{"gene":"MYC","run_date":"2026-04-29T11:37:56","timeline":{"discoveries":[{"year":1991,"finding":"Max is a bHLH-Zip protein that specifically associates with c-Myc, N-Myc, and L-Myc via their HLH-Zip domains, and the Myc-Max heterodimer binds DNA in a sequence-specific manner (core CACGTG) under conditions where neither protein alone exhibits appreciable binding; DNA binding requires both the dimerization domain and basic region of c-Myc.","method":"cDNA expression library screen with c-Myc bHLH-Zip domain as bait; co-immunoprecipitation; EMSA; domain mutagenesis","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 — original reconstitution of heterodimer DNA binding, multiple orthogonal methods, foundational discovery replicated across labs","pmids":["2006410"],"is_preprint":false},{"year":1992,"finding":"c-Myc protein is a nuclear phosphoprotein that binds DNA with a consensus sequence PuACCACGTGCTC; purified full-length c-Myc from eukaryotic cells (baculovirus and CHO) exists in two forms: c-Myc alone and a complex with a copurifying 26–29 kDa protein, the latter binding DNA with higher affinity.","method":"Protein purification to near homogeneity; SELEX (systematic evolution of ligands by exponential enrichment); EMSA","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro biochemical purification and SELEX with rigorous characterization of DNA-binding specificity","pmids":["1587829"],"is_preprint":false},{"year":1992,"finding":"Constitutive c-Myc expression in Rat-1 fibroblasts induces apoptosis; domains of c-Myc required for apoptosis induction overlap with those required for cotransformation, autoregulation, and inhibition of differentiation; higher c-Myc levels increase susceptibility to apoptosis upon serum deprivation, and deregulated c-Myc induces apoptosis at multiple cell cycle points.","method":"Conditional c-Myc expression in Rat-1 fibroblasts; cell death assays; domain-deletion mutant analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — clean gain-of-function with defined phenotypic readout, replicated in multiple conditions, foundational paper >3000 citations","pmids":["1555236"],"is_preprint":false},{"year":1990,"finding":"c-Myc protein negatively autoregulates its own gene transcription at the level of transcriptional initiation; the extent of suppression is proportional to cellular c-Myc concentration; autoregulation requires c-Myc protein plus additional trans-acting factors.","method":"Transient transfection of activated c-myc/v-myc into Rat-1 fibroblasts; run-on transcription assays; dose-response analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — direct measurement of transcriptional initiation, dose-dependence established, replicated across multiple cell lines","pmids":["2182320"],"is_preprint":false},{"year":1993,"finding":"The ornithine decarboxylase (ODC) gene is a direct transcriptional target of c-Myc; c-Myc transactivates ODC through conserved CACGTG repeats in intron 1; transactivation requires neither the leucine zipper of c-Myc nor its HLH domain, suggesting Myc may interact with transcription factors other than Max for ODC regulation.","method":"Promoter-reporter cotransfection assays; deletion and point mutagenesis of c-myc; heterologous promoter constructs","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple mutant constructs and reporter assays; systematic domain dissection","pmids":["8356088"],"is_preprint":false},{"year":1993,"finding":"Yin-yang 1 (YY1) binds two sites in the murine c-myc promoter (at −260 and −390 bp from P1) and strongly activates c-myc transcription from both major initiation sites; YY1 is identical to the previously described common factor 1 (CF1) as shown by biochemical, immunological, and recombinant protein analyses.","method":"Protein purification; immunological cross-reactivity; recombinant YY1 binding assays; cotransfection reporter assays; overexpression in MEL cells measuring endogenous c-myc mRNA","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods establishing identity and functional role","pmids":["8246966"],"is_preprint":false},{"year":1994,"finding":"c-Myc-induced apoptosis in quiescent mouse fibroblasts is mediated by p53; activation of c-Myc stabilizes p53, and p53-null fibroblasts undergo cell cycle re-entry but not apoptosis upon c-Myc activation, establishing p53 as an epistatic mediator of c-Myc-induced apoptosis.","method":"Conditional c-Myc activation (MycER fusion) in wild-type vs. p53-null fibroblasts; p53 stabilization Western blot; apoptosis assays","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis using isogenic p53-null cells with clean conditional system","pmids":["8091232"],"is_preprint":false},{"year":1995,"finding":"c-Myc is O-GlcNAc glycosylated on serine/threonine residues within or near its N-terminal transcription activation/transformation domain; the modification was demonstrated by lectin binding, glycosidase/glycosyltransferase treatment, and direct sugar characterization on purified recombinant protein.","method":"Lectin affinity assay; glycosidase/glycosyltransferase treatment; purification from insect and CHO cells; deletion mutant analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — three independent biochemical methods on purified protein; domain mapping by deletion mutants","pmids":["7753821"],"is_preprint":false},{"year":1998,"finding":"c-MYC is a transcriptional target of the APC/beta-catenin/Tcf-4 signaling pathway; wild-type APC represses c-MYC expression, and beta-catenin activates it through Tcf-4 binding sites in the c-MYC promoter, explaining c-MYC overexpression in colorectal cancers with APC mutations.","method":"Reporter assays with Tcf-4 binding site mutants; APC and beta-catenin gain/loss-of-function; endogenous c-MYC mRNA measurement","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — multiple functional assays linking pathway components to c-MYC promoter, replicated widely","pmids":["9727977"],"is_preprint":false},{"year":1999,"finding":"c-Myc overexpression causes colcemid-treated cells to become polyploid by replicating DNA without chromosomal segregation (uncoupling DNA replication from mitosis), and induces CDK2 activity in a G1-like state; in primary fibroblasts with wild-type p53, this triggers massive apoptosis instead.","method":"c-Myc overexpression in human and rodent cell lines; colcemid spindle checkpoint assay; flow cytometry; CDK2 kinase assay; primary vs. immortalized fibroblast comparison","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — defined molecular mechanism (CDK2 activation) with multiple cell-type comparisons and biochemical readout","pmids":["10409725"],"is_preprint":false},{"year":2000,"finding":"CDK4 is a direct transcriptional target of c-MYC; c-MYC induces CDK4 mRNA through four conserved c-MYC binding sites in the CDK4 promoter; c-MYC-deficient RAT1 cells show delayed cell-cycle progression and impaired CDK4 induction, which is partially rescued by ectopic CDK4 expression.","method":"SAGE; promoter analysis with ChIP; c-MYC-deficient cell lines; CDK4 rescue experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — direct promoter binding plus genetic rescue establishes pathway position","pmids":["10688915"],"is_preprint":false},{"year":2001,"finding":"C/EBPalpha directly represses c-Myc transcription through an E2F binding site in the c-Myc promoter; this repression is required for granulocytic differentiation, as stable ectopic c-Myc expression (from a C/EBPalpha-unresponsive promoter) blocks myeloid differentiation.","method":"Representational difference analysis; oligonucleotide array; promoter deletion/mutation analysis; stable c-Myc overexpression in C/EBPalpha-inducible myeloid cells; differentiation assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — genetic rescue/epistasis with direct promoter mapping; multiple independent screens","pmids":["11340171"],"is_preprint":false},{"year":2001,"finding":"c-Myc represses the PDGF beta-receptor by binding NF-Y subunits YB and YC (but not YA) through its Myc homology boxes (not the bHLHZip domain), without displacing NF-Y from DNA; instead, Myc represses NF-YC transactivation activity.","method":"Co-immunoprecipitation from cotransfected COS-1 cells; GST pulldown with in vitro translated c-Myc; luciferase reporter with CCAAT mutants; GAL4-NF-YC transactivation assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro pulldown plus cellular co-IP plus domain mutagenesis plus functional reporter assays","pmids":["11282029"],"is_preprint":false},{"year":2001,"finding":"PDGF-induced c-myc expression is Src-dependent and proceeds through Vav2 activation of a Rac-dependent pathway, independently of Ras and the MAP kinase pathway.","method":"Dominant-negative and constitutively active signaling constructs; Src inhibition; c-myc promoter-reporter assays; epistasis analysis in NIH3T3 cells","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — epistasis with multiple pathway components, clean promoter readout","pmids":["11389443"],"is_preprint":false},{"year":2001,"finding":"c-myc is a downstream target of TGF-beta/Smad signaling; Smad3 and E2F-4 directly bind a composite TIE/E2F element in the c-myc promoter; TGF-beta signaling suppresses c-myc transcription by dissociating p300 from E2F-4 without competing for DNA binding.","method":"EMSA; co-immunoprecipitation; promoter mutation analysis; chromatin-level timing experiments; p300 co-IP","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — direct protein-DNA binding shown by EMSA, co-IP for p300 dissociation, promoter mutagenesis","pmids":["11689553"],"is_preprint":false},{"year":2002,"finding":"c-Myc can induce DNA damage and reactive oxygen species (ROS) prior to S phase in normal human fibroblasts; deregulated c-Myc partially disables the p53-mediated DNA damage response; antioxidant treatment reduces ROS, DNA damage, and p53 activation, linking c-Myc to genomic instability via oxidative stress.","method":"Conditional c-Myc activation; comet assay; ROS measurement; gene expression arrays; antioxidant rescue; clonogenic survival","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods including antioxidant rescue establishing causal chain","pmids":["12049739"],"is_preprint":false},{"year":2004,"finding":"Fbw7, the substrate recognition subunit of the SCF(Fbw7) ubiquitin ligase, promotes proteasome-dependent c-Myc degradation in vivo and c-Myc ubiquitination in vitro; this interaction and degradation require GSK3-mediated phosphorylation of c-Myc on threonine-58 (T58); T58 is the most frequent site of c-myc mutation in lymphoma.","method":"In vivo degradation assay; in vitro ubiquitination reconstitution; T58 phospho-mutant analysis; tumor cell line comparison","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstituted ubiquitination plus in vivo degradation with phospho-mutants","pmids":["15150404"],"is_preprint":false},{"year":2005,"finding":"c-Myc directly activates expression of the miR-17-92 cluster (six miRNAs on chromosome 13) by binding to the locus, as shown by chromatin immunoprecipitation; two members of the cluster, miR-17-5p and miR-20a, negatively regulate E2F1 translation, revealing a mechanism by which c-Myc simultaneously activates E2F1 transcription and limits its translation.","method":"Chromatin immunoprecipitation (ChIP); miRNA overexpression and knockdown; E2F1 protein/mRNA measurement; luciferase reporter assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — direct ChIP evidence for c-Myc binding plus functional miRNA assays; widely replicated","pmids":["15944709"],"is_preprint":false},{"year":2004,"finding":"AID (activation-induced cytidine deaminase), the enzyme that initiates immunoglobulin class switch recombination, is essential for c-myc/IgH chromosome translocations in IL6-transgenic mice, directly linking aberrant CSR to oncogenic chromosome translocations.","method":"AID-knockout mouse model crossed with IL6-transgenic mice; cytogenetic analysis of c-myc/IgH translocations","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genetic epistasis in vivo with isogenic AID-null background","pmids":["15315756"],"is_preprint":false},{"year":2007,"finding":"c-Myc has a direct, non-transcriptional role in DNA replication initiation: it interacts with pre-replicative complex components, localizes to early DNA synthesis sites, and its depletion from mammalian cells and Xenopus cell-free extracts (devoid of RNA transcription) impairs DNA replication; c-Myc overexpression increases replication origin activity, causing DNA damage and checkpoint activation.","method":"Co-immunoprecipitation with pre-RC components; DNA fiber assay; Xenopus cell-free replication extracts (transcription-independent); c-Myc depletion (siRNA); origin firing analysis; DNA damage markers","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1 — transcription-independent Xenopus extract system plus mammalian reconstitution; multiple orthogonal methods","pmids":["17597761"],"is_preprint":false},{"year":2007,"finding":"Ribosomal protein L11 (a c-Myc transcriptional target) forms a negative feedback loop by binding to Myc box II (MBII) of c-Myc, competing with the coactivator TRRAP, and reducing histone H4 acetylation at c-Myc target gene promoters; L11 overexpression inhibits c-Myc transcriptional activity and cell proliferation, while L11 knockdown increases both.","method":"Co-immunoprecipitation; ChIP; siRNA knockdown; overexpression; histone acetylation assays; cell proliferation assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP, ChIP, and bidirectional genetic manipulation","pmids":["17599065"],"is_preprint":false},{"year":2008,"finding":"Pim-1 and Pim-2 kinases stabilize c-Myc protein in vivo; Pim-2 directly phosphorylates c-Myc on Ser329 to stabilize it; Pim-1 preferentially mediates decreased Thr58 phosphorylation and increased Ser62 phosphorylation; both kinases enhance c-Myc transcriptional activity and transforming ability through c-Myc stabilization.","method":"In vivo co-expression stabilization assays; in vitro kinase assay; phospho-site mutant analysis; transcriptional reporter assays; transformation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — direct kinase assay on Ser329 plus genetic mutant analysis plus functional transformation readout","pmids":["18438430"],"is_preprint":false},{"year":2009,"finding":"c-Myc transcriptionally represses miR-23a and miR-23b, resulting in elevated mitochondrial glutaminase expression, thereby upregulating glutamine catabolism in cancer cells; this establishes c-Myc as a regulator of glutamine metabolism through miRNA-mediated control.","method":"ChIP showing c-Myc binding to miR-23a/b promoters; miRNA overexpression/knockdown; glutaminase protein measurement; metabolic flux assays","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — direct ChIP evidence plus functional metabolic assays; replicated across two cancer cell models","pmids":["19219026"],"is_preprint":false},{"year":2009,"finding":"c-Myc upregulates hnRNP proteins (PTB/hnRNPI, hnRNPA1, hnRNPA2) at the transcriptional level, which then bind repressively to sequences flanking PKM exon 9 to promote PKM2 isoform splicing, thereby promoting aerobic glycolysis (Warburg effect) in cancer cells.","method":"c-Myc ChIP on hnRNP promoters; overexpression/knockdown of hnRNPs; RT-PCR for PKM splicing; RNA-protein binding assays; glioma tissue correlation","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 — direct ChIP plus functional splicing mechanism elucidated with multiple orthogonal methods","pmids":["20010808"],"is_preprint":false},{"year":2010,"finding":"c-Myc regulates RNA Pol II promoter-proximal pause release rather than Pol II recruitment at its target genes; c-Myc is a key regulator of transcriptional pause release in mammalian cells, and this mechanism explains amplification of gene expression by c-Myc.","method":"ChIP-seq for Pol II and c-Myc; GRO-seq (global run-on sequencing); comparison of Pol II occupancy at promoters vs. gene bodies","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genome-wide sequencing-based mechanistic analysis; defined molecular mechanism","pmids":["20434984"],"is_preprint":false},{"year":2010,"finding":"MYC directly binds the promoters of CD47 and PD-L1 genes and activates their transcription; MYC inactivation reduces CD47 and PD-L1 mRNA and protein levels and enhances the antitumor immune response; enforced CD47 or PD-L1 expression rescues tumor growth after MYC inactivation.","method":"ChIP showing MYC binding to Cd47 and Pd-l1 promoters; conditional MYC inactivation in mouse tumors; rescue experiments with enforced CD47/PD-L1 expression; immune cell analysis","journal":"Science","confidence":"High","confidence_rationale":"Tier 2 — direct ChIP plus genetic rescue demonstrating epistasis","pmids":["26966191"],"is_preprint":false},{"year":2012,"finding":"In tumor cells with elevated c-Myc, the transcription factor accumulates at promoters of already-active genes and causes transcriptional amplification—increasing transcript levels from the existing gene expression program rather than activating new target genes.","method":"ChIP-seq; RNA-seq; GRO-seq; comparison of high vs. low Myc-expressing tumor cells; genome-wide occupancy analysis","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 — genome-wide multi-method analysis defining mechanism of transcriptional amplification","pmids":["23021215"],"is_preprint":false},{"year":2013,"finding":"Ribosomal protein S14 (RPS14) binds to the Myc homology box II (MBII) and C-terminal bHLH-LZ domains of c-Myc, inhibits c-Myc transcriptional activity by preventing recruitment of c-Myc and TRRAP to target promoters, and promotes c-Myc mRNA degradation through an Argonaute2/miRNA pathway.","method":"Co-immunoprecipitation; domain-mapping pulldown; ChIP; siRNA knockdown; RT-qPCR; Argonaute2 epistasis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with domain mapping, direct ChIP for TRRAP displacement, dual mechanism demonstrated","pmids":["23775087"],"is_preprint":false},{"year":2014,"finding":"USP37 is a deubiquitinating enzyme that directly interacts with and deubiquitinates c-Myc in a DUB-activity-dependent manner, stabilizing c-Myc and enhancing cell proliferation and the Warburg effect; USP37 depletion promotes c-Myc proteasomal degradation.","method":"USP screening; co-immunoprecipitation; in vivo and in vitro deubiquitination assays; catalytic mutant (DUB-inactive) analysis; cell proliferation assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro DUB assay plus catalytic mutant validation plus cellular co-IP","pmids":["25284584"],"is_preprint":false},{"year":2014,"finding":"The lncRNA PCGEM1 physically interacts with c-Myc, promotes its chromatin recruitment, and enhances c-Myc transactivation activity; PCGEM1 regulates multiple metabolic pathways in prostate cancer cells predominantly through c-Myc coactivation.","method":"RNA immunoprecipitation; co-immunoprecipitation; ChIP; promoter-reporter assays; PCGEM1 domain mapping for c-Myc binding","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 3 — single lab; Co-IP and ChIP without in vitro reconstitution of the complex","pmids":["25512540"],"is_preprint":false},{"year":2014,"finding":"SerRS (seryl-tRNA synthetase) antagonizes c-Myc at the VEGFA promoter by two mechanisms: (1) direct competition blocking c-Myc from binding the promoter, and (2) recruiting SIRT2 histone deacetylase to erase c-Myc-promoted histone acetylation; nuclear localization of SerRS (conferred by a vertebrate-specific domain) is required for this anti-angiogenic activity.","method":"In vitro competition assays; ChIP; SIRT2 co-immunoprecipitation; histone acetylation assays; nuclear localization mutants; zebrafish vascular phenotype rescue","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro competition reconstituted, supported by ChIP and co-IP, functional in vivo validation","pmids":["24940000"],"is_preprint":false},{"year":2014,"finding":"PP2A (protein phosphatase 2A) dephosphorylates c-MYC at Ser62, destabilizing it; PP2A inhibitors SET (I2PP2A) and CIP2A are overexpressed in breast cancer and stabilize c-MYC by preventing PP2A-mediated dephosphorylation of S62; antagonizing SET with OP449 reduces S62 phosphorylation and c-MYC activity.","method":"PP2A inhibitor knockdown; phospho-specific Western blot for pS62-c-MYC; in vitro and in vivo tumor models; OP449 treatment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — bidirectional genetic manipulation of PP2A regulators with phospho-specific readout; in vitro and in vivo validation","pmids":["24927563"],"is_preprint":false},{"year":2015,"finding":"HBXIP interacts directly with c-Myc through leucine zippers, recruits the lncRNA Hotair as a scaffold, and the Hotair-bound histone demethylase LSD1 mediates c-Myc-dependent transcriptional activation of target genes; silencing HBXIP, Hotair, or LSD1 blocks c-Myc-enhanced cancer cell growth.","method":"Co-immunoprecipitation; RNA immunoprecipitation; ChIP; siRNA knockdown; in vitro and xenograft tumor growth assays","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 3 — multiple Co-IPs and ChIPs from single lab; no in vitro reconstitution of the multiprotein complex","pmids":["26719542"],"is_preprint":false},{"year":2016,"finding":"CRY2 functions as a component of an FBXL3-containing E3 ubiquitin ligase that recruits T58-phosphorylated c-MYC for ubiquitylation and degradation; CRY1 cannot substitute for CRY2 in this process; this mechanism provides circadian control of c-MYC protein turnover.","method":"Co-immunoprecipitation; in vivo ubiquitylation assay; T58 phospho-mutant analysis; CRY1/CRY2 knockout comparison; proteasome inhibitor experiments","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 — E3 complex reconstituted by Co-IP, phospho-degron mapping, CRY1 vs CRY2 specificity established","pmids":["27840026"],"is_preprint":false},{"year":2016,"finding":"ELL (eleven-nineteen lysine-rich leukaemia) functions as a direct E3 ubiquitin ligase for c-Myc, with UbcH8 as the conjugating enzyme; Cys595 of ELL is the catalytic active site; ELL-mediated c-Myc ubiquitination and degradation inhibits c-Myc transcriptional activity and suppresses tumor growth in xenograft models.","method":"In vivo ubiquitination assay; active-site mutagenesis (C595A); Co-immunoprecipitation; c-Myc reporter assays; xenograft tumor growth","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — catalytic site mutagenesis defines mechanism; in vivo functional validation","pmids":["27009366"],"is_preprint":false},{"year":2018,"finding":"SENP1 is a c-Myc deSUMOylating enzyme: it interacts with c-Myc, deSUMOylates it in cells and in vitro, and stabilizes it; catalytically inactive SENP1(C603S) cannot stabilize c-Myc; SENP1-mediated deSUMOylation reduces c-Myc polyubiquitination (SUMOylation promotes degradation) while promoting monoubiquitination and pS62/pT58 phosphorylation.","method":"Co-immunoprecipitation; in vitro deSUMOylation assay; catalytic mutant (C603S); ubiquitination assay; phospho-specific Western blot; cell cycle analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay plus catalytic mutant plus cellular validation; mechanistic crosstalk with ubiquitination established","pmids":["30305424"],"is_preprint":false},{"year":2018,"finding":"Eya3 co-opts the PP2A-B55α holoenzyme (its Ser/Thr phosphatase activity is not intrinsic to Eya3 but arises from interaction with PP2A-B55α) to dephosphorylate pT58 on c-Myc, stabilizing c-Myc; this opposes the canonical PP2A-B56α-mediated dephosphorylation of pS62 that destabilizes c-Myc.","method":"Co-immunoprecipitation of PP2A-B55α with Eya3; phosphatase assay; phospho-specific Western blot (pT58 vs pS62); c-Myc stability assay; xenograft metastasis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 — in vitro phosphatase assay, phospho-site specificity, Co-IP, functional in vivo validation; mechanistically distinguishes B55α vs B56α subunit specificity","pmids":["29535359"],"is_preprint":false},{"year":2009,"finding":"c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase; WRN (Werner helicase) is required to support c-Myc-driven S-phase, as WRN depletion in c-Myc-overexpressing cells increases DNA damage at replication sites and activates an ATR-CHK1-CHK2-p53 'replication stress' pathway, leading to rapid senescence; p53 depletion rescues senescence.","method":"BrdU pulse-chase S-phase kinetics; c-Myc overexpression; WRN siRNA; DNA damage markers (γH2AX); ATR/CHK1/CHK2/p53 pathway analysis; senescence assay","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — multiple genetic epistasis experiments with mechanistic pathway identification","pmids":["19554081"],"is_preprint":false},{"year":2009,"finding":"p53 represses c-Myc post-transcriptionally by transcriptionally inducing miR-145 (via a p53 response element in the miR-145 promoter); miR-145 directly targets c-Myc mRNA; blockade of miR-145 reverses p53-mediated c-Myc repression.","method":"Promoter-reporter assay with p53 RE; miR-145 overexpression/anti-miR; 3'UTR luciferase reporter for c-Myc targeting; in vitro and in vivo tumor growth assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — direct p53 binding to miR-145 promoter plus miR-145 targeting of c-Myc 3'UTR validated by multiple methods","pmids":["19202062"],"is_preprint":false},{"year":2001,"finding":"Deregulated c-Myc expression in keratinocytes (K14.MYC2 transgenic mice) reduces beta1 integrin expression and depletes epidermal stem cells by 75%, impairing wound healing and keratinocyte migration; beta1 integrin is essential for both keratinocyte migration and stem cell maintenance.","method":"K14.MYC2 transgenic mouse model; label-retaining cell analysis for stem cells; beta1 integrin immunostaining; wound healing assays","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — defined in vivo loss-of-function model with quantified stem cell depletion and molecular (beta1 integrin) mechanism","pmids":["11381265"],"is_preprint":false},{"year":2024,"finding":"USP43 stabilizes c-Myc by deubiquitinating it specifically at K148 and K289 through its deubiquitinase activity; USP43 competes with FBXW7 for c-Myc binding; USP43/c-Myc form a positive feedback loop in bladder cancer promoting glycolysis and metastasis.","method":"siRNA DUB library screen; co-immunoprecipitation; in vivo deubiquitination assay with lysine-specific mutants; FBXW7 competition assay; metabolic assays","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1–2 — site-specific deubiquitination at K148/K289 established with lysine mutants, FBXW7 competition mechanism defined","pmids":["38218970"],"is_preprint":false},{"year":2007,"finding":"Myc broadly represses miRNA expression by directly binding miRNA promoters (ChIP), providing a widespread reprogramming of the miRNA transcriptome that contributes to B-cell lymphomagenesis; enforced expression of Myc-repressed miRNAs diminishes tumorigenic potential of lymphoma cells.","method":"Genome-wide ChIP for Myc at miRNA loci; miRNA profiling in human and mouse B-cell lymphoma models; miRNA overexpression functional assays","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — direct ChIP evidence at miRNA promoters plus functional rescue experiments; replicated in human and mouse models","pmids":["18066065"],"is_preprint":false},{"year":2010,"finding":"MYC and MYCN directly bind the mir-9-3 locus and activate miR-9 expression; miR-9 targets CDH1 (E-cadherin) mRNA, promoting cell motility and invasiveness; miR-9-mediated E-cadherin loss activates beta-catenin signaling, increasing VEGF expression and angiogenesis.","method":"ChIP for MYC/MYCN at mir-9-3 locus; miR-9 overexpression; 3'UTR luciferase reporter for CDH1; metastasis mouse model; sponge inhibition","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — direct ChIP, validated miRNA-target interaction, in vivo metastasis model","pmids":["20173740"],"is_preprint":false},{"year":2006,"finding":"Endogenous c-Myc and Max protein-protein interactions can be directly visualized and quantified at single-molecule resolution in individual cells using proximity ligation; IFN-gamma signaling and small-molecule inhibitors (10058-F4) specifically regulate these interactions in situ.","method":"Proximity ligation assay (PLA) with antibodies against endogenous Myc and Max; rolling-circle amplification for single-molecule detection; quantification in response to IFN-gamma and small molecule inhibitors","journal":"Nature methods","confidence":"High","confidence_rationale":"Tier 2 — direct visualization of endogenous protein-protein interaction in situ; validated by small-molecule disruption","pmids":["17072308"],"is_preprint":false},{"year":2008,"finding":"The small molecule 10058-F4 binds disordered c-Myc monomers and disrupts c-Myc-Max heterodimerization; the inhibitory effect depends on the Max isoform: p22 Max (which homodimerizes well) competes with c-Myc-Max heterodimerization, lowering the effective inhibitor concentration needed compared to p21 Max.","method":"Biophysical binding assays; EMSA for DNA binding; thermodynamic analysis of heterodimerization vs. homodimerization","journal":"Bioorganic & medicinal chemistry letters","confidence":"Medium","confidence_rationale":"Tier 3 — single lab biophysical characterization; no structural or mutagenesis validation","pmids":["19114306"],"is_preprint":false},{"year":2009,"finding":"The ASK1-JNK pathway promotes c-Myc protein stability through phosphorylation at Ser62 and Ser71, which is required for c-Myc-dependent apoptosis; ASK1 signaling attenuates degradation of already-ubiquitinated c-Myc without affecting the ubiquitination process itself.","method":"ASK1 overexpression; phospho-site mutant c-Myc constructs (Ser62, Ser71); ubiquitination assay; proteasome inhibitor analysis; apoptosis assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 — single lab; phospho-mutant analysis without in vitro kinase reconstitution for ASK1 directly phosphorylating these sites","pmids":["11243879"],"is_preprint":false},{"year":2009,"finding":"c-Myc overexpression accelerates S-phase, and c-Myc-deficient fibroblasts exhibit prolonged S-phase; WRN helicase depletion in c-Myc-overexpressing cells causes DNA damage at active replication forks and activates ATR-CHK1-CHK2-p53 senescence pathway, suggesting WRN repairs aberrant replication structures caused by c-Myc-driven accelerated replication.","method":"BrdU pulse-chase S-phase kinetics; c-Myc overexpression and knockout fibroblasts; WRN siRNA; DNA damage markers at replication sites; senescence assay; p53 knockdown rescue","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — quantitative S-phase measurement, genetic epistasis with multiple pathway components","pmids":["19554081"],"is_preprint":false}],"current_model":"c-Myc is a nuclear bHLH-Zip transcription factor that heterodimerizes with Max to bind E-box (CACGTG) sequences and regulate transcription of a vast target gene network (~15% of all genes) governing cell proliferation, growth, apoptosis, metabolism (including glutamine catabolism and aerobic glycolysis via miR-23a/b repression and hnRNP upregulation), and DNA replication initiation (via direct interaction with the pre-replicative complex); its activity and stability are tightly controlled by a phosphorylation switch (GSK3-mediated pT58 promotes FBXW7/SCF-mediated proteasomal degradation, while Ras/ERK-mediated pS62 stabilizes it, with PP2A-B56α dephosphorylating pS62 to destabilize and PP2A-B55α/Eya3 dephosphorylating pT58 to stabilize), multiple ubiquitin ligases (FBXW7, ELL) and deubiquitinases (USP28, USP37, USP43, USP36), SUMO modification (reversed by SENP1), O-GlcNAc glycosylation of its transactivation domain, negative feedback from ribosomal proteins L11 and S14 that compete with TRRAP coactivator, and negative autoregulation of its own promoter; c-Myc also directly controls immune evasion through transcriptional activation of CD47 and PD-L1, and its overexpression causes genomic instability through ROS induction, replication stress, and mitotic checkpoint override."},"narrative":{"teleology":[{"year":1990,"claim":"Establishing that c-Myc negatively autoregulates its own promoter in a dose-dependent manner revealed an intrinsic feedback mechanism constraining its expression, setting the stage for understanding how deregulation (e.g., translocation) bypasses this control.","evidence":"Transfection of activated c-myc constructs into Rat-1 fibroblasts with run-on transcription measurements","pmids":["2182320"],"confidence":"High","gaps":["trans-acting cofactors required for autorepression were not identified","mechanism of promoter sensing (cis elements) not defined"]},{"year":1991,"claim":"Identification of Max as the obligate heterodimerization partner for c-Myc DNA binding resolved how c-Myc, which binds DNA poorly alone, achieves sequence-specific E-box recognition, establishing the Myc-Max paradigm central to all subsequent target gene studies.","evidence":"cDNA expression library screen with bHLH-Zip bait; co-immunoprecipitation; EMSA with purified proteins","pmids":["2006410","1587829"],"confidence":"High","gaps":["structural basis of heterodimerization not yet resolved","role of Max homodimers as antagonists not yet explored"]},{"year":1992,"claim":"Demonstrating that constitutive c-Myc expression induces apoptosis—using the same domains required for transformation—established the dual nature of c-Myc as both an oncogene and a pro-apoptotic factor, resolving the paradox of why tumors must disable apoptosis for Myc-driven oncogenesis.","evidence":"Conditional c-Myc expression in Rat-1 fibroblasts with domain-deletion mutants and serum deprivation","pmids":["1555236"],"confidence":"High","gaps":["downstream apoptotic effectors not identified at this stage","cell-type dependence of apoptotic threshold not established"]},{"year":1994,"claim":"Placing p53 as an epistatic mediator of c-Myc-induced apoptosis explained how p53 loss cooperates with Myc in tumorigenesis by disconnecting the apoptotic failsafe from proliferative signaling.","evidence":"Conditional MycER activation in wild-type vs. p53-null fibroblasts; p53 protein stabilization","pmids":["8091232"],"confidence":"High","gaps":["mechanism by which c-Myc stabilizes p53 protein not defined","alternative p53-independent apoptotic pathways not excluded"]},{"year":1993,"claim":"Identification of ODC as a direct c-Myc transcriptional target through intronic E-boxes provided the first concrete downstream effector linking c-Myc to a specific biosynthetic pathway (polyamine synthesis).","evidence":"Promoter-reporter cotransfection with E-box mutagenesis and c-Myc domain deletions","pmids":["8356088"],"confidence":"High","gaps":["physiological relevance of the HLH/Zip-independent transactivation not clarified","contribution of ODC to Myc-driven transformation not tested genetically"]},{"year":1998,"claim":"Discovery that c-MYC is a direct transcriptional target of the APC/β-catenin/Tcf-4 pathway connected Wnt signaling to proliferative gene expression and explained c-MYC overexpression in APC-mutant colorectal cancers.","evidence":"Reporter assays with Tcf-4 binding site mutants; APC/β-catenin gain- and loss-of-function in colorectal cancer cells","pmids":["9727977"],"confidence":"High","gaps":["quantitative contribution of Wnt-driven MYC vs. other MYC-activating signals in vivo unknown","chromatin-level mechanism of Tcf-4 activation not addressed"]},{"year":2000,"claim":"Identifying CDK4 as a direct c-Myc target, with genetic rescue of c-Myc-deficient cell-cycle defects by ectopic CDK4, established a concrete mechanism for c-Myc-driven G1/S progression.","evidence":"SAGE; ChIP at CDK4 promoter; c-MYC-null RAT1 cells; CDK4 rescue","pmids":["10688915"],"confidence":"High","gaps":["relative contribution of CDK4 vs. other Myc targets to proliferation not quantified"]},{"year":2001,"claim":"Multiple studies converged to define how upstream signals regulate c-myc transcription—C/EBPα represses it via E2F sites during myeloid differentiation, Smad3/E2F-4 mediate TGF-β repression by displacing p300, and Src-Vav2-Rac activates it downstream of PDGF—revealing c-myc as an integrating node for diverse signaling pathways.","evidence":"Promoter deletion/mutation analyses; Co-IP for p300 dissociation; dominant-negative signaling epistasis in multiple cell types","pmids":["11340171","11689553","11389443"],"confidence":"High","gaps":["chromatin remodeling events at the c-myc promoter during signal integration not resolved","combinatorial regulation by simultaneous signals not tested"]},{"year":2001,"claim":"Demonstration that c-Myc represses the PDGFβR gene by binding NF-Y subunits (YB/YC) through Myc homology boxes—without displacing NF-Y from DNA—revealed an E-box-independent repression mechanism expanding c-Myc's regulatory repertoire.","evidence":"GST pulldown; cellular Co-IP; GAL4-NF-YC transactivation assay with CCAAT mutants","pmids":["11282029"],"confidence":"High","gaps":["genome-wide extent of Myc-NF-Y repression not determined","structural basis of Myc-NF-Y interaction unknown"]},{"year":2002,"claim":"Showing that deregulated c-Myc induces reactive oxygen species and DNA damage prior to S-phase, rescuable by antioxidants, established a direct mechanistic link between c-Myc overexpression and genomic instability independent of replication.","evidence":"Conditional c-Myc activation; comet assay; ROS measurement; antioxidant rescue in human fibroblasts","pmids":["12049739"],"confidence":"High","gaps":["source of ROS (mitochondrial vs. enzymatic) not identified","specific oxidized DNA lesions not characterized"]},{"year":2004,"claim":"Reconstitution of SCF(FBXW7)-mediated c-Myc ubiquitination dependent on GSK3-phosphorylated Thr58 defined the core phospho-degron controlling c-Myc turnover and explained why T58 is a lymphoma hotspot mutation.","evidence":"In vitro ubiquitination reconstitution with FBXW7; T58 phospho-mutant analysis; in vivo degradation assays","pmids":["15150404"],"confidence":"High","gaps":["relative contribution of FBXW7 vs. other E3 ligases to steady-state Myc turnover not quantified"]},{"year":2005,"claim":"Discovery that c-Myc directly activates the miR-17-92 cluster, whose products repress E2F1 translation, uncovered a feedforward loop whereby c-Myc fine-tunes its own proliferative output through miRNA regulation.","evidence":"ChIP for c-Myc at miR-17-92 locus; miRNA overexpression/knockdown; E2F1 3'UTR reporters","pmids":["15944709"],"confidence":"High","gaps":["individual contributions of the six cluster miRNAs not fully delineated"]},{"year":2007,"claim":"Three parallel advances redefined c-Myc's scope: (1) a direct, transcription-independent role in DNA replication initiation via pre-RC interaction, (2) a ribosomal protein L11 negative feedback loop competing with TRRAP, and (3) genome-wide miRNA repression as a mechanism of lymphomagenesis.","evidence":"Xenopus cell-free replication extracts (transcription-free); Co-IP with pre-RC components; L11-MBII binding and TRRAP competition by ChIP; genome-wide miRNA ChIP in lymphoma models","pmids":["17597761","17599065","18066065"],"confidence":"High","gaps":["structural basis of Myc-pre-RC interaction unknown","whether L11 feedback operates at all Myc target promoters not tested","selectivity of Myc-mediated miRNA repression vs. activation not fully explained"]},{"year":2008,"claim":"Identification of Pim kinases as stabilizers of c-Myc—Pim-2 directly phosphorylating Ser329 and Pim-1 modulating the Thr58/Ser62 balance—added a cooperative oncogenic kinase axis to the phospho-degron switch.","evidence":"In vitro kinase assay; phospho-site mutants; transformation assays","pmids":["18438430"],"confidence":"High","gaps":["in vivo Pim-Myc axis contribution vs. PI3K/ERK not dissected genetically"]},{"year":2009,"claim":"Multiple discoveries linked c-Myc to metabolic reprogramming and replication stress: miR-23a/b repression upregulated glutaminase for glutamine catabolism, WRN helicase was shown essential for resolving c-Myc-driven replication stress, and p53-induced miR-145 was identified as a post-transcriptional suppressor of c-Myc, closing a p53-Myc regulatory loop.","evidence":"ChIP at miR-23a/b promoters with metabolic flux assays; BrdU pulse-chase with WRN siRNA and DNA damage markers; miR-145 3'UTR reporters with p53 RE validation","pmids":["19219026","19554081","19202062"],"confidence":"High","gaps":["relative importance of glutaminase vs. other metabolic targets in Myc-driven tumors not established","how WRN is specifically recruited to Myc-induced aberrant structures unknown"]},{"year":2010,"claim":"Genome-wide studies revealed that c-Myc acts primarily through Pol II pause release rather than recruitment, and at high levels amplifies the existing transcriptional program rather than activating novel targets—fundamentally reframing c-Myc as a universal transcriptional amplifier.","evidence":"ChIP-seq/GRO-seq for Pol II and c-Myc; RNA-seq comparing high vs. low Myc tumor cells","pmids":["20434984","23021215"],"confidence":"High","gaps":["how pause-release mechanism relates to Myc-mediated repression not reconciled","whether amplifier model applies to all tissues and contexts untested"]},{"year":2010,"claim":"Identification of miR-9 as a direct MYC/MYCN target that represses E-cadherin to promote invasiveness and angiogenesis via β-catenin/VEGF signaling established a miRNA-mediated mechanism for Myc-driven metastasis.","evidence":"ChIP at mir-9-3; CDH1 3'UTR reporter; metastasis mouse model","pmids":["20173740"],"confidence":"High","gaps":["contribution of miR-9 vs. other EMT-promoting Myc targets to metastasis not quantified"]},{"year":2014,"claim":"Multiple new layers of c-Myc regulation were defined: USP37 as a deubiquitinase stabilizing c-Myc, PP2A-B56α/SET/CIP2A axis controlling pS62 dephosphorylation, and SerRS competing with c-Myc at the VEGFA promoter while recruiting SIRT2 to erase Myc-driven histone acetylation.","evidence":"In vitro DUB assay with catalytic mutant; phospho-specific Western blots with PP2A inhibitor manipulation; in vitro promoter competition and ChIP with SIRT2 Co-IP","pmids":["25284584","24927563","24940000"],"confidence":"High","gaps":["USP37 substrate specificity beyond Myc not determined","physiological contexts where SerRS-Myc competition is rate-limiting unknown"]},{"year":2016,"claim":"Discovery that CRY2 recruits T58-phosphorylated c-Myc to an FBXL3-containing E3 ligase provided a circadian dimension to Myc turnover, while ELL was identified as a novel E3 ligase with a catalytic cysteine (C595) for direct Myc ubiquitination.","evidence":"Co-IP with T58 phospho-mutant and CRY1/CRY2 knockout comparison; ELL C595A catalytic mutant ubiquitination assay; xenograft validation","pmids":["27840026","27009366"],"confidence":"High","gaps":["circadian oscillation of Myc protein in normal tissues not directly measured","ELL's relative contribution to total Myc ubiquitination unclear"]},{"year":2016,"claim":"MYC was shown to directly activate transcription of immune checkpoint genes CD47 and PD-L1, with genetic rescue demonstrating these are necessary for tumor immune evasion—establishing MYC as a direct link between oncogene activation and immunosuppression.","evidence":"ChIP at Cd47/Pd-l1 promoters; conditional MYC inactivation in mouse tumors; enforced CD47/PD-L1 rescue","pmids":["26966191"],"confidence":"High","gaps":["whether MYC-driven immune evasion operates in all tumor types not tested","mechanism of MYC-dependent regulation of other immune modulators not explored"]},{"year":2018,"claim":"Two studies dissected opposing phosphatase arms controlling Myc stability: PP2A-B55α/Eya3 dephosphorylates pT58 to stabilize Myc, while PP2A-B56α dephosphorylates pS62 to destabilize it; separately, SENP1 was shown to deSUMOylate c-Myc, reducing polyubiquitination and stabilizing the protein, revealing SUMO-ubiquitin crosstalk in Myc turnover.","evidence":"In vitro phosphatase assays with B-subunit specificity; SENP1 catalytic mutant (C603S); phospho-specific and ubiquitin/SUMO blots","pmids":["29535359","30305424"],"confidence":"High","gaps":["spatial regulation of PP2A holoenzyme assembly on Myc not addressed","SUMO acceptor lysines on Myc not mapped"]},{"year":2024,"claim":"USP43 was identified as a deubiquitinase targeting specific lysines (K148 and K289) on c-Myc and competing with FBXW7 for binding, forming a positive feedback loop that drives glycolysis and metastasis in bladder cancer.","evidence":"siRNA DUB library screen; lysine-specific mutant deubiquitination assays; FBXW7 competition Co-IP; metabolic and metastasis assays","pmids":["38218970"],"confidence":"High","gaps":["whether USP43-Myc axis is relevant beyond bladder cancer not tested","structural basis of FBXW7/USP43 competition unknown"]},{"year":null,"claim":"Despite detailed mapping of individual regulatory inputs, a quantitative integrated model of how phosphorylation, ubiquitination, SUMOylation, and O-GlcNAcylation combinatorially determine c-Myc half-life in specific tissues remains unbuilt; the structural basis of c-Myc's intrinsically disordered transactivation domain interactions with diverse partners (TRRAP, L11, NF-Y, pre-RC) also awaits high-resolution characterization.","evidence":"","pmids":[],"confidence":"Low","gaps":["no integrated quantitative model of multi-PTM crosstalk on Myc stability","no high-resolution structure of full-length Myc-Max in complex with chromatin or coactivators","tissue-specific variation in Myc regulatory network topology not systematically mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,4,17,24,26]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3,4,10,11,17,22,23,24,25,26,41,42]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,7,19,24,26]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[9,10,19,37,46]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,13,14]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[2,6]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[16,28,33,34,35,40]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[22,23]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[25]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[19]},{"term_id":"R-HSA-9909396","term_label":"Circadian clock","supporting_discovery_ids":[33]}],"complexes":["Myc-Max heterodimer"],"partners":["MAX","FBXW7","TRRAP","RPL11","RPS14","USP37","USP43","SENP1"],"other_free_text":[]},"mechanistic_narrative":"MYC encodes a nuclear bHLH-Zip transcription factor that heterodimerizes with Max to bind E-box sequences (CACGTG) and functions as a master regulator of cell proliferation, apoptosis, metabolism, and immune evasion [PMID:2006410, PMID:1587829]. Rather than activating a discrete set of target genes, c-Myc amplifies the existing transcriptional program of active genes by promoting RNA Pol II pause release, directly inducing cell-cycle drivers (CDK4, E2F1 via miR-17-92), metabolic reprogramming enzymes (glutaminase via miR-23a/b repression, PKM2 via hnRNP upregulation), and immune checkpoint ligands CD47 and PD-L1 [PMID:20434984, PMID:23021215, PMID:10688915, PMID:19219026, PMID:20010808, PMID:26966191]. c-Myc protein stability is governed by an integrated phospho-degron switch—GSK3-mediated Thr58 phosphorylation targets c-Myc to SCF(FBXW7)- and CRY2/FBXL3-dependent ubiquitination and degradation, while Ser62 phosphorylation by ERK/Pim kinases stabilizes the protein, with opposing PP2A holoenzymes (B56α dephosphorylating pS62 to destabilize; B55α/Eya3 dephosphorylating pT58 to stabilize) and multiple deubiquitinases (USP28, USP37, USP43) and SENP1-mediated deSUMOylation providing additional layers of control [PMID:15150404, PMID:27840026, PMID:24927563, PMID:29535359, PMID:25284584, PMID:38218970, PMID:30305424]. Beyond transcription, c-Myc directly participates in DNA replication initiation by interacting with pre-replicative complex components, and its deregulation causes replication stress, reactive oxygen species, and genomic instability that are counteracted by p53-dependent apoptosis [PMID:17597761, PMID:12049739, PMID:1555236, PMID:8091232]."},"prefetch_data":{"uniprot":{"accession":"P01106","full_name":"Myc proto-oncogene protein","aliases":["Class E basic helix-loop-helix protein 39","bHLHe39","Proto-oncogene c-Myc","Transcription factor p64"],"length_aa":454,"mass_kda":50.6,"function":"Transcription factor that binds DNA in a non-specific manner, yet also specifically recognizes the core sequence 5'-CAC[GA]TG-3' (PubMed:24940000, PubMed:25956029). Activates the transcription of growth-related genes (PubMed:24940000, PubMed:25956029). Binds to the VEGFA promoter, promoting VEGFA production and subsequent sprouting angiogenesis (PubMed:24940000, PubMed:25956029). Regulator of somatic reprogramming, controls self-renewal of embryonic stem cells (By similarity). Functions with TAF6L to activate target gene expression through RNA polymerase II pause release (By similarity). Positively regulates transcription of HNRNPA1, HNRNPA2 and PTBP1 which in turn regulate splicing of pyruvate kinase PKM by binding repressively to sequences flanking PKM exon 9, inhibiting exon 9 inclusion and resulting in exon 10 inclusion and production of the PKM M2 isoform (PubMed:20010808)","subcellular_location":"Nucleus, nucleoplasm; Nucleus, nucleolus; Nucleus; Cytoplasm; Chromosome","url":"https://www.uniprot.org/uniprotkb/P01106/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/MYC","classification":"Common Essential","n_dependent_lines":1152,"n_total_lines":1208,"dependency_fraction":0.9536423841059603},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"TRRAP","stoichiometry":10.0},{"gene":"H2AFZ","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/MYC","total_profiled":1310},"omim":[{"mim_id":"621385","title":"NUCLEOLAR PROTEIN 12; NOL12","url":"https://www.omim.org/entry/621385"},{"mim_id":"621322","title":"FOXO-INDUCED LONG NONCODING RNA 1; FILNC1","url":"https://www.omim.org/entry/621322"},{"mim_id":"621230","title":"FOLLICULAR LYMPHOMA, SUSCEPTIBILITY TO, 2; FL2","url":"https://www.omim.org/entry/621230"},{"mim_id":"621176","title":"MYC-INDUCIBLE LONG NONCODING RNA INACTIVATING p53; MILIP","url":"https://www.omim.org/entry/621176"},{"mim_id":"621075","title":"TRANSMEMBRANE PROTEIN 45B; TMEM45B","url":"https://www.omim.org/entry/621075"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"adipose tissue","ntpm":250.6},{"tissue":"skin 1","ntpm":277.4}],"url":"https://www.proteinatlas.org/search/MYC"},"hgnc":{"alias_symbol":["c-Myc","bHLHe39","MYCC"],"prev_symbol":[]},"alphafold":{"accession":"P01106","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P01106","model_url":"https://alphafold.ebi.ac.uk/files/AF-P01106-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P01106-F1-predicted_aligned_error_v6.png","plddt_mean":60.41},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MYC","jax_strain_url":"https://www.jax.org/strain/search?query=MYC"},"sequence":{"accession":"P01106","fasta_url":"https://rest.uniprot.org/uniprotkb/P01106.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P01106/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P01106"}},"corpus_meta":[{"pmid":"9727977","id":"PMC_9727977","title":"Identification of c-MYC as a target of the APC pathway.","date":"1998","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/9727977","citation_count":4046,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"1555236","id":"PMC_1555236","title":"Induction of apoptosis in fibroblasts by c-myc protein.","date":"1992","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/1555236","citation_count":3030,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"15944709","id":"PMC_15944709","title":"c-Myc-regulated microRNAs modulate E2F1 expression.","date":"2005","source":"Nature","url":"https://pubmed.ncbi.nlm.nih.gov/15944709","citation_count":2308,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"23021215","id":"PMC_23021215","title":"Transcriptional amplification in tumor cells with elevated c-Myc.","date":"2012","source":"Cell","url":"https://pubmed.ncbi.nlm.nih.gov/23021215","citation_count":1227,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"16904903","id":"PMC_16904903","title":"The c-Myc target gene network.","date":"2006","source":"Seminars in cancer biology","url":"https://pubmed.ncbi.nlm.nih.gov/16904903","citation_count":960,"is_preprint":false,"source_track":"pubmed_title"},{"pmid":"12360279","id":"PMC_12360279","title":"c-MYC: more than just a matter of life and death.","date":"2002","source":"Nature reviews. 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 \"finding\": \"c-Myc protein drives apoptosis in fibroblasts; regions required for apoptosis overlap with those required for cotransformation, autoregulation, and inhibition of differentiation, and apoptosis is induced in cells growth-arrested by multiple means.\",\n      \"method\": \"Conditional c-myc expression in Rat-1 fibroblasts, serum deprivation, deletion mutant analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean loss/gain-of-function with defined apoptotic phenotype, domain mapping, highly cited\",\n      \"pmids\": [\"1555236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"c-Myc directly binds and activates transcription of the miR-17-92 cluster (chromosome 13); two miRNAs from this cluster (miR-17-5p and miR-20a) negatively regulate E2F1 translation, revealing a dual mechanism whereby c-Myc simultaneously activates E2F1 transcription and limits its translation.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), microRNA expression profiling, luciferase reporter assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal ChIP plus functional reporter assays, multiple orthogonal methods\",\n      \"pmids\": [\"15944709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In tumor cells with elevated c-Myc, the transcription factor accumulates at promoters of already-active genes and causes transcriptional amplification (increased output of the existing gene expression program) rather than activating a new set of target genes.\",\n      \"method\": \"ChIP-seq, global run-on sequencing (GRO-seq), quantitative RNA analysis in tumor vs. normal cells\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide ChIP-seq plus nascent transcription assays, replicated across multiple cell lines\",\n      \"pmids\": [\"23021215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"c-Myc is a transcriptional activator of the ornithine decarboxylase (ODC) gene through conserved CACGTG (E-box) repeats in ODC intron 1; transactivation requires specific c-Myc domains but not the leucine zipper.\",\n      \"method\": \"Reporter gene assays with deletion/point mutants of ODC promoter and c-Myc\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — promoter mutagenesis plus functional transactivation assay, highly cited\",\n      \"pmids\": [\"8356088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"c-Myc-induced apoptosis is mediated by p53; activation of c-Myc in quiescent wild-type p53 fibroblasts stabilizes p53 and induces apoptosis, whereas p53-null fibroblasts undergo cell cycle re-entry but not apoptosis upon c-Myc activation.\",\n      \"method\": \"Conditional c-Myc activation (c-Myc-ER fusion) in wild-type vs. p53-null mouse fibroblasts\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis with clean isogenic comparison, highly cited\",\n      \"pmids\": [\"8091232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-Myc has a direct, non-transcriptional role in DNA replication initiation: it interacts with the pre-replicative complex, localizes to early DNA synthesis sites, and depletion from mammalian cells and from cell-free Xenopus extracts (devoid of RNA transcription) impairs replication; overexpression increases replication origin activity, causing DNA damage and checkpoint activation.\",\n      \"method\": \"Co-immunoprecipitation with pre-RC components, DNA fiber assays, siRNA depletion in human/mouse cells, Xenopus cell-free extract reconstitution\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cell-free reconstitution in transcription-free Xenopus extracts plus multiple orthogonal methods across species\",\n      \"pmids\": [\"17597761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CDK4 is a direct transcriptional target of c-MYC; c-MYC binds four conserved E-boxes in the CDK4 promoter to rapidly induce CDK4 mRNA, and ectopic CDK4 expression partially rescues the cell-cycle progression defect of c-MYC-deficient cells.\",\n      \"method\": \"SAGE, promoter-reporter assays with E-box mutants, ChIP, genetic rescue experiment in c-MYC-deficient cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including genetic rescue epistasis\",\n      \"pmids\": [\"10688915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CRY2, as a component of an FBXL3-containing E3 ubiquitin ligase, recruits T58-phosphorylated c-MYC for ubiquitylation and degradation; CRY1 cannot substitute for CRY2 in this function.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitylation assays, CRY1/CRY2 isoform-specific mutagenesis, genetic knockdown/knockout\",\n      \"journal\": \"Molecular Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, in vivo ubiquitylation assay, isoform specificity demonstrated\",\n      \"pmids\": [\"27840026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"c-Myc protein stability is regulated through the ubiquitin-proteasome pathway controlled by sequential phosphorylation events at Ser62 and Thr58, involving Ras/Raf-activated kinases, Pin1 prolyl isomerase, and PP2A phosphatase.\",\n      \"method\": \"Protein stability assays, phosphorylation site mutagenesis, kinase/phosphatase inhibitor studies\",\n      \"journal\": \"Cell Cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — mechanistic framework review with primary data references, multiple pathway components identified\",\n      \"pmids\": [\"15467447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Pim-1 and Pim-2 kinases stabilize c-Myc by phosphorylating it at Ser329 (novel site) and by modulating Thr58/Ser62 phosphorylation, thereby enhancing c-Myc transcriptional activity and transforming potential.\",\n      \"method\": \"In vitro kinase assays, phosphomimetic/phosphodeficient mutants, co-expression and knockdown studies, transformation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro kinase assay plus mutagenesis and functional rescue\",\n      \"pmids\": [\"18438430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"c-Myc is modified by O-linked N-acetylglucosamine (O-GlcNAc) glycosylation, with modification sites mapping to the N-terminal transcriptional activation/transformation domain.\",\n      \"method\": \"Lectin binding mobility-shift assay, glycosidase/glycosyltransferase treatment, lectin affinity chromatography, deletion mutant mapping using purified recombinant protein\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — three independent biochemical methods on purified recombinant protein\",\n      \"pmids\": [\"7753821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ribosomal protein L11 binds to the Myc box II (MBII) domain of c-Myc, competes with the coactivator TRRAP, reduces histone H4 acetylation at c-Myc target gene promoters, and inhibits c-Myc-induced transcription and cell proliferation in a negative feedback loop (L11 is itself a c-Myc transcriptional target).\",\n      \"method\": \"Co-immunoprecipitation, ChIP, siRNA knockdown, promoter reporter assays\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ChIP, domain mapping, feedback loop established by multiple methods\",\n      \"pmids\": [\"17599065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP37 deubiquitinates c-Myc directly via physical interaction, stabilizing c-Myc protein in a DUB-activity-dependent manner; USP37 regulates cell proliferation and the Warburg effect through c-Myc.\",\n      \"method\": \"Co-immunoprecipitation, in vivo/in vitro deubiquitination assays, siRNA knockdown, catalytically inactive mutant\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — direct deubiquitination assay with catalytic mutant control plus binding and functional studies\",\n      \"pmids\": [\"25284584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SENP1 is a c-Myc deSUMOylating enzyme that interacts with and deSUMOylates c-Myc in cells and in vitro; SENP1-mediated deSUMOylation reduces c-Myc polyubiquitination and promotes its stability, while SUMOylation promotes c-Myc degradation through the proteasome.\",\n      \"method\": \"Co-immunoprecipitation, in vitro deSUMOylation assay, catalytically inactive mutant (C603S), ubiquitination assays, siRNA knockdown\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay with catalytic mutant, multiple orthogonal biochemical methods\",\n      \"pmids\": [\"30305424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ELL functions as an E3 ubiquitin ligase targeting c-Myc for proteasomal degradation, with UbcH8 as the cognate E2; Cysteine 595 is the catalytic active site of ELL's E3 ligase activity.\",\n      \"method\": \"Ubiquitination assays, active-site mutagenesis (C595A), co-immunoprecipitation, proliferation and xenograft assays\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — active-site mutagenesis with reconstituted ubiquitination assay\",\n      \"pmids\": [\"27009366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Eya3 interacts directly with the PP2A-B55α holoenzyme and redirects its phosphatase activity to dephosphorylate pT58 on c-Myc, stabilizing c-Myc; this contrasts with PP2A-B56α which dephosphorylates pS62 and destabilizes c-Myc.\",\n      \"method\": \"Co-immunoprecipitation, phosphatase activity assays, c-Myc stability measurements, xenograft tumor models\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct biochemical interaction and phosphatase assay with isoform specificity demonstrated\",\n      \"pmids\": [\"29535359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"c-Myc purified from eukaryotic cells binds specifically to a CACGTG core sequence (with optimal 12-base consensus PuACCACGTGCTC); c-Myc also forms a higher-affinity complex with a copurifying 26-29 kDa protein (Max) that binds the same consensus.\",\n      \"method\": \"Biochemical purification from baculovirus/insect cells and CHO cells, SELEX (systematic selection of binding sequences), gel mobility shift assay\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical purification and SELEX with sequence-specific DNA binding demonstrated\",\n      \"pmids\": [\"1587829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"c-Myc is a nuclear phosphoprotein that heterodimerizes with Max and binds specifically to a core DNA sequence (CAC(G/A)TG) to regulate gene transcription.\",\n      \"method\": \"Nuclear localization experiments, DNA-binding assays, dimerization studies with Max\",\n      \"journal\": \"FASEB Journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — review synthesizing primary biochemical data but not primary experimental paper\",\n      \"pmids\": [\"1521738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"c-Myc negatively autoregulates its own transcription at the level of transcriptional initiation; suppression is proportional to c-Myc protein concentration and requires additional trans-acting factors.\",\n      \"method\": \"Stable transfection of activated c-myc/v-myc in Rat-1 fibroblasts, RNA quantification, dose-response analysis\",\n      \"journal\": \"The EMBO Journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — quantitative transcriptional analysis with dose-response, multiple constructs\",\n      \"pmids\": [\"2182320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"YY1 (Yin-yang 1) binds two sites in the c-myc promoter (~−260 and −390 from P1) and activates c-myc transcription from both major promoters; YY1 was purified and identified as identical to CF1 (common factor 1).\",\n      \"method\": \"Protein purification, immunological cross-reactivity, recombinant YY1 binding assay, cotransfection reporter assays, analysis of endogenous c-myc mRNA in MEL cells\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — protein purification, recombinant binding assay, functional reporter assays\",\n      \"pmids\": [\"8246966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"C/EBPα represses c-Myc transcription through an E2F binding site in the c-Myc promoter during granulopoiesis; stable expression of c-Myc from a promoter not responsive to C/EBPα blocks myeloid differentiation, establishing C/EBPα→c-Myc repression as essential for entry into the granulocytic pathway.\",\n      \"method\": \"Representational difference analysis, oligonucleotide arrays, promoter mutagenesis, stable transfection with constitutive c-Myc, differentiation assays\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis (rescue by constitutive c-Myc), promoter mutagenesis, multiple screens\",\n      \"pmids\": [\"11340171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"TGF-β signaling represses c-myc transcription through a Smad3/E2F-4 complex binding to a composite TIE/E2F element in the c-myc promoter; TGF-β reduces p300 co-immunoprecipitating with E2F-4 without displacing E2F-4 from the promoter.\",\n      \"method\": \"Promoter reporter assays with nucleotide substitution mutants, electrophoretic mobility shift assays, co-immunoprecipitation, ChIP-like binding assays\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — promoter mutagenesis, EMSA, Co-IP, multiple orthogonal methods\",\n      \"pmids\": [\"11689553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"c-Myc overexpression causes colcemid-treated cells to replicate DNA without chromosomal segregation (polyploidy) by activating CDK2 activity in a G1-like state, uncoupling DNA replication from mitosis.\",\n      \"method\": \"c-Myc overexpression in human and rodent cells, colcemid treatment, flow cytometry, CDK2 activity assays\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — biochemical (CDK2 activity) plus cellular (flow cytometry polyploidy) with mechanistic link\",\n      \"pmids\": [\"10409725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-Myc overexpression accelerates S-phase in primary human fibroblasts; WRN helicase is required to support c-Myc-driven S-phase—WRN depletion in c-Myc-overexpressing cells causes DNA damage at replication sites, activating ATR/CHK1/CHK2/p53-dependent senescence.\",\n      \"method\": \"BrdU pulse-chase S-phase timing, DNA fiber assays, siRNA knockdown of WRN, γ-H2AX staining, epistasis with p53 knockdown\",\n      \"journal\": \"PloS One\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods, epistasis with p53 knockdown confirming pathway\",\n      \"pmids\": [\"19554081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-MYC represses p21 through a transcriptional cascade: c-MYC directly induces AP4 (TFAP4), which binds recognition motifs near the p21 promoter and represses p21, blocking p53/DNA-damage- and TGFβ/Smad-mediated p21 induction.\",\n      \"method\": \"ChIP, reporter assays, AP4 knockdown/overexpression, epistasis experiments\",\n      \"journal\": \"Cell Cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP plus functional epistasis, but primarily a review/commentary article summarizing primary findings\",\n      \"pmids\": [\"19270520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"c-Myc binds NF-Y subunits YB and YC through its Myc homology boxes (not the bHLHZip domain) and represses PDGF β-receptor transcription by inhibiting NF-Y transcriptional activation without affecting NF-Y DNA binding.\",\n      \"method\": \"Co-immunoprecipitation from cotransfected cells, GST pulldown with in vitro translated c-Myc, reporter assays with domain mutants, EMSA\",\n      \"journal\": \"Journal of Cell Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — GST pulldown plus Co-IP plus functional reporter with domain mutants\",\n      \"pmids\": [\"11282029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HBXIP interacts with c-Myc through leucine zippers and recruits lncRNA Hotair and LSD1 histone demethylase to c-Myc target gene promoters to mediate transcriptional activation; silencing any component (HBXIP, Hotair, or LSD1) blocks c-Myc-dependent target gene induction.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, RNAi knockdown, in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Cancer Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ChIP, RNAi epistasis, domain mapping\",\n      \"pmids\": [\"26719542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The lncRNA PCGEM1 physically interacts with c-Myc, promotes its chromatin recruitment, and enhances its transactivation activity to regulate multiple metabolic pathways (glycolysis, pentose phosphate pathway, nucleotide/lipid biosynthesis) in prostate cancer cells.\",\n      \"method\": \"RNA immunoprecipitation, co-immunoprecipitation, ChIP, reporter assays, metabolic flux assays (seahorse)\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — RNA-IP, Co-IP, ChIP, functional metabolic assays, c-Myc binding domain on PCGEM1 identified\",\n      \"pmids\": [\"25512540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ribosomal protein S14 (RPS14) interacts with the MBII and bHLH-LZ domains of c-Myc, inhibits c-Myc transcriptional activity by preventing recruitment of c-Myc and TRRAP to target gene promoters, and promotes c-Myc mRNA turnover through an Ago2/microRNA-mediated pathway.\",\n      \"method\": \"Co-immunoprecipitation, domain mapping, ChIP, siRNA knockdown, mRNA stability assays\",\n      \"journal\": \"The Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — domain-specific Co-IP, ChIP, multiple regulatory mechanisms demonstrated\",\n      \"pmids\": [\"23775087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SerRS (seryl-tRNA synthetase) antagonizes c-Myc at the VEGFA promoter through a tandem mechanism: nuclear SerRS directly competes with c-Myc for promoter binding and recruits SIRT2 histone deacetylase to erase c-Myc-promoted histone acetylation.\",\n      \"method\": \"In vitro competition assays, ChIP, co-immunoprecipitation, zebrafish genetic model, cell-based reporter assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — direct competition assay, ChIP, genetic validation in zebrafish model\",\n      \"pmids\": [\"24940000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"TIP49 (but not TRRAP) binds the c-Myc transactivation domain and modulates c-Myc-mediated apoptosis; TIP49 also binds E2F1 and modulates both its transforming and apoptotic activities.\",\n      \"method\": \"Co-immunoprecipitation, dominant-negative TIP49 and TRRAP expression, apoptosis and transformation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single Co-IP with functional follow-up, cofactor specificity demonstrated\",\n      \"pmids\": [\"12185582\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"c-Myc protein stability is aberrantly prolonged in leukemia cell lines and primary ALL samples through abnormal phosphorylation at T58 and S62 and decreased affinity for GSK3β (which normally phosphorylates T58 to promote degradation), without c-myc gene mutation.\",\n      \"method\": \"Protein half-life measurement, phospho-specific immunoprecipitation, GSK3β binding assay, sequencing\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — quantitative stability assay plus biochemical phosphorylation and kinase-binding analysis\",\n      \"pmids\": [\"16855632\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Deregulated c-Myc expression reduces β1 integrin expression and depletes epidermal stem cells in vivo, impairing wound healing keratinocyte migration; the effect is demonstrated in K14.MYC2 transgenic mice.\",\n      \"method\": \"Transgenic mouse model (K14.MYC2), label-retaining stem cell analysis, β1 integrin immunostaining, wound healing assays\",\n      \"journal\": \"Nature Genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — in vivo transgenic model with quantitative stem cell and functional migration readouts\",\n      \"pmids\": [\"11381265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PDGF induces c-myc expression in a Src-dependent, Ras/MAPK-independent manner via a Vav2/Rac signaling pathway downstream of Src.\",\n      \"method\": \"Reporter assays, dominant-negative and activated Rho GTPase constructs, Src inhibition, epistasis analysis\",\n      \"journal\": \"Nature Cell Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis using multiple pathway components with reporter readout\",\n      \"pmids\": [\"11389443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ASK1-JNK signaling phosphorylates c-Myc at Ser62 and Ser71, enhancing c-Myc protein stability and its proapoptotic activity by attenuating degradation of ubiquitinated c-Myc without affecting ubiquitination itself.\",\n      \"method\": \"ASK1 overexpression, phosphomimetic/phosphodeficient Ser62/71 mutants, ubiquitination assays, protein stability measurements\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — phosphorylation site mutagenesis with ubiquitination and stability assays, single lab\",\n      \"pmids\": [\"11243879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PP2A dephosphorylates c-MYC at Ser62, destabilizing it; endogenous PP2A inhibitors SET and CIP2A prevent this dephosphorylation, maintaining c-MYC S62 phosphorylation and stability in breast cancer.\",\n      \"method\": \"Knockdown of SET/CIP2A, pharmacological PP2A activation (OP449), phospho-S62 c-MYC immunoblotting, in vitro and xenograft tumor models\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic and pharmacological approaches with defined phosphorylation site readout, in vivo validation\",\n      \"pmids\": [\"24927563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"CircECE1 (a circular RNA) physically interacts with c-Myc to competitively inhibit SPOP-mediated c-Myc ubiquitination and degradation; stabilized c-Myc then represses TXNIP transcription, activating the Warburg effect in osteosarcoma.\",\n      \"method\": \"RNA immunoprecipitation, co-immunoprecipitation, competitive binding assay, seahorse metabolic assay, in vivo xenograft\",\n      \"journal\": \"Molecular Cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — RNA-IP and Co-IP with functional follow-up, single lab\",\n      \"pmids\": [\"33106166\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP43 stabilizes c-Myc by deubiquitinating it at K148 and K289, interfering with FBXW7 access; USP43 and c-Myc form a positive feedback loop contributing to glycolytic metabolism and metastasis in bladder cancer.\",\n      \"method\": \"siRNA library screen, co-immunoprecipitation, in vivo deubiquitination assay with site-specific K148/K289 mutants, FBXW7 competition assay\",\n      \"journal\": \"Cell Death & Disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — site-specific mutagenesis of ubiquitination sites plus in vivo deubiquitination assay and competition assay\",\n      \"pmids\": [\"38218970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Genome-wide ChIP analysis shows c-Myc preferentially binds promoter-associated E-boxes (CACGTG) located within CpG islands in an open, pre-acetylated chromatin state; c-Myc further enhances histone acetylation at these sites; overexpressed c-Myc also binds lower-affinity and non-E-box sequences.\",\n      \"method\": \"Quantitative ChIP with genomic array (ChIP-on-chip), bioinformatics analysis of ~700 loci in human cells\",\n      \"journal\": \"Genes & Development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — large-scale quantitative ChIP across the genome with multiple cell lines\",\n      \"pmids\": [\"12695333\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"c-Myc inhibits MyoD- and myogenin-initiated skeletal muscle differentiation independently of Id; neither myogenin alone nor myogenin plus MyoD can bypass the c-Myc block to myogenesis.\",\n      \"method\": \"Transient and stable cotransfection in NIH 3T3 cells, conditional c-myc induction, differentiation assays\",\n      \"journal\": \"Molecular and Cellular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis between c-Myc, MyoD, myogenin, and Id using conditional expression system\",\n      \"pmids\": [\"1850105\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"c-MYC is a nuclear bHLH-LZ transcription factor that heterodimerizes with Max to bind E-box sequences (CACGTG) and both activate (e.g., ODC, CDK4, miR-17-92, ribosome biogenesis genes) and repress (e.g., p21, PDGF-β-receptor) target genes, thereby driving cell proliferation and—under limiting growth conditions—apoptosis (via p53 stabilization and death-receptor pathways); its protein stability is tightly regulated by sequential phosphorylation at S62 (stabilizing, by Ras/Raf/ERK, Pim kinases, ASK1-JNK) and T58 (destabilizing, by GSK3β), with PP2A-B56α dephosphorylating pS62 to promote degradation and PP2A-B55α/Eya3 dephosphorylating pT58 to stabilize it, while ubiquitin ligases (FBXW7, FBXL3/CRY2, ELL) promote proteasomal degradation and deubiquitinases (USP28, USP37, USP43, SENP1) oppose it; additionally, c-Myc has a direct non-transcriptional role in DNA replication initiation through interaction with the pre-replicative complex, and its expression is controlled upstream by the APC/β-catenin/TCF-4, TGF-β/Smad3, C/EBPα, Ras/Raf, PDGF/Src/Vav2/Rac, and circadian (CRY2/FBXL3) pathways.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"Max is a bHLH-Zip protein that specifically associates with c-Myc, N-Myc, and L-Myc via their HLH-Zip domains, and the Myc-Max heterodimer binds DNA in a sequence-specific manner (core CACGTG) under conditions where neither protein alone exhibits appreciable binding; DNA binding requires both the dimerization domain and basic region of c-Myc.\",\n      \"method\": \"cDNA expression library screen with c-Myc bHLH-Zip domain as bait; co-immunoprecipitation; EMSA; domain mutagenesis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — original reconstitution of heterodimer DNA binding, multiple orthogonal methods, foundational discovery replicated across labs\",\n      \"pmids\": [\"2006410\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"c-Myc protein is a nuclear phosphoprotein that binds DNA with a consensus sequence PuACCACGTGCTC; purified full-length c-Myc from eukaryotic cells (baculovirus and CHO) exists in two forms: c-Myc alone and a complex with a copurifying 26–29 kDa protein, the latter binding DNA with higher affinity.\",\n      \"method\": \"Protein purification to near homogeneity; SELEX (systematic evolution of ligands by exponential enrichment); EMSA\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical purification and SELEX with rigorous characterization of DNA-binding specificity\",\n      \"pmids\": [\"1587829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Constitutive c-Myc expression in Rat-1 fibroblasts induces apoptosis; domains of c-Myc required for apoptosis induction overlap with those required for cotransformation, autoregulation, and inhibition of differentiation; higher c-Myc levels increase susceptibility to apoptosis upon serum deprivation, and deregulated c-Myc induces apoptosis at multiple cell cycle points.\",\n      \"method\": \"Conditional c-Myc expression in Rat-1 fibroblasts; cell death assays; domain-deletion mutant analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean gain-of-function with defined phenotypic readout, replicated in multiple conditions, foundational paper >3000 citations\",\n      \"pmids\": [\"1555236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"c-Myc protein negatively autoregulates its own gene transcription at the level of transcriptional initiation; the extent of suppression is proportional to cellular c-Myc concentration; autoregulation requires c-Myc protein plus additional trans-acting factors.\",\n      \"method\": \"Transient transfection of activated c-myc/v-myc into Rat-1 fibroblasts; run-on transcription assays; dose-response analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct measurement of transcriptional initiation, dose-dependence established, replicated across multiple cell lines\",\n      \"pmids\": [\"2182320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"The ornithine decarboxylase (ODC) gene is a direct transcriptional target of c-Myc; c-Myc transactivates ODC through conserved CACGTG repeats in intron 1; transactivation requires neither the leucine zipper of c-Myc nor its HLH domain, suggesting Myc may interact with transcription factors other than Max for ODC regulation.\",\n      \"method\": \"Promoter-reporter cotransfection assays; deletion and point mutagenesis of c-myc; heterologous promoter constructs\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple mutant constructs and reporter assays; systematic domain dissection\",\n      \"pmids\": [\"8356088\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Yin-yang 1 (YY1) binds two sites in the murine c-myc promoter (at −260 and −390 bp from P1) and strongly activates c-myc transcription from both major initiation sites; YY1 is identical to the previously described common factor 1 (CF1) as shown by biochemical, immunological, and recombinant protein analyses.\",\n      \"method\": \"Protein purification; immunological cross-reactivity; recombinant YY1 binding assays; cotransfection reporter assays; overexpression in MEL cells measuring endogenous c-myc mRNA\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods establishing identity and functional role\",\n      \"pmids\": [\"8246966\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"c-Myc-induced apoptosis in quiescent mouse fibroblasts is mediated by p53; activation of c-Myc stabilizes p53, and p53-null fibroblasts undergo cell cycle re-entry but not apoptosis upon c-Myc activation, establishing p53 as an epistatic mediator of c-Myc-induced apoptosis.\",\n      \"method\": \"Conditional c-Myc activation (MycER fusion) in wild-type vs. p53-null fibroblasts; p53 stabilization Western blot; apoptosis assays\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis using isogenic p53-null cells with clean conditional system\",\n      \"pmids\": [\"8091232\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"c-Myc is O-GlcNAc glycosylated on serine/threonine residues within or near its N-terminal transcription activation/transformation domain; the modification was demonstrated by lectin binding, glycosidase/glycosyltransferase treatment, and direct sugar characterization on purified recombinant protein.\",\n      \"method\": \"Lectin affinity assay; glycosidase/glycosyltransferase treatment; purification from insect and CHO cells; deletion mutant analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — three independent biochemical methods on purified protein; domain mapping by deletion mutants\",\n      \"pmids\": [\"7753821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"c-MYC is a transcriptional target of the APC/beta-catenin/Tcf-4 signaling pathway; wild-type APC represses c-MYC expression, and beta-catenin activates it through Tcf-4 binding sites in the c-MYC promoter, explaining c-MYC overexpression in colorectal cancers with APC mutations.\",\n      \"method\": \"Reporter assays with Tcf-4 binding site mutants; APC and beta-catenin gain/loss-of-function; endogenous c-MYC mRNA measurement\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple functional assays linking pathway components to c-MYC promoter, replicated widely\",\n      \"pmids\": [\"9727977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"c-Myc overexpression causes colcemid-treated cells to become polyploid by replicating DNA without chromosomal segregation (uncoupling DNA replication from mitosis), and induces CDK2 activity in a G1-like state; in primary fibroblasts with wild-type p53, this triggers massive apoptosis instead.\",\n      \"method\": \"c-Myc overexpression in human and rodent cell lines; colcemid spindle checkpoint assay; flow cytometry; CDK2 kinase assay; primary vs. immortalized fibroblast comparison\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — defined molecular mechanism (CDK2 activation) with multiple cell-type comparisons and biochemical readout\",\n      \"pmids\": [\"10409725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"CDK4 is a direct transcriptional target of c-MYC; c-MYC induces CDK4 mRNA through four conserved c-MYC binding sites in the CDK4 promoter; c-MYC-deficient RAT1 cells show delayed cell-cycle progression and impaired CDK4 induction, which is partially rescued by ectopic CDK4 expression.\",\n      \"method\": \"SAGE; promoter analysis with ChIP; c-MYC-deficient cell lines; CDK4 rescue experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct promoter binding plus genetic rescue establishes pathway position\",\n      \"pmids\": [\"10688915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"C/EBPalpha directly represses c-Myc transcription through an E2F binding site in the c-Myc promoter; this repression is required for granulocytic differentiation, as stable ectopic c-Myc expression (from a C/EBPalpha-unresponsive promoter) blocks myeloid differentiation.\",\n      \"method\": \"Representational difference analysis; oligonucleotide array; promoter deletion/mutation analysis; stable c-Myc overexpression in C/EBPalpha-inducible myeloid cells; differentiation assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic rescue/epistasis with direct promoter mapping; multiple independent screens\",\n      \"pmids\": [\"11340171\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"c-Myc represses the PDGF beta-receptor by binding NF-Y subunits YB and YC (but not YA) through its Myc homology boxes (not the bHLHZip domain), without displacing NF-Y from DNA; instead, Myc represses NF-YC transactivation activity.\",\n      \"method\": \"Co-immunoprecipitation from cotransfected COS-1 cells; GST pulldown with in vitro translated c-Myc; luciferase reporter with CCAAT mutants; GAL4-NF-YC transactivation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro pulldown plus cellular co-IP plus domain mutagenesis plus functional reporter assays\",\n      \"pmids\": [\"11282029\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PDGF-induced c-myc expression is Src-dependent and proceeds through Vav2 activation of a Rac-dependent pathway, independently of Ras and the MAP kinase pathway.\",\n      \"method\": \"Dominant-negative and constitutively active signaling constructs; Src inhibition; c-myc promoter-reporter assays; epistasis analysis in NIH3T3 cells\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis with multiple pathway components, clean promoter readout\",\n      \"pmids\": [\"11389443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"c-myc is a downstream target of TGF-beta/Smad signaling; Smad3 and E2F-4 directly bind a composite TIE/E2F element in the c-myc promoter; TGF-beta signaling suppresses c-myc transcription by dissociating p300 from E2F-4 without competing for DNA binding.\",\n      \"method\": \"EMSA; co-immunoprecipitation; promoter mutation analysis; chromatin-level timing experiments; p300 co-IP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct protein-DNA binding shown by EMSA, co-IP for p300 dissociation, promoter mutagenesis\",\n      \"pmids\": [\"11689553\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"c-Myc can induce DNA damage and reactive oxygen species (ROS) prior to S phase in normal human fibroblasts; deregulated c-Myc partially disables the p53-mediated DNA damage response; antioxidant treatment reduces ROS, DNA damage, and p53 activation, linking c-Myc to genomic instability via oxidative stress.\",\n      \"method\": \"Conditional c-Myc activation; comet assay; ROS measurement; gene expression arrays; antioxidant rescue; clonogenic survival\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods including antioxidant rescue establishing causal chain\",\n      \"pmids\": [\"12049739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Fbw7, the substrate recognition subunit of the SCF(Fbw7) ubiquitin ligase, promotes proteasome-dependent c-Myc degradation in vivo and c-Myc ubiquitination in vitro; this interaction and degradation require GSK3-mediated phosphorylation of c-Myc on threonine-58 (T58); T58 is the most frequent site of c-myc mutation in lymphoma.\",\n      \"method\": \"In vivo degradation assay; in vitro ubiquitination reconstitution; T58 phospho-mutant analysis; tumor cell line comparison\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstituted ubiquitination plus in vivo degradation with phospho-mutants\",\n      \"pmids\": [\"15150404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"c-Myc directly activates expression of the miR-17-92 cluster (six miRNAs on chromosome 13) by binding to the locus, as shown by chromatin immunoprecipitation; two members of the cluster, miR-17-5p and miR-20a, negatively regulate E2F1 translation, revealing a mechanism by which c-Myc simultaneously activates E2F1 transcription and limits its translation.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP); miRNA overexpression and knockdown; E2F1 protein/mRNA measurement; luciferase reporter assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct ChIP evidence for c-Myc binding plus functional miRNA assays; widely replicated\",\n      \"pmids\": [\"15944709\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"AID (activation-induced cytidine deaminase), the enzyme that initiates immunoglobulin class switch recombination, is essential for c-myc/IgH chromosome translocations in IL6-transgenic mice, directly linking aberrant CSR to oncogenic chromosome translocations.\",\n      \"method\": \"AID-knockout mouse model crossed with IL6-transgenic mice; cytogenetic analysis of c-myc/IgH translocations\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis in vivo with isogenic AID-null background\",\n      \"pmids\": [\"15315756\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"c-Myc has a direct, non-transcriptional role in DNA replication initiation: it interacts with pre-replicative complex components, localizes to early DNA synthesis sites, and its depletion from mammalian cells and Xenopus cell-free extracts (devoid of RNA transcription) impairs DNA replication; c-Myc overexpression increases replication origin activity, causing DNA damage and checkpoint activation.\",\n      \"method\": \"Co-immunoprecipitation with pre-RC components; DNA fiber assay; Xenopus cell-free replication extracts (transcription-independent); c-Myc depletion (siRNA); origin firing analysis; DNA damage markers\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — transcription-independent Xenopus extract system plus mammalian reconstitution; multiple orthogonal methods\",\n      \"pmids\": [\"17597761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Ribosomal protein L11 (a c-Myc transcriptional target) forms a negative feedback loop by binding to Myc box II (MBII) of c-Myc, competing with the coactivator TRRAP, and reducing histone H4 acetylation at c-Myc target gene promoters; L11 overexpression inhibits c-Myc transcriptional activity and cell proliferation, while L11 knockdown increases both.\",\n      \"method\": \"Co-immunoprecipitation; ChIP; siRNA knockdown; overexpression; histone acetylation assays; cell proliferation assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP, ChIP, and bidirectional genetic manipulation\",\n      \"pmids\": [\"17599065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Pim-1 and Pim-2 kinases stabilize c-Myc protein in vivo; Pim-2 directly phosphorylates c-Myc on Ser329 to stabilize it; Pim-1 preferentially mediates decreased Thr58 phosphorylation and increased Ser62 phosphorylation; both kinases enhance c-Myc transcriptional activity and transforming ability through c-Myc stabilization.\",\n      \"method\": \"In vivo co-expression stabilization assays; in vitro kinase assay; phospho-site mutant analysis; transcriptional reporter assays; transformation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — direct kinase assay on Ser329 plus genetic mutant analysis plus functional transformation readout\",\n      \"pmids\": [\"18438430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-Myc transcriptionally represses miR-23a and miR-23b, resulting in elevated mitochondrial glutaminase expression, thereby upregulating glutamine catabolism in cancer cells; this establishes c-Myc as a regulator of glutamine metabolism through miRNA-mediated control.\",\n      \"method\": \"ChIP showing c-Myc binding to miR-23a/b promoters; miRNA overexpression/knockdown; glutaminase protein measurement; metabolic flux assays\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct ChIP evidence plus functional metabolic assays; replicated across two cancer cell models\",\n      \"pmids\": [\"19219026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-Myc upregulates hnRNP proteins (PTB/hnRNPI, hnRNPA1, hnRNPA2) at the transcriptional level, which then bind repressively to sequences flanking PKM exon 9 to promote PKM2 isoform splicing, thereby promoting aerobic glycolysis (Warburg effect) in cancer cells.\",\n      \"method\": \"c-Myc ChIP on hnRNP promoters; overexpression/knockdown of hnRNPs; RT-PCR for PKM splicing; RNA-protein binding assays; glioma tissue correlation\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct ChIP plus functional splicing mechanism elucidated with multiple orthogonal methods\",\n      \"pmids\": [\"20010808\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"c-Myc regulates RNA Pol II promoter-proximal pause release rather than Pol II recruitment at its target genes; c-Myc is a key regulator of transcriptional pause release in mammalian cells, and this mechanism explains amplification of gene expression by c-Myc.\",\n      \"method\": \"ChIP-seq for Pol II and c-Myc; GRO-seq (global run-on sequencing); comparison of Pol II occupancy at promoters vs. gene bodies\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide sequencing-based mechanistic analysis; defined molecular mechanism\",\n      \"pmids\": [\"20434984\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MYC directly binds the promoters of CD47 and PD-L1 genes and activates their transcription; MYC inactivation reduces CD47 and PD-L1 mRNA and protein levels and enhances the antitumor immune response; enforced CD47 or PD-L1 expression rescues tumor growth after MYC inactivation.\",\n      \"method\": \"ChIP showing MYC binding to Cd47 and Pd-l1 promoters; conditional MYC inactivation in mouse tumors; rescue experiments with enforced CD47/PD-L1 expression; immune cell analysis\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct ChIP plus genetic rescue demonstrating epistasis\",\n      \"pmids\": [\"26966191\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"In tumor cells with elevated c-Myc, the transcription factor accumulates at promoters of already-active genes and causes transcriptional amplification—increasing transcript levels from the existing gene expression program rather than activating new target genes.\",\n      \"method\": \"ChIP-seq; RNA-seq; GRO-seq; comparison of high vs. low Myc-expressing tumor cells; genome-wide occupancy analysis\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — genome-wide multi-method analysis defining mechanism of transcriptional amplification\",\n      \"pmids\": [\"23021215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Ribosomal protein S14 (RPS14) binds to the Myc homology box II (MBII) and C-terminal bHLH-LZ domains of c-Myc, inhibits c-Myc transcriptional activity by preventing recruitment of c-Myc and TRRAP to target promoters, and promotes c-Myc mRNA degradation through an Argonaute2/miRNA pathway.\",\n      \"method\": \"Co-immunoprecipitation; domain-mapping pulldown; ChIP; siRNA knockdown; RT-qPCR; Argonaute2 epistasis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with domain mapping, direct ChIP for TRRAP displacement, dual mechanism demonstrated\",\n      \"pmids\": [\"23775087\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"USP37 is a deubiquitinating enzyme that directly interacts with and deubiquitinates c-Myc in a DUB-activity-dependent manner, stabilizing c-Myc and enhancing cell proliferation and the Warburg effect; USP37 depletion promotes c-Myc proteasomal degradation.\",\n      \"method\": \"USP screening; co-immunoprecipitation; in vivo and in vitro deubiquitination assays; catalytic mutant (DUB-inactive) analysis; cell proliferation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro DUB assay plus catalytic mutant validation plus cellular co-IP\",\n      \"pmids\": [\"25284584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The lncRNA PCGEM1 physically interacts with c-Myc, promotes its chromatin recruitment, and enhances c-Myc transactivation activity; PCGEM1 regulates multiple metabolic pathways in prostate cancer cells predominantly through c-Myc coactivation.\",\n      \"method\": \"RNA immunoprecipitation; co-immunoprecipitation; ChIP; promoter-reporter assays; PCGEM1 domain mapping for c-Myc binding\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab; Co-IP and ChIP without in vitro reconstitution of the complex\",\n      \"pmids\": [\"25512540\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SerRS (seryl-tRNA synthetase) antagonizes c-Myc at the VEGFA promoter by two mechanisms: (1) direct competition blocking c-Myc from binding the promoter, and (2) recruiting SIRT2 histone deacetylase to erase c-Myc-promoted histone acetylation; nuclear localization of SerRS (conferred by a vertebrate-specific domain) is required for this anti-angiogenic activity.\",\n      \"method\": \"In vitro competition assays; ChIP; SIRT2 co-immunoprecipitation; histone acetylation assays; nuclear localization mutants; zebrafish vascular phenotype rescue\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro competition reconstituted, supported by ChIP and co-IP, functional in vivo validation\",\n      \"pmids\": [\"24940000\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PP2A (protein phosphatase 2A) dephosphorylates c-MYC at Ser62, destabilizing it; PP2A inhibitors SET (I2PP2A) and CIP2A are overexpressed in breast cancer and stabilize c-MYC by preventing PP2A-mediated dephosphorylation of S62; antagonizing SET with OP449 reduces S62 phosphorylation and c-MYC activity.\",\n      \"method\": \"PP2A inhibitor knockdown; phospho-specific Western blot for pS62-c-MYC; in vitro and in vivo tumor models; OP449 treatment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — bidirectional genetic manipulation of PP2A regulators with phospho-specific readout; in vitro and in vivo validation\",\n      \"pmids\": [\"24927563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HBXIP interacts directly with c-Myc through leucine zippers, recruits the lncRNA Hotair as a scaffold, and the Hotair-bound histone demethylase LSD1 mediates c-Myc-dependent transcriptional activation of target genes; silencing HBXIP, Hotair, or LSD1 blocks c-Myc-enhanced cancer cell growth.\",\n      \"method\": \"Co-immunoprecipitation; RNA immunoprecipitation; ChIP; siRNA knockdown; in vitro and xenograft tumor growth assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — multiple Co-IPs and ChIPs from single lab; no in vitro reconstitution of the multiprotein complex\",\n      \"pmids\": [\"26719542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CRY2 functions as a component of an FBXL3-containing E3 ubiquitin ligase that recruits T58-phosphorylated c-MYC for ubiquitylation and degradation; CRY1 cannot substitute for CRY2 in this process; this mechanism provides circadian control of c-MYC protein turnover.\",\n      \"method\": \"Co-immunoprecipitation; in vivo ubiquitylation assay; T58 phospho-mutant analysis; CRY1/CRY2 knockout comparison; proteasome inhibitor experiments\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — E3 complex reconstituted by Co-IP, phospho-degron mapping, CRY1 vs CRY2 specificity established\",\n      \"pmids\": [\"27840026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ELL (eleven-nineteen lysine-rich leukaemia) functions as a direct E3 ubiquitin ligase for c-Myc, with UbcH8 as the conjugating enzyme; Cys595 of ELL is the catalytic active site; ELL-mediated c-Myc ubiquitination and degradation inhibits c-Myc transcriptional activity and suppresses tumor growth in xenograft models.\",\n      \"method\": \"In vivo ubiquitination assay; active-site mutagenesis (C595A); Co-immunoprecipitation; c-Myc reporter assays; xenograft tumor growth\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — catalytic site mutagenesis defines mechanism; in vivo functional validation\",\n      \"pmids\": [\"27009366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SENP1 is a c-Myc deSUMOylating enzyme: it interacts with c-Myc, deSUMOylates it in cells and in vitro, and stabilizes it; catalytically inactive SENP1(C603S) cannot stabilize c-Myc; SENP1-mediated deSUMOylation reduces c-Myc polyubiquitination (SUMOylation promotes degradation) while promoting monoubiquitination and pS62/pT58 phosphorylation.\",\n      \"method\": \"Co-immunoprecipitation; in vitro deSUMOylation assay; catalytic mutant (C603S); ubiquitination assay; phospho-specific Western blot; cell cycle analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay plus catalytic mutant plus cellular validation; mechanistic crosstalk with ubiquitination established\",\n      \"pmids\": [\"30305424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Eya3 co-opts the PP2A-B55α holoenzyme (its Ser/Thr phosphatase activity is not intrinsic to Eya3 but arises from interaction with PP2A-B55α) to dephosphorylate pT58 on c-Myc, stabilizing c-Myc; this opposes the canonical PP2A-B56α-mediated dephosphorylation of pS62 that destabilizes c-Myc.\",\n      \"method\": \"Co-immunoprecipitation of PP2A-B55α with Eya3; phosphatase assay; phospho-specific Western blot (pT58 vs pS62); c-Myc stability assay; xenograft metastasis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — in vitro phosphatase assay, phospho-site specificity, Co-IP, functional in vivo validation; mechanistically distinguishes B55α vs B56α subunit specificity\",\n      \"pmids\": [\"29535359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-Myc overexpression in primary human fibroblasts markedly accelerates S-phase; WRN (Werner helicase) is required to support c-Myc-driven S-phase, as WRN depletion in c-Myc-overexpressing cells increases DNA damage at replication sites and activates an ATR-CHK1-CHK2-p53 'replication stress' pathway, leading to rapid senescence; p53 depletion rescues senescence.\",\n      \"method\": \"BrdU pulse-chase S-phase kinetics; c-Myc overexpression; WRN siRNA; DNA damage markers (γH2AX); ATR/CHK1/CHK2/p53 pathway analysis; senescence assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple genetic epistasis experiments with mechanistic pathway identification\",\n      \"pmids\": [\"19554081\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"p53 represses c-Myc post-transcriptionally by transcriptionally inducing miR-145 (via a p53 response element in the miR-145 promoter); miR-145 directly targets c-Myc mRNA; blockade of miR-145 reverses p53-mediated c-Myc repression.\",\n      \"method\": \"Promoter-reporter assay with p53 RE; miR-145 overexpression/anti-miR; 3'UTR luciferase reporter for c-Myc targeting; in vitro and in vivo tumor growth assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct p53 binding to miR-145 promoter plus miR-145 targeting of c-Myc 3'UTR validated by multiple methods\",\n      \"pmids\": [\"19202062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Deregulated c-Myc expression in keratinocytes (K14.MYC2 transgenic mice) reduces beta1 integrin expression and depletes epidermal stem cells by 75%, impairing wound healing and keratinocyte migration; beta1 integrin is essential for both keratinocyte migration and stem cell maintenance.\",\n      \"method\": \"K14.MYC2 transgenic mouse model; label-retaining cell analysis for stem cells; beta1 integrin immunostaining; wound healing assays\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — defined in vivo loss-of-function model with quantified stem cell depletion and molecular (beta1 integrin) mechanism\",\n      \"pmids\": [\"11381265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"USP43 stabilizes c-Myc by deubiquitinating it specifically at K148 and K289 through its deubiquitinase activity; USP43 competes with FBXW7 for c-Myc binding; USP43/c-Myc form a positive feedback loop in bladder cancer promoting glycolysis and metastasis.\",\n      \"method\": \"siRNA DUB library screen; co-immunoprecipitation; in vivo deubiquitination assay with lysine-specific mutants; FBXW7 competition assay; metabolic assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 — site-specific deubiquitination at K148/K289 established with lysine mutants, FBXW7 competition mechanism defined\",\n      \"pmids\": [\"38218970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Myc broadly represses miRNA expression by directly binding miRNA promoters (ChIP), providing a widespread reprogramming of the miRNA transcriptome that contributes to B-cell lymphomagenesis; enforced expression of Myc-repressed miRNAs diminishes tumorigenic potential of lymphoma cells.\",\n      \"method\": \"Genome-wide ChIP for Myc at miRNA loci; miRNA profiling in human and mouse B-cell lymphoma models; miRNA overexpression functional assays\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct ChIP evidence at miRNA promoters plus functional rescue experiments; replicated in human and mouse models\",\n      \"pmids\": [\"18066065\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"MYC and MYCN directly bind the mir-9-3 locus and activate miR-9 expression; miR-9 targets CDH1 (E-cadherin) mRNA, promoting cell motility and invasiveness; miR-9-mediated E-cadherin loss activates beta-catenin signaling, increasing VEGF expression and angiogenesis.\",\n      \"method\": \"ChIP for MYC/MYCN at mir-9-3 locus; miR-9 overexpression; 3'UTR luciferase reporter for CDH1; metastasis mouse model; sponge inhibition\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct ChIP, validated miRNA-target interaction, in vivo metastasis model\",\n      \"pmids\": [\"20173740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Endogenous c-Myc and Max protein-protein interactions can be directly visualized and quantified at single-molecule resolution in individual cells using proximity ligation; IFN-gamma signaling and small-molecule inhibitors (10058-F4) specifically regulate these interactions in situ.\",\n      \"method\": \"Proximity ligation assay (PLA) with antibodies against endogenous Myc and Max; rolling-circle amplification for single-molecule detection; quantification in response to IFN-gamma and small molecule inhibitors\",\n      \"journal\": \"Nature methods\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct visualization of endogenous protein-protein interaction in situ; validated by small-molecule disruption\",\n      \"pmids\": [\"17072308\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The small molecule 10058-F4 binds disordered c-Myc monomers and disrupts c-Myc-Max heterodimerization; the inhibitory effect depends on the Max isoform: p22 Max (which homodimerizes well) competes with c-Myc-Max heterodimerization, lowering the effective inhibitor concentration needed compared to p21 Max.\",\n      \"method\": \"Biophysical binding assays; EMSA for DNA binding; thermodynamic analysis of heterodimerization vs. homodimerization\",\n      \"journal\": \"Bioorganic & medicinal chemistry letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab biophysical characterization; no structural or mutagenesis validation\",\n      \"pmids\": [\"19114306\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The ASK1-JNK pathway promotes c-Myc protein stability through phosphorylation at Ser62 and Ser71, which is required for c-Myc-dependent apoptosis; ASK1 signaling attenuates degradation of already-ubiquitinated c-Myc without affecting the ubiquitination process itself.\",\n      \"method\": \"ASK1 overexpression; phospho-site mutant c-Myc constructs (Ser62, Ser71); ubiquitination assay; proteasome inhibitor analysis; apoptosis assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single lab; phospho-mutant analysis without in vitro kinase reconstitution for ASK1 directly phosphorylating these sites\",\n      \"pmids\": [\"11243879\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"c-Myc overexpression accelerates S-phase, and c-Myc-deficient fibroblasts exhibit prolonged S-phase; WRN helicase depletion in c-Myc-overexpressing cells causes DNA damage at active replication forks and activates ATR-CHK1-CHK2-p53 senescence pathway, suggesting WRN repairs aberrant replication structures caused by c-Myc-driven accelerated replication.\",\n      \"method\": \"BrdU pulse-chase S-phase kinetics; c-Myc overexpression and knockout fibroblasts; WRN siRNA; DNA damage markers at replication sites; senescence assay; p53 knockdown rescue\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — quantitative S-phase measurement, genetic epistasis with multiple pathway components\",\n      \"pmids\": [\"19554081\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"c-Myc is a nuclear bHLH-Zip transcription factor that heterodimerizes with Max to bind E-box (CACGTG) sequences and regulate transcription of a vast target gene network (~15% of all genes) governing cell proliferation, growth, apoptosis, metabolism (including glutamine catabolism and aerobic glycolysis via miR-23a/b repression and hnRNP upregulation), and DNA replication initiation (via direct interaction with the pre-replicative complex); its activity and stability are tightly controlled by a phosphorylation switch (GSK3-mediated pT58 promotes FBXW7/SCF-mediated proteasomal degradation, while Ras/ERK-mediated pS62 stabilizes it, with PP2A-B56α dephosphorylating pS62 to destabilize and PP2A-B55α/Eya3 dephosphorylating pT58 to stabilize), multiple ubiquitin ligases (FBXW7, ELL) and deubiquitinases (USP28, USP37, USP43, USP36), SUMO modification (reversed by SENP1), O-GlcNAc glycosylation of its transactivation domain, negative feedback from ribosomal proteins L11 and S14 that compete with TRRAP coactivator, and negative autoregulation of its own promoter; c-Myc also directly controls immune evasion through transcriptional activation of CD47 and PD-L1, and its overexpression causes genomic instability through ROS induction, replication stress, and mitotic checkpoint override.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MYC encodes the c-Myc transcription factor, a master regulator of cell proliferation, apoptosis, differentiation, and metabolism that heterodimerizes with Max through its bHLH-LZ domain to bind E-box sequences (CACGTG) at promoters in open chromatin, where it amplifies the existing transcriptional program of active genes and directly induces targets including ODC, CDK4, and the miR-17-92 cluster while repressing p21 (via AP4) and PDGF-β receptor (via NF-Y inhibition) [PMID:1587829, PMID:23021215, PMID:8356088, PMID:10688915, PMID:15944709, PMID:11282029]. c-Myc protein stability is governed by sequential phosphorylation at Ser62 (stabilizing; by Ras/ERK, Pim kinases, ASK1-JNK) and Thr58 (destabilizing; by GSK3β), with PP2A-B56α dephosphorylating pS62 to promote degradation and PP2A-B55α/Eya3 dephosphorylating pT58 to stabilize it, while multiple E3 ligases (FBXW7, CRY2/FBXL3, ELL) and deubiquitinases (USP28, USP37, USP43) and the deSUMOylase SENP1 further tune c-Myc turnover [PMID:15467447, PMID:18438430, PMID:27840026, PMID:27009366, PMID:25284584, PMID:38218970, PMID:30305424, PMID:29535359, PMID:24927563]. Beyond transcription, c-Myc has a direct non-transcriptional role in DNA replication initiation through interaction with the pre-replicative complex, and its overexpression increases replication origin firing, causing replication stress and DNA damage [PMID:17597761, PMID:19554081]. Under growth-limiting conditions, c-Myc triggers p53-dependent apoptosis, and deregulated c-Myc expression blocks differentiation programs including myogenesis and granulopoiesis and depletes epidermal stem cells [PMID:1555236, PMID:8091232, PMID:1850105, PMID:11340171, PMID:11381265].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Establishing that c-Myc negatively autoregulates its own transcription answered how cells buffer against fluctuations in MYC expression and revealed a dose-dependent feedback mechanism at the transcriptional level.\",\n      \"evidence\": \"Stable transfection of activated c-myc/v-myc in Rat-1 fibroblasts with quantitative RNA analysis\",\n      \"pmids\": [\"2182320\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trans-acting factors required for autorepression were not identified\", \"Whether autoregulation operates in all cell types was not established\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Demonstrating that c-Myc blocks MyoD/myogenin-driven myogenesis independently of Id established c-Myc as a dominant inhibitor of terminal differentiation programs, linking proliferation to differentiation suppression.\",\n      \"evidence\": \"Conditional c-myc induction with cotransfection of MyoD and myogenin in NIH 3T3 cells\",\n      \"pmids\": [\"1850105\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular mechanism by which c-Myc antagonizes myogenic factors was not resolved\", \"Whether this extends to other lineage-specific differentiation was not tested\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Identification of the c-Myc/Max heterodimer as the DNA-binding species recognizing the CACGTG E-box consensus resolved how c-Myc achieves sequence-specific transcriptional control and established the obligate partnership with Max.\",\n      \"evidence\": \"Biochemical purification from baculovirus/insect cells, SELEX, gel mobility shift assays; nuclear localization and dimerization studies\",\n      \"pmids\": [\"1587829\", \"1521738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Max-independent activities might operate was unknown\", \"Structural basis of heterodimer selectivity over homodimer was not determined\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Discovery that c-Myc drives apoptosis in growth-arrested fibroblasts — with the apoptotic domains overlapping transformation/autoregulation domains — revealed a dual-outcome model in which c-Myc promotes either proliferation or death depending on context.\",\n      \"evidence\": \"Conditional c-myc expression in Rat-1 fibroblasts under serum deprivation, deletion mutant domain mapping\",\n      \"pmids\": [\"1555236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The downstream apoptotic pathway was unknown\", \"Whether apoptosis required transcriptional activation was unclear\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Genetic epistasis showing that c-Myc-induced apoptosis requires p53 — Myc activation stabilizes p53 and p53-null cells are resistant — defined the central apoptotic pathway downstream of c-Myc.\",\n      \"evidence\": \"Conditional c-Myc-ER activation in wild-type vs. p53-null mouse fibroblasts\",\n      \"pmids\": [\"8091232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How c-Myc stabilizes p53 (direct vs. indirect) was not determined\", \"p53-independent apoptotic pathways were not excluded in other cell types\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identification of ODC as a direct c-Myc target through intronic E-boxes provided the first validated endogenous transcriptional target and confirmed E-box dependence of transactivation.\",\n      \"evidence\": \"Reporter gene assays with ODC E-box mutants and c-Myc domain deletion/point mutants\",\n      \"pmids\": [\"8356088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide target scope was unknown\", \"Whether all targets use intronic vs. promoter E-boxes was unresolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Placing c-MYC as a direct transcriptional target of the APC/β-catenin/Tcf-4 pathway connected Wnt signaling to proliferative control and explained why APC loss in colorectal cancer upregulates c-MYC.\",\n      \"evidence\": \"Reporter gene assays with Tcf-4 binding site mutants, expression manipulation of APC and β-catenin\",\n      \"pmids\": [\"9727977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin context of Tcf-4-mediated activation was not explored\", \"Contribution relative to other c-MYC inducers in vivo was unclear\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying CDK4 as a direct c-Myc transcriptional target whose ectopic expression partially rescues c-Myc-null proliferation defects established a key effector link between c-Myc and cell-cycle entry.\",\n      \"evidence\": \"SAGE, promoter-reporter assays with E-box mutants, ChIP, genetic rescue in c-MYC-deficient cells\",\n      \"pmids\": [\"10688915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"CDK4 rescue was partial, implying additional essential targets\", \"Quantitative contribution of CDK4 vs. other targets was not measured\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Multiple upstream pathways converging on c-MYC transcription were delineated — C/EBPα repression via E2F sites (linking differentiation to c-MYC silencing), TGF-β/Smad3 repression through a TIE/E2F element, and PDGF/Src/Vav2/Rac induction — revealing c-MYC as an integrator node for diverse proliferative and differentiation signals.\",\n      \"evidence\": \"Promoter mutagenesis, EMSA, Co-IP, reporter assays, epistasis with dominant-negative pathway components across multiple labs\",\n      \"pmids\": [\"11340171\", \"11689553\", \"11389443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Combinatorial integration of simultaneous signals at the c-MYC promoter was not modeled\", \"Whether all pathways operate in the same cell types was not established\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showing that c-Myc represses PDGF-β receptor transcription by binding NF-Y through Myc boxes (not bHLH-LZ) established a non-E-box mechanism for c-Myc-mediated gene repression.\",\n      \"evidence\": \"GST pulldown, Co-IP, reporter assays with domain mutants, EMSA\",\n      \"pmids\": [\"11282029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of NF-Y-dependent repression across other repressed targets was unknown\", \"Whether repression requires additional cofactors was not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Genome-wide ChIP-on-chip revealed that c-Myc preferentially binds promoter-proximal E-boxes in CpG islands within pre-acetylated chromatin and further enhances histone acetylation, clarifying how c-Myc selects target loci genome-wide.\",\n      \"evidence\": \"Quantitative ChIP with genomic arrays (~700 loci) in human cells\",\n      \"pmids\": [\"12695333\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether c-Myc can pioneer closed chromatin was not addressed\", \"Low-affinity and non-E-box binding detected at high c-Myc levels was mechanistically uncharacterized\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Elucidation of the S62/T58 phosphorylation switch — Ras/Raf phosphorylates S62 to stabilize c-Myc, GSK3β phosphorylates T58 to prime degradation, and PP2A dephosphorylates pS62 — established the core post-translational mechanism controlling c-Myc turnover.\",\n      \"evidence\": \"Protein stability assays, phosphorylation site mutagenesis, kinase/phosphatase inhibitor studies\",\n      \"pmids\": [\"15467447\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct reconstitution of the full pathway in a cell-free system was not shown\", \"The role of Pin1 prolyl isomerase in the switch was not fully resolved biochemically\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Discovery that c-Myc directly activates the miR-17-92 cluster, whose products (miR-17-5p, miR-20a) repress E2F1 translation, uncovered a regulatory circuit in which c-Myc simultaneously activates E2F1 transcription and restrains its protein output — demonstrating microRNA-mediated fine-tuning of oncogenic programs.\",\n      \"evidence\": \"ChIP, microRNA expression profiling, luciferase reporter assays\",\n      \"pmids\": [\"15944709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Quantitative balance between transcriptional activation and translational repression of E2F1 was not measured\", \"Other miR-17-92 targets contributing to c-Myc biology were not systematically evaluated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstration that c-Myc interacts with the pre-replicative complex and is required for DNA replication in transcription-free Xenopus extracts established a direct, non-transcriptional role in replication initiation — expanding c-Myc function beyond transcription factor activity.\",\n      \"evidence\": \"Co-IP with pre-RC components, DNA fiber assays, siRNA in mammalian cells, Xenopus cell-free extract reconstitution\",\n      \"pmids\": [\"17597761\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which pre-RC subunit directly contacts c-Myc was not determined\", \"Whether excess origin firing fully accounts for c-Myc-induced genomic instability was unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identification of ribosomal protein L11 as a c-Myc inhibitor that competes with TRRAP for MBII binding and suppresses histone acetylation at target promoters established a ribosome biogenesis–linked negative feedback loop on c-Myc transcriptional activity.\",\n      \"evidence\": \"Co-IP, ChIP, siRNA knockdown, promoter reporter assays\",\n      \"pmids\": [\"17599065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether L11 inhibition is activated by ribosomal stress was not tested\", \"Structural basis of L11–MBII interaction was not resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Pim kinases were shown to stabilize c-Myc via phosphorylation at Ser329 and modulation of the T58/S62 axis, adding a new kinase input and phosphorylation site to c-Myc stability regulation.\",\n      \"evidence\": \"In vitro kinase assays, phosphomimetic/phosphodeficient mutants, co-expression and knockdown studies, transformation assays\",\n      \"pmids\": [\"18438430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How Ser329 phosphorylation mechanistically prevents degradation was not resolved\", \"Relative contribution of Pim vs. Ras/ERK to S62 phosphorylation in vivo was not quantified\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Genome-wide nascent transcription analysis revealed that elevated c-Myc does not activate a new gene set but amplifies the output of already-active genes — redefining c-Myc as a general transcriptional amplifier rather than a conventional sequence-specific activator of select targets.\",\n      \"evidence\": \"ChIP-seq, GRO-seq, quantitative RNA analysis across tumor and normal cell lines\",\n      \"pmids\": [\"23021215\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether amplification is uniform across all active genes or selective was debated\", \"Molecular mechanism of amplification (e.g., pause release, elongation, initiation) was not fully resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Multiple layers of c-Myc stability regulation were uncovered including deubiquitination by USP37, PP2A/SET/CIP2A antagonism at pS62, and lncRNA PCGEM1-mediated enhancement of c-Myc chromatin recruitment and metabolic gene activation — expanding both post-translational and RNA-based regulatory mechanisms.\",\n      \"evidence\": \"In vivo/in vitro deubiquitination assays with catalytic mutant (USP37); PP2A inhibitor knockdown and pharmacological activation (SET/CIP2A); RNA-IP, ChIP, metabolic flux assays (PCGEM1)\",\n      \"pmids\": [\"25284584\", \"24927563\", \"25512540\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of USP37 vs. other DUBs to steady-state c-Myc levels was not determined\", \"PCGEM1 mechanism was characterized only in prostate cancer cells\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Two new E3 ubiquitin ligases targeting c-Myc were identified: CRY2/FBXL3 recognizing pT58-c-Myc (linking circadian clock to c-Myc turnover) and ELL acting through its catalytic C595 residue with UbcH8 — diversifying the ubiquitin ligase landscape controlling c-Myc degradation.\",\n      \"evidence\": \"Co-IP, ubiquitylation assays, isoform-specific mutagenesis (CRY2 vs. CRY1), active-site mutagenesis (ELL C595A)\",\n      \"pmids\": [\"27840026\", \"27009366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How CRY2/FBXL3 and FBXW7 are differentially deployed was not resolved\", \"ELL's E3 ligase function has not been broadly confirmed outside this study\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Two opposing post-translational modifications were mechanistically resolved: Eya3 redirects PP2A-B55α to dephosphorylate pT58 (stabilizing c-Myc), and SENP1 deSUMOylates c-Myc to block SUMOylation-dependent ubiquitination — revealing that both phosphatase subunit specificity and SUMO cross-talk control c-Myc levels.\",\n      \"evidence\": \"Co-IP, phosphatase activity assays with PP2A subunit specificity (Eya3); in vitro deSUMOylation with catalytically inactive SENP1 C603S, ubiquitination assays (SENP1)\",\n      \"pmids\": [\"29535359\", \"30305424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SUMOylation sites on c-Myc overlap with or are distinct from ubiquitination sites was not fully mapped\", \"Physiological contexts in which Eya3/B55α stabilization dominates were not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"USP43 was identified as a c-Myc deubiquitinase acting at K148 and K289, directly competing with FBXW7 access, and forming a positive feedback loop with c-Myc in bladder cancer — establishing site-specific deubiquitination as a mechanism to evade FBXW7-mediated degradation.\",\n      \"evidence\": \"siRNA library screen, Co-IP, in vivo deubiquitination assay with K148/K289 mutants, FBXW7 competition assay\",\n      \"pmids\": [\"38218970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether USP43 acts on the same or different ubiquitin chain types as FBXW7 was not resolved\", \"Generalizability beyond bladder cancer was not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How c-Myc's transcriptional amplifier function, non-transcriptional replication role, and multilayered post-translational regulation are quantitatively integrated in vivo to determine cell fate (proliferation vs. apoptosis vs. differentiation) remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of c-Myc in complex with Max and DNA at atomic resolution from the timeline\", \"The relative contribution of each E3 ligase and DUB to c-Myc turnover in normal vs. tumor contexts is not quantified\", \"How transcriptional amplification is mechanistically achieved (pause release, initiation, elongation) is debated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [4, 17, 18, 39]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 3, 4, 7, 12, 26, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [18, 39]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [6, 39]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 3, 4, 7, 12, 26, 39]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [7, 23, 24]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [6, 24]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 5]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [0, 22, 34]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9, 10, 13, 14, 15, 16, 36, 38]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [21, 33, 40]}\n    ],\n    \"complexes\": [\n      \"Myc/Max heterodimer\"\n    ],\n    \"partners\": [\n      \"MAX\",\n      \"FBXW7\",\n      \"TRRAP\",\n      \"RPL11\",\n      \"RPS14\",\n      \"USP37\",\n      \"USP43\",\n      \"SENP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"MYC encodes a nuclear bHLH-Zip transcription factor that heterodimerizes with Max to bind E-box sequences (CACGTG) and functions as a master regulator of cell proliferation, apoptosis, metabolism, and immune evasion [PMID:2006410, PMID:1587829]. Rather than activating a discrete set of target genes, c-Myc amplifies the existing transcriptional program of active genes by promoting RNA Pol II pause release, directly inducing cell-cycle drivers (CDK4, E2F1 via miR-17-92), metabolic reprogramming enzymes (glutaminase via miR-23a/b repression, PKM2 via hnRNP upregulation), and immune checkpoint ligands CD47 and PD-L1 [PMID:20434984, PMID:23021215, PMID:10688915, PMID:19219026, PMID:20010808, PMID:26966191]. c-Myc protein stability is governed by an integrated phospho-degron switch—GSK3-mediated Thr58 phosphorylation targets c-Myc to SCF(FBXW7)- and CRY2/FBXL3-dependent ubiquitination and degradation, while Ser62 phosphorylation by ERK/Pim kinases stabilizes the protein, with opposing PP2A holoenzymes (B56α dephosphorylating pS62 to destabilize; B55α/Eya3 dephosphorylating pT58 to stabilize) and multiple deubiquitinases (USP28, USP37, USP43) and SENP1-mediated deSUMOylation providing additional layers of control [PMID:15150404, PMID:27840026, PMID:24927563, PMID:29535359, PMID:25284584, PMID:38218970, PMID:30305424]. Beyond transcription, c-Myc directly participates in DNA replication initiation by interacting with pre-replicative complex components, and its deregulation causes replication stress, reactive oxygen species, and genomic instability that are counteracted by p53-dependent apoptosis [PMID:17597761, PMID:12049739, PMID:1555236, PMID:8091232].\",\n  \"teleology\": [\n    {\n      \"year\": 1990,\n      \"claim\": \"Establishing that c-Myc negatively autoregulates its own promoter in a dose-dependent manner revealed an intrinsic feedback mechanism constraining its expression, setting the stage for understanding how deregulation (e.g., translocation) bypasses this control.\",\n      \"evidence\": \"Transfection of activated c-myc constructs into Rat-1 fibroblasts with run-on transcription measurements\",\n      \"pmids\": [\"2182320\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"trans-acting cofactors required for autorepression were not identified\", \"mechanism of promoter sensing (cis elements) not defined\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Identification of Max as the obligate heterodimerization partner for c-Myc DNA binding resolved how c-Myc, which binds DNA poorly alone, achieves sequence-specific E-box recognition, establishing the Myc-Max paradigm central to all subsequent target gene studies.\",\n      \"evidence\": \"cDNA expression library screen with bHLH-Zip bait; co-immunoprecipitation; EMSA with purified proteins\",\n      \"pmids\": [\"2006410\", \"1587829\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis of heterodimerization not yet resolved\", \"role of Max homodimers as antagonists not yet explored\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Demonstrating that constitutive c-Myc expression induces apoptosis—using the same domains required for transformation—established the dual nature of c-Myc as both an oncogene and a pro-apoptotic factor, resolving the paradox of why tumors must disable apoptosis for Myc-driven oncogenesis.\",\n      \"evidence\": \"Conditional c-Myc expression in Rat-1 fibroblasts with domain-deletion mutants and serum deprivation\",\n      \"pmids\": [\"1555236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"downstream apoptotic effectors not identified at this stage\", \"cell-type dependence of apoptotic threshold not established\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Placing p53 as an epistatic mediator of c-Myc-induced apoptosis explained how p53 loss cooperates with Myc in tumorigenesis by disconnecting the apoptotic failsafe from proliferative signaling.\",\n      \"evidence\": \"Conditional MycER activation in wild-type vs. p53-null fibroblasts; p53 protein stabilization\",\n      \"pmids\": [\"8091232\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"mechanism by which c-Myc stabilizes p53 protein not defined\", \"alternative p53-independent apoptotic pathways not excluded\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identification of ODC as a direct c-Myc transcriptional target through intronic E-boxes provided the first concrete downstream effector linking c-Myc to a specific biosynthetic pathway (polyamine synthesis).\",\n      \"evidence\": \"Promoter-reporter cotransfection with E-box mutagenesis and c-Myc domain deletions\",\n      \"pmids\": [\"8356088\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"physiological relevance of the HLH/Zip-independent transactivation not clarified\", \"contribution of ODC to Myc-driven transformation not tested genetically\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Discovery that c-MYC is a direct transcriptional target of the APC/β-catenin/Tcf-4 pathway connected Wnt signaling to proliferative gene expression and explained c-MYC overexpression in APC-mutant colorectal cancers.\",\n      \"evidence\": \"Reporter assays with Tcf-4 binding site mutants; APC/β-catenin gain- and loss-of-function in colorectal cancer cells\",\n      \"pmids\": [\"9727977\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"quantitative contribution of Wnt-driven MYC vs. other MYC-activating signals in vivo unknown\", \"chromatin-level mechanism of Tcf-4 activation not addressed\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying CDK4 as a direct c-Myc target, with genetic rescue of c-Myc-deficient cell-cycle defects by ectopic CDK4, established a concrete mechanism for c-Myc-driven G1/S progression.\",\n      \"evidence\": \"SAGE; ChIP at CDK4 promoter; c-MYC-null RAT1 cells; CDK4 rescue\",\n      \"pmids\": [\"10688915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"relative contribution of CDK4 vs. other Myc targets to proliferation not quantified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Multiple studies converged to define how upstream signals regulate c-myc transcription—C/EBPα represses it via E2F sites during myeloid differentiation, Smad3/E2F-4 mediate TGF-β repression by displacing p300, and Src-Vav2-Rac activates it downstream of PDGF—revealing c-myc as an integrating node for diverse signaling pathways.\",\n      \"evidence\": \"Promoter deletion/mutation analyses; Co-IP for p300 dissociation; dominant-negative signaling epistasis in multiple cell types\",\n      \"pmids\": [\"11340171\", \"11689553\", \"11389443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"chromatin remodeling events at the c-myc promoter during signal integration not resolved\", \"combinatorial regulation by simultaneous signals not tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstration that c-Myc represses the PDGFβR gene by binding NF-Y subunits (YB/YC) through Myc homology boxes—without displacing NF-Y from DNA—revealed an E-box-independent repression mechanism expanding c-Myc's regulatory repertoire.\",\n      \"evidence\": \"GST pulldown; cellular Co-IP; GAL4-NF-YC transactivation assay with CCAAT mutants\",\n      \"pmids\": [\"11282029\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"genome-wide extent of Myc-NF-Y repression not determined\", \"structural basis of Myc-NF-Y interaction unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Showing that deregulated c-Myc induces reactive oxygen species and DNA damage prior to S-phase, rescuable by antioxidants, established a direct mechanistic link between c-Myc overexpression and genomic instability independent of replication.\",\n      \"evidence\": \"Conditional c-Myc activation; comet assay; ROS measurement; antioxidant rescue in human fibroblasts\",\n      \"pmids\": [\"12049739\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"source of ROS (mitochondrial vs. enzymatic) not identified\", \"specific oxidized DNA lesions not characterized\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Reconstitution of SCF(FBXW7)-mediated c-Myc ubiquitination dependent on GSK3-phosphorylated Thr58 defined the core phospho-degron controlling c-Myc turnover and explained why T58 is a lymphoma hotspot mutation.\",\n      \"evidence\": \"In vitro ubiquitination reconstitution with FBXW7; T58 phospho-mutant analysis; in vivo degradation assays\",\n      \"pmids\": [\"15150404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"relative contribution of FBXW7 vs. other E3 ligases to steady-state Myc turnover not quantified\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Discovery that c-Myc directly activates the miR-17-92 cluster, whose products repress E2F1 translation, uncovered a feedforward loop whereby c-Myc fine-tunes its own proliferative output through miRNA regulation.\",\n      \"evidence\": \"ChIP for c-Myc at miR-17-92 locus; miRNA overexpression/knockdown; E2F1 3'UTR reporters\",\n      \"pmids\": [\"15944709\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"individual contributions of the six cluster miRNAs not fully delineated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Three parallel advances redefined c-Myc's scope: (1) a direct, transcription-independent role in DNA replication initiation via pre-RC interaction, (2) a ribosomal protein L11 negative feedback loop competing with TRRAP, and (3) genome-wide miRNA repression as a mechanism of lymphomagenesis.\",\n      \"evidence\": \"Xenopus cell-free replication extracts (transcription-free); Co-IP with pre-RC components; L11-MBII binding and TRRAP competition by ChIP; genome-wide miRNA ChIP in lymphoma models\",\n      \"pmids\": [\"17597761\", \"17599065\", \"18066065\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"structural basis of Myc-pre-RC interaction unknown\", \"whether L11 feedback operates at all Myc target promoters not tested\", \"selectivity of Myc-mediated miRNA repression vs. activation not fully explained\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Identification of Pim kinases as stabilizers of c-Myc—Pim-2 directly phosphorylating Ser329 and Pim-1 modulating the Thr58/Ser62 balance—added a cooperative oncogenic kinase axis to the phospho-degron switch.\",\n      \"evidence\": \"In vitro kinase assay; phospho-site mutants; transformation assays\",\n      \"pmids\": [\"18438430\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"in vivo Pim-Myc axis contribution vs. PI3K/ERK not dissected genetically\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Multiple discoveries linked c-Myc to metabolic reprogramming and replication stress: miR-23a/b repression upregulated glutaminase for glutamine catabolism, WRN helicase was shown essential for resolving c-Myc-driven replication stress, and p53-induced miR-145 was identified as a post-transcriptional suppressor of c-Myc, closing a p53-Myc regulatory loop.\",\n      \"evidence\": \"ChIP at miR-23a/b promoters with metabolic flux assays; BrdU pulse-chase with WRN siRNA and DNA damage markers; miR-145 3'UTR reporters with p53 RE validation\",\n      \"pmids\": [\"19219026\", \"19554081\", \"19202062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"relative importance of glutaminase vs. other metabolic targets in Myc-driven tumors not established\", \"how WRN is specifically recruited to Myc-induced aberrant structures unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Genome-wide studies revealed that c-Myc acts primarily through Pol II pause release rather than recruitment, and at high levels amplifies the existing transcriptional program rather than activating novel targets—fundamentally reframing c-Myc as a universal transcriptional amplifier.\",\n      \"evidence\": \"ChIP-seq/GRO-seq for Pol II and c-Myc; RNA-seq comparing high vs. low Myc tumor cells\",\n      \"pmids\": [\"20434984\", \"23021215\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"how pause-release mechanism relates to Myc-mediated repression not reconciled\", \"whether amplifier model applies to all tissues and contexts untested\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identification of miR-9 as a direct MYC/MYCN target that represses E-cadherin to promote invasiveness and angiogenesis via β-catenin/VEGF signaling established a miRNA-mediated mechanism for Myc-driven metastasis.\",\n      \"evidence\": \"ChIP at mir-9-3; CDH1 3'UTR reporter; metastasis mouse model\",\n      \"pmids\": [\"20173740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"contribution of miR-9 vs. other EMT-promoting Myc targets to metastasis not quantified\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Multiple new layers of c-Myc regulation were defined: USP37 as a deubiquitinase stabilizing c-Myc, PP2A-B56α/SET/CIP2A axis controlling pS62 dephosphorylation, and SerRS competing with c-Myc at the VEGFA promoter while recruiting SIRT2 to erase Myc-driven histone acetylation.\",\n      \"evidence\": \"In vitro DUB assay with catalytic mutant; phospho-specific Western blots with PP2A inhibitor manipulation; in vitro promoter competition and ChIP with SIRT2 Co-IP\",\n      \"pmids\": [\"25284584\", \"24927563\", \"24940000\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"USP37 substrate specificity beyond Myc not determined\", \"physiological contexts where SerRS-Myc competition is rate-limiting unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Discovery that CRY2 recruits T58-phosphorylated c-Myc to an FBXL3-containing E3 ligase provided a circadian dimension to Myc turnover, while ELL was identified as a novel E3 ligase with a catalytic cysteine (C595) for direct Myc ubiquitination.\",\n      \"evidence\": \"Co-IP with T58 phospho-mutant and CRY1/CRY2 knockout comparison; ELL C595A catalytic mutant ubiquitination assay; xenograft validation\",\n      \"pmids\": [\"27840026\", \"27009366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"circadian oscillation of Myc protein in normal tissues not directly measured\", \"ELL's relative contribution to total Myc ubiquitination unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"MYC was shown to directly activate transcription of immune checkpoint genes CD47 and PD-L1, with genetic rescue demonstrating these are necessary for tumor immune evasion—establishing MYC as a direct link between oncogene activation and immunosuppression.\",\n      \"evidence\": \"ChIP at Cd47/Pd-l1 promoters; conditional MYC inactivation in mouse tumors; enforced CD47/PD-L1 rescue\",\n      \"pmids\": [\"26966191\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"whether MYC-driven immune evasion operates in all tumor types not tested\", \"mechanism of MYC-dependent regulation of other immune modulators not explored\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Two studies dissected opposing phosphatase arms controlling Myc stability: PP2A-B55α/Eya3 dephosphorylates pT58 to stabilize Myc, while PP2A-B56α dephosphorylates pS62 to destabilize it; separately, SENP1 was shown to deSUMOylate c-Myc, reducing polyubiquitination and stabilizing the protein, revealing SUMO-ubiquitin crosstalk in Myc turnover.\",\n      \"evidence\": \"In vitro phosphatase assays with B-subunit specificity; SENP1 catalytic mutant (C603S); phospho-specific and ubiquitin/SUMO blots\",\n      \"pmids\": [\"29535359\", \"30305424\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"spatial regulation of PP2A holoenzyme assembly on Myc not addressed\", \"SUMO acceptor lysines on Myc not mapped\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"USP43 was identified as a deubiquitinase targeting specific lysines (K148 and K289) on c-Myc and competing with FBXW7 for binding, forming a positive feedback loop that drives glycolysis and metastasis in bladder cancer.\",\n      \"evidence\": \"siRNA DUB library screen; lysine-specific mutant deubiquitination assays; FBXW7 competition Co-IP; metabolic and metastasis assays\",\n      \"pmids\": [\"38218970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"whether USP43-Myc axis is relevant beyond bladder cancer not tested\", \"structural basis of FBXW7/USP43 competition unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Despite detailed mapping of individual regulatory inputs, a quantitative integrated model of how phosphorylation, ubiquitination, SUMOylation, and O-GlcNAcylation combinatorially determine c-Myc half-life in specific tissues remains unbuilt; the structural basis of c-Myc's intrinsically disordered transactivation domain interactions with diverse partners (TRRAP, L11, NF-Y, pre-RC) also awaits high-resolution characterization.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"no integrated quantitative model of multi-PTM crosstalk on Myc stability\", \"no high-resolution structure of full-length Myc-Max in complex with chromatin or coactivators\", \"tissue-specific variation in Myc regulatory network topology not systematically mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 4, 17, 24, 26]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3, 4, 10, 11, 17, 22, 23, 24, 25, 26, 41, 42]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 7, 19, 24, 26]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0074160\", \"supporting_discovery_ids\": [3, 4, 10, 17, 22, 23, 24, 25, 26, 41, 42]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [9, 10, 19, 37, 46]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 13, 14]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [16, 28, 33, 34, 35, 40]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [22, 23]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [25]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"R-HSA-9909396\", \"supporting_discovery_ids\": [33]}\n    ],\n    \"complexes\": [\n      \"Myc-Max heterodimer\"\n    ],\n    \"partners\": [\n      \"MAX\",\n      \"FBXW7\",\n      \"TRRAP\",\n      \"RPL11\",\n      \"RPS14\",\n      \"USP37\",\n      \"USP43\",\n      \"SENP1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}