{"gene":"MAML1","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":2000,"finding":"MAML1 binds to the ankyrin repeat domain of all four mammalian NOTCH receptors, forms a ternary DNA-binding complex with the Notch intracellular domain (ICN) and RBP-Jκ (CSL), and amplifies NOTCH-induced transcription of HES1. MAML1 localizes to nuclear bodies.","method":"Co-immunoprecipitation, transcriptional reporter assays, nuclear localization imaging","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal binding assays and functional reporter assays in the founding paper, independently replicated across many subsequent studies","pmids":["11101851"],"is_preprint":false},{"year":2004,"finding":"Murine Maml1 encodes a nuclear protein that binds the ankyrin repeat domain of Notch receptors, forms a ternary complex with ICN and CSL, and enhances Notch-induced transcription of HES-1, confirming it as the functional murine ortholog of human MAML1.","method":"Co-immunoprecipitation, transcriptional reporter assays, nuclear localization","journal":"Gene","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal binding and functional assays, replicates human findings in mouse","pmids":["15019995"],"is_preprint":false},{"year":2006,"finding":"MAML1 interacts physically with MEF2C and functions as a potent co-transcriptional regulator for MEF2C-driven muscle-specific genes. MAML1 is required for MyoD-induced myogenic differentiation, and activation of Notch signaling recruits MAML1 away from MEF2C to the Notch transcriptional complex, thereby blocking pro-myogenic effects.","method":"Co-immunoprecipitation, RNAi knockdown, overexpression in C2C12 cells, Maml1-knockout mouse model","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP plus KO mouse model plus gain/loss-of-function cellular assays","pmids":["16510869"],"is_preprint":false},{"year":2006,"finding":"MAML1 is required downstream of Notch1 activation to suppress endothelial cell proliferation; a dominant-negative MAML1 mutant antagonizes Notch1-mediated suppression of the MAPK and PI3K/Akt pathways.","method":"Dominant-negative MAML1 overexpression, pathway inhibitor assays, proliferation assays in endothelial cells","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — dominant-negative approach with pathway readout, single lab, single study","pmids":["16571776"],"is_preprint":false},{"year":2007,"finding":"MAML1 physically interacts with p53 via its N-terminal region binding to the p53 DNA-binding domain, associates with native p53-response element promoters by ChIP, stabilizes p53 protein, enhances p53 phosphorylation/acetylation upon DNA damage, and functions as a transcriptional coactivator for p53 independently of its Notch coactivator role.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), overexpression/RNAi, p53-reporter assays, C. elegans genetic epistasis (lag-3 RNAi)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ChIP, gain/loss-of-function, and in vivo epistasis in C. elegans, multiple orthogonal methods single lab","pmids":["17317671"],"is_preprint":false},{"year":2007,"finding":"p300 acetylates MAML1 at conserved N-terminal lysine residues; a proline repeat motif (PXPAAPAP) in the MAML1 N-terminus mediates direct interaction with p300 and enhances MAML1 transcriptional activity. The MAML1 N-terminal domain also directly interacts with histones, and the p300-MAML1 complex acetylates histone H3 and H4 tails in chromatin.","method":"In vitro acetylation assay, co-immunoprecipitation, mutagenesis of lysine/proline residues, chromatin HAT assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of acetylation, mutagenesis of substrates, and binding domain mapping, single lab","pmids":["17300219"],"is_preprint":false},{"year":2007,"finding":"Maml1 deficiency specifically impairs Notch2-dependent marginal zone B-cell development while minimally affecting T-cell development, demonstrating that MAML1 is required for Notch2 signaling in a receptor-specific context in vivo.","method":"Maml1-knockout mouse, hematopoietic chimeras, B-cell and T-cell developmental analysis","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular phenotype, gene-dosage correlation, hematopoietic chimera controls","pmids":["17699740"],"is_preprint":false},{"year":2009,"finding":"MAML1 potentiates p300 autoacetylation and directly enhances p300 HAT activity, coinciding with translocation of MAML1, p300, and acetylated histones to nuclear bodies.","method":"In vitro HAT assay, autoacetylation assay, nuclear body imaging","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro biochemical reconstitution of HAT stimulation plus cell imaging, single lab","pmids":["19304754"],"is_preprint":false},{"year":2009,"finding":"Active GSK3β directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity. MAML1 translocates GSK3β to nuclear bodies (requires full-length MAML1). GSK3 inhibition further enhances MAML1-dependent histone acetylation.","method":"Co-immunoprecipitation, transcriptional reporter assays, nuclear body imaging, GSK3 inhibitor treatment","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and functional outcome demonstrated with inhibitor validation, single lab","pmids":["19740771"],"is_preprint":false},{"year":2010,"finding":"MAML1 is SUMOylated at Lys217 and Lys299 by UBC9 (E2 enzyme); PIAS1 (E3 ligase) stimulates SUMOylation; SENP1 reverses it. SUMOylation represses MAML1 transcriptional activity by enhancing its interaction with HDAC7. Mutation of both lysines abolishes SUMOylation and strongly increases MAML1-activated transcription.","method":"In vitro SUMOylation assay, site-directed mutagenesis, co-immunoprecipitation (HDAC7 interaction), transcriptional reporter assays","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution of SUMOylation, mutagenesis of acceptor sites, identification of writer/eraser/reader enzymes","pmids":["20203086"],"is_preprint":false},{"year":2010,"finding":"MAML1 co-activates the NF-κB subunit RelA (p65) and promotes degradation of IκBα, thereby modulating NF-κB signaling. Maml1-deficient MEFs show impaired TNFα-induced NF-κB responses, and Maml1-null mice exhibit spontaneous hepatocyte apoptosis in vivo.","method":"Co-immunoprecipitation, NF-κB reporter assays, Maml1-KO MEFs and mice, TNFα cytotoxicity assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO MEFs, KO mouse phenotype, and direct binding/reporter assays across multiple methods","pmids":["20231278"],"is_preprint":false},{"year":2010,"finding":"Addition of NOTCH intracellular domains and MAML1 to CSL does not detectably alter the DNA binding site preferences of CSL, supporting the conclusion that MAML1 promotes transcriptional activation without changing CSL's preferred DNA binding specificity.","method":"Protein-binding microarrays (PBMs) with purified CSL, ICN, and MAML1 proteins","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified proteins on microarrays; single lab, negative mechanistic finding confirmed rigorously","pmids":["21124806"],"is_preprint":false},{"year":2011,"finding":"MamL1 and MamL3 are collectively essential for Notch signaling in vivo: MamL1/MamL3 double-null mice die during early organogenesis with classic pan-Notch defects, while single nulls are viable, demonstrating functional redundancy between MamL1 and MamL3 in Notch-dependent developmental processes.","method":"MamL1 and MamL3 single- and double-knockout mouse generation, embryological and molecular phenotyping (lunatic fringe expression)","journal":"Development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis via double-KO rescue/exacerbation, replicated across multiple developmental readouts","pmids":["22069191"],"is_preprint":false},{"year":2011,"finding":"MAML1-mediated potentiation of p300 autoacetylation enhances p300-dependent acetylation of the Notch1 intracellular domain at conserved C-terminal NLS lysines. This MAML1/p300-dependent acetylation of Notch1 ICD decreases Notch1 ICD ubiquitination. CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300.","method":"Cell-based and in vitro acetylation assays, co-immunoprecipitation, ubiquitination assays, CDK8 overexpression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro and cellular acetylation/ubiquitination assays, single lab, mechanistic chain partly inferred","pmids":["22100894"],"is_preprint":false},{"year":2012,"finding":"Cutaneous papillomavirus E6 oncoproteins (BPV-1, HPV-1, HPV-8) bind an acidic LXXLL motif at the MAML1 C-terminus, repress MAML1 transactivation, and inhibit NOTCH-responsive promoters. BPV-1 E6 is found in a complex with MAML1 in stably transformed cells.","method":"Proteomic pulldown, co-immunoprecipitation, transcriptional reporter assays, LXXLL motif mapping","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteomic identification confirmed by Co-IP and functional reporter assays across multiple E6 proteins","pmids":["22249263"],"is_preprint":false},{"year":2012,"finding":"MAML1 physically interacts with EGR1 and acts cooperatively to activate EGR1-regulated promoters (including EGR1 and p300 own promoters). MAML1 strongly induces p300-mediated acetylation of EGR1 and increases EGR1 protein expression in embryonic kidney cells.","method":"Co-immunoprecipitation, transcriptional reporter assays, acetylation assay, overexpression in HEK cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and functional reporter plus acetylation assay, single lab, two orthogonal methods","pmids":["23029358"],"is_preprint":false},{"year":2013,"finding":"MAML1 enhances the transcriptional activity of Runx2 in a Notch-independent manner (N-terminal Notch-binding domain deletion mutant retains activity; Notch inhibition does not affect Runx2 activation). MAML1 loss in mice impairs chondrocyte maturation and results in shorter bone lengths.","method":"Luciferase reporter assay, MAML1 deletion mutants, Maml1-KO mouse embryo analysis, alkaline phosphatase assay","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mapping by deletion mutants, KO mouse phenotype, and multiple cellular assays in one study","pmids":["23326237"],"is_preprint":false},{"year":2013,"finding":"Snail suppresses NOTCH1 ICD-mediated transcription by physically interacting with NICD and competing with MAML1 for inclusion in the NICD/RBPJk transcription complex.","method":"Co-immunoprecipitation, transcriptional reporter assays, competition binding assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP and reporter assay, single lab, single study","pmids":["23454378"],"is_preprint":false},{"year":2015,"finding":"p53 associates with the Notch transcriptional complex via MAML1 (p53-NICD-MAML1 complex detected by co-IP and far-Western); formation of this complex is dependent on MAML1 (blocked by dominant-negative MAML1). In MCF-7 cells, this association results in inhibition of Notch-dependent transcription.","method":"Co-immunoprecipitation, far-Western blotting, chromatin immunoprecipitation (ChIP), dominant-negative MAML1","journal":"Journal of cellular physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, far-Western, and ChIP in one study, single lab","pmids":["26033683"],"is_preprint":false},{"year":2017,"finding":"MAML1 interacts with Gli family transcription factors (Gli1, Gli2) and functions as a potent transcriptional coactivator of Shh/Gli target genes. Maml1 silencing reduces Gli target gene expression and impairs cerebellar granule cell progenitor (GCP) proliferation; Maml1-null mice show compromised Shh pathway activity and impaired cerebellum development.","method":"Co-immunoprecipitation, transcriptional reporter assays, RNAi/siRNA knockdown, Maml1-KO mouse MEFs and GCPs","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding plus KO mouse in vivo phenotype plus cellular assays across multiple methods","pmids":["28726779"],"is_preprint":false},{"year":2020,"finding":"MAML1 and MAML2 are required for YAP/TAZ nuclear localization and transcriptional activity. Ectopic MAML1 expression induces nuclear translocation of YAP/TAZ; MAML1 depletion causes cytoplasmic retention. Mutation of the MAML1 nuclear localization signal or its YAP/TAZ-interacting region abolishes this effect. MAML1 mRNA is regulated by miR-30c in a cell-density-dependent manner.","method":"Overexpression and siRNA knockdown with YAP/TAZ localization imaging, domain mutagenesis, co-immunoprecipitation, in vivo tumor models","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — gain/loss-of-function with imaging readout, domain mutagenesis, and in vivo validation, multiple orthogonal methods","pmids":["32482852"],"is_preprint":false},{"year":2020,"finding":"MAML1 residues 151–350 are functionally essential for Notch-dependent transcriptional induction of both HES4 (promoter-driven) and DTX1 (enhancer-driven) target genes, as shown by add-back of MAML1 variants in MAML1-knockout cells. Fusion of the Notch-binding region of MAML1 to the p300 HAT domain rescues HES4 but not DTX1 expression, indicating that MAML1 has an additional recruitment activity beyond p300 HAT recruitment for enhancer-driven targets.","method":"MAML1 CRISPR knockout, add-back of deletion/truncation variants, HAT domain fusion constructs, H3K27ac ChIP","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR KO with domain add-back mutagenesis, ChIP, and functional fusion rescue, multiple orthogonal methods","pmids":["32179552"],"is_preprint":false},{"year":2025,"finding":"MAML1 interacts with the E3 ubiquitin ligase Itch via a PPQY motif and promotes K63-linked self-ubiquitylation of Itch, thereby deregulating Itch activity. Loss of MAML1 stabilizes Itch and suppresses Notch1 and Gli1 protein levels; MAML1 upregulation enhances Notch1 and Gli1 expression. Thus MAML1 acts as a post-translational regulator of Itch in addition to its transcriptional coactivator role.","method":"Co-immunoprecipitation, ubiquitylation assay (K63-linkage), Maml1-KO mouse model, domain mutagenesis (PPQY motif), in vivo tumor models","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct binding via domain mutagenesis, biochemical ubiquitylation assay, and KO mouse confirmation, multiple orthogonal methods","pmids":["41272291"],"is_preprint":false},{"year":2025,"finding":"MAML1 interacts with STAT3 and enhances STAT3 acetylation in a p300-dependent manner to promote hepatocellular carcinoma progression. YAP transcriptionally regulates MAML1 expression by directly binding its promoter, forming a YAP-MAML1-STAT3 signaling axis.","method":"Co-immunoprecipitation, acetylation assay, STAT3 inhibitor rescue experiments, ChIP for YAP binding to MAML1 promoter, knockdown/overexpression","journal":"Experimental hematology & oncology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding, functional acetylation assay, and promoter ChIP, single lab","pmids":["41345959"],"is_preprint":false},{"year":2026,"finding":"MAML1 activity in endocardial cells depends on liquid-liquid phase separation (LLPS) to form nuclear condensates required for efficient interaction with the NOTCH1 intracellular domain and downstream transcriptional activation. Patient-derived CHD-associated charge-altering variants (e.g., Q401K) within the intrinsically disordered region 2 of MAML1 abolish LLPS and downregulate Notch signaling. PKN2 kinase phosphorylates MAML1 at Ser314, destabilizing condensates and attenuating Notch transcriptional output.","method":"Knock-in mouse model (Q401K), endocardium-specific KO mouse, CRISPR-edited human heart organoids, biochemical LLPS assays, mass spectrometry (PTM identification), microscopy of condensates, echocardiography","journal":"Circulation","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro LLPS reconstitution, mass spectrometry identification of phosphorylation site, KI and KO mouse models with human organoid replication, multiple orthogonal methods","pmids":["42246060"],"is_preprint":false}],"current_model":"MAML1 is a transcriptional coactivator that forms ternary complexes with Notch intracellular domains and the CSL DNA-binding protein to activate Notch target genes; it also independently coactivates p53, MEF2C, NF-κB (RelA), β-catenin, EGR1, Runx2, Gli/Shh pathway transcription factors, STAT3, and YAP/TAZ nuclear localization, with its activity modulated by p300-mediated acetylation (of histones, MAML1 itself, and Notch1 ICD), SUMOylation (via UBC9/PIAS1, reversed by SENP1, recruiting HDAC7), GSK3β-mediated repression, CDK8, and PKN2-mediated phosphorylation at Ser314 that disrupts liquid-liquid phase separation condensates required for Notch transcriptional output; additionally, MAML1 post-translationally inhibits the E3 ubiquitin ligase Itch by promoting its K63-linked self-ubiquitylation, thereby stabilizing Notch1 and Gli1, and cutaneous papillomavirus E6 oncoproteins repress MAML1 by binding its C-terminal LXXLL motif."},"narrative":{"mechanistic_narrative":"MAML1 is a nuclear transcriptional coactivator that serves as the obligate third subunit of the canonical Notch transcriptional complex, binding the ankyrin repeat domain of all four mammalian Notch receptors and assembling a ternary DNA-binding complex with the Notch intracellular domain and CSL/RBP-Jκ to amplify Notch target-gene transcription such as HES1 [PMID:11101851, PMID:15019995]; addition of Notch ICD and MAML1 does not alter CSL's intrinsic DNA-binding preference, indicating MAML1 works as an activation module rather than a specificity factor [PMID:21124806]. In vivo MAML1 is required for receptor- and context-specific Notch outputs, including Notch2-dependent marginal zone B-cell development [PMID:17699740], and it acts redundantly with MAML3 such that combined loss produces pan-Notch developmental lethality [PMID:22069191]. Mechanistically, MAML1 couples Notch to chromatin through p300: an N-terminal proline motif recruits p300, MAML1 potentiates p300 autoacetylation and HAT activity, and the MAML1/p300 complex acetylates histone H3/H4 tails and the Notch1 ICD itself, the latter reducing Notch1 ubiquitination [PMID:17300219, PMID:19304754, PMID:22100894]. For enhancer-driven Notch targets, MAML1 contributes a recruitment activity beyond p300 HAT delivery, mapping to an essential central region (residues ~151–350) [PMID:32179552]. Beyond Notch, MAML1 functions as an independent coactivator for MEF2C-driven myogenesis [PMID:16510869], p53 [PMID:17317671], NF-κB/RelA [PMID:20231278], EGR1 [PMID:23029358], Runx2-dependent chondrocyte and bone maturation [PMID:23326237], Gli/Shh signaling in cerebellar development [PMID:28726779], and STAT3 in hepatocellular carcinoma [PMID:41345959], and it is required for YAP/TAZ nuclear localization and activity [PMID:32482852]. MAML1 activity is heavily regulated by post-translational modification: SUMOylation at Lys217/Lys299 (via UBC9/PIAS1, reversed by SENP1) recruits HDAC7 to repress it [PMID:20203086], GSK3β binding represses its activity [PMID:19740771], and PKN2-mediated phosphorylation at Ser314 destabilizes the liquid-liquid phase-separated nuclear condensates required for efficient Notch1 ICD engagement and transcriptional output [PMID:42246060]. MAML1 also acts post-translationally to inhibit the E3 ligase Itch by promoting Itch K63-linked self-ubiquitylation, thereby stabilizing Notch1 and Gli1 [PMID:41272291]. Charge-altering variants in a MAML1 intrinsically disordered region that abolish phase separation are associated with congenital heart disease [PMID:42246060], and cutaneous papillomavirus E6 oncoproteins repress MAML1 by binding its C-terminal LXXLL motif [PMID:22249263].","teleology":[{"year":2000,"claim":"Established the founding mechanistic identity of MAML1 as the missing coactivator that converts the CSL/Notch ICD DNA-binding platform into an active transcription complex.","evidence":"Co-IP, reporter assays, and nuclear localization imaging defining ternary complex with Notch ICD and RBP-Jκ in mammalian cells","pmids":["11101851"],"confidence":"High","gaps":["Did not resolve how the ternary complex recruits the general transcription machinery","No structural detail of the MAML1–ankyrin interface"]},{"year":2004,"claim":"Confirmed functional conservation of the Notch coactivator role in the murine ortholog, enabling in vivo genetic dissection.","evidence":"Co-IP, reporter assays, and localization of murine Maml1","pmids":["15019995"],"confidence":"High","gaps":["Did not address receptor-specific or tissue-specific requirements"]},{"year":2006,"claim":"Showed MAML1 is not Notch-exclusive but a shared, limiting coactivator partitioned between MEF2C and Notch, linking it to myogenic differentiation.","evidence":"Co-IP, RNAi, overexpression in C2C12, and Maml1-KO mouse","pmids":["16510869"],"confidence":"High","gaps":["Quantitative basis of competitive partitioning between MEF2C and Notch unresolved"]},{"year":2006,"claim":"Placed MAML1 downstream of Notch1 in controlling endothelial proliferation via MAPK and PI3K/Akt suppression.","evidence":"Dominant-negative MAML1 and pathway inhibitor proliferation assays in endothelial cells","pmids":["16571776"],"confidence":"Medium","gaps":["Dominant-negative approach only; no direct binding to pathway components","Single lab, single study"]},{"year":2007,"claim":"Demonstrated a Notch-independent coactivator function for p53, broadening MAML1 from a Notch-specific factor to a multi-pathway hub.","evidence":"Co-IP, ChIP, gain/loss-of-function reporter assays, and C. elegans epistasis","pmids":["17317671"],"confidence":"High","gaps":["Mechanism of p53 stabilization not fully defined"]},{"year":2007,"claim":"Defined the biochemical link between MAML1 and chromatin by mapping p300 recruitment and showing the complex acetylates histones and MAML1 itself.","evidence":"In vitro acetylation, mutagenesis of lysine/proline residues, chromatin HAT assay","pmids":["17300219"],"confidence":"High","gaps":["Functional consequence of MAML1 autoacetylation at target genes not resolved"]},{"year":2007,"claim":"Showed receptor-specific in vivo requirement, establishing MAML1 as essential for Notch2-driven marginal zone B-cell development.","evidence":"Maml1-KO mouse and hematopoietic chimeras with B/T-cell phenotyping","pmids":["17699740"],"confidence":"High","gaps":["Molecular basis of receptor selectivity unexplained"]},{"year":2009,"claim":"Showed MAML1 actively stimulates p300 enzymatic output, not merely recruits it, coupling coactivation to nuclear body translocation.","evidence":"In vitro HAT and autoacetylation assays plus nuclear body imaging","pmids":["19304754"],"confidence":"High","gaps":["Nature and composition of the nuclear bodies undefined at this stage"]},{"year":2009,"claim":"Identified GSK3β as a direct repressor of MAML1 activity, adding a kinase-based regulatory layer.","evidence":"Co-IP, reporter assays, GSK3 inhibitor treatment, nuclear body imaging","pmids":["19740771"],"confidence":"Medium","gaps":["GSK3β phosphorylation site on MAML1 not mapped","Single lab"]},{"year":2010,"claim":"Defined a complete SUMOylation regulatory circuit that represses MAML1 via HDAC7 recruitment.","evidence":"In vitro SUMOylation, acceptor-site mutagenesis, HDAC7 Co-IP, reporter assays","pmids":["20203086"],"confidence":"High","gaps":["Signals controlling the SUMOylation/de-SUMOylation balance in vivo unknown"]},{"year":2010,"claim":"Extended MAML1 coactivation to NF-κB/RelA and linked its loss to hepatocyte survival in vivo.","evidence":"Co-IP, NF-κB reporter assays, Maml1-KO MEFs and mice, TNFα cytotoxicity","pmids":["20231278"],"confidence":"High","gaps":["Mechanism of IκBα degradation promotion not detailed"]},{"year":2010,"claim":"Clarified that MAML1 activates transcription without reprogramming CSL DNA-binding specificity.","evidence":"Protein-binding microarrays with purified CSL, ICN, and MAML1","pmids":["21124806"],"confidence":"Medium","gaps":["In vitro array context; chromatin-level effects on site selection not tested"]},{"year":2011,"claim":"Demonstrated genetic redundancy with MAML3 for global Notch-dependent development.","evidence":"Single- and double-KO mice with developmental and molecular phenotyping","pmids":["22069191"],"confidence":"High","gaps":["Determinants of paralog specificity vs. redundancy across tissues unresolved"]},{"year":2011,"claim":"Connected MAML1/p300 acetylation directly to Notch1 ICD stability and identified CDK8 as an opposing regulator.","evidence":"Cellular and in vitro acetylation/ubiquitination assays plus CDK8 overexpression","pmids":["22100894"],"confidence":"Medium","gaps":["Causal chain between ICD acetylation and reduced ubiquitination partly inferred","Single lab"]},{"year":2012,"claim":"Identified a viral repression mechanism via E6 binding to the MAML1 C-terminal LXXLL motif.","evidence":"Proteomic pulldown, Co-IP, reporter assays, motif mapping across multiple E6 proteins","pmids":["22249263"],"confidence":"High","gaps":["Structural detail of the E6–LXXLL interaction not resolved"]},{"year":2012,"claim":"Added EGR1 as a MAML1 partner with feed-forward activation of EGR1 and p300 promoters.","evidence":"Co-IP, reporter assays, acetylation assay in HEK cells","pmids":["23029358"],"confidence":"Medium","gaps":["In vivo relevance of the EGR1 circuit not tested","Single lab"]},{"year":2013,"claim":"Established a Notch-independent role for MAML1 in Runx2-driven skeletal maturation through domain mapping and KO phenotype.","evidence":"Reporter assays with deletion mutants, Maml1-KO mouse embryos, ALP assays","pmids":["23326237"],"confidence":"High","gaps":["Whether p300 acetylation underlies Runx2 coactivation not addressed"]},{"year":2013,"claim":"Revealed a competitive antagonism mechanism in which Snail displaces MAML1 from the Notch complex.","evidence":"Co-IP, reporter assays, competition binding assay","pmids":["23454378"],"confidence":"Medium","gaps":["Physiological contexts of Snail competition not defined","Single lab"]},{"year":2015,"claim":"Showed MAML1 can bridge p53 into the Notch complex to repress Notch transcription, illustrating cross-pathway regulation through a shared coactivator.","evidence":"Co-IP, far-Western, ChIP, dominant-negative MAML1 in MCF-7 cells","pmids":["26033683"],"confidence":"Medium","gaps":["Generalizability beyond MCF-7 cells untested","Single lab"]},{"year":2017,"claim":"Established MAML1 as a Gli/Shh coactivator essential for cerebellar GCP proliferation and development.","evidence":"Co-IP, reporter assays, RNAi, Maml1-KO MEFs and GCPs","pmids":["28726779"],"confidence":"High","gaps":["Whether Gli coactivation uses the same p300 mechanism not resolved"]},{"year":2020,"claim":"Identified an unexpected requirement for MAML1 (and MAML2) in YAP/TAZ nuclear localization, expanding its role into Hippo pathway output.","evidence":"Overexpression/knockdown with localization imaging, domain mutagenesis, Co-IP, in vivo tumor models","pmids":["32482852"],"confidence":"High","gaps":["Mechanism by which MAML1 promotes YAP/TAZ import not fully defined"]},{"year":2020,"claim":"Mapped an essential central region and showed MAML1 provides recruitment activity distinct from p300 HAT delivery for enhancer-driven Notch targets.","evidence":"CRISPR KO with add-back variants, HAT-domain fusion rescue, H3K27ac ChIP","pmids":["32179552"],"confidence":"High","gaps":["Identity of the additional enhancer recruitment factor not determined"]},{"year":2025,"claim":"Uncovered a post-translational, non-transcriptional function in which MAML1 inhibits Itch to stabilize Notch1 and Gli1.","evidence":"Co-IP, K63-linkage ubiquitylation assay, PPQY motif mutagenesis, Maml1-KO and tumor models","pmids":["41272291"],"confidence":"High","gaps":["How MAML1 selects Itch among HECT ligases not addressed"]},{"year":2025,"claim":"Defined a YAP-MAML1-STAT3 axis driving hepatocellular carcinoma via p300-dependent STAT3 acetylation.","evidence":"Co-IP, acetylation assay, STAT3 inhibitor rescue, YAP-promoter ChIP, knockdown/overexpression","pmids":["41345959"],"confidence":"Medium","gaps":["Direct vs. indirect MAML1–STAT3 contact not fully resolved","Single lab"]},{"year":2026,"claim":"Showed MAML1 forms liquid-liquid phase-separated condensates required for Notch1 ICD engagement, with PKN2 phosphorylation at Ser314 and CHD-associated IDR variants disrupting condensates and Notch output.","evidence":"Knock-in and endocardium-specific KO mice, human heart organoids, in vitro LLPS assays, mass spectrometry, condensate microscopy","pmids":["42246060"],"confidence":"High","gaps":["Whether condensate formation governs MAML1's non-Notch functions unknown","Composition of MAML1 condensates beyond Notch components undefined"]},{"year":null,"claim":"It remains unresolved how MAML1 integrates its many independent coactivator partnerships and its post-translational Itch-regulatory role into coordinated cellular decisions, and whether phase separation underlies coactivation of pathways beyond Notch.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking competitive partner partitioning, PTM regulation, and condensate behavior","Structural basis of the diverse partner interfaces unresolved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,4,10,16,19]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[4,11]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[5,13,22]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,22]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0,7,24]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,12,16,19]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,4,10,21]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[0,19,20]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[5,7]}],"complexes":["Notch/CSL/MAML1 ternary transcription complex","MAML1/p300 coactivator complex"],"partners":["NOTCH1","RBPJ","EP300","MEF2C","TP53","GLI1","ITCH","YAP1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q92585","full_name":"Mastermind-like protein 1","aliases":[],"length_aa":1016,"mass_kda":108.1,"function":"Acts as a transcriptional coactivator for NOTCH proteins. Has been shown to amplify NOTCH-induced transcription of HES1. Enhances phosphorylation and proteolytic turnover of the NOTCH intracellular domain in the nucleus through interaction with CDK8. Binds to CREBBP/CBP which promotes nucleosome acetylation at NOTCH enhancers and activates transcription. Induces phosphorylation and localization of CREBBP to nuclear foci. 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MAML1 localizes to nuclear bodies.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, nuclear localization imaging\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal binding assays and functional reporter assays in the founding paper, independently replicated across many subsequent studies\",\n      \"pmids\": [\"11101851\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Murine Maml1 encodes a nuclear protein that binds the ankyrin repeat domain of Notch receptors, forms a ternary complex with ICN and CSL, and enhances Notch-induced transcription of HES-1, confirming it as the functional murine ortholog of human MAML1.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, nuclear localization\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal binding and functional assays, replicates human findings in mouse\",\n      \"pmids\": [\"15019995\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MAML1 interacts physically with MEF2C and functions as a potent co-transcriptional regulator for MEF2C-driven muscle-specific genes. MAML1 is required for MyoD-induced myogenic differentiation, and activation of Notch signaling recruits MAML1 away from MEF2C to the Notch transcriptional complex, thereby blocking pro-myogenic effects.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, overexpression in C2C12 cells, Maml1-knockout mouse model\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP plus KO mouse model plus gain/loss-of-function cellular assays\",\n      \"pmids\": [\"16510869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"MAML1 is required downstream of Notch1 activation to suppress endothelial cell proliferation; a dominant-negative MAML1 mutant antagonizes Notch1-mediated suppression of the MAPK and PI3K/Akt pathways.\",\n      \"method\": \"Dominant-negative MAML1 overexpression, pathway inhibitor assays, proliferation assays in endothelial cells\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — dominant-negative approach with pathway readout, single lab, single study\",\n      \"pmids\": [\"16571776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MAML1 physically interacts with p53 via its N-terminal region binding to the p53 DNA-binding domain, associates with native p53-response element promoters by ChIP, stabilizes p53 protein, enhances p53 phosphorylation/acetylation upon DNA damage, and functions as a transcriptional coactivator for p53 independently of its Notch coactivator role.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (ChIP), overexpression/RNAi, p53-reporter assays, C. elegans genetic epistasis (lag-3 RNAi)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ChIP, gain/loss-of-function, and in vivo epistasis in C. elegans, multiple orthogonal methods single lab\",\n      \"pmids\": [\"17317671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"p300 acetylates MAML1 at conserved N-terminal lysine residues; a proline repeat motif (PXPAAPAP) in the MAML1 N-terminus mediates direct interaction with p300 and enhances MAML1 transcriptional activity. The MAML1 N-terminal domain also directly interacts with histones, and the p300-MAML1 complex acetylates histone H3 and H4 tails in chromatin.\",\n      \"method\": \"In vitro acetylation assay, co-immunoprecipitation, mutagenesis of lysine/proline residues, chromatin HAT assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of acetylation, mutagenesis of substrates, and binding domain mapping, single lab\",\n      \"pmids\": [\"17300219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Maml1 deficiency specifically impairs Notch2-dependent marginal zone B-cell development while minimally affecting T-cell development, demonstrating that MAML1 is required for Notch2 signaling in a receptor-specific context in vivo.\",\n      \"method\": \"Maml1-knockout mouse, hematopoietic chimeras, B-cell and T-cell developmental analysis\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular phenotype, gene-dosage correlation, hematopoietic chimera controls\",\n      \"pmids\": [\"17699740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"MAML1 potentiates p300 autoacetylation and directly enhances p300 HAT activity, coinciding with translocation of MAML1, p300, and acetylated histones to nuclear bodies.\",\n      \"method\": \"In vitro HAT assay, autoacetylation assay, nuclear body imaging\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro biochemical reconstitution of HAT stimulation plus cell imaging, single lab\",\n      \"pmids\": [\"19304754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Active GSK3β directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity. MAML1 translocates GSK3β to nuclear bodies (requires full-length MAML1). GSK3 inhibition further enhances MAML1-dependent histone acetylation.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, nuclear body imaging, GSK3 inhibitor treatment\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and functional outcome demonstrated with inhibitor validation, single lab\",\n      \"pmids\": [\"19740771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MAML1 is SUMOylated at Lys217 and Lys299 by UBC9 (E2 enzyme); PIAS1 (E3 ligase) stimulates SUMOylation; SENP1 reverses it. SUMOylation represses MAML1 transcriptional activity by enhancing its interaction with HDAC7. Mutation of both lysines abolishes SUMOylation and strongly increases MAML1-activated transcription.\",\n      \"method\": \"In vitro SUMOylation assay, site-directed mutagenesis, co-immunoprecipitation (HDAC7 interaction), transcriptional reporter assays\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution of SUMOylation, mutagenesis of acceptor sites, identification of writer/eraser/reader enzymes\",\n      \"pmids\": [\"20203086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MAML1 co-activates the NF-κB subunit RelA (p65) and promotes degradation of IκBα, thereby modulating NF-κB signaling. Maml1-deficient MEFs show impaired TNFα-induced NF-κB responses, and Maml1-null mice exhibit spontaneous hepatocyte apoptosis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, NF-κB reporter assays, Maml1-KO MEFs and mice, TNFα cytotoxicity assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO MEFs, KO mouse phenotype, and direct binding/reporter assays across multiple methods\",\n      \"pmids\": [\"20231278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Addition of NOTCH intracellular domains and MAML1 to CSL does not detectably alter the DNA binding site preferences of CSL, supporting the conclusion that MAML1 promotes transcriptional activation without changing CSL's preferred DNA binding specificity.\",\n      \"method\": \"Protein-binding microarrays (PBMs) with purified CSL, ICN, and MAML1 proteins\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified proteins on microarrays; single lab, negative mechanistic finding confirmed rigorously\",\n      \"pmids\": [\"21124806\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MamL1 and MamL3 are collectively essential for Notch signaling in vivo: MamL1/MamL3 double-null mice die during early organogenesis with classic pan-Notch defects, while single nulls are viable, demonstrating functional redundancy between MamL1 and MamL3 in Notch-dependent developmental processes.\",\n      \"method\": \"MamL1 and MamL3 single- and double-knockout mouse generation, embryological and molecular phenotyping (lunatic fringe expression)\",\n      \"journal\": \"Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis via double-KO rescue/exacerbation, replicated across multiple developmental readouts\",\n      \"pmids\": [\"22069191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"MAML1-mediated potentiation of p300 autoacetylation enhances p300-dependent acetylation of the Notch1 intracellular domain at conserved C-terminal NLS lysines. This MAML1/p300-dependent acetylation of Notch1 ICD decreases Notch1 ICD ubiquitination. CDK8 inhibits Notch acetylation and Notch transcription enhanced by p300.\",\n      \"method\": \"Cell-based and in vitro acetylation assays, co-immunoprecipitation, ubiquitination assays, CDK8 overexpression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro and cellular acetylation/ubiquitination assays, single lab, mechanistic chain partly inferred\",\n      \"pmids\": [\"22100894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cutaneous papillomavirus E6 oncoproteins (BPV-1, HPV-1, HPV-8) bind an acidic LXXLL motif at the MAML1 C-terminus, repress MAML1 transactivation, and inhibit NOTCH-responsive promoters. BPV-1 E6 is found in a complex with MAML1 in stably transformed cells.\",\n      \"method\": \"Proteomic pulldown, co-immunoprecipitation, transcriptional reporter assays, LXXLL motif mapping\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteomic identification confirmed by Co-IP and functional reporter assays across multiple E6 proteins\",\n      \"pmids\": [\"22249263\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MAML1 physically interacts with EGR1 and acts cooperatively to activate EGR1-regulated promoters (including EGR1 and p300 own promoters). MAML1 strongly induces p300-mediated acetylation of EGR1 and increases EGR1 protein expression in embryonic kidney cells.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, acetylation assay, overexpression in HEK cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and functional reporter plus acetylation assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"23029358\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"MAML1 enhances the transcriptional activity of Runx2 in a Notch-independent manner (N-terminal Notch-binding domain deletion mutant retains activity; Notch inhibition does not affect Runx2 activation). MAML1 loss in mice impairs chondrocyte maturation and results in shorter bone lengths.\",\n      \"method\": \"Luciferase reporter assay, MAML1 deletion mutants, Maml1-KO mouse embryo analysis, alkaline phosphatase assay\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mapping by deletion mutants, KO mouse phenotype, and multiple cellular assays in one study\",\n      \"pmids\": [\"23326237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Snail suppresses NOTCH1 ICD-mediated transcription by physically interacting with NICD and competing with MAML1 for inclusion in the NICD/RBPJk transcription complex.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, competition binding assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP and reporter assay, single lab, single study\",\n      \"pmids\": [\"23454378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"p53 associates with the Notch transcriptional complex via MAML1 (p53-NICD-MAML1 complex detected by co-IP and far-Western); formation of this complex is dependent on MAML1 (blocked by dominant-negative MAML1). In MCF-7 cells, this association results in inhibition of Notch-dependent transcription.\",\n      \"method\": \"Co-immunoprecipitation, far-Western blotting, chromatin immunoprecipitation (ChIP), dominant-negative MAML1\",\n      \"journal\": \"Journal of cellular physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, far-Western, and ChIP in one study, single lab\",\n      \"pmids\": [\"26033683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAML1 interacts with Gli family transcription factors (Gli1, Gli2) and functions as a potent transcriptional coactivator of Shh/Gli target genes. Maml1 silencing reduces Gli target gene expression and impairs cerebellar granule cell progenitor (GCP) proliferation; Maml1-null mice show compromised Shh pathway activity and impaired cerebellum development.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, RNAi/siRNA knockdown, Maml1-KO mouse MEFs and GCPs\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding plus KO mouse in vivo phenotype plus cellular assays across multiple methods\",\n      \"pmids\": [\"28726779\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MAML1 and MAML2 are required for YAP/TAZ nuclear localization and transcriptional activity. Ectopic MAML1 expression induces nuclear translocation of YAP/TAZ; MAML1 depletion causes cytoplasmic retention. Mutation of the MAML1 nuclear localization signal or its YAP/TAZ-interacting region abolishes this effect. MAML1 mRNA is regulated by miR-30c in a cell-density-dependent manner.\",\n      \"method\": \"Overexpression and siRNA knockdown with YAP/TAZ localization imaging, domain mutagenesis, co-immunoprecipitation, in vivo tumor models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gain/loss-of-function with imaging readout, domain mutagenesis, and in vivo validation, multiple orthogonal methods\",\n      \"pmids\": [\"32482852\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MAML1 residues 151–350 are functionally essential for Notch-dependent transcriptional induction of both HES4 (promoter-driven) and DTX1 (enhancer-driven) target genes, as shown by add-back of MAML1 variants in MAML1-knockout cells. Fusion of the Notch-binding region of MAML1 to the p300 HAT domain rescues HES4 but not DTX1 expression, indicating that MAML1 has an additional recruitment activity beyond p300 HAT recruitment for enhancer-driven targets.\",\n      \"method\": \"MAML1 CRISPR knockout, add-back of deletion/truncation variants, HAT domain fusion constructs, H3K27ac ChIP\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR KO with domain add-back mutagenesis, ChIP, and functional fusion rescue, multiple orthogonal methods\",\n      \"pmids\": [\"32179552\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MAML1 interacts with the E3 ubiquitin ligase Itch via a PPQY motif and promotes K63-linked self-ubiquitylation of Itch, thereby deregulating Itch activity. Loss of MAML1 stabilizes Itch and suppresses Notch1 and Gli1 protein levels; MAML1 upregulation enhances Notch1 and Gli1 expression. Thus MAML1 acts as a post-translational regulator of Itch in addition to its transcriptional coactivator role.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assay (K63-linkage), Maml1-KO mouse model, domain mutagenesis (PPQY motif), in vivo tumor models\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct binding via domain mutagenesis, biochemical ubiquitylation assay, and KO mouse confirmation, multiple orthogonal methods\",\n      \"pmids\": [\"41272291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MAML1 interacts with STAT3 and enhances STAT3 acetylation in a p300-dependent manner to promote hepatocellular carcinoma progression. YAP transcriptionally regulates MAML1 expression by directly binding its promoter, forming a YAP-MAML1-STAT3 signaling axis.\",\n      \"method\": \"Co-immunoprecipitation, acetylation assay, STAT3 inhibitor rescue experiments, ChIP for YAP binding to MAML1 promoter, knockdown/overexpression\",\n      \"journal\": \"Experimental hematology & oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding, functional acetylation assay, and promoter ChIP, single lab\",\n      \"pmids\": [\"41345959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"MAML1 activity in endocardial cells depends on liquid-liquid phase separation (LLPS) to form nuclear condensates required for efficient interaction with the NOTCH1 intracellular domain and downstream transcriptional activation. Patient-derived CHD-associated charge-altering variants (e.g., Q401K) within the intrinsically disordered region 2 of MAML1 abolish LLPS and downregulate Notch signaling. PKN2 kinase phosphorylates MAML1 at Ser314, destabilizing condensates and attenuating Notch transcriptional output.\",\n      \"method\": \"Knock-in mouse model (Q401K), endocardium-specific KO mouse, CRISPR-edited human heart organoids, biochemical LLPS assays, mass spectrometry (PTM identification), microscopy of condensates, echocardiography\",\n      \"journal\": \"Circulation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro LLPS reconstitution, mass spectrometry identification of phosphorylation site, KI and KO mouse models with human organoid replication, multiple orthogonal methods\",\n      \"pmids\": [\"42246060\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAML1 is a transcriptional coactivator that forms ternary complexes with Notch intracellular domains and the CSL DNA-binding protein to activate Notch target genes; it also independently coactivates p53, MEF2C, NF-κB (RelA), β-catenin, EGR1, Runx2, Gli/Shh pathway transcription factors, STAT3, and YAP/TAZ nuclear localization, with its activity modulated by p300-mediated acetylation (of histones, MAML1 itself, and Notch1 ICD), SUMOylation (via UBC9/PIAS1, reversed by SENP1, recruiting HDAC7), GSK3β-mediated repression, CDK8, and PKN2-mediated phosphorylation at Ser314 that disrupts liquid-liquid phase separation condensates required for Notch transcriptional output; additionally, MAML1 post-translationally inhibits the E3 ubiquitin ligase Itch by promoting its K63-linked self-ubiquitylation, thereby stabilizing Notch1 and Gli1, and cutaneous papillomavirus E6 oncoproteins repress MAML1 by binding its C-terminal LXXLL motif.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAML1 is a nuclear transcriptional coactivator that serves as the obligate third subunit of the canonical Notch transcriptional complex, binding the ankyrin repeat domain of all four mammalian Notch receptors and assembling a ternary DNA-binding complex with the Notch intracellular domain and CSL/RBP-Jκ to amplify Notch target-gene transcription such as HES1 [#0, #1]; addition of Notch ICD and MAML1 does not alter CSL's intrinsic DNA-binding preference, indicating MAML1 works as an activation module rather than a specificity factor [#11]. In vivo MAML1 is required for receptor- and context-specific Notch outputs, including Notch2-dependent marginal zone B-cell development [#6], and it acts redundantly with MAML3 such that combined loss produces pan-Notch developmental lethality [#12]. Mechanistically, MAML1 couples Notch to chromatin through p300: an N-terminal proline motif recruits p300, MAML1 potentiates p300 autoacetylation and HAT activity, and the MAML1/p300 complex acetylates histone H3/H4 tails and the Notch1 ICD itself, the latter reducing Notch1 ubiquitination [#5, #7, #13]. For enhancer-driven Notch targets, MAML1 contributes a recruitment activity beyond p300 HAT delivery, mapping to an essential central region (residues ~151–350) [#21]. Beyond Notch, MAML1 functions as an independent coactivator for MEF2C-driven myogenesis [#2], p53 [#4], NF-κB/RelA [#10], EGR1 [#15], Runx2-dependent chondrocyte and bone maturation [#16], Gli/Shh signaling in cerebellar development [#19], and STAT3 in hepatocellular carcinoma [#23], and it is required for YAP/TAZ nuclear localization and activity [#20]. MAML1 activity is heavily regulated by post-translational modification: SUMOylation at Lys217/Lys299 (via UBC9/PIAS1, reversed by SENP1) recruits HDAC7 to repress it [#9], GSK3β binding represses its activity [#8], and PKN2-mediated phosphorylation at Ser314 destabilizes the liquid-liquid phase-separated nuclear condensates required for efficient Notch1 ICD engagement and transcriptional output [#24]. MAML1 also acts post-translationally to inhibit the E3 ligase Itch by promoting Itch K63-linked self-ubiquitylation, thereby stabilizing Notch1 and Gli1 [#22]. Charge-altering variants in a MAML1 intrinsically disordered region that abolish phase separation are associated with congenital heart disease [#24], and cutaneous papillomavirus E6 oncoproteins repress MAML1 by binding its C-terminal LXXLL motif [#14].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Established the founding mechanistic identity of MAML1 as the missing coactivator that converts the CSL/Notch ICD DNA-binding platform into an active transcription complex.\",\n      \"evidence\": \"Co-IP, reporter assays, and nuclear localization imaging defining ternary complex with Notch ICD and RBP-Jκ in mammalian cells\",\n      \"pmids\": [\"11101851\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve how the ternary complex recruits the general transcription machinery\", \"No structural detail of the MAML1–ankyrin interface\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Confirmed functional conservation of the Notch coactivator role in the murine ortholog, enabling in vivo genetic dissection.\",\n      \"evidence\": \"Co-IP, reporter assays, and localization of murine Maml1\",\n      \"pmids\": [\"15019995\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address receptor-specific or tissue-specific requirements\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Showed MAML1 is not Notch-exclusive but a shared, limiting coactivator partitioned between MEF2C and Notch, linking it to myogenic differentiation.\",\n      \"evidence\": \"Co-IP, RNAi, overexpression in C2C12, and Maml1-KO mouse\",\n      \"pmids\": [\"16510869\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative basis of competitive partitioning between MEF2C and Notch unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed MAML1 downstream of Notch1 in controlling endothelial proliferation via MAPK and PI3K/Akt suppression.\",\n      \"evidence\": \"Dominant-negative MAML1 and pathway inhibitor proliferation assays in endothelial cells\",\n      \"pmids\": [\"16571776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Dominant-negative approach only; no direct binding to pathway components\", \"Single lab, single study\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated a Notch-independent coactivator function for p53, broadening MAML1 from a Notch-specific factor to a multi-pathway hub.\",\n      \"evidence\": \"Co-IP, ChIP, gain/loss-of-function reporter assays, and C. elegans epistasis\",\n      \"pmids\": [\"17317671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of p53 stabilization not fully defined\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the biochemical link between MAML1 and chromatin by mapping p300 recruitment and showing the complex acetylates histones and MAML1 itself.\",\n      \"evidence\": \"In vitro acetylation, mutagenesis of lysine/proline residues, chromatin HAT assay\",\n      \"pmids\": [\"17300219\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of MAML1 autoacetylation at target genes not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Showed receptor-specific in vivo requirement, establishing MAML1 as essential for Notch2-driven marginal zone B-cell development.\",\n      \"evidence\": \"Maml1-KO mouse and hematopoietic chimeras with B/T-cell phenotyping\",\n      \"pmids\": [\"17699740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of receptor selectivity unexplained\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Showed MAML1 actively stimulates p300 enzymatic output, not merely recruits it, coupling coactivation to nuclear body translocation.\",\n      \"evidence\": \"In vitro HAT and autoacetylation assays plus nuclear body imaging\",\n      \"pmids\": [\"19304754\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature and composition of the nuclear bodies undefined at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified GSK3β as a direct repressor of MAML1 activity, adding a kinase-based regulatory layer.\",\n      \"evidence\": \"Co-IP, reporter assays, GSK3 inhibitor treatment, nuclear body imaging\",\n      \"pmids\": [\"19740771\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"GSK3β phosphorylation site on MAML1 not mapped\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined a complete SUMOylation regulatory circuit that represses MAML1 via HDAC7 recruitment.\",\n      \"evidence\": \"In vitro SUMOylation, acceptor-site mutagenesis, HDAC7 Co-IP, reporter assays\",\n      \"pmids\": [\"20203086\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Signals controlling the SUMOylation/de-SUMOylation balance in vivo unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Extended MAML1 coactivation to NF-κB/RelA and linked its loss to hepatocyte survival in vivo.\",\n      \"evidence\": \"Co-IP, NF-κB reporter assays, Maml1-KO MEFs and mice, TNFα cytotoxicity\",\n      \"pmids\": [\"20231278\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of IκBα degradation promotion not detailed\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Clarified that MAML1 activates transcription without reprogramming CSL DNA-binding specificity.\",\n      \"evidence\": \"Protein-binding microarrays with purified CSL, ICN, and MAML1\",\n      \"pmids\": [\"21124806\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro array context; chromatin-level effects on site selection not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated genetic redundancy with MAML3 for global Notch-dependent development.\",\n      \"evidence\": \"Single- and double-KO mice with developmental and molecular phenotyping\",\n      \"pmids\": [\"22069191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Determinants of paralog specificity vs. redundancy across tissues unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Connected MAML1/p300 acetylation directly to Notch1 ICD stability and identified CDK8 as an opposing regulator.\",\n      \"evidence\": \"Cellular and in vitro acetylation/ubiquitination assays plus CDK8 overexpression\",\n      \"pmids\": [\"22100894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causal chain between ICD acetylation and reduced ubiquitination partly inferred\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified a viral repression mechanism via E6 binding to the MAML1 C-terminal LXXLL motif.\",\n      \"evidence\": \"Proteomic pulldown, Co-IP, reporter assays, motif mapping across multiple E6 proteins\",\n      \"pmids\": [\"22249263\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural detail of the E6–LXXLL interaction not resolved\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Added EGR1 as a MAML1 partner with feed-forward activation of EGR1 and p300 promoters.\",\n      \"evidence\": \"Co-IP, reporter assays, acetylation assay in HEK cells\",\n      \"pmids\": [\"23029358\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of the EGR1 circuit not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established a Notch-independent role for MAML1 in Runx2-driven skeletal maturation through domain mapping and KO phenotype.\",\n      \"evidence\": \"Reporter assays with deletion mutants, Maml1-KO mouse embryos, ALP assays\",\n      \"pmids\": [\"23326237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether p300 acetylation underlies Runx2 coactivation not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed a competitive antagonism mechanism in which Snail displaces MAML1 from the Notch complex.\",\n      \"evidence\": \"Co-IP, reporter assays, competition binding assay\",\n      \"pmids\": [\"23454378\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological contexts of Snail competition not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed MAML1 can bridge p53 into the Notch complex to repress Notch transcription, illustrating cross-pathway regulation through a shared coactivator.\",\n      \"evidence\": \"Co-IP, far-Western, ChIP, dominant-negative MAML1 in MCF-7 cells\",\n      \"pmids\": [\"26033683\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Generalizability beyond MCF-7 cells untested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established MAML1 as a Gli/Shh coactivator essential for cerebellar GCP proliferation and development.\",\n      \"evidence\": \"Co-IP, reporter assays, RNAi, Maml1-KO MEFs and GCPs\",\n      \"pmids\": [\"28726779\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether Gli coactivation uses the same p300 mechanism not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified an unexpected requirement for MAML1 (and MAML2) in YAP/TAZ nuclear localization, expanding its role into Hippo pathway output.\",\n      \"evidence\": \"Overexpression/knockdown with localization imaging, domain mutagenesis, Co-IP, in vivo tumor models\",\n      \"pmids\": [\"32482852\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MAML1 promotes YAP/TAZ import not fully defined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapped an essential central region and showed MAML1 provides recruitment activity distinct from p300 HAT delivery for enhancer-driven Notch targets.\",\n      \"evidence\": \"CRISPR KO with add-back variants, HAT-domain fusion rescue, H3K27ac ChIP\",\n      \"pmids\": [\"32179552\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the additional enhancer recruitment factor not determined\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Uncovered a post-translational, non-transcriptional function in which MAML1 inhibits Itch to stabilize Notch1 and Gli1.\",\n      \"evidence\": \"Co-IP, K63-linkage ubiquitylation assay, PPQY motif mutagenesis, Maml1-KO and tumor models\",\n      \"pmids\": [\"41272291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How MAML1 selects Itch among HECT ligases not addressed\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a YAP-MAML1-STAT3 axis driving hepatocellular carcinoma via p300-dependent STAT3 acetylation.\",\n      \"evidence\": \"Co-IP, acetylation assay, STAT3 inhibitor rescue, YAP-promoter ChIP, knockdown/overexpression\",\n      \"pmids\": [\"41345959\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs. indirect MAML1–STAT3 contact not fully resolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Showed MAML1 forms liquid-liquid phase-separated condensates required for Notch1 ICD engagement, with PKN2 phosphorylation at Ser314 and CHD-associated IDR variants disrupting condensates and Notch output.\",\n      \"evidence\": \"Knock-in and endocardium-specific KO mice, human heart organoids, in vitro LLPS assays, mass spectrometry, condensate microscopy\",\n      \"pmids\": [\"42246060\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether condensate formation governs MAML1's non-Notch functions unknown\", \"Composition of MAML1 condensates beyond Notch components undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how MAML1 integrates its many independent coactivator partnerships and its post-translational Itch-regulatory role into coordinated cellular decisions, and whether phase separation underlies coactivation of pathways beyond Notch.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying model linking competitive partner partitioning, PTM regulation, and condensate behavior\", \"Structural basis of the diverse partner interfaces unresolved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 4, 10, 16, 19]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [4, 11]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [5, 13, 22]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 22]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0, 7, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 12, 16, 19]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 4, 10, 21]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 19, 20]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [5, 7]}\n    ],\n    \"complexes\": [\"Notch/CSL/MAML1 ternary transcription complex\", \"MAML1/p300 coactivator complex\"],\n    \"partners\": [\"NOTCH1\", \"RBPJ\", \"EP300\", \"MEF2C\", \"TP53\", \"GLI1\", \"ITCH\", \"YAP1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}