{"gene":"MAX","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1992,"finding":"Myc and Max associate in vivo: coimmunoprecipitation with anti-Myc and anti-Max antibodies showed that essentially all newly synthesized Myc is complexed with Max in cells; the complex possesses specific DNA-binding activity for CACGTG-containing oligonucleotides; Max is a stable nuclear phosphoprotein whose expression level is constant across quiescent, mitogen-stimulated, and cycling cells, whereas Myc is rapidly degraded during/after association with Max.","method":"Coimmunoprecipitation, DNA binding assay, metabolic labeling, subcellular fractionation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal co-IP with anti-Myc and anti-Max antibodies, replicated across multiple labs in the same era","pmids":["1730411"],"is_preprint":false},{"year":1992,"finding":"Max functional domains mapped: Max requires intact HLH and leucine zipper motifs for intracellular interaction with c-Myc; a nuclear localization signal (PQSRKKLR) was mapped to the carboxy-terminal region of Max; Max lacks a transcriptional activation domain functional in CHO cells, suggesting it acts as a cofactor or transcriptional repressor.","method":"Fusion protein expression in cultured cells, deletion analysis, coimmunoprecipitation, reporter gene assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — direct mutagenesis/deletion mapping of functional domains plus in-cell interaction assays, replicated conceptually across multiple studies","pmids":["1730412"],"is_preprint":false},{"year":1992,"finding":"Max overexpression represses transcription of a CACGTG-containing reporter gene in mammalian cells, and this repression is relieved by co-expression of c-Myc; repression requires the Max DNA-binding domain, while relief requires Myc dimerization and transactivation domains.","method":"Transient transfection reporter gene assay in mammalian cells, domain deletion analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — functional reporter assay with domain mutants, independently replicated by multiple labs","pmids":["1406956"],"is_preprint":false},{"year":1992,"finding":"Max homodimers and c-Myc/Max heterodimers both bind CACGTG-containing DNA sequences using a dimeric structure (demonstrated by chemical and photo-cross-linking); full-length c-Myc alone cannot bind DNA but does so in complex with Max.","method":"Chemical and photo-cross-linking, electrophoretic mobility shift assay (EMSA), bacterially produced proteins","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with cross-linking, replicated across multiple studies","pmids":["1730412"],"is_preprint":false},{"year":1992,"finding":"Casein kinase II (CKII) phosphorylates Max homodimers in vitro and inhibits their DNA-binding activity in an ATP-dependent manner; this inhibition maps to a CKII phosphorylation site in the amino terminus of Max; Myc/Max heterodimer DNA-binding activity is not inhibited by CKII phosphorylation; Max is phosphorylated in NIH-3T3 cells and can bind DNA after phosphatase treatment or heterodimerization with Myc.","method":"In vitro kinase assay with purified bovine CKII, EMSA, deletion analysis and site-directed mutagenesis, phosphatase treatment, immunoprecipitation from cells","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus cell-based phosphorylation, replicated and extended in subsequent studies","pmids":["1737614"],"is_preprint":false},{"year":1992,"finding":"An alternatively spliced mRNA encodes delta-Max, a truncated form lacking the C-terminal nuclear localization signal and putative regulatory domain; delta-Max retains binding to CACGTG in complex with c-Myc but lacks nuclear localization; in a Myc-Ras cotransformation assay, full-length Max suppresses transformation whereas delta-Max enhances it, indicating the two isoforms have opposing regulatory effects on c-Myc function.","method":"cDNA cloning, Myc-Ras cotransformation assay in rat embryo fibroblasts","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional transformation assay in cells, single lab, two methods (molecular cloning + biological assay)","pmids":["1566084"],"is_preprint":false},{"year":1992,"finding":"c-Myc and Max homodimers bend DNA in opposite orientations when bound to CACGTG, as measured by circular permutation and phasing analysis; c-Myc-Max heterodimers cause a smaller bend in an orientation similar to Max homodimers; no specific DNA unwinding was detected.","method":"Circular permutation assay, phasing analysis, EMSA","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biophysical assay, single lab","pmids":["1323849"],"is_preprint":false},{"year":1992,"finding":"In cell extracts virtually all c-Myc is associated with Max in heterodimeric complexes; c-Myc cannot bind CACGTG in the absence of Max, whereas both Max alone and c-Myc/Max bind the same DNA sequence.","method":"Cell extract EMSA, immunoprecipitation","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell extract binding studies and immunoprecipitation, consistent with multiple independent studies","pmids":["1501888"],"is_preprint":false},{"year":1993,"finding":"Mad (a bHLH-Zip protein) binds Max in vitro to form a sequence-specific DNA binding complex with properties similar to Myc-Max; Mad does not homodimerize efficiently and does not associate with Myc; Mad-Max and Myc-Max heterocomplexes are both favored over Max homodimers; CKII phosphorylation does not affect heterodimer DNA binding; in vivo transactivation assays show Myc-Max and Mad-Max have opposing transcriptional activities, with Mad-Max functioning as a repressor.","method":"Lambda gt11 library screen with radiolabeled Max protein, in vitro binding, EMSA, CKII phosphorylation assay, transactivation assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution plus cell-based functional assay, multiple orthogonal methods","pmids":["8425218"],"is_preprint":false},{"year":1993,"finding":"Mxi1 (Mad family bHLH-Zip protein) specifically interacts with Max to form heterodimers that bind Myc-Max consensus recognition sites (CACGTG); Mxi1-Max heterodimers do not stimulate transcription; proposed mechanism of indirect Myc inhibition by sequestering Max and competing at target sites.","method":"Yeast interaction trap, DNA binding assay","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus DNA binding reconstitution, replicated across labs","pmids":["8425219"],"is_preprint":false},{"year":1993,"finding":"Mad:Max heterocomplexes accumulate in vivo in human myeloid cells undergoing macrophage differentiation (TPA-treated U937): undifferentiated cells show only Myc:Max complexes, but within 2 hours of TPA treatment Mad:Max complexes appear, and by 48 hours only Mad:Max complexes are detectable; Mad is a nuclear phosphoprotein with a very short half-life (~15–30 min).","method":"Coimmunoprecipitation from cell lysates, pulse-chase metabolic labeling, subcellular fractionation","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo co-IP at multiple time points, consistent with the differentiation-dependent switch model","pmids":["8224841"],"is_preprint":false},{"year":1993,"finding":"c-Myc/Max and Max/Max dimers have distinct DNA-binding preferences at flanking nucleotides: the c-Myc/Max dimer fails to bind the core CACGTG when flanked by 5'T or 3'A, while Max/Max homodimers bind such sequences readily; inappropriate flanking sequences preclude Myc transactivation in vivo.","method":"Site-selection protocol, EMSA, transactivation assay","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro site selection plus in vivo reporter assay, single lab","pmids":["8265351"],"is_preprint":false},{"year":1993,"finding":"Two major in vivo CKII phosphorylation sites in Max identified as Ser-2 and Ser-11; phosphorylation of these sites increases both the on- and off-rates of Max homodimer and Myc/Max heterodimer DNA binding; the shorter Max isoform (p21) dissociates from DNA faster than p22; these kinetic differences could allow different Max complexes to exchange on DNA in response to changing growth conditions.","method":"In vitro kinase assay, site-directed mutagenesis, DNA binding kinetics measurements","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro phosphorylation plus mutagenesis plus quantitative kinetics, single lab with multiple orthogonal methods","pmids":["8247525"],"is_preprint":false},{"year":1993,"finding":"The c-Myc/Max protein complex directly binds the CACGTG element in the human ornithine decarboxylase (ODC) promoter, as shown by EMSA with anti-Myc/anti-Max antibodies and purified recombinant proteins; co-transfection of c-Myc and/or Max enhances ODC promoter-driven reporter expression; antisense c-Myc oligomers reduce endogenous ODC mRNA.","method":"EMSA with antibody supershift, methylation interference, transient transfection, antisense oligonucleotide treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA supershift plus reporter assay, single lab with multiple orthogonal methods","pmids":["8262968"],"is_preprint":false},{"year":1993,"finding":"Max and c-myc proteins show distinct patterns of DNA binding across related CACGTG-containing oligonucleotides; the nine-amino-acid N-terminal insertion distinguishing Max(long) from Max(short) serves a regulatory function affecting DNA sequence recognition; phosphorylation of Max(long) in reticulocyte lysates strongly affects its DNA binding, whereas Max(short) is unaffected.","method":"Recombinant protein EMSA, phosphorylation in reticulocyte lysate","journal":"Proceedings of the National Academy of Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding studies with multiple Myc and Max variants, single lab","pmids":["8430110"],"is_preprint":false},{"year":1994,"finding":"Max homodimers can bind CACGTG sequences in nucleosomal DNA, whereas truncated c-Myc homodimers cannot; modifying the c-Myc dimerization interface or changing its partner to Max enables nucleosomal DNA binding; domains beyond the basic region influence nucleosome binding.","method":"In vitro nucleosome binding assay with reconstituted chromatin templates","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution on nucleosomal templates, single lab","pmids":["8196648"],"is_preprint":false},{"year":1996,"finding":"Drosophila homologs dMyc and dMax heterodimerize, recognize the same CACGTG DNA sequence as vertebrate Myc/Max, and activate transcription; dMyc is likely encoded by the diminutive (dm) locus whose loss causes small body size and female sterility.","method":"cDNA cloning, heterodimerization assay, DNA binding assay, transcription activation assay, genetic mapping","journal":"Science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional reconstitution in fly system, consistent with vertebrate mechanism","pmids":["8929412"],"is_preprint":false},{"year":1997,"finding":"Max(L) isoform is much more effective than Max(S) at homodimeric DNA binding and can repress a c-Myc-responsive reporter gene; Max(L)-overexpressing NIH3T3 cells grow more slowly, have higher growth factor requirements, and show accelerated apoptosis after growth factor deprivation compared to Max(S)-overexpressing or control cells.","method":"In vitro DNA binding assay, stable cell line overexpression, reporter gene assay, growth assay, apoptosis assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cell-based functional studies with defined isoforms, single lab, multiple readouts","pmids":["9211884"],"is_preprint":false},{"year":1997,"finding":"Mnt is a novel Max-binding protein that interacts with Max in vivo; Mnt:Max complexes repress transcription from CACGTG-containing promoters and suppress Myc-dependent activation; transcriptional repression maps to a 13-amino-acid N-terminal SID that mediates interaction with mSin3 corepressor; deletion of the SID converts Mnt from a repressor/Myc-suppressor to an activator/cooperating oncogene.","method":"Coimmunoprecipitation, reporter gene assay, deletion mutagenesis, Myc-Ras cotransformation assay","journal":"Current topics in microbiology and immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo co-IP plus functional mutagenesis, single lab","pmids":["9308234"],"is_preprint":false},{"year":1997,"finding":"Max alpha-helical content increases upon dimerization and upon binding to CACGTG-containing double-stranded DNA; Max exists as a monomer at low protein concentration and as a dimer at high protein concentration; both dimerization and DNA binding favor increased alpha-helical structure.","method":"Circular dichroism spectroscopy, sedimentation equilibrium","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — biophysical characterization in solution, single lab","pmids":["9399572"],"is_preprint":false},{"year":1999,"finding":"Mlx, a novel Max-like bHLHZip protein, forms heterodimers with Mad1 and Mad4 (but not with Myc or other Mad family members) and binds CACGTG; Mad1:Mlx heterodimer repression requires dimerization, DNA binding, and recruitment of the mSin3A-HDAC corepressor complex.","method":"Protein interaction screening, coimmunoprecipitation, EMSA, reporter gene assay, mSin3A co-IP","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple binding and functional assays, single lab","pmids":["10593926"],"is_preprint":false},{"year":1999,"finding":"Adrenomedullin and CGRP induce Max expression in quiescent rat endothelial cells, which rescues serum deprivation-induced apoptosis; antisense knockdown of Max blocks both adrenomedullin-induced Max upregulation and its cell survival effect; Max overexpression alone rescues apoptosis; adrenomedullin-induced Max negatively regulates E-box-driven transcription (e.g., preproendothelin-1 promoter).","method":"Real-time quantitative PCR, transfection of Max-expressing plasmid, antisense oligodeoxynucleotide knockdown, reporter gene assay","journal":"Molecular endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple functional methods linking Max expression to apoptosis rescue, single lab","pmids":["10446908"],"is_preprint":false},{"year":2003,"finding":"X-ray crystal structures of Myc-Max and Mad-Max bHLHZ heterodimers bound to the CACGTG E-box determined at 1.9 Å and 2.0 Å resolution; both heterodimers are quasisymmetric and resemble the Max homodimer; structural differences in coiled-coil leucine zipper regions explain preferential homo- and heteromeric dimerization; the Myc-Max heterodimer (but not Mad-Max) dimerizes into a bivalent heterotetramer, explaining how Myc upregulates genes with widely separated E-boxes.","method":"X-ray crystallography","journal":"Cell","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structures of multiple complexes, landmark study providing structural mechanistic insight","pmids":["12553908"],"is_preprint":false},{"year":2004,"finding":"Binding constants of Max/Max and Myc/Max dimers to E-box DNA are similar (K~7×10^6 M^-1); Max/Max dimer formation is kinetically easier than Myc/Max dimer formation for truncated b/HLH/Zip proteins, but domains outside b/HLH/Zip are important for physiological transcriptional regulation; curcuminoid 004 inhibits Max/Max-DNA binding with a dissociation constant of ~9 μM by competing with DNA.","method":"Surface plasmon resonance kinetics, fluorescence-based binding assay, EMSA with cell extract","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — quantitative in vitro binding kinetics, single lab","pmids":["14980448"],"is_preprint":false},{"year":2005,"finding":"Mnt-Max to c-Myc-Max complex switching regulates cell cycle entry: c-Myc induction during G0-to-S transition causes a transient decrease in Mnt-Max complexes and a switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes; Mnt overexpression suppresses cell cycle entry; Cre-lox deletion of both Mnt and c-Myc rescues the cell cycle block caused by c-Myc ablation alone.","method":"Coimmunoprecipitation, chromatin immunoprecipitation (ChIP), cell cycle analysis, Cre-lox genetic epistasis in MEFs","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods including genetic epistasis (double KO rescue), ChIP, and co-IP","pmids":["15866886"],"is_preprint":false},{"year":2005,"finding":"The complete p21 Max gene product has unstructured N- and C-terminal regions flanking the folded bHLH-LZ; p21 Max homodimerizes with an apparent KD ~7×10^-6 M at 37°C (10–100× weaker than the isolated bHLH-LZ alone, due to electrostatic repulsions); a double-mutant p21 Max forms a highly stable dimer (KD ~3×10^-10 M) with a higher DNA-complex melting temperature.","method":"Circular dichroism, NMR, analytical ultracentrifugation (sedimentation equilibrium), fluorescence-based dimerization assay","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple biophysical methods on full-length protein, single lab","pmids":["16171389"],"is_preprint":false},{"year":2007,"finding":"Max is acetylated in vivo at Lys-57, Lys-144, and Lys-145; acetylation is stimulated by HDAC inhibitors and by p300 overexpression; the p300 HAT directly acetylates Max in vitro at these three residues; the three acetylated lysines are important for Max nuclear localization and for Max-mediated suppression of Myc transactivation.","method":"Mass spectrometry identification of modification sites, in vitro acetylation assay with p300, cellular acetylation assay with HDAC inhibitors and p300 overexpression, functional reporter assay, nuclear localization assay","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — MS identification plus in vitro enzymatic assay plus functional cellular readout, single lab with multiple orthogonal methods","pmids":["17217336"],"is_preprint":false},{"year":2008,"finding":"In Drosophila, many biological activities of Myc do not require Max association: control of endoreplication and cell competition are Max-independent or only partially Max-dependent; a Myc mutant unable to interact with Max retains substantial biological activity; Myc controls RNA polymerase III transcription independently of Max.","method":"Drosophila genetics (Max loss-of-function and reduction-of-function mutations), Myc dimerization-mutant analysis, RNA Pol III transcription assay","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic loss-of-function in whole organism with multiple biological readouts and mechanistic follow-up on RNA Pol III","pmids":["19165923"],"is_preprint":false},{"year":2011,"finding":"Germline mutations in MAX (including loss-of-function mutations) cause hereditary pheochromocytoma; absence of MAX protein in tumors and loss of heterozygosity by uniparental disomy confirm MAX as a tumor suppressor; the rat pheochromocytoma cell line PC12 also lacks functional MAX.","method":"Exome sequencing, validation sequencing, immunohistochemistry for MAX protein, LOH analysis","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genomic sequencing plus protein loss validated in independent tumors, replicated across 59 additional cases","pmids":["21685915"],"is_preprint":false},{"year":2014,"finding":"Hypoxia in endothelial cells induces alternative splicing of MAX, producing two isoforms: isoform C (degraded by nonsense-mediated decay) and isoform E (encodes a highly unstable protein whose instability is conferred by 36 isoform-specific amino acids); both splicing events are unproductive and serve to downregulate wild-type MAX protein under hypoxia.","method":"RT-PCR isoform detection, protein stability assay, NMD inhibition, heterologous protein stability reporter","journal":"FEBS letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — molecular characterization of splicing outcomes with functional stability assays, single lab","pmids":["25451222"],"is_preprint":false},{"year":2017,"finding":"MAX is an epigenetic sensor of 5-carboxylcytosine (5caC): MAX exhibits greatest affinity for unmodified C or 5caC in the E-box CpG, and much reduced affinity for 5mC, 5hmC, or 5fC forms; crystal structure of MAX with 5caC-modified E-box revealed that Arg36 recognizes 5caC via a 5caC-Arg-Guanine triad; mutations of Arg35 or Arg36 abolish DNA binding while Arg60 mutation reduces binding but retains 5caC preference; MAX alterations in multiple myeloma cluster at Arg35, Arg36, and Arg60.","method":"Quantitative binding assays, X-ray crystallography of MAX–DNA complex, in vitro mutagenesis, analysis of >800 primary myeloma genomes","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus in vitro mutagenesis plus quantitative binding, multiple orthogonal methods in a single study","pmids":["27903915"],"is_preprint":false},{"year":2017,"finding":"MAX inactivation (hemizygous or homozygous mutations) occurs in ~21% of GISTs and is an early event; loss of MAX protein expression is associated with p16 silencing (without p16 coding sequence deletion); re-introduction of MAX restores p16 expression and inhibits GIST proliferation.","method":"Next-generation sequencing, immunohistochemistry, MAX re-expression/rescue experiment in GIST cells with p16 and proliferation readouts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function validated by rescue experiment with defined molecular (p16) and proliferation phenotypes","pmids":["28270683"],"is_preprint":false},{"year":2019,"finding":"B-cell-specific deletion of Max completely abrogates Eµ-Myc-driven lymphomagenesis while having only a modest effect on normal B-cell development; Max loss globally down-regulates Myc-activated genes in premalignant Eµ-Myc cells; Max loss leads to significant reduction in MYC protein levels and down-regulation of direct transcriptional targets including regulators of MYC stability; this MYC protein destabilization by Max loss is also observed in multiple cell lines treated with MYC-MAX dimerization inhibitors.","method":"B-cell-specific Cre-lox Max deletion, Eµ-Myc lymphoma model, RNA-seq, ChIP-seq, western blot for MYC protein levels","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional genetic knockout in mouse model with multiple orthogonal molecular readouts (RNA-seq, ChIP-seq, protein levels)","pmids":["31395740"],"is_preprint":false},{"year":2022,"finding":"The disordered MAX N-terminus interacts with the MYC:MAX DNA-binding domain (DBD) via electrostatic interactions, competitive with DNA binding; this intramolecular interaction accelerates DNA binding kinetics of MYC:MAX and MAX:MAX dimers while providing E-box specificity; Casein Kinase 2-mediated phosphorylation of two serines in the MAX N-terminus further enhances these effects.","method":"NMR spectroscopy (interaction mapping), Surface Plasmon Resonance (DNA binding kinetics), Casein Kinase 2 in vitro phosphorylation","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR mapping plus SPR quantitative kinetics plus in vitro kinase assay, single lab with multiple orthogonal methods","pmids":["36174765"],"is_preprint":false}],"current_model":"MAX is a constitutively expressed, nuclear bHLH-leucine zipper protein that functions as the obligate dimerization hub of the MYC/MAX/MAD transcriptional network: it homodimerizes or heterodimerizes with MYC family oncoproteins (activating transcription at CACGTG E-box elements) and with MAD/MXD, MXI1, MNT, and MLX family repressors (recruiting mSin3-HDAC to silence the same targets), and the balance among these competing complexes governs cell proliferation, differentiation, apoptosis, and metabolic gene expression; MAX activity is regulated post-translationally by CKII phosphorylation of N-terminal serines (which modulates DNA-binding kinetics), by p300-mediated acetylation of nuclear-localization lysines, by alternative splicing that produces isoforms with distinct transcriptional and growth-regulatory properties, by hypoxia-induced unproductive splicing that degrades MAX, and by an intramolecular interaction between the disordered MAX N-terminus and the folded DNA-binding domain; structurally, MAX homodimers and Myc-MAX/Mad-MAX heterodimers bind the E-box as quasi-symmetric dimers whose leucine-zipper coiled-coil differences dictate partner preference, and MAX acts as a direct epigenetic sensor through Arg36-mediated recognition of 5-carboxylcytosine in the E-box CpG; loss-of-function mutations in MAX cause hereditary pheochromocytoma and occur early in GISTs, establishing MAX as a tumor suppressor that constrains MYC protein stability and oncogenic transcriptional programs."},"narrative":{"mechanistic_narrative":"MAX is a constitutively expressed nuclear bHLH-leucine zipper phosphoprotein that serves as the obligate dimerization hub of the MYC transcriptional network, with virtually all newly synthesized Myc found complexed with the stable, constant Max partner in cells [PMID:1730411, PMID:1501888]. Max requires intact HLH and leucine zipper motifs to interact with c-Myc and carries a C-terminal nuclear localization signal, but lacks an intrinsic activation domain, functioning instead as a cofactor that on its own represses CACGTG (E-box) reporters until Myc co-expression relieves repression [PMID:1730412, PMID:1406956]. Max homodimers and Myc-Max heterodimers both bind E-box DNA as dimers, while full-length c-Myc cannot bind DNA without Max [PMID:1730412, PMID:1501888]; crystal structures of Myc-Max and Mad-Max heterodimers show quasisymmetric architectures resembling the Max homodimer, with coiled-coil leucine-zipper differences dictating partner preference and a Myc-Max-specific bivalent heterotetramer linking distant E-boxes [PMID:12553908]. Max additionally heterodimerizes with the repressive partners Mad, Mxi1, Mnt, and (via the Max-like protein Mlx) Mad1/Mad4, which silence the same E-box targets by recruiting the mSin3A-HDAC corepressor complex, so the balance among competing Max complexes governs proliferation, differentiation, and apoptosis [PMID:8425218, PMID:8425219, PMID:9308234, PMID:10593926]; differentiation drives a switch from Myc:Max to Mad:Max complexes, and Mnt:Max-to-Myc:Max switching controls cell-cycle entry [PMID:8224841, PMID:15866886]. Max activity is tuned post-translationally by CKII phosphorylation of N-terminal serines Ser-2/Ser-11, which alters DNA-binding kinetics, by p300-mediated acetylation of lysines required for nuclear localization and Myc suppression, by an intramolecular interaction between the disordered N-terminus and the DNA-binding domain that accelerates binding and confers E-box specificity, and by alternative-splicing isoforms with opposing growth-regulatory properties [PMID:1737614, PMID:8247525, PMID:17217336, PMID:36174765, PMID:1566084, PMID:9211884]. Max acts as a direct epigenetic sensor through Arg36-mediated recognition of 5-carboxylcytosine in the E-box CpG [PMID:27903915]. Germline loss-of-function mutations in MAX cause hereditary pheochromocytoma, and MAX inactivation is an early event in gastrointestinal stromal tumors, establishing MAX as a tumor suppressor that constrains MYC protein stability and oncogenic transcription [PMID:21685915, PMID:28270683, PMID:31395740].","teleology":[{"year":1992,"claim":"Established that Myc, an oncoprotein unable to bind DNA alone, acts only through an obligate partner — identifying Max as that constitutive nuclear partner and defining the heterodimeric DNA-binding unit.","evidence":"Reciprocal co-IP, DNA-binding assays, metabolic labeling and fractionation; cross-linking EMSA with bacterial proteins","pmids":["1730411","1730412","1501888","1323849"],"confidence":"High","gaps":["Did not define the full repertoire of Max partners beyond Myc","Did not establish in vivo transcriptional consequences genome-wide"]},{"year":1992,"claim":"Mapped Max functional architecture and showed Max alone represses E-box reporters relieved by Myc, establishing Max as a partner-dependent regulatory switch rather than an independent activator.","evidence":"Deletion/domain mapping, NLS localization, transient reporter assays with domain mutants","pmids":["1730412","1406956"],"confidence":"High","gaps":["Mechanism of repression by Max homodimers not defined at this stage","No corepressor identified yet"]},{"year":1992,"claim":"Defined post-translational and isoform-level control of Max DNA binding, showing CKII phosphorylation differentially regulates Max homodimers versus Myc:Max heterodimers and that splice variants have opposing transforming effects.","evidence":"In vitro CKII kinase assays with mutagenesis and phosphatase treatment; cDNA cloning and Myc-Ras cotransformation","pmids":["1737614","1566084"],"confidence":"High","gaps":["Physiological signal controlling CKII activity on Max not identified","delta-Max evidence is single-lab functional assay"]},{"year":1993,"claim":"Discovered the repressive arm of the network — Mad and Mxi1 form Max heterodimers at the same E-box sites — establishing competition among activating and repressing Max complexes as the regulatory logic.","evidence":"Library screen with radiolabeled Max, yeast interaction trap, EMSA, transactivation assays; in vivo co-IP during myeloid differentiation","pmids":["8425218","8425219","8224841"],"confidence":"High","gaps":["Corepressor machinery not yet identified in these reports","Direct target genes of repression not enumerated"]},{"year":1993,"claim":"Refined DNA recognition rules and kinetic tuning, showing flanking-sequence preferences distinguish Myc:Max from Max:Max binding and that CKII phosphorylation at Ser-2/Ser-11 governs binding on/off rates.","evidence":"Site-selection, EMSA, transactivation assays; in vitro phosphorylation with binding kinetics; identification of a direct target (ODC promoter)","pmids":["8265351","8247525","8262968","8430110"],"confidence":"Medium","gaps":["Genome-wide validation of flanking-sequence rules lacking","ODC target shown in single-lab reporter context"]},{"year":1994,"claim":"Showed Max homodimers, unlike truncated Myc homodimers, can engage nucleosomal E-boxes, implicating partner choice in chromatin access.","evidence":"In vitro nucleosome binding with reconstituted chromatin templates","pmids":["8196648"],"confidence":"Medium","gaps":["Not tested with full-length Myc:Max in vivo","Chromatin remodeling requirements not addressed"]},{"year":1996,"claim":"Demonstrated evolutionary conservation of the Myc/Max heterodimer and its E-box function in Drosophila, supporting a conserved transcriptional mechanism.","evidence":"cDNA cloning, heterodimerization, DNA binding, transcription assays, genetic mapping","pmids":["8929412"],"confidence":"Medium","gaps":["Did not address Max-independent Myc functions later revealed"]},{"year":1997,"claim":"Linked Max isoform identity and additional repressors (Mnt) to growth and apoptotic outcomes, and defined the SID-mSin3 repression module of the network.","evidence":"Stable overexpression growth/apoptosis assays, in vitro DNA binding, reporter assays; co-IP and SID deletion mutagenesis for Mnt","pmids":["9211884","9308234"],"confidence":"Medium","gaps":["Single-lab functional studies","Endogenous balance of isoforms in vivo not quantified"]},{"year":1999,"claim":"Defined the Mad-Mlx repression branch requiring mSin3A-HDAC recruitment and connected Max induction to cell-survival signaling, broadening the network's physiological reach.","evidence":"Interaction screening, co-IP, EMSA, reporter assays, mSin3A co-IP; qPCR, plasmid/antisense manipulation and apoptosis rescue in endothelial cells","pmids":["10593926","10446908"],"confidence":"Medium","gaps":["Single-lab studies","Mechanism linking adrenomedullin signaling to Max transcription not resolved"]},{"year":2003,"claim":"Provided the structural basis for partner discrimination and Myc-specific gene activation, showing leucine-zipper differences dictate dimer preference and a Myc-Max heterotetramer bridges distant E-boxes.","evidence":"X-ray crystallography of Myc-Max and Mad-Max bHLHZ:E-box complexes at 1.9–2.0 Å","pmids":["12553908"],"confidence":"High","gaps":["Full-length disordered regions not in the crystallized constructs","Tetramer functional role inferred, not directly tested in vivo here"]},{"year":2004,"claim":"Quantified the comparable E-box affinities of Max:Max and Myc:Max and identified small-molecule competition with Max-DNA binding.","evidence":"Surface plasmon resonance, fluorescence binding, EMSA; curcuminoid inhibitor characterization","pmids":["14980448"],"confidence":"Medium","gaps":["Truncated constructs used","Inhibitor specificity/cellular efficacy not established"]},{"year":2005,"claim":"Connected network complex switching to cell-cycle entry and defined the biophysical consequences of the full-length disordered Max termini on dimerization.","evidence":"Co-IP, ChIP, Cre-lox Mnt/c-Myc double-knockout epistasis in MEFs; CD/NMR/AUC on full-length p21 Max","pmids":["15866886","16171389"],"confidence":"High","gaps":["Mechanistic basis of electrostatic weakening of full-length dimer biology in vivo not tested","Switching kinetics on individual targets not resolved"]},{"year":2007,"claim":"Established acetylation as a regulatory layer controlling Max nuclear localization and its suppression of Myc, identifying p300 as the modifying enzyme.","evidence":"MS site mapping, in vitro p300 acetylation, cellular HDAC-inhibitor/p300 assays, reporter and localization readouts","pmids":["17217336"],"confidence":"High","gaps":["Deacetylase for Max not identified","Dynamics of acetylation across cell states not addressed"]},{"year":2008,"claim":"Revealed that key Myc activities can proceed without Max, redefining Max as essential for some but not all Myc functions.","evidence":"Drosophila Max loss-of-function genetics, Myc dimerization-mutant analysis, RNA Pol III transcription assay","pmids":["19165923"],"confidence":"High","gaps":["Extent of Max-independent Myc activity in mammals not quantified here","Alternative Myc partners for these activities not identified"]},{"year":2011,"claim":"Established MAX as a tumor suppressor by identifying causative germline loss-of-function mutations in hereditary pheochromocytoma with confirmed protein loss and LOH.","evidence":"Exome and validation sequencing, MAX IHC, LOH/uniparental disomy analysis","pmids":["21685915"],"confidence":"High","gaps":["Mechanism linking MAX loss to pheochromocytoma transformation not fully defined here","Tissue-specificity of the tumor suppressor effect unexplained"]},{"year":2014,"claim":"Identified hypoxia-induced unproductive splicing as a mechanism to downregulate MAX protein, linking environmental stress to network output.","evidence":"RT-PCR isoform detection, protein stability and NMD-inhibition assays, heterologous stability reporter","pmids":["25451222"],"confidence":"Medium","gaps":["Single-lab study","Transcriptional consequences of hypoxic MAX loss not mapped genome-wide"]},{"year":2017,"claim":"Defined MAX as a direct epigenetic sensor of cytosine carboxylation and characterized its DNA-binding arginines as cancer mutation hotspots, and showed MAX loss is an early, reversible driver in GIST.","evidence":"Quantitative binding, X-ray crystallography of MAX–5caC E-box, mutagenesis, myeloma genome analysis; NGS, IHC and MAX re-expression rescue with p16/proliferation readouts in GIST","pmids":["27903915","28270683"],"confidence":"High","gaps":["In vivo prevalence and dynamics of 5caC at MAX-bound E-boxes not established","Mechanism by which MAX loss silences p16 not fully defined"]},{"year":2019,"claim":"Demonstrated that MAX is required for MYC-driven lymphomagenesis and that MAX loss destabilizes MYC protein, mechanistically linking the dimerization hub to MYC oncoprotein stability.","evidence":"B-cell-specific Cre-lox Max deletion in Eµ-Myc mice, RNA-seq, ChIP-seq, western blot; dimerization-inhibitor treatment of cell lines","pmids":["31395740"],"confidence":"High","gaps":["Identity of the MYC-stability regulators downstream of MAX not fully resolved","Whether MYC destabilization fully accounts for tumor suppression unclear"]},{"year":2022,"claim":"Resolved how the disordered MAX N-terminus regulates the folded DNA-binding domain, showing an intramolecular, DNA-competitive electrostatic interaction that accelerates binding and confers E-box specificity, enhanced by CKII phosphorylation.","evidence":"NMR interaction mapping, SPR DNA-binding kinetics, in vitro CKII phosphorylation","pmids":["36174765"],"confidence":"High","gaps":["In vivo relevance of the intramolecular regulation not tested","Interplay with acetylation not examined"]},{"year":null,"claim":"How the competing Max complexes are quantitatively partitioned at endogenous loci in real time, and how this partitioning integrates phosphorylation, acetylation, splicing, and 5caC sensing to set proliferative versus tumor-suppressive output, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated quantitative model of complex competition at single loci","Signal-to-PTM linkages for Max regulation incomplete","Deacetylase and full degradation machinery for Max not identified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3,7,22,30]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,8,9,18]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,2]},{"term_id":"GO:0140299","term_label":"molecular sensor activity","supporting_discovery_ids":[30]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,26]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0,22]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,8,13,22]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[24,17]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[28,31,32,30]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[18,20,26]}],"complexes":["Myc-MAX heterodimer","Mad-MAX heterodimer","Mnt-MAX heterodimer","MAX homodimer"],"partners":["MYC","MXD1","MXI1","MNT","MLX","EP300","CSNK2A1","SIN3A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P61244","full_name":"Protein max","aliases":["Class D basic helix-loop-helix protein 4","bHLHd4","Myc-associated factor X"],"length_aa":160,"mass_kda":18.3,"function":"Transcription regulator. 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Prospects of Genome Editing in Soybean (Glycine max).","date":"2020","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/33193504","citation_count":28,"is_preprint":false},{"pmid":"14980448","id":"PMC_14980448","title":"Determination of binding constant of transcription factor myc-max/max-max and E-box DNA: the effect of inhibitors on the binding.","date":"2004","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/14980448","citation_count":26,"is_preprint":false},{"pmid":"29626511","id":"PMC_29626511","title":"RNA sequencing analysis of salt tolerance in soybean (Glycine max).","date":"2018","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/29626511","citation_count":25,"is_preprint":false},{"pmid":"10446446","id":"PMC_10446446","title":"Analysis of the Max-binding protein MNT in human medulloblastomas.","date":"1999","source":"International journal of 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biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/18309275","citation_count":21,"is_preprint":false},{"pmid":"17217336","id":"PMC_17217336","title":"Max is acetylated by p300 at several nuclear localization residues.","date":"2007","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/17217336","citation_count":21,"is_preprint":false},{"pmid":"30741632","id":"PMC_30741632","title":"LncEGFL7OS regulates human angiogenesis by interacting with MAX at the EGFL7/miR-126 locus.","date":"2019","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/30741632","citation_count":20,"is_preprint":false},{"pmid":"16171389","id":"PMC_16171389","title":"Structural and thermodynamical characterization of the complete p21 gene product of Max.","date":"2005","source":"Biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/16171389","citation_count":19,"is_preprint":false},{"pmid":"24833227","id":"PMC_24833227","title":"Changes in RNA Splicing in Developing Soybean (Glycine max) Embryos.","date":"2013","source":"Biology","url":"https://pubmed.ncbi.nlm.nih.gov/24833227","citation_count":19,"is_preprint":false},{"pmid":"25617765","id":"PMC_25617765","title":"Functional analysis of duplicated Symbiosis Receptor Kinase (SymRK) genes during nodulation and mycorrhizal infection in soybean (Glycine max).","date":"2015","source":"Journal of plant physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25617765","citation_count":19,"is_preprint":false},{"pmid":"30126925","id":"PMC_30126925","title":"Functional interplay between c-Myc and Max in B lymphocyte differentiation.","date":"2018","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/30126925","citation_count":18,"is_preprint":false},{"pmid":"30381476","id":"PMC_30381476","title":"Deciphering V̇O: limits of the genetic approach.","date":"2018","source":"The Journal of experimental 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sciences","url":"https://pubmed.ncbi.nlm.nih.gov/33066688","citation_count":17,"is_preprint":false},{"pmid":"29621573","id":"PMC_29621573","title":"Genomic, molecular evolution, and expression analysis of NOX genes in soybean (Glycine max).","date":"2018","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/29621573","citation_count":16,"is_preprint":false},{"pmid":"29428202","id":"PMC_29428202","title":"PET-SUV Max and Upstaging of Lung Cancer.","date":"2018","source":"Heart, lung & circulation","url":"https://pubmed.ncbi.nlm.nih.gov/29428202","citation_count":15,"is_preprint":false},{"pmid":"25451222","id":"PMC_25451222","title":"Hypoxia reduces MAX expression in endothelial cells by unproductive splicing.","date":"2014","source":"FEBS letters","url":"https://pubmed.ncbi.nlm.nih.gov/25451222","citation_count":15,"is_preprint":false},{"pmid":"31497394","id":"PMC_31497394","title":"Genome-wide identification and expression analysis of the VQ gene family in soybean (Glycine max).","date":"2019","source":"PeerJ","url":"https://pubmed.ncbi.nlm.nih.gov/31497394","citation_count":15,"is_preprint":false},{"pmid":"12406560","id":"PMC_12406560","title":"Unsaturated fatty acids bind Myc-Max transcription factor and inhibit Myc-Max-DNA complex formation.","date":"2002","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/12406560","citation_count":15,"is_preprint":false},{"pmid":"25659749","id":"PMC_25659749","title":"Ectopic phytocystatin expression increases nodule numbers and influences the responses of soybean (Glycine max) to nitrogen deficiency.","date":"2015","source":"Phytochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/25659749","citation_count":15,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":44906,"output_tokens":8210,"usd":0.128934,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":17949,"output_tokens":5937,"usd":0.119085,"stage2_stop_reason":"end_turn"},"total_usd":0.248019,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1992,\n      \"finding\": \"Myc and Max associate in vivo: coimmunoprecipitation with anti-Myc and anti-Max antibodies showed that essentially all newly synthesized Myc is complexed with Max in cells; the complex possesses specific DNA-binding activity for CACGTG-containing oligonucleotides; Max is a stable nuclear phosphoprotein whose expression level is constant across quiescent, mitogen-stimulated, and cycling cells, whereas Myc is rapidly degraded during/after association with Max.\",\n      \"method\": \"Coimmunoprecipitation, DNA binding assay, metabolic labeling, subcellular fractionation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal co-IP with anti-Myc and anti-Max antibodies, replicated across multiple labs in the same era\",\n      \"pmids\": [\"1730411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Max functional domains mapped: Max requires intact HLH and leucine zipper motifs for intracellular interaction with c-Myc; a nuclear localization signal (PQSRKKLR) was mapped to the carboxy-terminal region of Max; Max lacks a transcriptional activation domain functional in CHO cells, suggesting it acts as a cofactor or transcriptional repressor.\",\n      \"method\": \"Fusion protein expression in cultured cells, deletion analysis, coimmunoprecipitation, reporter gene assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — direct mutagenesis/deletion mapping of functional domains plus in-cell interaction assays, replicated conceptually across multiple studies\",\n      \"pmids\": [\"1730412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Max overexpression represses transcription of a CACGTG-containing reporter gene in mammalian cells, and this repression is relieved by co-expression of c-Myc; repression requires the Max DNA-binding domain, while relief requires Myc dimerization and transactivation domains.\",\n      \"method\": \"Transient transfection reporter gene assay in mammalian cells, domain deletion analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — functional reporter assay with domain mutants, independently replicated by multiple labs\",\n      \"pmids\": [\"1406956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Max homodimers and c-Myc/Max heterodimers both bind CACGTG-containing DNA sequences using a dimeric structure (demonstrated by chemical and photo-cross-linking); full-length c-Myc alone cannot bind DNA but does so in complex with Max.\",\n      \"method\": \"Chemical and photo-cross-linking, electrophoretic mobility shift assay (EMSA), bacterially produced proteins\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with cross-linking, replicated across multiple studies\",\n      \"pmids\": [\"1730412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"Casein kinase II (CKII) phosphorylates Max homodimers in vitro and inhibits their DNA-binding activity in an ATP-dependent manner; this inhibition maps to a CKII phosphorylation site in the amino terminus of Max; Myc/Max heterodimer DNA-binding activity is not inhibited by CKII phosphorylation; Max is phosphorylated in NIH-3T3 cells and can bind DNA after phosphatase treatment or heterodimerization with Myc.\",\n      \"method\": \"In vitro kinase assay with purified bovine CKII, EMSA, deletion analysis and site-directed mutagenesis, phosphatase treatment, immunoprecipitation from cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus mutagenesis plus cell-based phosphorylation, replicated and extended in subsequent studies\",\n      \"pmids\": [\"1737614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"An alternatively spliced mRNA encodes delta-Max, a truncated form lacking the C-terminal nuclear localization signal and putative regulatory domain; delta-Max retains binding to CACGTG in complex with c-Myc but lacks nuclear localization; in a Myc-Ras cotransformation assay, full-length Max suppresses transformation whereas delta-Max enhances it, indicating the two isoforms have opposing regulatory effects on c-Myc function.\",\n      \"method\": \"cDNA cloning, Myc-Ras cotransformation assay in rat embryo fibroblasts\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional transformation assay in cells, single lab, two methods (molecular cloning + biological assay)\",\n      \"pmids\": [\"1566084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"c-Myc and Max homodimers bend DNA in opposite orientations when bound to CACGTG, as measured by circular permutation and phasing analysis; c-Myc-Max heterodimers cause a smaller bend in an orientation similar to Max homodimers; no specific DNA unwinding was detected.\",\n      \"method\": \"Circular permutation assay, phasing analysis, EMSA\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biophysical assay, single lab\",\n      \"pmids\": [\"1323849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"In cell extracts virtually all c-Myc is associated with Max in heterodimeric complexes; c-Myc cannot bind CACGTG in the absence of Max, whereas both Max alone and c-Myc/Max bind the same DNA sequence.\",\n      \"method\": \"Cell extract EMSA, immunoprecipitation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell extract binding studies and immunoprecipitation, consistent with multiple independent studies\",\n      \"pmids\": [\"1501888\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Mad (a bHLH-Zip protein) binds Max in vitro to form a sequence-specific DNA binding complex with properties similar to Myc-Max; Mad does not homodimerize efficiently and does not associate with Myc; Mad-Max and Myc-Max heterocomplexes are both favored over Max homodimers; CKII phosphorylation does not affect heterodimer DNA binding; in vivo transactivation assays show Myc-Max and Mad-Max have opposing transcriptional activities, with Mad-Max functioning as a repressor.\",\n      \"method\": \"Lambda gt11 library screen with radiolabeled Max protein, in vitro binding, EMSA, CKII phosphorylation assay, transactivation assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution plus cell-based functional assay, multiple orthogonal methods\",\n      \"pmids\": [\"8425218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Mxi1 (Mad family bHLH-Zip protein) specifically interacts with Max to form heterodimers that bind Myc-Max consensus recognition sites (CACGTG); Mxi1-Max heterodimers do not stimulate transcription; proposed mechanism of indirect Myc inhibition by sequestering Max and competing at target sites.\",\n      \"method\": \"Yeast interaction trap, DNA binding assay\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus DNA binding reconstitution, replicated across labs\",\n      \"pmids\": [\"8425219\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Mad:Max heterocomplexes accumulate in vivo in human myeloid cells undergoing macrophage differentiation (TPA-treated U937): undifferentiated cells show only Myc:Max complexes, but within 2 hours of TPA treatment Mad:Max complexes appear, and by 48 hours only Mad:Max complexes are detectable; Mad is a nuclear phosphoprotein with a very short half-life (~15–30 min).\",\n      \"method\": \"Coimmunoprecipitation from cell lysates, pulse-chase metabolic labeling, subcellular fractionation\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo co-IP at multiple time points, consistent with the differentiation-dependent switch model\",\n      \"pmids\": [\"8224841\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"c-Myc/Max and Max/Max dimers have distinct DNA-binding preferences at flanking nucleotides: the c-Myc/Max dimer fails to bind the core CACGTG when flanked by 5'T or 3'A, while Max/Max homodimers bind such sequences readily; inappropriate flanking sequences preclude Myc transactivation in vivo.\",\n      \"method\": \"Site-selection protocol, EMSA, transactivation assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro site selection plus in vivo reporter assay, single lab\",\n      \"pmids\": [\"8265351\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Two major in vivo CKII phosphorylation sites in Max identified as Ser-2 and Ser-11; phosphorylation of these sites increases both the on- and off-rates of Max homodimer and Myc/Max heterodimer DNA binding; the shorter Max isoform (p21) dissociates from DNA faster than p22; these kinetic differences could allow different Max complexes to exchange on DNA in response to changing growth conditions.\",\n      \"method\": \"In vitro kinase assay, site-directed mutagenesis, DNA binding kinetics measurements\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro phosphorylation plus mutagenesis plus quantitative kinetics, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8247525\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"The c-Myc/Max protein complex directly binds the CACGTG element in the human ornithine decarboxylase (ODC) promoter, as shown by EMSA with anti-Myc/anti-Max antibodies and purified recombinant proteins; co-transfection of c-Myc and/or Max enhances ODC promoter-driven reporter expression; antisense c-Myc oligomers reduce endogenous ODC mRNA.\",\n      \"method\": \"EMSA with antibody supershift, methylation interference, transient transfection, antisense oligonucleotide treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA supershift plus reporter assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8262968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"Max and c-myc proteins show distinct patterns of DNA binding across related CACGTG-containing oligonucleotides; the nine-amino-acid N-terminal insertion distinguishing Max(long) from Max(short) serves a regulatory function affecting DNA sequence recognition; phosphorylation of Max(long) in reticulocyte lysates strongly affects its DNA binding, whereas Max(short) is unaffected.\",\n      \"method\": \"Recombinant protein EMSA, phosphorylation in reticulocyte lysate\",\n      \"journal\": \"Proceedings of the National Academy of Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding studies with multiple Myc and Max variants, single lab\",\n      \"pmids\": [\"8430110\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Max homodimers can bind CACGTG sequences in nucleosomal DNA, whereas truncated c-Myc homodimers cannot; modifying the c-Myc dimerization interface or changing its partner to Max enables nucleosomal DNA binding; domains beyond the basic region influence nucleosome binding.\",\n      \"method\": \"In vitro nucleosome binding assay with reconstituted chromatin templates\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution on nucleosomal templates, single lab\",\n      \"pmids\": [\"8196648\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Drosophila homologs dMyc and dMax heterodimerize, recognize the same CACGTG DNA sequence as vertebrate Myc/Max, and activate transcription; dMyc is likely encoded by the diminutive (dm) locus whose loss causes small body size and female sterility.\",\n      \"method\": \"cDNA cloning, heterodimerization assay, DNA binding assay, transcription activation assay, genetic mapping\",\n      \"journal\": \"Science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional reconstitution in fly system, consistent with vertebrate mechanism\",\n      \"pmids\": [\"8929412\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Max(L) isoform is much more effective than Max(S) at homodimeric DNA binding and can repress a c-Myc-responsive reporter gene; Max(L)-overexpressing NIH3T3 cells grow more slowly, have higher growth factor requirements, and show accelerated apoptosis after growth factor deprivation compared to Max(S)-overexpressing or control cells.\",\n      \"method\": \"In vitro DNA binding assay, stable cell line overexpression, reporter gene assay, growth assay, apoptosis assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cell-based functional studies with defined isoforms, single lab, multiple readouts\",\n      \"pmids\": [\"9211884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Mnt is a novel Max-binding protein that interacts with Max in vivo; Mnt:Max complexes repress transcription from CACGTG-containing promoters and suppress Myc-dependent activation; transcriptional repression maps to a 13-amino-acid N-terminal SID that mediates interaction with mSin3 corepressor; deletion of the SID converts Mnt from a repressor/Myc-suppressor to an activator/cooperating oncogene.\",\n      \"method\": \"Coimmunoprecipitation, reporter gene assay, deletion mutagenesis, Myc-Ras cotransformation assay\",\n      \"journal\": \"Current topics in microbiology and immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo co-IP plus functional mutagenesis, single lab\",\n      \"pmids\": [\"9308234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Max alpha-helical content increases upon dimerization and upon binding to CACGTG-containing double-stranded DNA; Max exists as a monomer at low protein concentration and as a dimer at high protein concentration; both dimerization and DNA binding favor increased alpha-helical structure.\",\n      \"method\": \"Circular dichroism spectroscopy, sedimentation equilibrium\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — biophysical characterization in solution, single lab\",\n      \"pmids\": [\"9399572\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Mlx, a novel Max-like bHLHZip protein, forms heterodimers with Mad1 and Mad4 (but not with Myc or other Mad family members) and binds CACGTG; Mad1:Mlx heterodimer repression requires dimerization, DNA binding, and recruitment of the mSin3A-HDAC corepressor complex.\",\n      \"method\": \"Protein interaction screening, coimmunoprecipitation, EMSA, reporter gene assay, mSin3A co-IP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple binding and functional assays, single lab\",\n      \"pmids\": [\"10593926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Adrenomedullin and CGRP induce Max expression in quiescent rat endothelial cells, which rescues serum deprivation-induced apoptosis; antisense knockdown of Max blocks both adrenomedullin-induced Max upregulation and its cell survival effect; Max overexpression alone rescues apoptosis; adrenomedullin-induced Max negatively regulates E-box-driven transcription (e.g., preproendothelin-1 promoter).\",\n      \"method\": \"Real-time quantitative PCR, transfection of Max-expressing plasmid, antisense oligodeoxynucleotide knockdown, reporter gene assay\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple functional methods linking Max expression to apoptosis rescue, single lab\",\n      \"pmids\": [\"10446908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"X-ray crystal structures of Myc-Max and Mad-Max bHLHZ heterodimers bound to the CACGTG E-box determined at 1.9 Å and 2.0 Å resolution; both heterodimers are quasisymmetric and resemble the Max homodimer; structural differences in coiled-coil leucine zipper regions explain preferential homo- and heteromeric dimerization; the Myc-Max heterodimer (but not Mad-Max) dimerizes into a bivalent heterotetramer, explaining how Myc upregulates genes with widely separated E-boxes.\",\n      \"method\": \"X-ray crystallography\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structures of multiple complexes, landmark study providing structural mechanistic insight\",\n      \"pmids\": [\"12553908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Binding constants of Max/Max and Myc/Max dimers to E-box DNA are similar (K~7×10^6 M^-1); Max/Max dimer formation is kinetically easier than Myc/Max dimer formation for truncated b/HLH/Zip proteins, but domains outside b/HLH/Zip are important for physiological transcriptional regulation; curcuminoid 004 inhibits Max/Max-DNA binding with a dissociation constant of ~9 μM by competing with DNA.\",\n      \"method\": \"Surface plasmon resonance kinetics, fluorescence-based binding assay, EMSA with cell extract\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — quantitative in vitro binding kinetics, single lab\",\n      \"pmids\": [\"14980448\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Mnt-Max to c-Myc-Max complex switching regulates cell cycle entry: c-Myc induction during G0-to-S transition causes a transient decrease in Mnt-Max complexes and a switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes; Mnt overexpression suppresses cell cycle entry; Cre-lox deletion of both Mnt and c-Myc rescues the cell cycle block caused by c-Myc ablation alone.\",\n      \"method\": \"Coimmunoprecipitation, chromatin immunoprecipitation (ChIP), cell cycle analysis, Cre-lox genetic epistasis in MEFs\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods including genetic epistasis (double KO rescue), ChIP, and co-IP\",\n      \"pmids\": [\"15866886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The complete p21 Max gene product has unstructured N- and C-terminal regions flanking the folded bHLH-LZ; p21 Max homodimerizes with an apparent KD ~7×10^-6 M at 37°C (10–100× weaker than the isolated bHLH-LZ alone, due to electrostatic repulsions); a double-mutant p21 Max forms a highly stable dimer (KD ~3×10^-10 M) with a higher DNA-complex melting temperature.\",\n      \"method\": \"Circular dichroism, NMR, analytical ultracentrifugation (sedimentation equilibrium), fluorescence-based dimerization assay\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple biophysical methods on full-length protein, single lab\",\n      \"pmids\": [\"16171389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Max is acetylated in vivo at Lys-57, Lys-144, and Lys-145; acetylation is stimulated by HDAC inhibitors and by p300 overexpression; the p300 HAT directly acetylates Max in vitro at these three residues; the three acetylated lysines are important for Max nuclear localization and for Max-mediated suppression of Myc transactivation.\",\n      \"method\": \"Mass spectrometry identification of modification sites, in vitro acetylation assay with p300, cellular acetylation assay with HDAC inhibitors and p300 overexpression, functional reporter assay, nuclear localization assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — MS identification plus in vitro enzymatic assay plus functional cellular readout, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"17217336\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"In Drosophila, many biological activities of Myc do not require Max association: control of endoreplication and cell competition are Max-independent or only partially Max-dependent; a Myc mutant unable to interact with Max retains substantial biological activity; Myc controls RNA polymerase III transcription independently of Max.\",\n      \"method\": \"Drosophila genetics (Max loss-of-function and reduction-of-function mutations), Myc dimerization-mutant analysis, RNA Pol III transcription assay\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic loss-of-function in whole organism with multiple biological readouts and mechanistic follow-up on RNA Pol III\",\n      \"pmids\": [\"19165923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Germline mutations in MAX (including loss-of-function mutations) cause hereditary pheochromocytoma; absence of MAX protein in tumors and loss of heterozygosity by uniparental disomy confirm MAX as a tumor suppressor; the rat pheochromocytoma cell line PC12 also lacks functional MAX.\",\n      \"method\": \"Exome sequencing, validation sequencing, immunohistochemistry for MAX protein, LOH analysis\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genomic sequencing plus protein loss validated in independent tumors, replicated across 59 additional cases\",\n      \"pmids\": [\"21685915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Hypoxia in endothelial cells induces alternative splicing of MAX, producing two isoforms: isoform C (degraded by nonsense-mediated decay) and isoform E (encodes a highly unstable protein whose instability is conferred by 36 isoform-specific amino acids); both splicing events are unproductive and serve to downregulate wild-type MAX protein under hypoxia.\",\n      \"method\": \"RT-PCR isoform detection, protein stability assay, NMD inhibition, heterologous protein stability reporter\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — molecular characterization of splicing outcomes with functional stability assays, single lab\",\n      \"pmids\": [\"25451222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAX is an epigenetic sensor of 5-carboxylcytosine (5caC): MAX exhibits greatest affinity for unmodified C or 5caC in the E-box CpG, and much reduced affinity for 5mC, 5hmC, or 5fC forms; crystal structure of MAX with 5caC-modified E-box revealed that Arg36 recognizes 5caC via a 5caC-Arg-Guanine triad; mutations of Arg35 or Arg36 abolish DNA binding while Arg60 mutation reduces binding but retains 5caC preference; MAX alterations in multiple myeloma cluster at Arg35, Arg36, and Arg60.\",\n      \"method\": \"Quantitative binding assays, X-ray crystallography of MAX–DNA complex, in vitro mutagenesis, analysis of >800 primary myeloma genomes\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus in vitro mutagenesis plus quantitative binding, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"27903915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"MAX inactivation (hemizygous or homozygous mutations) occurs in ~21% of GISTs and is an early event; loss of MAX protein expression is associated with p16 silencing (without p16 coding sequence deletion); re-introduction of MAX restores p16 expression and inhibits GIST proliferation.\",\n      \"method\": \"Next-generation sequencing, immunohistochemistry, MAX re-expression/rescue experiment in GIST cells with p16 and proliferation readouts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function validated by rescue experiment with defined molecular (p16) and proliferation phenotypes\",\n      \"pmids\": [\"28270683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"B-cell-specific deletion of Max completely abrogates Eµ-Myc-driven lymphomagenesis while having only a modest effect on normal B-cell development; Max loss globally down-regulates Myc-activated genes in premalignant Eµ-Myc cells; Max loss leads to significant reduction in MYC protein levels and down-regulation of direct transcriptional targets including regulators of MYC stability; this MYC protein destabilization by Max loss is also observed in multiple cell lines treated with MYC-MAX dimerization inhibitors.\",\n      \"method\": \"B-cell-specific Cre-lox Max deletion, Eµ-Myc lymphoma model, RNA-seq, ChIP-seq, western blot for MYC protein levels\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional genetic knockout in mouse model with multiple orthogonal molecular readouts (RNA-seq, ChIP-seq, protein levels)\",\n      \"pmids\": [\"31395740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The disordered MAX N-terminus interacts with the MYC:MAX DNA-binding domain (DBD) via electrostatic interactions, competitive with DNA binding; this intramolecular interaction accelerates DNA binding kinetics of MYC:MAX and MAX:MAX dimers while providing E-box specificity; Casein Kinase 2-mediated phosphorylation of two serines in the MAX N-terminus further enhances these effects.\",\n      \"method\": \"NMR spectroscopy (interaction mapping), Surface Plasmon Resonance (DNA binding kinetics), Casein Kinase 2 in vitro phosphorylation\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR mapping plus SPR quantitative kinetics plus in vitro kinase assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"36174765\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MAX is a constitutively expressed, nuclear bHLH-leucine zipper protein that functions as the obligate dimerization hub of the MYC/MAX/MAD transcriptional network: it homodimerizes or heterodimerizes with MYC family oncoproteins (activating transcription at CACGTG E-box elements) and with MAD/MXD, MXI1, MNT, and MLX family repressors (recruiting mSin3-HDAC to silence the same targets), and the balance among these competing complexes governs cell proliferation, differentiation, apoptosis, and metabolic gene expression; MAX activity is regulated post-translationally by CKII phosphorylation of N-terminal serines (which modulates DNA-binding kinetics), by p300-mediated acetylation of nuclear-localization lysines, by alternative splicing that produces isoforms with distinct transcriptional and growth-regulatory properties, by hypoxia-induced unproductive splicing that degrades MAX, and by an intramolecular interaction between the disordered MAX N-terminus and the folded DNA-binding domain; structurally, MAX homodimers and Myc-MAX/Mad-MAX heterodimers bind the E-box as quasi-symmetric dimers whose leucine-zipper coiled-coil differences dictate partner preference, and MAX acts as a direct epigenetic sensor through Arg36-mediated recognition of 5-carboxylcytosine in the E-box CpG; loss-of-function mutations in MAX cause hereditary pheochromocytoma and occur early in GISTs, establishing MAX as a tumor suppressor that constrains MYC protein stability and oncogenic transcriptional programs.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MAX is a constitutively expressed nuclear bHLH-leucine zipper phosphoprotein that serves as the obligate dimerization hub of the MYC transcriptional network, with virtually all newly synthesized Myc found complexed with the stable, constant Max partner in cells [#0, #7]. Max requires intact HLH and leucine zipper motifs to interact with c-Myc and carries a C-terminal nuclear localization signal, but lacks an intrinsic activation domain, functioning instead as a cofactor that on its own represses CACGTG (E-box) reporters until Myc co-expression relieves repression [#1, #2]. Max homodimers and Myc-Max heterodimers both bind E-box DNA as dimers, while full-length c-Myc cannot bind DNA without Max [#3, #7]; crystal structures of Myc-Max and Mad-Max heterodimers show quasisymmetric architectures resembling the Max homodimer, with coiled-coil leucine-zipper differences dictating partner preference and a Myc-Max-specific bivalent heterotetramer linking distant E-boxes [#22]. Max additionally heterodimerizes with the repressive partners Mad, Mxi1, Mnt, and (via the Max-like protein Mlx) Mad1/Mad4, which silence the same E-box targets by recruiting the mSin3A-HDAC corepressor complex, so the balance among competing Max complexes governs proliferation, differentiation, and apoptosis [#8, #9, #18, #20]; differentiation drives a switch from Myc:Max to Mad:Max complexes, and Mnt:Max-to-Myc:Max switching controls cell-cycle entry [#10, #24]. Max activity is tuned post-translationally by CKII phosphorylation of N-terminal serines Ser-2/Ser-11, which alters DNA-binding kinetics, by p300-mediated acetylation of lysines required for nuclear localization and Myc suppression, by an intramolecular interaction between the disordered N-terminus and the DNA-binding domain that accelerates binding and confers E-box specificity, and by alternative-splicing isoforms with opposing growth-regulatory properties [#4, #12, #26, #33, #5, #17]. Max acts as a direct epigenetic sensor through Arg36-mediated recognition of 5-carboxylcytosine in the E-box CpG [#30]. Germline loss-of-function mutations in MAX cause hereditary pheochromocytoma, and MAX inactivation is an early event in gastrointestinal stromal tumors, establishing MAX as a tumor suppressor that constrains MYC protein stability and oncogenic transcription [#28, #31, #32].\",\n  \"teleology\": [\n    {\n      \"year\": 1992,\n      \"claim\": \"Established that Myc, an oncoprotein unable to bind DNA alone, acts only through an obligate partner — identifying Max as that constitutive nuclear partner and defining the heterodimeric DNA-binding unit.\",\n      \"evidence\": \"Reciprocal co-IP, DNA-binding assays, metabolic labeling and fractionation; cross-linking EMSA with bacterial proteins\",\n      \"pmids\": [\"1730411\", \"1730412\", \"1501888\", \"1323849\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the full repertoire of Max partners beyond Myc\", \"Did not establish in vivo transcriptional consequences genome-wide\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Mapped Max functional architecture and showed Max alone represses E-box reporters relieved by Myc, establishing Max as a partner-dependent regulatory switch rather than an independent activator.\",\n      \"evidence\": \"Deletion/domain mapping, NLS localization, transient reporter assays with domain mutants\",\n      \"pmids\": [\"1730412\", \"1406956\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of repression by Max homodimers not defined at this stage\", \"No corepressor identified yet\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Defined post-translational and isoform-level control of Max DNA binding, showing CKII phosphorylation differentially regulates Max homodimers versus Myc:Max heterodimers and that splice variants have opposing transforming effects.\",\n      \"evidence\": \"In vitro CKII kinase assays with mutagenesis and phosphatase treatment; cDNA cloning and Myc-Ras cotransformation\",\n      \"pmids\": [\"1737614\", \"1566084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological signal controlling CKII activity on Max not identified\", \"delta-Max evidence is single-lab functional assay\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Discovered the repressive arm of the network — Mad and Mxi1 form Max heterodimers at the same E-box sites — establishing competition among activating and repressing Max complexes as the regulatory logic.\",\n      \"evidence\": \"Library screen with radiolabeled Max, yeast interaction trap, EMSA, transactivation assays; in vivo co-IP during myeloid differentiation\",\n      \"pmids\": [\"8425218\", \"8425219\", \"8224841\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Corepressor machinery not yet identified in these reports\", \"Direct target genes of repression not enumerated\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Refined DNA recognition rules and kinetic tuning, showing flanking-sequence preferences distinguish Myc:Max from Max:Max binding and that CKII phosphorylation at Ser-2/Ser-11 governs binding on/off rates.\",\n      \"evidence\": \"Site-selection, EMSA, transactivation assays; in vitro phosphorylation with binding kinetics; identification of a direct target (ODC promoter)\",\n      \"pmids\": [\"8265351\", \"8247525\", \"8262968\", \"8430110\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genome-wide validation of flanking-sequence rules lacking\", \"ODC target shown in single-lab reporter context\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Showed Max homodimers, unlike truncated Myc homodimers, can engage nucleosomal E-boxes, implicating partner choice in chromatin access.\",\n      \"evidence\": \"In vitro nucleosome binding with reconstituted chromatin templates\",\n      \"pmids\": [\"8196648\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Not tested with full-length Myc:Max in vivo\", \"Chromatin remodeling requirements not addressed\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Demonstrated evolutionary conservation of the Myc/Max heterodimer and its E-box function in Drosophila, supporting a conserved transcriptional mechanism.\",\n      \"evidence\": \"cDNA cloning, heterodimerization, DNA binding, transcription assays, genetic mapping\",\n      \"pmids\": [\"8929412\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address Max-independent Myc functions later revealed\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Linked Max isoform identity and additional repressors (Mnt) to growth and apoptotic outcomes, and defined the SID-mSin3 repression module of the network.\",\n      \"evidence\": \"Stable overexpression growth/apoptosis assays, in vitro DNA binding, reporter assays; co-IP and SID deletion mutagenesis for Mnt\",\n      \"pmids\": [\"9211884\", \"9308234\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab functional studies\", \"Endogenous balance of isoforms in vivo not quantified\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Defined the Mad-Mlx repression branch requiring mSin3A-HDAC recruitment and connected Max induction to cell-survival signaling, broadening the network's physiological reach.\",\n      \"evidence\": \"Interaction screening, co-IP, EMSA, reporter assays, mSin3A co-IP; qPCR, plasmid/antisense manipulation and apoptosis rescue in endothelial cells\",\n      \"pmids\": [\"10593926\", \"10446908\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab studies\", \"Mechanism linking adrenomedullin signaling to Max transcription not resolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Provided the structural basis for partner discrimination and Myc-specific gene activation, showing leucine-zipper differences dictate dimer preference and a Myc-Max heterotetramer bridges distant E-boxes.\",\n      \"evidence\": \"X-ray crystallography of Myc-Max and Mad-Max bHLHZ:E-box complexes at 1.9–2.0 Å\",\n      \"pmids\": [\"12553908\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-length disordered regions not in the crystallized constructs\", \"Tetramer functional role inferred, not directly tested in vivo here\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Quantified the comparable E-box affinities of Max:Max and Myc:Max and identified small-molecule competition with Max-DNA binding.\",\n      \"evidence\": \"Surface plasmon resonance, fluorescence binding, EMSA; curcuminoid inhibitor characterization\",\n      \"pmids\": [\"14980448\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Truncated constructs used\", \"Inhibitor specificity/cellular efficacy not established\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected network complex switching to cell-cycle entry and defined the biophysical consequences of the full-length disordered Max termini on dimerization.\",\n      \"evidence\": \"Co-IP, ChIP, Cre-lox Mnt/c-Myc double-knockout epistasis in MEFs; CD/NMR/AUC on full-length p21 Max\",\n      \"pmids\": [\"15866886\", \"16171389\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis of electrostatic weakening of full-length dimer biology in vivo not tested\", \"Switching kinetics on individual targets not resolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Established acetylation as a regulatory layer controlling Max nuclear localization and its suppression of Myc, identifying p300 as the modifying enzyme.\",\n      \"evidence\": \"MS site mapping, in vitro p300 acetylation, cellular HDAC-inhibitor/p300 assays, reporter and localization readouts\",\n      \"pmids\": [\"17217336\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Deacetylase for Max not identified\", \"Dynamics of acetylation across cell states not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealed that key Myc activities can proceed without Max, redefining Max as essential for some but not all Myc functions.\",\n      \"evidence\": \"Drosophila Max loss-of-function genetics, Myc dimerization-mutant analysis, RNA Pol III transcription assay\",\n      \"pmids\": [\"19165923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Extent of Max-independent Myc activity in mammals not quantified here\", \"Alternative Myc partners for these activities not identified\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Established MAX as a tumor suppressor by identifying causative germline loss-of-function mutations in hereditary pheochromocytoma with confirmed protein loss and LOH.\",\n      \"evidence\": \"Exome and validation sequencing, MAX IHC, LOH/uniparental disomy analysis\",\n      \"pmids\": [\"21685915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking MAX loss to pheochromocytoma transformation not fully defined here\", \"Tissue-specificity of the tumor suppressor effect unexplained\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identified hypoxia-induced unproductive splicing as a mechanism to downregulate MAX protein, linking environmental stress to network output.\",\n      \"evidence\": \"RT-PCR isoform detection, protein stability and NMD-inhibition assays, heterologous stability reporter\",\n      \"pmids\": [\"25451222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Transcriptional consequences of hypoxic MAX loss not mapped genome-wide\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined MAX as a direct epigenetic sensor of cytosine carboxylation and characterized its DNA-binding arginines as cancer mutation hotspots, and showed MAX loss is an early, reversible driver in GIST.\",\n      \"evidence\": \"Quantitative binding, X-ray crystallography of MAX–5caC E-box, mutagenesis, myeloma genome analysis; NGS, IHC and MAX re-expression rescue with p16/proliferation readouts in GIST\",\n      \"pmids\": [\"27903915\", \"28270683\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo prevalence and dynamics of 5caC at MAX-bound E-boxes not established\", \"Mechanism by which MAX loss silences p16 not fully defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Demonstrated that MAX is required for MYC-driven lymphomagenesis and that MAX loss destabilizes MYC protein, mechanistically linking the dimerization hub to MYC oncoprotein stability.\",\n      \"evidence\": \"B-cell-specific Cre-lox Max deletion in Eµ-Myc mice, RNA-seq, ChIP-seq, western blot; dimerization-inhibitor treatment of cell lines\",\n      \"pmids\": [\"31395740\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the MYC-stability regulators downstream of MAX not fully resolved\", \"Whether MYC destabilization fully accounts for tumor suppression unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved how the disordered MAX N-terminus regulates the folded DNA-binding domain, showing an intramolecular, DNA-competitive electrostatic interaction that accelerates binding and confers E-box specificity, enhanced by CKII phosphorylation.\",\n      \"evidence\": \"NMR interaction mapping, SPR DNA-binding kinetics, in vitro CKII phosphorylation\",\n      \"pmids\": [\"36174765\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of the intramolecular regulation not tested\", \"Interplay with acetylation not examined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the competing Max complexes are quantitatively partitioned at endogenous loci in real time, and how this partitioning integrates phosphorylation, acetylation, splicing, and 5caC sensing to set proliferative versus tumor-suppressive output, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated quantitative model of complex competition at single loci\", \"Signal-to-PTM linkages for Max regulation incomplete\", \"Deacetylase and full degradation machinery for Max not identified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3, 7, 22, 30]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 8, 9, 18]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0140299\", \"supporting_discovery_ids\": [30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 26]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0, 22]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 8, 13, 22]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [24, 17]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [28, 31, 32, 30]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [18, 20, 26]}\n    ],\n    \"complexes\": [\n      \"Myc-MAX heterodimer\",\n      \"Mad-MAX heterodimer\",\n      \"Mnt-MAX heterodimer\",\n      \"MAX homodimer\"\n    ],\n    \"partners\": [\n      \"MYC\",\n      \"MXD1\",\n      \"MXI1\",\n      \"MNT\",\n      \"MLX\",\n      \"EP300\",\n      \"CSNK2A1\",\n      \"SIN3A\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}