.","method":"Knock-in transgenic mouse model, histology, whole-mount analysis","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — in vivo gain-of-function with defined vascular phenotype; 227 citations","pmids":["11344305"],"is_preprint":false},{"year":2002,"finding":"Constitutively active Notch4 inhibits VEGF-induced angiogenesis and endothelial sprouting in vitro and in vivo by promoting beta1-integrin activation (high-affinity conformation) and increased adhesion to collagen, without increasing surface expression of beta1-integrins.","method":"In vitro endothelial sprouting assay, chick CAM assay, flow cytometry for integrin activation, function-activating antibodies","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal methods including in vitro and in vivo assays with mechanistic dissection; 140 citations","pmids":["11909975"],"is_preprint":false},{"year":2003,"finding":"Constitutively active Notch4 intracellular domain inhibits endothelial apoptosis via two distinct pathways: (1) RBP-Jkappa-dependent inhibition of JNK-mediated apoptosis, and (2) RBP-Jkappa-independent upregulation of Bcl-2.","method":"Overexpression of Notch4-ICD and deletion mutants, RBP-Jkappa reporter assays, apoptosis assays, Western blot","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods dissecting RBP-Jkappa-dependent and -independent mechanisms with mutant analysis","pmids":["14701863"],"is_preprint":false},{"year":2004,"finding":"Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats but not the RAM domain or C-terminal region, and involves both RBP-Jkappa-dependent and -independent signaling; the ankyrin repeats alone are sufficient to partially inhibit sprouting and upregulate RBP-Jkappa target genes.","method":"Deletion mutant analysis, quantitative endothelial sprouting assay, RBP-Jkappa reporter assays","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1 — systematic domain deletion mutagenesis with functional readout and transcriptional reporter assays","pmids":["15187023"],"is_preprint":false},{"year":2005,"finding":"Notch4 intracellular domain (ICD) binds Smad3 (with higher affinity than Smad2 or Smad4) via the Smad3 MH2 domain, independent of the RAM23 region, and inhibits TGF-beta/Activin-Smad signaling, rendering cells resistant to TGF-beta-induced growth inhibition.","method":"Co-immunoprecipitation, deletion analysis, TGF-beta reporter luciferase assays, RT-PCR, Western blot, gamma-secretase inhibitor treatment","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical binding assay with domain mapping and functional transcriptional reporter validation","pmids":["16007227"],"is_preprint":false},{"year":2005,"finding":"NOTCH4 transcription in vascular endothelial cells is controlled by AP-1 complexes occupying the NOTCH4 chromatin; intron 1 or upstream sequences are required for endothelial-specific expression in vivo, and vascular angiogenic factors activate AP-1 to reprogram NOTCH4 from a repressed to transcriptionally active state.","method":"Chromatin immunoprecipitation (ChIP), transgenic mouse reporter assays, RNA-FISH, transfection assays, histone modification analysis","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 1-2 — multiple orthogonal chromatin and functional assays establishing AP-1 as direct regulator","pmids":["15684396"],"is_preprint":false},{"year":2005,"finding":"In adult mouse endothelium, constitutively active Notch4 (int3) causes arteriovenous malformations with blood vessel enlargement, AV shunting, arterialization (ectopic venous ephrinB2 expression, increased smooth muscle cells, upregulated Notch signaling), and these defects are fully reversible upon repression of int3 expression.","method":"Tetracycline-repressible endothelial transgenic system, histology, immunofluorescence","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — reversible gain-of-function in vivo with multiple mechanistic markers; 164 citations","pmids":["15994223"],"is_preprint":false},{"year":2006,"finding":"VEGF upregulates DLL4 and presenilin, leading to activation of Notch4, which in turn upregulates ephrinB2 and downregulates EphB4 in venous endothelial cells; pharmacological inhibition of presenilin or soluble DLL4 blocks VEGF-induced Notch4 activation and venous endothelial differentiation and migration.","method":"In vitro endothelial cell assays, Western blot, immunohistochemistry in transgenic tumor model, pharmacological inhibition","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple assays but mechanistic dissection relies partly on pharmacological inhibitors","pmids":["16951162"],"is_preprint":false},{"year":2007,"finding":"Glucocorticoid receptor (GR) and AP-1 synergistically activate NOTCH4 transcription in endothelial cells via a composite response element (imperfect GRE + AP-1 motif) in the NOTCH4 promoter; AP-1 stabilizes GR occupancy, and together they reprogram NOTCH4 chromatin from repressed to active in multipotent cells.","method":"ChIP assay, promoter reporter assays, composite element mutagenesis, histone modification analysis","journal":"Molecular and Cellular Biology","confidence":"High","confidence_rationale":"Tier 1 — reconstituted transcriptional mechanism with ChIP and functional mutagenesis","pmids":["17220278"],"is_preprint":false},{"year":2008,"finding":"Constitutively active Notch4 expressed in brain endothelium from birth causes hallmarks of brain arteriovenous malformations including cerebral AV shunting, vessel enlargement, hemorrhage, and neuronal cell death; repression of Notch4 resolves ataxia and reverses disease, indicating Notch4 is required to sustain as well as initiate the AVM phenotype.","method":"Tetracycline-repressible endothelial-specific transgenic system, MRI, histology, behavioral assays","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — reversible in vivo gain-of-function with multiple phenotypic readouts; 113 citations","pmids":["18667694"],"is_preprint":false},{"year":2008,"finding":"Notch4 knockdown in endothelial cells increases VCAM-1 expression and promotes apoptosis, while Notch4 and Hes1 are required to maintain EC quiescence and for endothelial injury repair; impaired Notch4/Hes1 activity is associated with transplant arteriosclerosis.","method":"siRNA knockdown, CBF1 reporter assays, adhesion molecule expression, apoptosis assays, allograft model","journal":"Arteriosclerosis, Thrombosis, and Vascular Biology","confidence":"Medium","confidence_rationale":"Tier 2-3 — loss-of-function with defined cellular phenotypes but mechanism not fully reconstituted","pmids":["18802018"],"is_preprint":false},{"year":2008,"finding":"Notch4 signaling in mammary glands is mediated primarily through interaction of the intracellular domain with RBP-Jkappa (CBF1): conditional Rbpj knockout rescues the mammary gland developmental arrest induced by Wap-Int3, but mammary tumor development occurs independently of RBP-Jkappa interaction.","method":"Conditional Rbpj knockout in mammary glands, Wap-Cre system, mammary transplantation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis via conditional knockout dissecting RBP-Jkappa-dependent and -independent functions","pmids":["18836481"],"is_preprint":false},{"year":2010,"finding":"Notch4 signaling activity is 8-fold higher in breast cancer stem cell-enriched populations compared with differentiated cells (versus 4-fold lower for Notch1); pharmacological or genetic inhibition of Notch4 completely inhibits tumor initiation in vivo, implicating Notch4 as the primary Notch receptor maintaining breast cancer stem cell activity.","method":"Stem cell enrichment (anoikis-resistance, ESA+/CD44+/CD24low), Notch activity reporter assays, GSI and siRNA inhibition, in vivo tumor formation assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with in vitro and in vivo functional readouts; 420 citations","pmids":["20068161"],"is_preprint":false},{"year":2010,"finding":"Notch4 is specifically required for Nodal expression in aggressive melanoma cells via an RBPJ-dependent Nodal enhancer element, placing Notch4 upstream of Nodal in the signaling cascade; Notch4-mediated vasculogenic mimicry and anchorage-independent growth are partly dependent on Nodal.","method":"Notch4 knockdown, RBPJ-dependent reporter assays, expression correlation in multiple cell lines, rescue experiments","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2-3 — reporter assay plus KD/rescue experiments establishing pathway hierarchy","pmids":["21159651"],"is_preprint":false},{"year":2012,"finding":"Normalization of Notch4 signaling in mouse brain converts large-caliber high-flow AV shunts to capillary-like vessels and reverses tissue hypoxia; this structural regression requires restoration of EphB4 receptor expression by venous ECs and occurs via vessel narrowing without loss of ECs.","method":"4D two-photon imaging through cranial windows, Notch4* repression, EphB4 expression analysis, blood flow measurement","journal":"Science Translational Medicine","confidence":"High","confidence_rationale":"Tier 2 — reversible in vivo Notch4 manipulation with mechanistic identification of EphB4 requirement","pmids":["22261032"],"is_preprint":false},{"year":2013,"finding":"PKCα overexpression selectively increases Notch4 (but not Notch1) expression via AP-1 occupancy at the Notch4 promoter; Notch4 intracellular domain promotes estrogen-independent, tamoxifen-resistant breast cancer growth and activates pathways associated with endocrine resistance.","method":"ChIP for AP-1 on Notch4 promoter, siRNA knockdown, Notch4-IC expression, gene expression profiling, in vivo xenograft with gamma-secretase inhibitor","journal":"Oncogenesis","confidence":"High","confidence_rationale":"Tier 2 — ChIP confirming AP-1 binding, combined with functional in vitro and in vivo validation","pmids":["23917222"],"is_preprint":false},{"year":2013,"finding":"YB-1 (Y-box binding protein-1) transcriptionally regulates Notch4: ChIP-on-chip shows 12-fold enrichment of YB-1 binding at the Notch4 promoter, YB-1 siRNA decreases Notch4 mRNA, and constitutively active YB-1 increases Notch4 mRNA. Upstream, RSK phosphorylates YB-1 (S102), and RSK inhibition with luteolin suppresses Notch4 levels and reduces tumor-initiating cells.","method":"ChIP-on-Chip, siRNA knockdown, overexpression constructs, in vitro kinase assay, Notch4 mRNA measurement","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1-2 — ChIP-on-Chip with orthogonal siRNA and overexpression validation establishing transcriptional mechanism","pmids":["23593654"],"is_preprint":false},{"year":2014,"finding":"NOTCH4 did not signal in response to canonical ligand activation in cultured cells, unlike other Notch receptors; furthermore, NOTCH4 inhibits signaling from NOTCH1 in cis (the first report of cis-inhibition by another Notch receptor), and this inhibition is mediated by the NOTCH4 extracellular domain. In vivo, Notch4 null mice show slightly delayed vessel growth in the retina.","method":"Notch signaling reporter assays in cultured cells, NOTCH4 knockout mouse generation (full coding region deletion), cis-inhibition constructs, retinal vascular imaging","journal":"Biochimica et Biophysica Acta","confidence":"High","confidence_rationale":"Tier 1-2 — cell-based signaling assays with genetic mouse model; novel mechanistic finding","pmids":["24667410"],"is_preprint":false},{"year":2014,"finding":"Brain AVMs arise from enlargement of pre-existing capillary-diameter microvessels accompanied by increased individual endothelial cell area (not increased EC number/proliferation); AV shunting begins promptly after Notch4* expression, and alterations in Notch signaling in ECs of all vessels (not arteries alone) drive AVM formation, indicating Notch4 functions in microvasculature/veins.","method":"Time-lapse two-photon imaging through cranial windows in Notch4* mice, EC area/number quantification, cell-type-specific Notch manipulation","journal":"PNAS","confidence":"High","confidence_rationale":"Tier 2 — real-time in vivo imaging with mechanistic dissection of cellular events","pmids":["25468970"],"is_preprint":false},{"year":2015,"finding":"AKT phosphorylates Notch4 intracellular domain (ICD) at four distinct sites in vitro and in vivo; this phosphorylation is regulated by growth factors and PI3K, creates binding sites for 14-3-3 proteins, and restricts phosphorylated Notch4-ICD to the cytoplasm, thereby negatively regulating Notch4 nuclear signaling.","method":"In vitro kinase assay, site-directed mutagenesis, co-immunoprecipitation with 14-3-3, PI3K inhibitor treatment, subcellular fractionation","journal":"Scientific Reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase reconstitution with in vivo validation and subcellular localization functional consequence","pmids":["25740432"],"is_preprint":false},{"year":2015,"finding":"JAG1-NOTCH4 activation by anti-estrogen treatment (tamoxifen/fulvestrant) increases breast cancer stem cell (BCSC) activity; targeting NOTCH4 reverses this increase and reduces BCSC activity in patient-derived xenograft tumors with acquired tamoxifen resistance.","method":"Patient-derived samples, PDX models, NOTCH4 targeting, ALDH1 activity, mammosphere assay","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 — human PDX models with mechanistic JAG1-NOTCH4 pathway identification; 160 citations","pmids":["26387946"],"is_preprint":false},{"year":2016,"finding":"Constitutively active Notch4 ICD (N4ICD) in melanoma cells triggers a mesenchymal-to-epithelial-like switch and suppresses invasion/migration; mechanistically, N4ICD induces Hey1 and Hey2 transcription factors which directly bind the promoters of Snail2 and Twist1 and repress their transcription, as shown by EMSA and luciferase assays.","method":"N4ICD overexpression, EMSA, luciferase reporter assays, invasion/migration assays, in vivo tumor growth assay","journal":"Cancer Research","confidence":"High","confidence_rationale":"Tier 1-2 — direct promoter binding shown by EMSA plus functional luciferase validation","pmids":["26801977"],"is_preprint":false},{"year":2017,"finding":"NOTCH4 transcriptionally activates HEY1 in HNSCC, and NOTCH4-HEY1 pathway activation promotes EMT (decreased E-cadherin, increased vimentin/fibronectin/Twist1/SOX2), proliferation, cisplatin resistance, and invasion.","method":"TCGA analysis, in vitro proliferation/apoptosis/cell-cycle assays, siRNA knockdown, expression analysis","journal":"Clinical Cancer Research","confidence":"Medium","confidence_rationale":"Tier 2-3 — functional KD with defined phenotypic readouts but limited mechanistic reconstitution","pmids":["29146722"],"is_preprint":false},{"year":2017,"finding":"NOTCH4 expression is negatively controlled by RUNX1 via a novel regulatory DNA element in the NOTCH4 locus; NOTCH4 inactivation by CRISPR-Cas9 in human iPSCs enhances megakaryopoiesis, establishing NOTCH4 as a RUNX1 target gene that negatively regulates megakaryocyte differentiation.","method":"Integrative genomic analysis of FPD-iPSCs, ChIP for RUNX1 binding, CRISPR-Cas9 NOTCH4 knockout, MK differentiation assays from iPSCs and CD34+ cells","journal":"Blood","confidence":"High","confidence_rationale":"Tier 1-2 — genomic binding data plus functional CRISPR knockout with defined differentiation phenotype","pmids":["29101237"],"is_preprint":false},{"year":2018,"finding":"Notch4 intracellular domain interacts with TAK1 and inhibits its activation; furthermore, Notch4-ICD prevents TRAF6 autoubiquitination and suppresses TRAF6-mediated TAK1 polyubiquitination, thereby negatively regulating M. tuberculosis-induced proinflammatory cytokine production.","method":"Co-immunoprecipitation, ubiquitination assays, Notch4-deficient mice, bacterial burden measurement","journal":"Journal of Infectious Diseases","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical mechanism (co-IP + ubiquitination assays) with in vivo genetic validation","pmids":["29228365"],"is_preprint":false},{"year":2020,"finding":"NOTCH4 transcriptionally upregulates SLUG and GAS1 by directly binding their promoters (confirmed by dual-luciferase reporter and ChIP assays), promoting EMT and quiescence in triple-negative breast cancer mesenchymal-like cancer stem cells.","method":"RNA-seq, dual-luciferase reporter assays, chromatin immunoprecipitation, stable overexpression and knockdown, mammosphere and chemoresistance assays","journal":"Theranostics","confidence":"High","confidence_rationale":"Tier 1-2 — direct promoter binding confirmed by ChIP and luciferase assays with functional validation","pmids":["32104513"],"is_preprint":false},{"year":2020,"finding":"Notch4 on lung tissue regulatory T cells (induced by IL-6/STAT3) subverts Treg cells into TH2 and TH17 effector T cells via Wnt and Hippo pathway-dependent mechanisms; Wnt activation induces GDF15 expression in Treg cells, which activates group 2 innate lymphoid cells in a feed-forward inflammatory loop.","method":"Treg-specific Notch4 conditional knockout mice, in vivo allergen challenge models, Wnt/Hippo pathway inhibitors, GDF15 measurement, cytokine assays","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional knockout with multiple pathway analyses; 104 citations","pmids":["32929274"],"is_preprint":false},{"year":2021,"finding":"Notch4 on circulating regulatory T cells suppresses IL-18-induced amphiregulin production (a tissue repair cytokine); deletion of Notch4 in Treg cells or anti-Notch4 antibody therapy normalizes dysregulated innate immunity and rescues disease morbidity in viral RNA analog and influenza H1N1 infection models.","method":"Treg-specific Notch4 conditional knockout, anti-Notch4 antibody therapy, amphiregulin measurement, humanized mice, IL-18 stimulation assays","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 — cell-type-specific conditional knockout plus therapeutic antibody recapitulation with defined mechanistic pathway","pmids":["33915108"],"is_preprint":false},{"year":2021,"finding":"DLL4 activation of Notch induces distinct subsets of Notch effectors and lymphatic genes in lymphatic endothelial cells (LECs), with Notch1 and Notch4 differentially regulating these genes; Notch4-null mice show increased closure of lymphangiogenic fronts, reduced vessel caliber, and reduced branching; Notch4 activation suppresses LEC migration more than Notch1, and loss of Notch4 does not affect canonical Notch signaling, establishing Notch4 as acting through a non-canonical pathway in lymphangiogenesis.","method":"Notch4 null mouse generation, embryonic lymphatic analysis, LEC migration wound assay, DNMAML dominant-negative canonical Notch inhibition, DLL4 activation assays","journal":"Angiogenesis","confidence":"High","confidence_rationale":"Tier 2 — genetic null mouse combined with functional cell assays and canonical pathway dissection","pmids":["34665379"],"is_preprint":false},{"year":2021,"finding":"NOTCH4 inhibits IFN-γ signaling in macrophages by favoring STAT3 over STAT1 phosphorylation (without affecting their expression), reducing STAT1-dependent transcription; NOTCH4 also reduces NF-κB transcriptional activity downstream of LPS/TLR4 signaling; HES1 mediates at least part of the STAT3 enhancement by NOTCH4.","method":"NOTCH4 overexpression in macrophages, STAT1/STAT3 phosphorylation assays, NF-κB reporter assays, cytokine measurement, gene expression analysis","journal":"Frontiers in Immunology","confidence":"Medium","confidence_rationale":"Tier 2-3 — overexpression system with multiple signaling readouts but limited loss-of-function validation","pmids":["34925319"],"is_preprint":false},{"year":2022,"finding":"JAG1-NOTCH4 signaling is activated by disturbed blood flow in endothelial cells; EC-specific Jag1 deletion in mice reduces atherosclerosis at sites of disturbed flow; single-cell RNA sequencing shows Jag1 suppresses EC subsets that proliferate and migrate, establishing JAG1-NOTCH4 as a mechanosensor pathway promoting atherosclerosis.","method":"Disturbed flow models (porcine/murine arteries and cultured human coronary artery EC), light-sheet imaging, EC-specific Jag1 conditional knockout, single-cell RNA sequencing","journal":"Science Advances","confidence":"High","confidence_rationale":"Tier 2 — multiple model systems including conditional knockout with scRNA-seq mechanistic characterization","pmids":["36044575"],"is_preprint":false},{"year":2022,"finding":"Notch4 promotes pulmonary artery smooth muscle cell proliferation and migration and inhibits apoptosis under hypoxia via interaction with ERK, JNK, and P38 MAPK; co-immunoprecipitation confirms physical interaction between Notch4 and ERK/JNK/P38; AAV1-mediated Notch4 silencing in rats reduces hypoxia-induced right ventricular systolic pressure and pulmonary vascular remodeling.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, cell viability/proliferation/apoptosis/migration assays, AAV1-mediated in vivo silencing in HPH rats","journal":"Respiratory Research","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical co-IP establishing interaction plus in vivo genetic validation with defined vascular phenotype","pmids":["35016680"],"is_preprint":false},{"year":2011,"finding":"PEA3 (an ETS transcription factor) activates NOTCH4 transcription in breast cancer cells; PEA3 recruitment to the NOTCH4 promoter is c-JUN/AP-1-dependent (unlike PEA3 recruitment to Notch-1 which is AP-1-independent), confirmed by ChIP and Notch4 luciferase reporter assays.","method":"ChIP for PEA3 on NOTCH4 promoter, siRNA knockdown, Notch4 luciferase reporter, c-Jun siRNA/TAM-67 epistasis","journal":"Breast Cancer Research","confidence":"High","confidence_rationale":"Tier 2 — direct promoter ChIP with epistasis experiments establishing c-JUN dependency","pmids":["21679465"],"is_preprint":false},{"year":1998,"finding":"The minimal domain of Notch4(int-3) required to inhibit mammary epithelial branching morphogenesis consists of the CBF-1 interaction domain and the cdc10 repeat domain; Wnt-1 can overcome Notch4-mediated inhibition of branching, demonstrating opposing roles of Notch and Wnt pathways in mammary morphogenesis.","method":"Notch4 deletion constructs transfected into TAC-2 mammary epithelial cells, 3D collagen gel branching morphogenesis assay, co-expression of Wnt-1","journal":"Developmental Biology","confidence":"High","confidence_rationale":"Tier 2 — systematic domain deletion analysis with functional morphogenesis assay and pathway epistasis","pmids":["9576833"],"is_preprint":false},{"year":2000,"finding":"Both constitutively active Notch4/int-3 and Jagged-1 induce microvessel-like structures in rat brain endothelial cells (RBE4) with morphological and biochemical brain endothelial properties; this correlates with induction of endogenous Notch4 and Jagged-1, while full-length Notch4 does not affect RBE4 cells.","method":"Transfection of RBE4 cells, morphological analysis, gene expression analysis of endogenous Notch4 and Jagged-1","journal":"Microvascular Research","confidence":"Medium","confidence_rationale":"Tier 3 — single-lab gain-of-function with morphological and molecular readouts","pmids":["10964583"],"is_preprint":false}],"current_model":"NOTCH4 is an endothelial cell-enriched transmembrane receptor that, upon activation (ligand-induced cleavage or truncation removing the extracellular domain), releases an intracellular domain (ICD) that translocates to the nucleus where it signals primarily through RBP-Jkappa/CBF1 to regulate vascular development, arteriovenous specification, and endothelial homeostasis; its nuclear localization is negatively regulated by AKT-mediated phosphorylation and 14-3-3 binding; it inhibits angiogenic sprouting via ankyrin repeat-dependent RBP-Jkappa-dependent and -independent mechanisms (including beta1-integrin activation), inhibits TGF-beta signaling by binding Smad3, and its transcription is controlled by AP-1, GR, PEA3/c-Jun, and RUNX1; on regulatory T cells, Notch4 suppresses amphiregulin and GDF15 production to promote inflammation via Wnt/Hippo pathways; and in cancer cells, the ICD directly transactivates downstream targets including SLUG, GAS1, and HEY1 to drive stem cell maintenance, EMT, and tumor progression."},"narrative":{"teleology":[{"year":1992,"claim":"Establishing that the intracellular domain of Notch4/int-3 is an autonomous oncogenic signal answered the foundational question of which portion of the protein drives transformation, showing that removal of the extracellular domain produces a constitutively active signaling moiety sufficient for mammary tumorigenesis.","evidence":"MMTV-driven transgenic mice expressing truncated int-3 ICD develop mammary adenocarcinomas; soft-agar transformation assay with ankyrin repeat-containing ICD in HC11 cells","pmids":["1372276","1312643"],"confidence":"High","gaps":["Ligand and cleavage events that activate full-length Notch4 in physiological settings were unknown","Downstream transcriptional targets of the ICD were not identified","Whether oncogenic activity required RBP-Jκ interaction was untested"]},{"year":1996,"claim":"Demonstrating endothelial-specific expression of Notch4 during development established the primary physiological context, shifting focus from mammary oncogenesis to vascular biology.","evidence":"In situ hybridization across embryonic and adult mouse tissues showing endothelial-restricted expression","pmids":["8681805"],"confidence":"High","gaps":["Functional requirement for Notch4 in endothelial biology was not yet tested","Whether Notch4 had non-redundant roles versus Notch1 in vasculature was unclear"]},{"year":1998,"claim":"Domain mapping revealed that the CBF-1 interaction domain plus ankyrin repeats constitute the minimal signaling unit for morphogenetic inhibition, and that Wnt-1 opposes Notch4, providing the first pathway epistasis data.","evidence":"Deletion constructs in TAC-2 mammary epithelial cells in 3D collagen gel branching morphogenesis assay with Wnt-1 co-expression","pmids":["9576833"],"confidence":"High","gaps":["Whether ankyrin repeats act through RBP-Jκ-dependent or -independent routes was unresolved","Wnt-Notch crosstalk mechanism was not defined biochemically"]},{"year":2001,"claim":"Endothelial-specific constitutive Notch4 activation in vivo proved that Notch4 signaling is sufficient to disrupt vascular remodeling, causing embryonic lethality with dilated, disorganized vessels — the first direct in vivo evidence of Notch4's vascular function.","evidence":"Flk1 promoter-driven activated Notch4 knock-in transgenic mice with whole-mount and histological vascular analysis","pmids":["11344305"],"confidence":"High","gaps":["Whether loss of Notch4 alone causes vascular defects was untested","Downstream effectors mediating vessel disorganization were unidentified"]},{"year":2002,"claim":"Identification of β1-integrin activation as a downstream effector explained how Notch4 inhibits angiogenic sprouting: by promoting integrin high-affinity conformation and increased adhesion rather than altering integrin expression.","evidence":"Endothelial sprouting assay, chick CAM assay, flow cytometry for integrin activation state","pmids":["11909975"],"confidence":"High","gaps":["The signaling intermediates connecting Notch4-ICD to integrin activation were not identified","Contribution of RBP-Jκ-dependent versus -independent pathways to integrin effects was unclear"]},{"year":2003,"claim":"Dissection of anti-apoptotic signaling revealed that Notch4-ICD inhibits endothelial cell death through two parallel mechanisms — RBP-Jκ-dependent JNK inhibition and RBP-Jκ-independent Bcl-2 upregulation — establishing bifurcating downstream pathways.","evidence":"Notch4-ICD and deletion mutant overexpression with RBP-Jκ reporter assays and apoptosis measurement","pmids":["14701863"],"confidence":"High","gaps":["Identity of the RBP-Jκ-independent pathway intermediates upstream of Bcl-2 was unknown","Relevance of anti-apoptotic function to in vivo vascular phenotypes was not demonstrated"]},{"year":2004,"claim":"Systematic domain deletion refined the sprouting inhibition mechanism to the ankyrin repeats as the minimal sufficient domain, operating through both RBP-Jκ-dependent and -independent routes.","evidence":"Quantitative endothelial sprouting assay with ankyrin repeat, RAM domain, and C-terminal deletion mutants plus RBP-Jκ reporters","pmids":["15187023"],"confidence":"High","gaps":["Direct binding partners of the ankyrin repeats mediating RBP-Jκ-independent effects were not identified"]},{"year":2005,"claim":"Three parallel advances resolved transcriptional regulation, TGF-β crosstalk, and in vivo AVM pathobiology: AP-1/GR control NOTCH4 endothelial transcription; Notch4-ICD binds Smad3 to block TGF-β signaling; and constitutive Notch4 in adult endothelium causes reversible arteriovenous malformations with venous arterialization.","evidence":"ChIP and promoter mutagenesis for AP-1/GR; co-IP and domain mapping for Smad3 binding; tetracycline-repressible endothelial transgenic system with histology and ephrinB2 expression","pmids":["15684396","16007227","15994223"],"confidence":"High","gaps":["How Smad3 binding is coordinated with RBP-Jκ interaction was unresolved","Upstream signals triggering Notch4 activation in AVM context were unknown","Whether AP-1-mediated transcription is required for AVM was not tested"]},{"year":2008,"claim":"Genetic epistasis using conditional Rbpj knockout demonstrated that Notch4-driven mammary developmental arrest requires RBP-Jκ, but Notch4-driven mammary tumorigenesis does not, definitively separating RBP-Jκ-dependent developmental from RBP-Jκ-independent oncogenic signaling. Simultaneously, brain-specific constitutive Notch4 was shown to cause cerebral AVMs with hemorrhage and neuronal death, reversible upon signal normalization.","evidence":"Conditional Rbpj knockout with Wap-Cre in mammary glands; tetracycline-repressible endothelial transgenic system in brain with MRI and behavioral assays","pmids":["18836481","18667694"],"confidence":"High","gaps":["The RBP-Jκ-independent effectors driving mammary tumorigenesis were unidentified","Whether human cerebral AVMs involve NOTCH4 mutations was not established"]},{"year":2010,"claim":"Discovery that Notch4 signaling is selectively enriched 8-fold in breast cancer stem cells (versus Notch1 being lower) and that Notch4 inhibition abolishes tumor initiation established Notch4 as the principal Notch receptor maintaining cancer stem cell activity.","evidence":"Stem cell enrichment, Notch reporter assays, GSI and siRNA inhibition, in vivo tumor formation in breast cancer models","pmids":["20068161"],"confidence":"High","gaps":["Specific Notch4 target genes in cancer stem cells were not identified at this point","Whether Notch4 stem cell function is RBP-Jκ-dependent was untested"]},{"year":2012,"claim":"Live imaging revealed that AVM regression upon Notch4 normalization occurs through vessel narrowing (not EC loss) and requires restoration of venous EphB4 expression, identifying EphB4 as a critical downstream mediator of AV identity.","evidence":"4D two-photon imaging through cranial windows in Notch4* mice with EphB4 expression analysis and blood flow measurement","pmids":["22261032"],"confidence":"High","gaps":["Whether EphB4 restoration is sufficient for AVM regression was not tested","Transcriptional mechanism linking Notch4 to EphB4 repression was not defined"]},{"year":2014,"claim":"A surprising finding that full-length NOTCH4 does not signal canonically in response to ligands but instead inhibits NOTCH1 in cis via its extracellular domain reframed Notch4 as both a signaling receptor (when cleaved) and a cis-inhibitor of Notch1, with Notch4-null mice showing only mild retinal vascular delay.","evidence":"Notch signaling reporter assays in cultured cells, full coding region Notch4 knockout mouse, cis-inhibition constructs, retinal vascular imaging","pmids":["24667410"],"confidence":"High","gaps":["In vivo significance of cis-inhibition in specific vascular beds was not established","Whether Notch4 inhibits other Notch paralogs beyond Notch1 was untested","Structural basis for cis-inhibition was not determined"]},{"year":2015,"claim":"AKT-mediated phosphorylation of Notch4-ICD at four sites creates 14-3-3 binding sites that sequester the ICD in the cytoplasm, establishing PI3K/AKT as a negative regulator of Notch4 nuclear signaling and linking growth factor signaling to Notch4 output.","evidence":"In vitro kinase assay, site-directed mutagenesis, co-IP with 14-3-3, PI3K inhibitor treatment, subcellular fractionation","pmids":["25740432"],"confidence":"High","gaps":["Whether AKT-mediated regulation operates in endothelial cells in vivo was not shown","The phosphatase(s) reversing this modification were not identified"]},{"year":2015,"claim":"JAG1 was identified as the activating ligand for NOTCH4 in anti-estrogen-treated breast cancer, and JAG1-NOTCH4 targeting reversed therapy-induced cancer stem cell expansion in patient-derived xenografts, establishing the ligand-receptor pair driving endocrine resistance.","evidence":"Patient-derived samples, PDX models, NOTCH4 targeting, ALDH1/mammosphere assays","pmids":["26387946"],"confidence":"High","gaps":["Whether JAG1-NOTCH4 signaling is ligand-cleavage-dependent or involves the cis-inhibition mode was not distinguished","Downstream transcriptional program in resistant cancer stem cells was incompletely defined"]},{"year":2016,"claim":"Direct target gene identification showed Notch4-ICD activates Hey1/Hey2 transcription factors that bind and repress Snail2 and Twist1 promoters, demonstrating Notch4 can suppress invasion in melanoma — context-dependent signaling opposite to its pro-EMT role in other cancers.","evidence":"EMSA and luciferase reporter assays for Hey1/Hey2 binding to Snail2/Twist1 promoters, N4ICD overexpression with invasion/migration assays","pmids":["26801977"],"confidence":"High","gaps":["Why Notch4 promotes EMT in breast cancer but suppresses invasion in melanoma was mechanistically unexplained","Whether chromatin context differs between these cell types was not addressed"]},{"year":2017,"claim":"Identification of RUNX1 as a negative transcriptional regulator of NOTCH4, with NOTCH4 knockout enhancing megakaryopoiesis, expanded Notch4's role beyond endothelium to hematopoietic lineage decisions.","evidence":"ChIP for RUNX1 at NOTCH4 locus, CRISPR-Cas9 NOTCH4 knockout in iPSCs, megakaryocyte differentiation assays","pmids":["29101237"],"confidence":"High","gaps":["Whether Notch4 affects megakaryocyte function beyond differentiation was unknown","The specific Notch4-dependent transcriptional program in megakaryocyte progenitors was not characterized"]},{"year":2018,"claim":"Notch4-ICD was shown to physically interact with TAK1 and block TRAF6 autoubiquitination, revealing a non-transcriptional cytoplasmic mechanism by which Notch4 suppresses innate immune activation during mycobacterial infection.","evidence":"Co-IP, ubiquitination assays, Notch4-deficient mice with M. tuberculosis infection and bacterial burden measurement","pmids":["29228365"],"confidence":"High","gaps":["Whether this cytoplasmic function is independent of nuclear Notch4 signaling was not fully dissected","Relevance to human tuberculosis immunity was not established"]},{"year":2020,"claim":"Two key advances: direct ChIP confirmation that NOTCH4-ICD binds SLUG and GAS1 promoters to maintain mesenchymal cancer stem cell quiescence in TNBC; and discovery that Notch4 on Tregs drives inflammatory reprogramming via Wnt/Hippo-dependent GDF15 production activating ILC2s.","evidence":"ChIP and dual-luciferase assays for SLUG/GAS1 promoters in TNBC; Treg-specific Notch4 conditional knockout in allergen challenge models with pathway inhibitors","pmids":["32104513","32929274"],"confidence":"High","gaps":["How Notch4 engages Wnt/Hippo pathways in Tregs was not mechanistically defined at the protein level","Whether GAS1-mediated quiescence and SLUG-driven EMT are coordinated or independent was unclear"]},{"year":2021,"claim":"Notch4 was demonstrated to regulate lymphangiogenesis through a non-canonical (RBP-Jκ-independent) pathway distinct from Notch1, and separately shown to suppress Treg-mediated tissue repair by inhibiting amphiregulin, with therapeutic anti-Notch4 antibody rescuing viral infection morbidity.","evidence":"Notch4-null mice with lymphatic vessel analysis and DNMAML canonical pathway control; Treg-specific Notch4 cKO and anti-Notch4 antibody in influenza model","pmids":["34665379","33915108"],"confidence":"High","gaps":["Identity of the non-canonical Notch4 effector in lymphangiogenesis was unknown","Whether anti-Notch4 therapy has off-target vascular effects was not assessed"]},{"year":2022,"claim":"JAG1-NOTCH4 was identified as a mechanosensitive pathway activated by disturbed blood flow to promote atherosclerosis, and separately Notch4 was found to physically interact with ERK/JNK/P38 MAPK to promote pulmonary artery smooth muscle cell proliferation under hypoxia.","evidence":"Disturbed flow models with EC-specific Jag1 cKO and scRNA-seq; co-IP of Notch4 with MAPK in PASMCs plus AAV1-mediated in vivo silencing in hypoxic pulmonary hypertension rats","pmids":["36044575","35016680"],"confidence":"High","gaps":["Whether NOTCH4 is the sole Notch receptor mediating flow-dependent atherogenesis was not resolved","Structural basis for Notch4-MAPK interaction was not determined","Whether the smooth muscle cell function is cleavage-dependent was not tested"]},{"year":null,"claim":"Major unresolved questions include: the structural basis for Notch4's unique properties (cis-inhibition, lack of canonical ligand response); the identity of non-canonical effectors in lymphangiogenesis and mammary tumorigenesis; whether and how full-length versus cleaved Notch4 functions are coordinated in vivo; and the therapeutic window for Notch4-targeted interventions given its opposing roles in vascular, immune, and cancer contexts.","evidence":"","pmids":[],"confidence":"Low","gaps":["No crystal structure of Notch4 extracellular or intracellular domains exists","Comprehensive interactome of Notch4-ICD in primary endothelial cells has not been determined","Context-dependent switching between pro- and anti-EMT effects lacks a unified mechanistic explanation"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6,14,24,25,28]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,20,27,32]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[22,27]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[0,20,33]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[5,6,22,28]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,5,6,7,14,20,22,29,30]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[3,9,12,17,21,31]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[27,29,30,32]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[1,2,15,18,23,25,28]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[8,11,19,26,35]}],"complexes":["RBP-Jκ/CBF1 transcription complex"],"partners":["RBPJ","SMAD3","JAG1","DLL4","TAK1","YWHAZ","MAPK1","TRAF6"],"other_free_text":[]},"mechanistic_narrative":"NOTCH4 is an endothelial cell-enriched transmembrane receptor that governs vascular development, arteriovenous specification, and immune cell regulation through both canonical (RBP-Jκ/CBF1-dependent) and non-canonical signaling pathways. Upon proteolytic release, the Notch4 intracellular domain (ICD) translocates to the nucleus — a process negatively regulated by AKT-mediated phosphorylation and 14-3-3 binding [PMID:25740432] — where it activates transcription of targets including HEY1, SLUG, GAS1, and ephrinB2 via ankyrin repeat-dependent interactions with RBP-Jκ, while also inhibiting TGF-β signaling through direct Smad3 binding and suppressing NF-κB/TAK1 signaling by blocking TRAF6 autoubiquitination [PMID:16007227, PMID:29228365, PMID:32104513]. Constitutive activation in endothelium causes arteriovenous malformations, enlarged vessels, and AV shunting that are fully reversible upon signal normalization through restoration of venous EphB4 expression [PMID:15994223, PMID:22261032], while in the immune system Notch4 on regulatory T cells subverts Treg function toward pro-inflammatory TH2/TH17 fates via Wnt/Hippo pathways and suppresses amphiregulin-mediated tissue repair [PMID:32929274, PMID:33915108]. NOTCH4 transcription is controlled by AP-1, glucocorticoid receptor, PEA3/c-Jun, RUNX1, and YB-1, and in cancer the ICD drives stem cell maintenance, EMT, and therapy resistance, particularly in breast cancer where Notch4 activity is selectively enriched in tumor-initiating populations and JAG1-NOTCH4 signaling mediates anti-estrogen resistance [PMID:15684396, PMID:20068161, PMID:26387946]."},"prefetch_data":{"uniprot":{"accession":"Q99466","full_name":"Neurogenic locus notch homolog protein 4","aliases":[],"length_aa":2003,"mass_kda":209.6,"function":"Functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 to regulate cell-fate determination. Upon ligand activation through the released notch intracellular domain (NICD) it forms a transcriptional activator complex with RBPJ/RBPSUH and activates genes of the enhancer of split locus. Affects the implementation of differentiation, proliferation and apoptotic programs. May regulate branching morphogenesis in the developing vascular system (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q99466/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NOTCH4","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NOTCH4","total_profiled":1310},"omim":[{"mim_id":"621120","title":"DELTA-LIKE NONCANONICAL NOTCH LIGAND 2; DLK2","url":"https://www.omim.org/entry/621120"},{"mim_id":"608991","title":"MASTERMIND-LIKE 3; MAML3","url":"https://www.omim.org/entry/608991"},{"mim_id":"608582","title":"EPIDERMAL GROWTH FACTOR-LIKE 7; EGFL7","url":"https://www.omim.org/entry/608582"},{"mim_id":"607537","title":"MASTERMIND-LIKE 2; MAML2","url":"https://www.omim.org/entry/607537"},{"mim_id":"607536","title":"CREB-REGULATED TRANSCRIPTION COACTIVATOR 1; CRTC1","url":"https://www.omim.org/entry/607536"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"adipose tissue","ntpm":22.4}],"url":"https://www.proteinatlas.org/search/NOTCH4"},"hgnc":{"alias_symbol":[],"prev_symbol":["INT3"]},"alphafold":{"accession":"Q99466","domains":[{"cath_id":"2.10.25.10","chopping":"160-193","consensus_level":"medium","plddt":69.1547,"start":160,"end":193},{"cath_id":"2.10.25.10","chopping":"465-519","consensus_level":"medium","plddt":73.9984,"start":465,"end":519},{"cath_id":"2.10.25.10","chopping":"671-768","consensus_level":"medium","plddt":62.1515,"start":671,"end":768},{"cath_id":"2.10.25.10","chopping":"841-882","consensus_level":"medium","plddt":64.9776,"start":841,"end":882},{"cath_id":"2.10.25.10","chopping":"983-1048","consensus_level":"medium","plddt":72.6082,"start":983,"end":1048},{"cath_id":"4.10.470.20","chopping":"1195-1343_1400-1436","consensus_level":"medium","plddt":74.7933,"start":1195,"end":1436},{"cath_id":"1.25.40.20","chopping":"1731-1821","consensus_level":"medium","plddt":91.8951,"start":1731,"end":1821}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99466","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99466-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99466-F1-predicted_aligned_error_v6.png","plddt_mean":61.78},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NOTCH4","jax_strain_url":"https://www.jax.org/strain/search?query=NOTCH4"},"sequence":{"accession":"Q99466","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99466.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99466/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99466"}},"corpus_meta":[{"pmid":"8681805","id":"PMC_8681805","title":"Notch4/int-3, 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foundational paper with 427 citations\",\n      \"pmids\": [\"8681805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Expression of the truncated int-3/Notch4 intracellular domain (encoding the intracellular portion homologous to Drosophila Notch) in transgenic mice causes mammary gland differentiation arrest and adenocarcinoma formation, establishing that the intracellular domain is a constitutively active oncogenic form.\",\n      \"method\": \"Transgenic mouse model, histology, MMTV promoter-driven expression\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain-of-function with defined phenotypic readout, replicated; 320 citations\",\n      \"pmids\": [\"1372276\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"The int-3/Notch4 intracellular domain protein contains six cdc10/ankyrin repeats homologous to Drosophila Notch, and expression of this domain in HC11 mouse mammary epithelial cells induces anchorage-independent growth in soft agar, demonstrating oncogenic transformation.\",\n      \"method\": \"In vitro soft-agar transformation assay, sequence analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vitro functional assay with defined molecular characterization; 237 citations\",\n      \"pmids\": [\"1312643\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Endothelial-specific expression of activated Notch4 under the Flk1 promoter in mice causes embryonic lethality with vascular patterning defects including restricted vasculature, dilated vessels, disorganized networks, and compromised vessel-wall integrity, implicating Notch4 in vessel remodeling.\",\n      \"method\": \"Knock-in transgenic mouse model, histology, whole-mount analysis\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo gain-of-function with defined vascular phenotype; 227 citations\",\n      \"pmids\": [\"11344305\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Constitutively active Notch4 inhibits VEGF-induced angiogenesis and endothelial sprouting in vitro and in vivo by promoting beta1-integrin activation (high-affinity conformation) and increased adhesion to collagen, without increasing surface expression of beta1-integrins.\",\n      \"method\": \"In vitro endothelial sprouting assay, chick CAM assay, flow cytometry for integrin activation, function-activating antibodies\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods including in vitro and in vivo assays with mechanistic dissection; 140 citations\",\n      \"pmids\": [\"11909975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Constitutively active Notch4 intracellular domain inhibits endothelial apoptosis via two distinct pathways: (1) RBP-Jkappa-dependent inhibition of JNK-mediated apoptosis, and (2) RBP-Jkappa-independent upregulation of Bcl-2.\",\n      \"method\": \"Overexpression of Notch4-ICD and deletion mutants, RBP-Jkappa reporter assays, apoptosis assays, Western blot\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods dissecting RBP-Jkappa-dependent and -independent mechanisms with mutant analysis\",\n      \"pmids\": [\"14701863\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Notch4-induced inhibition of endothelial sprouting requires the ankyrin repeats but not the RAM domain or C-terminal region, and involves both RBP-Jkappa-dependent and -independent signaling; the ankyrin repeats alone are sufficient to partially inhibit sprouting and upregulate RBP-Jkappa target genes.\",\n      \"method\": \"Deletion mutant analysis, quantitative endothelial sprouting assay, RBP-Jkappa reporter assays\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — systematic domain deletion mutagenesis with functional readout and transcriptional reporter assays\",\n      \"pmids\": [\"15187023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Notch4 intracellular domain (ICD) binds Smad3 (with higher affinity than Smad2 or Smad4) via the Smad3 MH2 domain, independent of the RAM23 region, and inhibits TGF-beta/Activin-Smad signaling, rendering cells resistant to TGF-beta-induced growth inhibition.\",\n      \"method\": \"Co-immunoprecipitation, deletion analysis, TGF-beta reporter luciferase assays, RT-PCR, Western blot, gamma-secretase inhibitor treatment\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical binding assay with domain mapping and functional transcriptional reporter validation\",\n      \"pmids\": [\"16007227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"NOTCH4 transcription in vascular endothelial cells is controlled by AP-1 complexes occupying the NOTCH4 chromatin; intron 1 or upstream sequences are required for endothelial-specific expression in vivo, and vascular angiogenic factors activate AP-1 to reprogram NOTCH4 from a repressed to transcriptionally active state.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), transgenic mouse reporter assays, RNA-FISH, transfection assays, histone modification analysis\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal chromatin and functional assays establishing AP-1 as direct regulator\",\n      \"pmids\": [\"15684396\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In adult mouse endothelium, constitutively active Notch4 (int3) causes arteriovenous malformations with blood vessel enlargement, AV shunting, arterialization (ectopic venous ephrinB2 expression, increased smooth muscle cells, upregulated Notch signaling), and these defects are fully reversible upon repression of int3 expression.\",\n      \"method\": \"Tetracycline-repressible endothelial transgenic system, histology, immunofluorescence\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reversible gain-of-function in vivo with multiple mechanistic markers; 164 citations\",\n      \"pmids\": [\"15994223\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"VEGF upregulates DLL4 and presenilin, leading to activation of Notch4, which in turn upregulates ephrinB2 and downregulates EphB4 in venous endothelial cells; pharmacological inhibition of presenilin or soluble DLL4 blocks VEGF-induced Notch4 activation and venous endothelial differentiation and migration.\",\n      \"method\": \"In vitro endothelial cell assays, Western blot, immunohistochemistry in transgenic tumor model, pharmacological inhibition\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple assays but mechanistic dissection relies partly on pharmacological inhibitors\",\n      \"pmids\": [\"16951162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Glucocorticoid receptor (GR) and AP-1 synergistically activate NOTCH4 transcription in endothelial cells via a composite response element (imperfect GRE + AP-1 motif) in the NOTCH4 promoter; AP-1 stabilizes GR occupancy, and together they reprogram NOTCH4 chromatin from repressed to active in multipotent cells.\",\n      \"method\": \"ChIP assay, promoter reporter assays, composite element mutagenesis, histone modification analysis\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstituted transcriptional mechanism with ChIP and functional mutagenesis\",\n      \"pmids\": [\"17220278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Constitutively active Notch4 expressed in brain endothelium from birth causes hallmarks of brain arteriovenous malformations including cerebral AV shunting, vessel enlargement, hemorrhage, and neuronal cell death; repression of Notch4 resolves ataxia and reverses disease, indicating Notch4 is required to sustain as well as initiate the AVM phenotype.\",\n      \"method\": \"Tetracycline-repressible endothelial-specific transgenic system, MRI, histology, behavioral assays\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reversible in vivo gain-of-function with multiple phenotypic readouts; 113 citations\",\n      \"pmids\": [\"18667694\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Notch4 knockdown in endothelial cells increases VCAM-1 expression and promotes apoptosis, while Notch4 and Hes1 are required to maintain EC quiescence and for endothelial injury repair; impaired Notch4/Hes1 activity is associated with transplant arteriosclerosis.\",\n      \"method\": \"siRNA knockdown, CBF1 reporter assays, adhesion molecule expression, apoptosis assays, allograft model\",\n      \"journal\": \"Arteriosclerosis, Thrombosis, and Vascular Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — loss-of-function with defined cellular phenotypes but mechanism not fully reconstituted\",\n      \"pmids\": [\"18802018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Notch4 signaling in mammary glands is mediated primarily through interaction of the intracellular domain with RBP-Jkappa (CBF1): conditional Rbpj knockout rescues the mammary gland developmental arrest induced by Wap-Int3, but mammary tumor development occurs independently of RBP-Jkappa interaction.\",\n      \"method\": \"Conditional Rbpj knockout in mammary glands, Wap-Cre system, mammary transplantation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis via conditional knockout dissecting RBP-Jkappa-dependent and -independent functions\",\n      \"pmids\": [\"18836481\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Notch4 signaling activity is 8-fold higher in breast cancer stem cell-enriched populations compared with differentiated cells (versus 4-fold lower for Notch1); pharmacological or genetic inhibition of Notch4 completely inhibits tumor initiation in vivo, implicating Notch4 as the primary Notch receptor maintaining breast cancer stem cell activity.\",\n      \"method\": \"Stem cell enrichment (anoikis-resistance, ESA+/CD44+/CD24low), Notch activity reporter assays, GSI and siRNA inhibition, in vivo tumor formation assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with in vitro and in vivo functional readouts; 420 citations\",\n      \"pmids\": [\"20068161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Notch4 is specifically required for Nodal expression in aggressive melanoma cells via an RBPJ-dependent Nodal enhancer element, placing Notch4 upstream of Nodal in the signaling cascade; Notch4-mediated vasculogenic mimicry and anchorage-independent growth are partly dependent on Nodal.\",\n      \"method\": \"Notch4 knockdown, RBPJ-dependent reporter assays, expression correlation in multiple cell lines, rescue experiments\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — reporter assay plus KD/rescue experiments establishing pathway hierarchy\",\n      \"pmids\": [\"21159651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Normalization of Notch4 signaling in mouse brain converts large-caliber high-flow AV shunts to capillary-like vessels and reverses tissue hypoxia; this structural regression requires restoration of EphB4 receptor expression by venous ECs and occurs via vessel narrowing without loss of ECs.\",\n      \"method\": \"4D two-photon imaging through cranial windows, Notch4* repression, EphB4 expression analysis, blood flow measurement\",\n      \"journal\": \"Science Translational Medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reversible in vivo Notch4 manipulation with mechanistic identification of EphB4 requirement\",\n      \"pmids\": [\"22261032\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PKCα overexpression selectively increases Notch4 (but not Notch1) expression via AP-1 occupancy at the Notch4 promoter; Notch4 intracellular domain promotes estrogen-independent, tamoxifen-resistant breast cancer growth and activates pathways associated with endocrine resistance.\",\n      \"method\": \"ChIP for AP-1 on Notch4 promoter, siRNA knockdown, Notch4-IC expression, gene expression profiling, in vivo xenograft with gamma-secretase inhibitor\",\n      \"journal\": \"Oncogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP confirming AP-1 binding, combined with functional in vitro and in vivo validation\",\n      \"pmids\": [\"23917222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"YB-1 (Y-box binding protein-1) transcriptionally regulates Notch4: ChIP-on-chip shows 12-fold enrichment of YB-1 binding at the Notch4 promoter, YB-1 siRNA decreases Notch4 mRNA, and constitutively active YB-1 increases Notch4 mRNA. Upstream, RSK phosphorylates YB-1 (S102), and RSK inhibition with luteolin suppresses Notch4 levels and reduces tumor-initiating cells.\",\n      \"method\": \"ChIP-on-Chip, siRNA knockdown, overexpression constructs, in vitro kinase assay, Notch4 mRNA measurement\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — ChIP-on-Chip with orthogonal siRNA and overexpression validation establishing transcriptional mechanism\",\n      \"pmids\": [\"23593654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NOTCH4 did not signal in response to canonical ligand activation in cultured cells, unlike other Notch receptors; furthermore, NOTCH4 inhibits signaling from NOTCH1 in cis (the first report of cis-inhibition by another Notch receptor), and this inhibition is mediated by the NOTCH4 extracellular domain. In vivo, Notch4 null mice show slightly delayed vessel growth in the retina.\",\n      \"method\": \"Notch signaling reporter assays in cultured cells, NOTCH4 knockout mouse generation (full coding region deletion), cis-inhibition constructs, retinal vascular imaging\",\n      \"journal\": \"Biochimica et Biophysica Acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — cell-based signaling assays with genetic mouse model; novel mechanistic finding\",\n      \"pmids\": [\"24667410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Brain AVMs arise from enlargement of pre-existing capillary-diameter microvessels accompanied by increased individual endothelial cell area (not increased EC number/proliferation); AV shunting begins promptly after Notch4* expression, and alterations in Notch signaling in ECs of all vessels (not arteries alone) drive AVM formation, indicating Notch4 functions in microvasculature/veins.\",\n      \"method\": \"Time-lapse two-photon imaging through cranial windows in Notch4* mice, EC area/number quantification, cell-type-specific Notch manipulation\",\n      \"journal\": \"PNAS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — real-time in vivo imaging with mechanistic dissection of cellular events\",\n      \"pmids\": [\"25468970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"AKT phosphorylates Notch4 intracellular domain (ICD) at four distinct sites in vitro and in vivo; this phosphorylation is regulated by growth factors and PI3K, creates binding sites for 14-3-3 proteins, and restricts phosphorylated Notch4-ICD to the cytoplasm, thereby negatively regulating Notch4 nuclear signaling.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, co-immunoprecipitation with 14-3-3, PI3K inhibitor treatment, subcellular fractionation\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase reconstitution with in vivo validation and subcellular localization functional consequence\",\n      \"pmids\": [\"25740432\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"JAG1-NOTCH4 activation by anti-estrogen treatment (tamoxifen/fulvestrant) increases breast cancer stem cell (BCSC) activity; targeting NOTCH4 reverses this increase and reduces BCSC activity in patient-derived xenograft tumors with acquired tamoxifen resistance.\",\n      \"method\": \"Patient-derived samples, PDX models, NOTCH4 targeting, ALDH1 activity, mammosphere assay\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — human PDX models with mechanistic JAG1-NOTCH4 pathway identification; 160 citations\",\n      \"pmids\": [\"26387946\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Constitutively active Notch4 ICD (N4ICD) in melanoma cells triggers a mesenchymal-to-epithelial-like switch and suppresses invasion/migration; mechanistically, N4ICD induces Hey1 and Hey2 transcription factors which directly bind the promoters of Snail2 and Twist1 and repress their transcription, as shown by EMSA and luciferase assays.\",\n      \"method\": \"N4ICD overexpression, EMSA, luciferase reporter assays, invasion/migration assays, in vivo tumor growth assay\",\n      \"journal\": \"Cancer Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct promoter binding shown by EMSA plus functional luciferase validation\",\n      \"pmids\": [\"26801977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NOTCH4 transcriptionally activates HEY1 in HNSCC, and NOTCH4-HEY1 pathway activation promotes EMT (decreased E-cadherin, increased vimentin/fibronectin/Twist1/SOX2), proliferation, cisplatin resistance, and invasion.\",\n      \"method\": \"TCGA analysis, in vitro proliferation/apoptosis/cell-cycle assays, siRNA knockdown, expression analysis\",\n      \"journal\": \"Clinical Cancer Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — functional KD with defined phenotypic readouts but limited mechanistic reconstitution\",\n      \"pmids\": [\"29146722\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NOTCH4 expression is negatively controlled by RUNX1 via a novel regulatory DNA element in the NOTCH4 locus; NOTCH4 inactivation by CRISPR-Cas9 in human iPSCs enhances megakaryopoiesis, establishing NOTCH4 as a RUNX1 target gene that negatively regulates megakaryocyte differentiation.\",\n      \"method\": \"Integrative genomic analysis of FPD-iPSCs, ChIP for RUNX1 binding, CRISPR-Cas9 NOTCH4 knockout, MK differentiation assays from iPSCs and CD34+ cells\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — genomic binding data plus functional CRISPR knockout with defined differentiation phenotype\",\n      \"pmids\": [\"29101237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Notch4 intracellular domain interacts with TAK1 and inhibits its activation; furthermore, Notch4-ICD prevents TRAF6 autoubiquitination and suppresses TRAF6-mediated TAK1 polyubiquitination, thereby negatively regulating M. tuberculosis-induced proinflammatory cytokine production.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays, Notch4-deficient mice, bacterial burden measurement\",\n      \"journal\": \"Journal of Infectious Diseases\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical mechanism (co-IP + ubiquitination assays) with in vivo genetic validation\",\n      \"pmids\": [\"29228365\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NOTCH4 transcriptionally upregulates SLUG and GAS1 by directly binding their promoters (confirmed by dual-luciferase reporter and ChIP assays), promoting EMT and quiescence in triple-negative breast cancer mesenchymal-like cancer stem cells.\",\n      \"method\": \"RNA-seq, dual-luciferase reporter assays, chromatin immunoprecipitation, stable overexpression and knockdown, mammosphere and chemoresistance assays\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct promoter binding confirmed by ChIP and luciferase assays with functional validation\",\n      \"pmids\": [\"32104513\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Notch4 on lung tissue regulatory T cells (induced by IL-6/STAT3) subverts Treg cells into TH2 and TH17 effector T cells via Wnt and Hippo pathway-dependent mechanisms; Wnt activation induces GDF15 expression in Treg cells, which activates group 2 innate lymphoid cells in a feed-forward inflammatory loop.\",\n      \"method\": \"Treg-specific Notch4 conditional knockout mice, in vivo allergen challenge models, Wnt/Hippo pathway inhibitors, GDF15 measurement, cytokine assays\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional knockout with multiple pathway analyses; 104 citations\",\n      \"pmids\": [\"32929274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Notch4 on circulating regulatory T cells suppresses IL-18-induced amphiregulin production (a tissue repair cytokine); deletion of Notch4 in Treg cells or anti-Notch4 antibody therapy normalizes dysregulated innate immunity and rescues disease morbidity in viral RNA analog and influenza H1N1 infection models.\",\n      \"method\": \"Treg-specific Notch4 conditional knockout, anti-Notch4 antibody therapy, amphiregulin measurement, humanized mice, IL-18 stimulation assays\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — cell-type-specific conditional knockout plus therapeutic antibody recapitulation with defined mechanistic pathway\",\n      \"pmids\": [\"33915108\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"DLL4 activation of Notch induces distinct subsets of Notch effectors and lymphatic genes in lymphatic endothelial cells (LECs), with Notch1 and Notch4 differentially regulating these genes; Notch4-null mice show increased closure of lymphangiogenic fronts, reduced vessel caliber, and reduced branching; Notch4 activation suppresses LEC migration more than Notch1, and loss of Notch4 does not affect canonical Notch signaling, establishing Notch4 as acting through a non-canonical pathway in lymphangiogenesis.\",\n      \"method\": \"Notch4 null mouse generation, embryonic lymphatic analysis, LEC migration wound assay, DNMAML dominant-negative canonical Notch inhibition, DLL4 activation assays\",\n      \"journal\": \"Angiogenesis\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic null mouse combined with functional cell assays and canonical pathway dissection\",\n      \"pmids\": [\"34665379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"NOTCH4 inhibits IFN-γ signaling in macrophages by favoring STAT3 over STAT1 phosphorylation (without affecting their expression), reducing STAT1-dependent transcription; NOTCH4 also reduces NF-κB transcriptional activity downstream of LPS/TLR4 signaling; HES1 mediates at least part of the STAT3 enhancement by NOTCH4.\",\n      \"method\": \"NOTCH4 overexpression in macrophages, STAT1/STAT3 phosphorylation assays, NF-κB reporter assays, cytokine measurement, gene expression analysis\",\n      \"journal\": \"Frontiers in Immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — overexpression system with multiple signaling readouts but limited loss-of-function validation\",\n      \"pmids\": [\"34925319\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"JAG1-NOTCH4 signaling is activated by disturbed blood flow in endothelial cells; EC-specific Jag1 deletion in mice reduces atherosclerosis at sites of disturbed flow; single-cell RNA sequencing shows Jag1 suppresses EC subsets that proliferate and migrate, establishing JAG1-NOTCH4 as a mechanosensor pathway promoting atherosclerosis.\",\n      \"method\": \"Disturbed flow models (porcine/murine arteries and cultured human coronary artery EC), light-sheet imaging, EC-specific Jag1 conditional knockout, single-cell RNA sequencing\",\n      \"journal\": \"Science Advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple model systems including conditional knockout with scRNA-seq mechanistic characterization\",\n      \"pmids\": [\"36044575\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Notch4 promotes pulmonary artery smooth muscle cell proliferation and migration and inhibits apoptosis under hypoxia via interaction with ERK, JNK, and P38 MAPK; co-immunoprecipitation confirms physical interaction between Notch4 and ERK/JNK/P38; AAV1-mediated Notch4 silencing in rats reduces hypoxia-induced right ventricular systolic pressure and pulmonary vascular remodeling.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, cell viability/proliferation/apoptosis/migration assays, AAV1-mediated in vivo silencing in HPH rats\",\n      \"journal\": \"Respiratory Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical co-IP establishing interaction plus in vivo genetic validation with defined vascular phenotype\",\n      \"pmids\": [\"35016680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PEA3 (an ETS transcription factor) activates NOTCH4 transcription in breast cancer cells; PEA3 recruitment to the NOTCH4 promoter is c-JUN/AP-1-dependent (unlike PEA3 recruitment to Notch-1 which is AP-1-independent), confirmed by ChIP and Notch4 luciferase reporter assays.\",\n      \"method\": \"ChIP for PEA3 on NOTCH4 promoter, siRNA knockdown, Notch4 luciferase reporter, c-Jun siRNA/TAM-67 epistasis\",\n      \"journal\": \"Breast Cancer Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter ChIP with epistasis experiments establishing c-JUN dependency\",\n      \"pmids\": [\"21679465\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The minimal domain of Notch4(int-3) required to inhibit mammary epithelial branching morphogenesis consists of the CBF-1 interaction domain and the cdc10 repeat domain; Wnt-1 can overcome Notch4-mediated inhibition of branching, demonstrating opposing roles of Notch and Wnt pathways in mammary morphogenesis.\",\n      \"method\": \"Notch4 deletion constructs transfected into TAC-2 mammary epithelial cells, 3D collagen gel branching morphogenesis assay, co-expression of Wnt-1\",\n      \"journal\": \"Developmental Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — systematic domain deletion analysis with functional morphogenesis assay and pathway epistasis\",\n      \"pmids\": [\"9576833\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Both constitutively active Notch4/int-3 and Jagged-1 induce microvessel-like structures in rat brain endothelial cells (RBE4) with morphological and biochemical brain endothelial properties; this correlates with induction of endogenous Notch4 and Jagged-1, while full-length Notch4 does not affect RBE4 cells.\",\n      \"method\": \"Transfection of RBE4 cells, morphological analysis, gene expression analysis of endogenous Notch4 and Jagged-1\",\n      \"journal\": \"Microvascular Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single-lab gain-of-function with morphological and molecular readouts\",\n      \"pmids\": [\"10964583\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NOTCH4 is an endothelial cell-enriched transmembrane receptor that, upon activation (ligand-induced cleavage or truncation removing the extracellular domain), releases an intracellular domain (ICD) that translocates to the nucleus where it signals primarily through RBP-Jkappa/CBF1 to regulate vascular development, arteriovenous specification, and endothelial homeostasis; its nuclear localization is negatively regulated by AKT-mediated phosphorylation and 14-3-3 binding; it inhibits angiogenic sprouting via ankyrin repeat-dependent RBP-Jkappa-dependent and -independent mechanisms (including beta1-integrin activation), inhibits TGF-beta signaling by binding Smad3, and its transcription is controlled by AP-1, GR, PEA3/c-Jun, and RUNX1; on regulatory T cells, Notch4 suppresses amphiregulin and GDF15 production to promote inflammation via Wnt/Hippo pathways; and in cancer cells, the ICD directly transactivates downstream targets including SLUG, GAS1, and HEY1 to drive stem cell maintenance, EMT, and tumor progression.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"NOTCH4 is an endothelial cell-enriched transmembrane receptor that governs vascular development, arteriovenous specification, and immune cell regulation through both canonical (RBP-Jκ/CBF1-dependent) and non-canonical signaling pathways. Upon proteolytic release, the Notch4 intracellular domain (ICD) translocates to the nucleus — a process negatively regulated by AKT-mediated phosphorylation and 14-3-3 binding [PMID:25740432] — where it activates transcription of targets including HEY1, SLUG, GAS1, and ephrinB2 via ankyrin repeat-dependent interactions with RBP-Jκ, while also inhibiting TGF-β signaling through direct Smad3 binding and suppressing NF-κB/TAK1 signaling by blocking TRAF6 autoubiquitination [PMID:16007227, PMID:29228365, PMID:32104513]. Constitutive activation in endothelium causes arteriovenous malformations, enlarged vessels, and AV shunting that are fully reversible upon signal normalization through restoration of venous EphB4 expression [PMID:15994223, PMID:22261032], while in the immune system Notch4 on regulatory T cells subverts Treg function toward pro-inflammatory TH2/TH17 fates via Wnt/Hippo pathways and suppresses amphiregulin-mediated tissue repair [PMID:32929274, PMID:33915108]. NOTCH4 transcription is controlled by AP-1, glucocorticoid receptor, PEA3/c-Jun, RUNX1, and YB-1, and in cancer the ICD drives stem cell maintenance, EMT, and therapy resistance, particularly in breast cancer where Notch4 activity is selectively enriched in tumor-initiating populations and JAG1-NOTCH4 signaling mediates anti-estrogen resistance [PMID:15684396, PMID:20068161, PMID:26387946].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Establishing that the intracellular domain of Notch4/int-3 is an autonomous oncogenic signal answered the foundational question of which portion of the protein drives transformation, showing that removal of the extracellular domain produces a constitutively active signaling moiety sufficient for mammary tumorigenesis.\",\n      \"evidence\": \"MMTV-driven transgenic mice expressing truncated int-3 ICD develop mammary adenocarcinomas; soft-agar transformation assay with ankyrin repeat-containing ICD in HC11 cells\",\n      \"pmids\": [\"1372276\", \"1312643\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Ligand and cleavage events that activate full-length Notch4 in physiological settings were unknown\", \"Downstream transcriptional targets of the ICD were not identified\", \"Whether oncogenic activity required RBP-Jκ interaction was untested\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrating endothelial-specific expression of Notch4 during development established the primary physiological context, shifting focus from mammary oncogenesis to vascular biology.\",\n      \"evidence\": \"In situ hybridization across embryonic and adult mouse tissues showing endothelial-restricted expression\",\n      \"pmids\": [\"8681805\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional requirement for Notch4 in endothelial biology was not yet tested\", \"Whether Notch4 had non-redundant roles versus Notch1 in vasculature was unclear\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Domain mapping revealed that the CBF-1 interaction domain plus ankyrin repeats constitute the minimal signaling unit for morphogenetic inhibition, and that Wnt-1 opposes Notch4, providing the first pathway epistasis data.\",\n      \"evidence\": \"Deletion constructs in TAC-2 mammary epithelial cells in 3D collagen gel branching morphogenesis assay with Wnt-1 co-expression\",\n      \"pmids\": [\"9576833\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ankyrin repeats act through RBP-Jκ-dependent or -independent routes was unresolved\", \"Wnt-Notch crosstalk mechanism was not defined biochemically\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Endothelial-specific constitutive Notch4 activation in vivo proved that Notch4 signaling is sufficient to disrupt vascular remodeling, causing embryonic lethality with dilated, disorganized vessels — the first direct in vivo evidence of Notch4's vascular function.\",\n      \"evidence\": \"Flk1 promoter-driven activated Notch4 knock-in transgenic mice with whole-mount and histological vascular analysis\",\n      \"pmids\": [\"11344305\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether loss of Notch4 alone causes vascular defects was untested\", \"Downstream effectors mediating vessel disorganization were unidentified\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identification of β1-integrin activation as a downstream effector explained how Notch4 inhibits angiogenic sprouting: by promoting integrin high-affinity conformation and increased adhesion rather than altering integrin expression.\",\n      \"evidence\": \"Endothelial sprouting assay, chick CAM assay, flow cytometry for integrin activation state\",\n      \"pmids\": [\"11909975\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The signaling intermediates connecting Notch4-ICD to integrin activation were not identified\", \"Contribution of RBP-Jκ-dependent versus -independent pathways to integrin effects was unclear\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Dissection of anti-apoptotic signaling revealed that Notch4-ICD inhibits endothelial cell death through two parallel mechanisms — RBP-Jκ-dependent JNK inhibition and RBP-Jκ-independent Bcl-2 upregulation — establishing bifurcating downstream pathways.\",\n      \"evidence\": \"Notch4-ICD and deletion mutant overexpression with RBP-Jκ reporter assays and apoptosis measurement\",\n      \"pmids\": [\"14701863\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the RBP-Jκ-independent pathway intermediates upstream of Bcl-2 was unknown\", \"Relevance of anti-apoptotic function to in vivo vascular phenotypes was not demonstrated\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Systematic domain deletion refined the sprouting inhibition mechanism to the ankyrin repeats as the minimal sufficient domain, operating through both RBP-Jκ-dependent and -independent routes.\",\n      \"evidence\": \"Quantitative endothelial sprouting assay with ankyrin repeat, RAM domain, and C-terminal deletion mutants plus RBP-Jκ reporters\",\n      \"pmids\": [\"15187023\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct binding partners of the ankyrin repeats mediating RBP-Jκ-independent effects were not identified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Three parallel advances resolved transcriptional regulation, TGF-β crosstalk, and in vivo AVM pathobiology: AP-1/GR control NOTCH4 endothelial transcription; Notch4-ICD binds Smad3 to block TGF-β signaling; and constitutive Notch4 in adult endothelium causes reversible arteriovenous malformations with venous arterialization.\",\n      \"evidence\": \"ChIP and promoter mutagenesis for AP-1/GR; co-IP and domain mapping for Smad3 binding; tetracycline-repressible endothelial transgenic system with histology and ephrinB2 expression\",\n      \"pmids\": [\"15684396\", \"16007227\", \"15994223\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Smad3 binding is coordinated with RBP-Jκ interaction was unresolved\", \"Upstream signals triggering Notch4 activation in AVM context were unknown\", \"Whether AP-1-mediated transcription is required for AVM was not tested\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Genetic epistasis using conditional Rbpj knockout demonstrated that Notch4-driven mammary developmental arrest requires RBP-Jκ, but Notch4-driven mammary tumorigenesis does not, definitively separating RBP-Jκ-dependent developmental from RBP-Jκ-independent oncogenic signaling. Simultaneously, brain-specific constitutive Notch4 was shown to cause cerebral AVMs with hemorrhage and neuronal death, reversible upon signal normalization.\",\n      \"evidence\": \"Conditional Rbpj knockout with Wap-Cre in mammary glands; tetracycline-repressible endothelial transgenic system in brain with MRI and behavioral assays\",\n      \"pmids\": [\"18836481\", \"18667694\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The RBP-Jκ-independent effectors driving mammary tumorigenesis were unidentified\", \"Whether human cerebral AVMs involve NOTCH4 mutations was not established\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Discovery that Notch4 signaling is selectively enriched 8-fold in breast cancer stem cells (versus Notch1 being lower) and that Notch4 inhibition abolishes tumor initiation established Notch4 as the principal Notch receptor maintaining cancer stem cell activity.\",\n      \"evidence\": \"Stem cell enrichment, Notch reporter assays, GSI and siRNA inhibition, in vivo tumor formation in breast cancer models\",\n      \"pmids\": [\"20068161\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific Notch4 target genes in cancer stem cells were not identified at this point\", \"Whether Notch4 stem cell function is RBP-Jκ-dependent was untested\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Live imaging revealed that AVM regression upon Notch4 normalization occurs through vessel narrowing (not EC loss) and requires restoration of venous EphB4 expression, identifying EphB4 as a critical downstream mediator of AV identity.\",\n      \"evidence\": \"4D two-photon imaging through cranial windows in Notch4* mice with EphB4 expression analysis and blood flow measurement\",\n      \"pmids\": [\"22261032\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether EphB4 restoration is sufficient for AVM regression was not tested\", \"Transcriptional mechanism linking Notch4 to EphB4 repression was not defined\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A surprising finding that full-length NOTCH4 does not signal canonically in response to ligands but instead inhibits NOTCH1 in cis via its extracellular domain reframed Notch4 as both a signaling receptor (when cleaved) and a cis-inhibitor of Notch1, with Notch4-null mice showing only mild retinal vascular delay.\",\n      \"evidence\": \"Notch signaling reporter assays in cultured cells, full coding region Notch4 knockout mouse, cis-inhibition constructs, retinal vascular imaging\",\n      \"pmids\": [\"24667410\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of cis-inhibition in specific vascular beds was not established\", \"Whether Notch4 inhibits other Notch paralogs beyond Notch1 was untested\", \"Structural basis for cis-inhibition was not determined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"AKT-mediated phosphorylation of Notch4-ICD at four sites creates 14-3-3 binding sites that sequester the ICD in the cytoplasm, establishing PI3K/AKT as a negative regulator of Notch4 nuclear signaling and linking growth factor signaling to Notch4 output.\",\n      \"evidence\": \"In vitro kinase assay, site-directed mutagenesis, co-IP with 14-3-3, PI3K inhibitor treatment, subcellular fractionation\",\n      \"pmids\": [\"25740432\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether AKT-mediated regulation operates in endothelial cells in vivo was not shown\", \"The phosphatase(s) reversing this modification were not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"JAG1 was identified as the activating ligand for NOTCH4 in anti-estrogen-treated breast cancer, and JAG1-NOTCH4 targeting reversed therapy-induced cancer stem cell expansion in patient-derived xenografts, establishing the ligand-receptor pair driving endocrine resistance.\",\n      \"evidence\": \"Patient-derived samples, PDX models, NOTCH4 targeting, ALDH1/mammosphere assays\",\n      \"pmids\": [\"26387946\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether JAG1-NOTCH4 signaling is ligand-cleavage-dependent or involves the cis-inhibition mode was not distinguished\", \"Downstream transcriptional program in resistant cancer stem cells was incompletely defined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Direct target gene identification showed Notch4-ICD activates Hey1/Hey2 transcription factors that bind and repress Snail2 and Twist1 promoters, demonstrating Notch4 can suppress invasion in melanoma — context-dependent signaling opposite to its pro-EMT role in other cancers.\",\n      \"evidence\": \"EMSA and luciferase reporter assays for Hey1/Hey2 binding to Snail2/Twist1 promoters, N4ICD overexpression with invasion/migration assays\",\n      \"pmids\": [\"26801977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why Notch4 promotes EMT in breast cancer but suppresses invasion in melanoma was mechanistically unexplained\", \"Whether chromatin context differs between these cell types was not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identification of RUNX1 as a negative transcriptional regulator of NOTCH4, with NOTCH4 knockout enhancing megakaryopoiesis, expanded Notch4's role beyond endothelium to hematopoietic lineage decisions.\",\n      \"evidence\": \"ChIP for RUNX1 at NOTCH4 locus, CRISPR-Cas9 NOTCH4 knockout in iPSCs, megakaryocyte differentiation assays\",\n      \"pmids\": [\"29101237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Notch4 affects megakaryocyte function beyond differentiation was unknown\", \"The specific Notch4-dependent transcriptional program in megakaryocyte progenitors was not characterized\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Notch4-ICD was shown to physically interact with TAK1 and block TRAF6 autoubiquitination, revealing a non-transcriptional cytoplasmic mechanism by which Notch4 suppresses innate immune activation during mycobacterial infection.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, Notch4-deficient mice with M. tuberculosis infection and bacterial burden measurement\",\n      \"pmids\": [\"29228365\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this cytoplasmic function is independent of nuclear Notch4 signaling was not fully dissected\", \"Relevance to human tuberculosis immunity was not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Two key advances: direct ChIP confirmation that NOTCH4-ICD binds SLUG and GAS1 promoters to maintain mesenchymal cancer stem cell quiescence in TNBC; and discovery that Notch4 on Tregs drives inflammatory reprogramming via Wnt/Hippo-dependent GDF15 production activating ILC2s.\",\n      \"evidence\": \"ChIP and dual-luciferase assays for SLUG/GAS1 promoters in TNBC; Treg-specific Notch4 conditional knockout in allergen challenge models with pathway inhibitors\",\n      \"pmids\": [\"32104513\", \"32929274\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Notch4 engages Wnt/Hippo pathways in Tregs was not mechanistically defined at the protein level\", \"Whether GAS1-mediated quiescence and SLUG-driven EMT are coordinated or independent was unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Notch4 was demonstrated to regulate lymphangiogenesis through a non-canonical (RBP-Jκ-independent) pathway distinct from Notch1, and separately shown to suppress Treg-mediated tissue repair by inhibiting amphiregulin, with therapeutic anti-Notch4 antibody rescuing viral infection morbidity.\",\n      \"evidence\": \"Notch4-null mice with lymphatic vessel analysis and DNMAML canonical pathway control; Treg-specific Notch4 cKO and anti-Notch4 antibody in influenza model\",\n      \"pmids\": [\"34665379\", \"33915108\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the non-canonical Notch4 effector in lymphangiogenesis was unknown\", \"Whether anti-Notch4 therapy has off-target vascular effects was not assessed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"JAG1-NOTCH4 was identified as a mechanosensitive pathway activated by disturbed blood flow to promote atherosclerosis, and separately Notch4 was found to physically interact with ERK/JNK/P38 MAPK to promote pulmonary artery smooth muscle cell proliferation under hypoxia.\",\n      \"evidence\": \"Disturbed flow models with EC-specific Jag1 cKO and scRNA-seq; co-IP of Notch4 with MAPK in PASMCs plus AAV1-mediated in vivo silencing in hypoxic pulmonary hypertension rats\",\n      \"pmids\": [\"36044575\", \"35016680\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether NOTCH4 is the sole Notch receptor mediating flow-dependent atherogenesis was not resolved\", \"Structural basis for Notch4-MAPK interaction was not determined\", \"Whether the smooth muscle cell function is cleavage-dependent was not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Major unresolved questions include: the structural basis for Notch4's unique properties (cis-inhibition, lack of canonical ligand response); the identity of non-canonical effectors in lymphangiogenesis and mammary tumorigenesis; whether and how full-length versus cleaved Notch4 functions are coordinated in vivo; and the therapeutic window for Notch4-targeted interventions given its opposing roles in vascular, immune, and cancer contexts.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No crystal structure of Notch4 extracellular or intracellular domains exists\", \"Comprehensive interactome of Notch4-ICD in primary endothelial cells has not been determined\", \"Context-dependent switching between pro- and anti-EMT effects lacks a unified mechanistic explanation\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6, 14, 24, 25, 28]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 20, 27, 32]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [22, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [0, 20, 33]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [5, 6, 22, 28]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 5, 6, 7, 14, 20, 22, 29, 30]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 9, 12, 17, 21, 31]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [27, 29, 30, 32]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 2, 15, 18, 23, 25, 28]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [8, 11, 19, 26, 35]}\n    ],\n    \"complexes\": [\n      \"RBP-Jκ/CBF1 transcription complex\"\n    ],\n    \"partners\": [\n      \"RBPJ\",\n      \"SMAD3\",\n      \"JAG1\",\n      \"DLL4\",\n      \"TAK1\",\n      \"YWHAZ\",\n      \"MAPK1\",\n      \"TRAF6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}