{"gene":"MXD4","run_date":"2026-06-10T05:19:51","timeline":{"discoveries":[{"year":1995,"finding":"MXD4 (Mad4) forms heterodimers with Max and binds CACGTG E-box sequences, represses transcription from CACGTG-containing promoters, interacts with mSin3, and suppresses c-Myc-dependent cell transformation in rat embryo fibroblast assays.","method":"Co-immunoprecipitation, reporter gene (transcriptional repression) assay, rat embryo fibroblast transformation assay, protein interaction studies","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding, transcription repression, transformation suppression) in a foundational study replicated across the field","pmids":["8521822"],"is_preprint":false},{"year":2003,"finding":"Mad4 transcription is repressed in proliferating cells by a complex containing c-Myc and Miz-1 bound to the initiator element of the Mad4 promoter; loss of this complex during differentiation activates Mad4 expression. Miz-1 alone activates the Mad4 promoter, and this activation is antagonized by c-Myc.","method":"Reporter gene assays in stably transfected MEL cells, deletion/mutation analysis of Mad4 core promoter, transient transfection assays, identification of initiator element requirement","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reporter assays with defined 49 nt core promoter, initiator element mutagenesis, and multiple functional readouts in a single focused study","pmids":["12418961"],"is_preprint":false},{"year":2011,"finding":"OX40 engagement on activated T cells increases MXD4 protein stability through a critical phosphorylation site in MXD4 that controls its stability, leading to nuclear translocation of MXD4; siRNA knockdown of MXD4 increased T-cell death, establishing that MXD4 upregulation contributes to OX40-mediated T-cell survival.","method":"Direct ex vivo analysis of antigen-stimulated murine T cells, protein stability assays, siRNA knockdown with cell death readout, phosphorylation site identification","journal":"European journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function (siRNA) with defined survival phenotype and phosphorylation site identified, single lab study","pmids":["21400495"],"is_preprint":false},{"year":2011,"finding":"Enforced expression of Mxd4 during early hematopoietic specification from embryonic stem cells severely impairs hematopoietic progenitor development by decreasing cell proliferation, increasing cells in G0/G1, and reducing cells in S phase, establishing MXD4 as a regulator of blood progenitor proliferation.","method":"Doxycycline-inducible gain-of-function in embryonic stem cells differentiated in vitro, cell cycle analysis (G0/G1 and S phase frequency)","journal":"Experimental hematology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — defined gain-of-function with cell cycle readout, single lab using two orthogonal methods","pmids":["21782766"],"is_preprint":false},{"year":2012,"finding":"Sin3B directly stabilizes Mad4 protein by protecting it from c-IAP1-mediated degradation, likely through direct binding of Sin3B to c-IAP1 rather than by disrupting Mad4–c-IAP1 interaction. Silencing of Sin3B reduces Mad4 levels, while co-expression of Sin3B stabilizes exogenous and endogenous Mad4. The E3 ligase activity of c-IAP1 is required for Mad4 downregulation (distinct from Mad1, which is a c-IAP1 substrate).","method":"siRNA knockdown, co-transfection/overexpression, co-immunoprecipitation, protein stability assays in GBM cell lines","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — co-IP and overexpression with functional outcome, multiple conditions tested, single lab","pmids":["22895069"],"is_preprint":false},{"year":2004,"finding":"Human liver-specific transcription factor TCP10L physically interacts with MAD4, as identified by yeast two-hybrid screen and confirmed by co-immunoprecipitation and subcellular co-localization experiments.","method":"Yeast two-hybrid screen, co-immunoprecipitation, subcellular localization experiments","journal":"Journal of biochemistry and molecular biology","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — yeast two-hybrid confirmed by reciprocal co-IP and localization, single lab with two orthogonal methods","pmids":["15469726"],"is_preprint":false},{"year":2022,"finding":"UHRF1 interacts with SAP30 (via residues G572 and F573 in its SRA domain) to repress MXD4 gene expression; depletion of UHRF1 or SAP30 de-represses MXD4, and further MXD4 knockdown rescues leukemogenesis by reactivating the MYC pathway, placing MXD4 downstream of the UHRF1-SAP30 epigenetic repressor complex.","method":"Co-immunoprecipitation, mutagenesis of UHRF1 SRA domain, siRNA/shRNA knockdown, leukemia PDX model, genetic epistasis (rescue experiment)","journal":"Cell research","confidence":"High","confidence_rationale":"Tier 2 / Strong — epistasis rescue experiment, domain mutagenesis, reciprocal Co-IP, and in vivo PDX model across multiple orthogonal approaches","pmids":["36302855"],"is_preprint":false},{"year":2022,"finding":"shRNA-mediated knockdown of MXD4/MAD4 in human keratinocyte precursors increases MYC expression and enhances proliferation and clonogenic potential of keratinocyte precursors, while preserving their functionality in 3D epidermis organoid generation, establishing MXD4 as a regulator of the MYC-dependent stemness balance in human epidermis.","method":"Stable shRNA knockdown in human keratinocytes, clonogenic assays, 3D organoid generation, MYC expression analysis","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean loss-of-function with defined phenotypic readout and MYC pathway link, single lab","pmids":["36007550"],"is_preprint":false},{"year":2025,"finding":"METTL16 installs m6A on MXD4 mRNA, reducing its stability and thereby decreasing MXD4 protein levels; this reduction promotes MYC-MAX complex formation and MYC target gene expression required for AML proliferation. Depletion of METTL16 stabilizes MXD4 mRNA, increases MXD4 protein, and suppresses AML cell growth, with MXD4 suppression rescuing MYC target gene expression.","method":"Transcriptome-wide m6A analysis, METTL16 genetic depletion and pharmacological inhibition, mRNA stability assays, MYC target gene expression rescue experiments in AML cells","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — transcriptome-wide m6A mapping combined with epistasis rescue, loss-of-function, and mRNA stability assays, single lab with multiple orthogonal methods","pmids":["40946103"],"is_preprint":false}],"current_model":"MXD4 is a bHLH-Zip transcription factor that heterodimerizes with Max to bind CACGTG E-box sequences, recruits mSin3 co-repressors, and transcriptionally represses MYC target genes to antagonize MYC-driven proliferation and promote differentiation; its expression is itself repressed in proliferating cells by a c-Myc–Miz-1 complex at its initiator element, and by the UHRF1–SAP30 epigenetic complex and METTL16-mediated m6A-dependent mRNA destabilization, while its protein stability is positively regulated by Sin3B (which protects it from c-IAP1-mediated degradation) and by a phosphorylation event downstream of OX40 signaling."},"narrative":{"mechanistic_narrative":"MXD4 (Mad4) is a transcriptional repressor that antagonizes MYC-driven proliferation: it heterodimerizes with Max, binds CACGTG E-box sequences to repress transcription from such promoters, interacts with mSin3, and suppresses c-Myc-dependent cell transformation [PMID:8521822]. This MYC-antagonist activity makes MXD4 a brake on proliferation across multiple cell systems—enforced expression impairs hematopoietic progenitor development by reducing proliferation and arresting cells in G0/G1 [PMID:21782766], while its loss in keratinocyte precursors elevates MYC and enhances proliferative and clonogenic capacity [PMID:36007550]. Because MXD4 opposes MYC, its levels are tightly held down in proliferating and malignant cells through several converging mechanisms: a c-Myc–Miz-1 complex bound to the MXD4 initiator element represses its transcription, with Miz-1 alone activating the promoter and c-Myc antagonizing this activation [PMID:12418961]; the UHRF1–SAP30 epigenetic complex represses MXD4 to sustain leukemogenesis via the MYC pathway [PMID:36302855]; and METTL16-mediated m6A deposition on MXD4 mRNA destabilizes the transcript, lowering MXD4 protein to favor MYC-MAX complex formation in AML [PMID:40946103]. MXD4 protein stability is also positively controlled, being protected from c-IAP1-mediated degradation by Sin3B [PMID:22895069] and increased through an OX40-driven phosphorylation event that promotes nuclear translocation and supports T-cell survival [PMID:21400495].","teleology":[{"year":1995,"claim":"Established MXD4's core molecular identity—whether it acts as a MYC antagonist—by showing it dimerizes with Max, binds E-boxes, represses transcription, and blocks Myc-driven transformation.","evidence":"Co-IP, reporter repression assays, and rat embryo fibroblast transformation assays","pmids":["8521822"],"confidence":"High","gaps":["Did not define endogenous target genes repressed by MXD4","Mechanism of mSin3 corepressor recruitment not detailed"]},{"year":2003,"claim":"Answered how MXD4 expression is held low in proliferating cells, showing a c-Myc–Miz-1 complex represses its promoter at the initiator element.","evidence":"Reporter and initiator-element mutagenesis assays in MEL cells","pmids":["12418961"],"confidence":"High","gaps":["Did not establish chromatin/epigenetic state at the endogenous promoter","In vivo relevance during differentiation not tested genetically"]},{"year":2004,"claim":"Identified a tissue-specific physical partner, TCP10L, expanding the MXD4 interactome beyond Max/mSin3.","evidence":"Yeast two-hybrid screen confirmed by co-IP and subcellular co-localization","pmids":["15469726"],"confidence":"Medium","gaps":["Functional consequence of the TCP10L–MXD4 interaction not determined","Single lab, no reciprocal validation in a physiological liver context"]},{"year":2011,"claim":"Showed MXD4 protein stability is an actively regulated node, with OX40 signaling driving a stabilizing phosphorylation that promotes nuclear translocation and T-cell survival.","evidence":"Ex vivo murine T-cell analysis, protein stability assays, siRNA knockdown with cell-death readout","pmids":["21400495"],"confidence":"Medium","gaps":["Kinase responsible for the phosphorylation not identified","Direct transcriptional targets driving survival not defined"]},{"year":2011,"claim":"Tested MXD4's cell-cycle function in hematopoiesis, demonstrating that gain-of-function impairs progenitor proliferation via G0/G1 arrest.","evidence":"Doxycycline-inducible gain-of-function in ES-derived hematopoietic cells with cell-cycle analysis","pmids":["21782766"],"confidence":"Medium","gaps":["Loss-of-function consequence in normal hematopoiesis not addressed","Direct E-box targets mediating the arrest not mapped"]},{"year":2012,"claim":"Resolved a post-translational control mechanism, showing Sin3B stabilizes MXD4 by protecting it from c-IAP1-mediated degradation.","evidence":"siRNA knockdown, co-transfection, co-IP, and protein stability assays in GBM cells","pmids":["22895069"],"confidence":"Medium","gaps":["Whether c-IAP1 directly ubiquitinates MXD4 versus an indirect route not fully resolved","In vivo relevance in glioma not established"]},{"year":2022,"claim":"Placed MXD4 as a functional effector downstream of the UHRF1–SAP30 repressor complex in leukemia, using epistasis to show its de-repression depends on this complex.","evidence":"Co-IP, SRA-domain mutagenesis, knockdown, leukemia PDX, and genetic rescue","pmids":["36302855"],"confidence":"High","gaps":["Direct UHRF1/SAP30 occupancy at the MXD4 locus not shown at base-pair resolution","Generality beyond the leukemia context untested"]},{"year":2022,"claim":"Defined MXD4 as a regulator of MYC-dependent stemness balance in human epidermis, with knockdown raising MYC and proliferative/clonogenic potential.","evidence":"shRNA knockdown in keratinocytes, clonogenic assays, and 3D organoid generation","pmids":["36007550"],"confidence":"Medium","gaps":["Direct MXD4 target genes in keratinocytes not identified","Mechanism linking MXD4 loss to MYC upregulation not dissected"]},{"year":2025,"claim":"Identified an RNA-level control of MXD4, showing METTL16-deposited m6A destabilizes MXD4 mRNA to favor MYC-MAX activity in AML.","evidence":"Transcriptome-wide m6A mapping, METTL16 depletion/inhibition, mRNA stability and MYC-target rescue assays in AML cells","pmids":["40946103"],"confidence":"High","gaps":["The m6A reader interpreting the MXD4 mark not identified","Whether this axis operates in non-AML contexts unknown"]},{"year":null,"claim":"The genome-wide set of MXD4 direct target genes and the structural basis of its corepressor recruitment remain undefined across the corpus.","evidence":"No discovery maps endogenous MXD4 binding sites or its repression complex architecture","pmids":[],"confidence":"Medium","gaps":["No ChIP-based genome-wide target map","No structural model of the MXD4–Max–mSin3 repressive complex"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,2]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3]}],"complexes":[],"partners":["MAX","SIN3A","SIN3B","TCP10L","BIRC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q14582","full_name":"Max dimerization protein 4","aliases":["Class C basic helix-loop-helix protein 12","bHLHc12","Max-associated protein 4","Max-interacting transcriptional repressor MAD4"],"length_aa":209,"mass_kda":23.5,"function":"Transcriptional repressor. Binds with MAX to form a sequence-specific DNA-binding protein complex which recognizes the core sequence 5'-CAC[GA]TG-3'. Antagonizes MYC transcriptional activity by competing for MAX and suppresses MYC dependent cell transformation (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q14582/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MXD4","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MXD4","total_profiled":1310},"omim":[{"mim_id":"620016","title":"MAX DIMERIZATION PROTEIN 4; MXD4","url":"https://www.omim.org/entry/620016"},{"mim_id":"608365","title":"T-COMPLEX PROTEIN 10-LIKE; TCP10L","url":"https://www.omim.org/entry/608365"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MXD4"},"hgnc":{"alias_symbol":["MAD4","MSTP149","MST149","bHLHc12"],"prev_symbol":[]},"alphafold":{"accession":"Q14582","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14582","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q14582-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q14582-F1-predicted_aligned_error_v6.png","plddt_mean":71.75},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MXD4","jax_strain_url":"https://www.jax.org/strain/search?query=MXD4"},"sequence":{"accession":"Q14582","fasta_url":"https://rest.uniprot.org/uniprotkb/Q14582.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q14582/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q14582"}},"corpus_meta":[{"pmid":"8521822","id":"PMC_8521822","title":"Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation.","date":"1995","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/8521822","citation_count":269,"is_preprint":false},{"pmid":"12090425","id":"PMC_12090425","title":"K-ras, p53, and DPC4 (MAD4) alterations in fine-needle aspirates of the pancreas: a molecular panel correlates with and supplements cytologic diagnosis.","date":"2002","source":"American journal of clinical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/12090425","citation_count":69,"is_preprint":false},{"pmid":"12418961","id":"PMC_12418961","title":"Mad4 is regulated by a transcriptional repressor complex that contains Miz-1 and c-Myc.","date":"2003","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/12418961","citation_count":42,"is_preprint":false},{"pmid":"30338611","id":"PMC_30338611","title":"Novel MXD4-NUTM1 fusion transcript identified in primary ovarian undifferentiated small round cell sarcoma.","date":"2018","source":"Genes, chromosomes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/30338611","citation_count":41,"is_preprint":false},{"pmid":"36302855","id":"PMC_36302855","title":"Targeting UHRF1-SAP30-MXD4 axis for leukemia initiating cell eradication in myeloid leukemia.","date":"2022","source":"Cell research","url":"https://pubmed.ncbi.nlm.nih.gov/36302855","citation_count":36,"is_preprint":false},{"pmid":"21400495","id":"PMC_21400495","title":"OX40 engagement stabilizes Mxd4 and Mnt protein levels in antigen-stimulated T cells leading to an increase in cell survival.","date":"2011","source":"European journal of immunology","url":"https://pubmed.ncbi.nlm.nih.gov/21400495","citation_count":16,"is_preprint":false},{"pmid":"21782766","id":"PMC_21782766","title":"The transcription factor Mxd4 controls the proliferation of the first blood precursors at the onset of hematopoietic development in vitro.","date":"2011","source":"Experimental hematology","url":"https://pubmed.ncbi.nlm.nih.gov/21782766","citation_count":10,"is_preprint":false},{"pmid":"22895069","id":"PMC_22895069","title":"Dissecting the complex regulation of Mad4 in glioblastoma multiforme cells.","date":"2012","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/22895069","citation_count":7,"is_preprint":false},{"pmid":"34487480","id":"PMC_34487480","title":"MiR-125b regulates the differentiation of hair follicles in Fine-wool Sheep and Cashmere goats by targeting MXD4 and FGFR2.","date":"2021","source":"Animal biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/34487480","citation_count":5,"is_preprint":false},{"pmid":"15469726","id":"PMC_15469726","title":"Human liver specific transcriptional factor TCP10L binds to MAD4.","date":"2004","source":"Journal of biochemistry and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/15469726","citation_count":5,"is_preprint":false},{"pmid":"38595053","id":"PMC_38595053","title":"Cytomorphology of metastatic colonic MXD4::NUTM1-rearranged sarcoma.","date":"2024","source":"Diagnostic cytopathology","url":"https://pubmed.ncbi.nlm.nih.gov/38595053","citation_count":4,"is_preprint":false},{"pmid":"40946103","id":"PMC_40946103","title":"METTL16-mediated inhibition of MXD4 promotes leukemia through activation of the MYC-MAX axis.","date":"2025","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/40946103","citation_count":3,"is_preprint":false},{"pmid":"10373021","id":"PMC_10373021","title":"Alternative splicing and embryonic expression of the Xenopus mad4 bHLH gene.","date":"1999","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/10373021","citation_count":3,"is_preprint":false},{"pmid":"36007550","id":"PMC_36007550","title":"MXD4/MAD4 Regulates Human Keratinocyte Precursor Fate.","date":"2022","source":"The Journal of investigative dermatology","url":"https://pubmed.ncbi.nlm.nih.gov/36007550","citation_count":2,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.09.11.675674","title":"Integrated Expression Analysis of C-MYC Oncogene-Associated Pathways in Gastric Adenocarcinoma and it’s Correlation with Clinicopathological Factors","date":"2025-09-16","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.11.675674","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9571,"output_tokens":2379,"usd":0.032199,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9433,"output_tokens":2931,"usd":0.06022,"stage2_stop_reason":"end_turn"},"total_usd":0.092419,"stage1_batch_id":"msgbatch_013a8chtv3AWyTqv4z4sEF4C","stage2_batch_id":"msgbatch_01AHsuW63vinsvuTgzmQ3n62","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1995,\n      \"finding\": \"MXD4 (Mad4) forms heterodimers with Max and binds CACGTG E-box sequences, represses transcription from CACGTG-containing promoters, interacts with mSin3, and suppresses c-Myc-dependent cell transformation in rat embryo fibroblast assays.\",\n      \"method\": \"Co-immunoprecipitation, reporter gene (transcriptional repression) assay, rat embryo fibroblast transformation assay, protein interaction studies\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding, transcription repression, transformation suppression) in a foundational study replicated across the field\",\n      \"pmids\": [\"8521822\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Mad4 transcription is repressed in proliferating cells by a complex containing c-Myc and Miz-1 bound to the initiator element of the Mad4 promoter; loss of this complex during differentiation activates Mad4 expression. Miz-1 alone activates the Mad4 promoter, and this activation is antagonized by c-Myc.\",\n      \"method\": \"Reporter gene assays in stably transfected MEL cells, deletion/mutation analysis of Mad4 core promoter, transient transfection assays, identification of initiator element requirement\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assays with defined 49 nt core promoter, initiator element mutagenesis, and multiple functional readouts in a single focused study\",\n      \"pmids\": [\"12418961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"OX40 engagement on activated T cells increases MXD4 protein stability through a critical phosphorylation site in MXD4 that controls its stability, leading to nuclear translocation of MXD4; siRNA knockdown of MXD4 increased T-cell death, establishing that MXD4 upregulation contributes to OX40-mediated T-cell survival.\",\n      \"method\": \"Direct ex vivo analysis of antigen-stimulated murine T cells, protein stability assays, siRNA knockdown with cell death readout, phosphorylation site identification\",\n      \"journal\": \"European journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function (siRNA) with defined survival phenotype and phosphorylation site identified, single lab study\",\n      \"pmids\": [\"21400495\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Enforced expression of Mxd4 during early hematopoietic specification from embryonic stem cells severely impairs hematopoietic progenitor development by decreasing cell proliferation, increasing cells in G0/G1, and reducing cells in S phase, establishing MXD4 as a regulator of blood progenitor proliferation.\",\n      \"method\": \"Doxycycline-inducible gain-of-function in embryonic stem cells differentiated in vitro, cell cycle analysis (G0/G1 and S phase frequency)\",\n      \"journal\": \"Experimental hematology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — defined gain-of-function with cell cycle readout, single lab using two orthogonal methods\",\n      \"pmids\": [\"21782766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Sin3B directly stabilizes Mad4 protein by protecting it from c-IAP1-mediated degradation, likely through direct binding of Sin3B to c-IAP1 rather than by disrupting Mad4–c-IAP1 interaction. Silencing of Sin3B reduces Mad4 levels, while co-expression of Sin3B stabilizes exogenous and endogenous Mad4. The E3 ligase activity of c-IAP1 is required for Mad4 downregulation (distinct from Mad1, which is a c-IAP1 substrate).\",\n      \"method\": \"siRNA knockdown, co-transfection/overexpression, co-immunoprecipitation, protein stability assays in GBM cell lines\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — co-IP and overexpression with functional outcome, multiple conditions tested, single lab\",\n      \"pmids\": [\"22895069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Human liver-specific transcription factor TCP10L physically interacts with MAD4, as identified by yeast two-hybrid screen and confirmed by co-immunoprecipitation and subcellular co-localization experiments.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, subcellular localization experiments\",\n      \"journal\": \"Journal of biochemistry and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — yeast two-hybrid confirmed by reciprocal co-IP and localization, single lab with two orthogonal methods\",\n      \"pmids\": [\"15469726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"UHRF1 interacts with SAP30 (via residues G572 and F573 in its SRA domain) to repress MXD4 gene expression; depletion of UHRF1 or SAP30 de-represses MXD4, and further MXD4 knockdown rescues leukemogenesis by reactivating the MYC pathway, placing MXD4 downstream of the UHRF1-SAP30 epigenetic repressor complex.\",\n      \"method\": \"Co-immunoprecipitation, mutagenesis of UHRF1 SRA domain, siRNA/shRNA knockdown, leukemia PDX model, genetic epistasis (rescue experiment)\",\n      \"journal\": \"Cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — epistasis rescue experiment, domain mutagenesis, reciprocal Co-IP, and in vivo PDX model across multiple orthogonal approaches\",\n      \"pmids\": [\"36302855\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"shRNA-mediated knockdown of MXD4/MAD4 in human keratinocyte precursors increases MYC expression and enhances proliferation and clonogenic potential of keratinocyte precursors, while preserving their functionality in 3D epidermis organoid generation, establishing MXD4 as a regulator of the MYC-dependent stemness balance in human epidermis.\",\n      \"method\": \"Stable shRNA knockdown in human keratinocytes, clonogenic assays, 3D organoid generation, MYC expression analysis\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean loss-of-function with defined phenotypic readout and MYC pathway link, single lab\",\n      \"pmids\": [\"36007550\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"METTL16 installs m6A on MXD4 mRNA, reducing its stability and thereby decreasing MXD4 protein levels; this reduction promotes MYC-MAX complex formation and MYC target gene expression required for AML proliferation. Depletion of METTL16 stabilizes MXD4 mRNA, increases MXD4 protein, and suppresses AML cell growth, with MXD4 suppression rescuing MYC target gene expression.\",\n      \"method\": \"Transcriptome-wide m6A analysis, METTL16 genetic depletion and pharmacological inhibition, mRNA stability assays, MYC target gene expression rescue experiments in AML cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transcriptome-wide m6A mapping combined with epistasis rescue, loss-of-function, and mRNA stability assays, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"40946103\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MXD4 is a bHLH-Zip transcription factor that heterodimerizes with Max to bind CACGTG E-box sequences, recruits mSin3 co-repressors, and transcriptionally represses MYC target genes to antagonize MYC-driven proliferation and promote differentiation; its expression is itself repressed in proliferating cells by a c-Myc–Miz-1 complex at its initiator element, and by the UHRF1–SAP30 epigenetic complex and METTL16-mediated m6A-dependent mRNA destabilization, while its protein stability is positively regulated by Sin3B (which protects it from c-IAP1-mediated degradation) and by a phosphorylation event downstream of OX40 signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MXD4 (Mad4) is a transcriptional repressor that antagonizes MYC-driven proliferation: it heterodimerizes with Max, binds CACGTG E-box sequences to repress transcription from such promoters, interacts with mSin3, and suppresses c-Myc-dependent cell transformation [#0]. This MYC-antagonist activity makes MXD4 a brake on proliferation across multiple cell systems\\u2014enforced expression impairs hematopoietic progenitor development by reducing proliferation and arresting cells in G0/G1 [#3], while its loss in keratinocyte precursors elevates MYC and enhances proliferative and clonogenic capacity [#7]. Because MXD4 opposes MYC, its levels are tightly held down in proliferating and malignant cells through several converging mechanisms: a c-Myc\\u2013Miz-1 complex bound to the MXD4 initiator element represses its transcription, with Miz-1 alone activating the promoter and c-Myc antagonizing this activation [#1]; the UHRF1\\u2013SAP30 epigenetic complex represses MXD4 to sustain leukemogenesis via the MYC pathway [#6]; and METTL16-mediated m6A deposition on MXD4 mRNA destabilizes the transcript, lowering MXD4 protein to favor MYC-MAX complex formation in AML [#8]. MXD4 protein stability is also positively controlled, being protected from c-IAP1-mediated degradation by Sin3B [#4] and increased through an OX40-driven phosphorylation event that promotes nuclear translocation and supports T-cell survival [#2].\",\n  \"teleology\": [\n    {\n      \"year\": 1995,\n      \"claim\": \"Established MXD4's core molecular identity\\u2014whether it acts as a MYC antagonist\\u2014by showing it dimerizes with Max, binds E-boxes, represses transcription, and blocks Myc-driven transformation.\",\n      \"evidence\": \"Co-IP, reporter repression assays, and rat embryo fibroblast transformation assays\",\n      \"pmids\": [\"8521822\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define endogenous target genes repressed by MXD4\", \"Mechanism of mSin3 corepressor recruitment not detailed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Answered how MXD4 expression is held low in proliferating cells, showing a c-Myc\\u2013Miz-1 complex represses its promoter at the initiator element.\",\n      \"evidence\": \"Reporter and initiator-element mutagenesis assays in MEL cells\",\n      \"pmids\": [\"12418961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish chromatin/epigenetic state at the endogenous promoter\", \"In vivo relevance during differentiation not tested genetically\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identified a tissue-specific physical partner, TCP10L, expanding the MXD4 interactome beyond Max/mSin3.\",\n      \"evidence\": \"Yeast two-hybrid screen confirmed by co-IP and subcellular co-localization\",\n      \"pmids\": [\"15469726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of the TCP10L\\u2013MXD4 interaction not determined\", \"Single lab, no reciprocal validation in a physiological liver context\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed MXD4 protein stability is an actively regulated node, with OX40 signaling driving a stabilizing phosphorylation that promotes nuclear translocation and T-cell survival.\",\n      \"evidence\": \"Ex vivo murine T-cell analysis, protein stability assays, siRNA knockdown with cell-death readout\",\n      \"pmids\": [\"21400495\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Kinase responsible for the phosphorylation not identified\", \"Direct transcriptional targets driving survival not defined\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Tested MXD4's cell-cycle function in hematopoiesis, demonstrating that gain-of-function impairs progenitor proliferation via G0/G1 arrest.\",\n      \"evidence\": \"Doxycycline-inducible gain-of-function in ES-derived hematopoietic cells with cell-cycle analysis\",\n      \"pmids\": [\"21782766\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Loss-of-function consequence in normal hematopoiesis not addressed\", \"Direct E-box targets mediating the arrest not mapped\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved a post-translational control mechanism, showing Sin3B stabilizes MXD4 by protecting it from c-IAP1-mediated degradation.\",\n      \"evidence\": \"siRNA knockdown, co-transfection, co-IP, and protein stability assays in GBM cells\",\n      \"pmids\": [\"22895069\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether c-IAP1 directly ubiquitinates MXD4 versus an indirect route not fully resolved\", \"In vivo relevance in glioma not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Placed MXD4 as a functional effector downstream of the UHRF1\\u2013SAP30 repressor complex in leukemia, using epistasis to show its de-repression depends on this complex.\",\n      \"evidence\": \"Co-IP, SRA-domain mutagenesis, knockdown, leukemia PDX, and genetic rescue\",\n      \"pmids\": [\"36302855\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct UHRF1/SAP30 occupancy at the MXD4 locus not shown at base-pair resolution\", \"Generality beyond the leukemia context untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined MXD4 as a regulator of MYC-dependent stemness balance in human epidermis, with knockdown raising MYC and proliferative/clonogenic potential.\",\n      \"evidence\": \"shRNA knockdown in keratinocytes, clonogenic assays, and 3D organoid generation\",\n      \"pmids\": [\"36007550\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct MXD4 target genes in keratinocytes not identified\", \"Mechanism linking MXD4 loss to MYC upregulation not dissected\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identified an RNA-level control of MXD4, showing METTL16-deposited m6A destabilizes MXD4 mRNA to favor MYC-MAX activity in AML.\",\n      \"evidence\": \"Transcriptome-wide m6A mapping, METTL16 depletion/inhibition, mRNA stability and MYC-target rescue assays in AML cells\",\n      \"pmids\": [\"40946103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The m6A reader interpreting the MXD4 mark not identified\", \"Whether this axis operates in non-AML contexts unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The genome-wide set of MXD4 direct target genes and the structural basis of its corepressor recruitment remain undefined across the corpus.\",\n      \"evidence\": \"No discovery maps endogenous MXD4 binding sites or its repression complex architecture\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No ChIP-based genome-wide target map\", \"No structural model of the MXD4\\u2013Max\\u2013mSin3 repressive complex\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MAX\", \"SIN3A\", \"SIN3B\", \"TCP10L\", \"BIRC2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":4,"faith_total":4,"faith_pct":100.0}}