| 1992 |
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. |
Coimmunoprecipitation, DNA binding assay, metabolic labeling, subcellular fractionation |
Genes & development |
High |
1730411
|
| 1992 |
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. |
Fusion protein expression in cultured cells, deletion analysis, coimmunoprecipitation, reporter gene assay |
Genes & development |
High |
1730412
|
| 1992 |
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. |
Transient transfection reporter gene assay in mammalian cells, domain deletion analysis |
Nature |
High |
1406956
|
| 1992 |
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. |
Chemical and photo-cross-linking, electrophoretic mobility shift assay (EMSA), bacterially produced proteins |
Genes & development |
High |
1730412
|
| 1992 |
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. |
In vitro kinase assay with purified bovine CKII, EMSA, deletion analysis and site-directed mutagenesis, phosphatase treatment, immunoprecipitation from cells |
Genes & development |
High |
1737614
|
| 1992 |
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. |
cDNA cloning, Myc-Ras cotransformation assay in rat embryo fibroblasts |
Science |
Medium |
1566084
|
| 1992 |
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. |
Circular permutation assay, phasing analysis, EMSA |
Proceedings of the National Academy of Sciences |
Medium |
1323849
|
| 1992 |
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. |
Cell extract EMSA, immunoprecipitation |
Oncogene |
High |
1501888
|
| 1993 |
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. |
Lambda gt11 library screen with radiolabeled Max protein, in vitro binding, EMSA, CKII phosphorylation assay, transactivation assay |
Cell |
High |
8425218
|
| 1993 |
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. |
Yeast interaction trap, DNA binding assay |
Cell |
High |
8425219
|
| 1993 |
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). |
Coimmunoprecipitation from cell lysates, pulse-chase metabolic labeling, subcellular fractionation |
Genes & development |
High |
8224841
|
| 1993 |
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. |
Site-selection protocol, EMSA, transactivation assay |
Nucleic acids research |
Medium |
8265351
|
| 1993 |
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. |
In vitro kinase assay, site-directed mutagenesis, DNA binding kinetics measurements |
Oncogene |
High |
8247525
|
| 1993 |
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. |
EMSA with antibody supershift, methylation interference, transient transfection, antisense oligonucleotide treatment |
The Journal of biological chemistry |
Medium |
8262968
|
| 1993 |
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. |
Recombinant protein EMSA, phosphorylation in reticulocyte lysate |
Proceedings of the National Academy of Sciences |
Medium |
8430110
|
| 1994 |
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. |
In vitro nucleosome binding assay with reconstituted chromatin templates |
Molecular and cellular biology |
Medium |
8196648
|
| 1996 |
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. |
cDNA cloning, heterodimerization assay, DNA binding assay, transcription activation assay, genetic mapping |
Science |
Medium |
8929412
|
| 1997 |
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. |
In vitro DNA binding assay, stable cell line overexpression, reporter gene assay, growth assay, apoptosis assay |
The Journal of biological chemistry |
Medium |
9211884
|
| 1997 |
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. |
Coimmunoprecipitation, reporter gene assay, deletion mutagenesis, Myc-Ras cotransformation assay |
Current topics in microbiology and immunology |
Medium |
9308234
|
| 1997 |
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. |
Circular dichroism spectroscopy, sedimentation equilibrium |
Journal of biochemistry |
Medium |
9399572
|
| 1999 |
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. |
Protein interaction screening, coimmunoprecipitation, EMSA, reporter gene assay, mSin3A co-IP |
The Journal of biological chemistry |
Medium |
10593926
|
| 1999 |
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). |
Real-time quantitative PCR, transfection of Max-expressing plasmid, antisense oligodeoxynucleotide knockdown, reporter gene assay |
Molecular endocrinology |
Medium |
10446908
|
| 2003 |
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. |
X-ray crystallography |
Cell |
High |
12553908
|
| 2004 |
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. |
Surface plasmon resonance kinetics, fluorescence-based binding assay, EMSA with cell extract |
Biochimica et biophysica acta |
Medium |
14980448
|
| 2005 |
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. |
Coimmunoprecipitation, chromatin immunoprecipitation (ChIP), cell cycle analysis, Cre-lox genetic epistasis in MEFs |
The Journal of cell biology |
High |
15866886
|
| 2005 |
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. |
Circular dichroism, NMR, analytical ultracentrifugation (sedimentation equilibrium), fluorescence-based dimerization assay |
Biochemistry |
Medium |
16171389
|
| 2007 |
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. |
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 |
The Biochemical journal |
High |
17217336
|
| 2008 |
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. |
Drosophila genetics (Max loss-of-function and reduction-of-function mutations), Myc dimerization-mutant analysis, RNA Pol III transcription assay |
Nature genetics |
High |
19165923
|
| 2011 |
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. |
Exome sequencing, validation sequencing, immunohistochemistry for MAX protein, LOH analysis |
Nature genetics |
High |
21685915
|
| 2014 |
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. |
RT-PCR isoform detection, protein stability assay, NMD inhibition, heterologous protein stability reporter |
FEBS letters |
Medium |
25451222
|
| 2017 |
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. |
Quantitative binding assays, X-ray crystallography of MAX–DNA complex, in vitro mutagenesis, analysis of >800 primary myeloma genomes |
Nucleic acids research |
High |
27903915
|
| 2017 |
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. |
Next-generation sequencing, immunohistochemistry, MAX re-expression/rescue experiment in GIST cells with p16 and proliferation readouts |
Nature communications |
High |
28270683
|
| 2019 |
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. |
B-cell-specific Cre-lox Max deletion, Eµ-Myc lymphoma model, RNA-seq, ChIP-seq, western blot for MYC protein levels |
Genes & development |
High |
31395740
|
| 2022 |
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. |
NMR spectroscopy (interaction mapping), Surface Plasmon Resonance (DNA binding kinetics), Casein Kinase 2 in vitro phosphorylation |
Journal of molecular biology |
High |
36174765
|