{"gene":"ID4","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1994,"finding":"ID4 protein contains an HLH dimerization domain but lacks the DNA-binding basic region, functioning as a dominant-negative inhibitor of bHLH transcription factors; ID4 inhibits DNA-binding of E47 homodimers and E47/MyoD heterodimers in vitro.","method":"In vitro DNA-binding assay (gel shift/EMSA) with recombinant proteins","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct in vitro reconstitution of inhibitory activity on E47 and MyoD heterodimers, foundational biochemical characterization replicated across multiple papers","pmids":["8139914"],"is_preprint":false},{"year":1995,"finding":"Enforced expression of ID4 (Id-4H) inhibits trans-activation of the muscle creatine kinase E-box enhancer by MyoD in transfection experiments, confirming dominant-negative function.","method":"Transient transfection reporter assay (E-box/MCK enhancer-luciferase) in mammalian cells","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — single lab, single method (reporter assay), but direct functional demonstration","pmids":["7665172"],"is_preprint":false},{"year":1998,"finding":"The human ID4 promoter is regulated by two functional elements: (1) an E-box bound by the bHLH-zip protein USF1 that stimulates transcription (enforced USF1 increases E-box-mediated promoter activity), and ID4 itself blocks this stimulation via heterodimerization with bHLH factors; (2) a downstream GA motif recognized by Sp1 and Sp3 that represses ID4 transcription ~20-fold.","method":"Transient transfection reporter assay, gel mobility shift assay (EMSA), transfection into Drosophila SL2 cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — multiple orthogonal methods (EMSA, reporter assay, heterologous cell system), single lab, direct cis-element and trans-factor identification","pmids":["9516472"],"is_preprint":false},{"year":1999,"finding":"The cAMP-dependent signaling pathway (activated by dibutyryl-cAMP, forskolin, or pentoxifylline) selectively and dramatically decreases Id4 gene expression in primary astrocytes; overexpression of Id4 in an astrocyte-derived cell line induces apoptosis (cells round up and die).","method":"Pharmacological treatment of primary astrocyte cultures, Northern blot, overexpression by transfection with morphological/apoptosis readout","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal approaches (pharmacological regulation and overexpression phenotype), single lab","pmids":["10066362"],"is_preprint":false},{"year":2000,"finding":"Enforced expression of Id4 in oligodendrocyte precursor cells (OPCs) stimulates proliferation and blocks differentiation induced by mitogen withdrawal or thyroid hormone; Id4 mRNA and protein decrease together under pro-differentiation conditions, indicating transcriptional control as part of a differentiation-timing mechanism.","method":"Retroviral overexpression in OPC cultures, differentiation induction by mitogen withdrawal and thyroid hormone treatment, Northern and Western blot for Id4","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal conditions (mitogen withdrawal, thyroid hormone), gain-of-function with specific cellular phenotypes, replicated across conditions","pmids":["10790366"],"is_preprint":false},{"year":2001,"finding":"Id4 expression in Sertoli cells is induced by FSH or cAMP (mimicking FSH via PKA pathway), whereas Id1 is downregulated by FSH; antisense to Id4 had no effect on FSH-mediated transferrin promoter activation in Sertoli cells, unlike Id1 and Id2.","method":"Immunoprecipitation with isoform-specific antibodies, RT-PCR, Northern blot, antisense oligonucleotide transfection with transferrin promoter reporter assay","journal":"Endocrinology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal approach (antisense + reporter) and protein detection methods, single lab, two orthogonal methods","pmids":["11316735"],"is_preprint":false},{"year":2001,"finding":"Ribozyme-mediated knockdown of ID4 upregulates BRCA1 expression in human ovarian cancer cells (identified by inverse genomics), and increased ID4 expression correlates with anchorage-independent growth; ID4 acts as a negative regulator of BRCA1 promoter activity.","method":"Randomized ribozyme gene library screen with EGFP reporter under BRCA1 promoter, target gene identification and modulation experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional genomic screen plus direct expression modulation experiments, single lab","pmids":["11136250"],"is_preprint":false},{"year":2003,"finding":"ID4 promoter hypermethylation silences ID4 expression in gastric cancer; treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (with or without trichostatin A) restores ID4 expression and is accompanied by promoter demethylation. ID4 promoter methylation significantly associates with hMLH1 methylation and microsatellite instability.","method":"Bisulfite genomic sequencing, methylation-specific PCR, RT-PCR, pharmacological demethylation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (bisulfite sequencing, MSP, pharmacological reactivation), single lab","pmids":["14534543"],"is_preprint":false},{"year":2003,"finding":"Id4 overexpression in HC11 mammary epithelial cells increases cell proliferation, inhibits lactogenic hormone-mediated differentiation (reduces beta-casein promoter activity and expression), and induces anchorage-independent growth in soft agar.","method":"Transient and stable transfection of Id4, proliferation assay, beta-casein promoter reporter assay, soft agar colony assay","journal":"The American journal of pathology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal readouts (proliferation, differentiation marker, soft agar), single lab","pmids":["14633621"],"is_preprint":false},{"year":2003,"finding":"Xenopus Id4 can inhibit the neurogenic activity of neurogenin and neuroD and the myogenic activity of MyoD in animal cap and in vivo overexpression assays, whereas Id2 inhibits only MyoD and neuroD, and Id3 blocks only neuroD—demonstrating differential substrate specificity among Id proteins.","method":"Animal cap explant assay, in vivo overexpression in Xenopus embryos, reporter assay","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple bHLH substrates tested, ortholog study with direct functional comparison, single lab","pmids":["14651922"],"is_preprint":false},{"year":2004,"finding":"Id4 is required for normal G1-S transition in early cortical progenitors; Id4-null mice show reduced proliferation of ventricular zone stem cells and premature neuronal differentiation, demonstrating that Id4 regulates neural progenitor proliferation and differentiation in vivo.","method":"Id4-knockout mouse analysis, BrdU incorporation, immunohistochemistry, in vitro neurosphere cultures","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function (knockout mouse) with multiple orthogonal readouts (BrdU, IHC, neurosphere), replicated in vitro and in vivo","pmids":["15469968"],"is_preprint":false},{"year":2005,"finding":"Id4 deficiency in mice leads to precocious oligodendrocyte differentiation both in vitro and in vivo; overexpression of Id4 in OPC progenitors prevents differentiation and decreases myelin gene expression (MBP, CGT but not PLP promoters), and Id4 differentially regulates myelin protein expression and subcellular distribution.","method":"Id4-knockout mouse analysis, overexpression via retroviral transduction, luciferase reporter assay for CGT/MBP/PLP promoters, immunohistochemistry","journal":"Glia","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function mouse plus gain-of-function with multiple reporter assays and in vivo/in vitro concordance","pmids":["16862533"],"is_preprint":false},{"year":2005,"finding":"Id4 deficiency causes premature neuronal differentiation resulting in a thicker neocortex with more early-born neurons; adult Id4-/- brains are smaller with depleted late-born cortical neurons and astrocytes, and neurosphere proliferation is impaired; BMP2-mediated astrocyte differentiation is accelerated in Id4-/- NPCs.","method":"Id4-knockout mouse analysis, immunohistochemistry, neurosphere culture, BMP2 treatment assay","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with multiple quantitative readouts in vivo and in vitro, replicated in neurosphere system","pmids":["15882580"],"is_preprint":false},{"year":2006,"finding":"All four ID genes (ID1-4) are neuronal targets repressed by MeCP2; chromatin immunoprecipitation confirmed MeCP2 binding near/within ID1, ID2, ID3 promoters; ID4 mRNA and protein are increased in Mecp2-deficient mouse brain.","method":"Chromatin immunoprecipitation (ChIP), quantitative RT-PCR, immunofluorescence, laser scanning cytometry in Mecp2-deficient mouse and human RTT brain","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus expression validation in multiple systems (mouse and human tissue), single lab","pmids":["16682435"],"is_preprint":false},{"year":2009,"finding":"The protein complex mutant p53-E2F1 assembles on specific regions of the ID4 promoter and positively controls ID4 transcription; ID4 protein then binds to and stabilizes mRNAs encoding pro-angiogenic factors IL8 and GRO-alpha, increasing angiogenic potential in mutant p53-expressing cancer cells.","method":"Chromatin immunoprecipitation (ChIP) of mutant p53-E2F1 on ID4 promoter; RNA immunoprecipitation (RIP) and mRNA stability assays for IL8/GRO-alpha; siRNA knockdown of mutant p53; angiogenesis assays","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ChIP for promoter occupancy, RNA pulldown for mRNA binding, functional angiogenesis assay; multiple orthogonal methods in single rigorous study","pmids":["19783986"],"is_preprint":false},{"year":2009,"finding":"Ectopic Id4 expression in DU145 prostate cancer cells increases apoptosis and decreases proliferation (S-phase arrest), associated with increased androgen receptor (AR), p21, p27, and p53 expression; Id4 promoter is hypermethylated in DU145 cells.","method":"Ectopic expression by transfection, cell cycle analysis (3H-thymidine, FACS), RT-PCR, real-time PCR, Western blot, immunocytochemistry, MSP","journal":"BMC cancer","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal readouts (cell cycle, apoptosis, molecular markers), single lab","pmids":["19500415"],"is_preprint":false},{"year":2010,"finding":"ID4 is required for adipocyte differentiation: Id4-/- mice have reduced body fat; MEFs from Id4-/- mice have reduced adipogenic potential with decreased C/EBPα and PPARγ expression; ID4 is induced during 3T3-L1 adipocyte differentiation.","method":"Id4-knockout mouse analysis, MEF isolation and adipogenic differentiation assay, Western blot for C/EBPα and PPARγ","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout plus MEF differentiation assay with molecular markers, in vitro and in vivo concordance","pmids":["20460371"],"is_preprint":false},{"year":2010,"finding":"Id4 promotes osteoblast differentiation via an Id4-Hes1-Hey2 mechanism: Id4 releases Hes1 from Hes1-Hey2 repressor complexes, and free Hes1 increases stability and transcriptional activity of Runx2, a key osteoblast differentiation factor. Loss of Id4 (Id4-/- mice) drastically reduces osteoblast differentiation and drives MSC differentiation toward adipocytes instead.","method":"Id4-knockout mouse analysis, ST2 cell line Id4 knockdown, genome-wide expression analysis, co-immunoprecipitation for Hes1-Hey2 interaction, Runx2 activity assays","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, knockdown, and biochemical interaction studies with mechanistic pathway placement (Id4-Hes1-Hey2-Runx2 axis)","pmids":["20628571"],"is_preprint":false},{"year":2011,"finding":"ID4 enhances SOX2 protein expression by suppressing miR-9* (which targets SOX2 3'-UTR), leading to SOX2-mediated induction of ABC transporters ABCC3 and ABCC6 and chemoresistance in glioma stem-like cells.","method":"shRNA knockdown, microRNA overexpression, luciferase 3'-UTR reporter assay, RT-PCR, Western blot, drug resistance assay","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — 3'-UTR reporter validation, shRNA knockdown of pathway components with rescue experiments, multiple orthogonal methods in single study","pmids":["21531766"],"is_preprint":false},{"year":2011,"finding":"ID4 regulates mammary gland ductal expansion by suppressing p38MAPK activity; p38MAPK is activated in Id4-null mammary cells and after siRNA-mediated knockdown; this p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands.","method":"Id4-knockout mouse mammary analysis, siRNA knockdown in transformed mammary cells, p38MAPK phosphorylation Western blot, p38MAPK inhibitor treatment","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO, siRNA KD, and pharmacological inhibitor rescue for pathway placement, multiple orthogonal approaches","pmids":["22069192"],"is_preprint":false},{"year":2013,"finding":"Id4 interacts directly with Twist1 (a bHLH transcription factor) to suppress Twist1-mediated MMP2 expression, thereby inhibiting invasiveness of glioblastoma cells; Id4 expression inversely correlates with MMP2 in GBM patient data.","method":"Co-immunoprecipitation of Id4 with Twist1, MMP2 reporter assay, invasion assay, siRNA knockdown, TCGA bioinformatic correlation","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction by Co-IP plus functional invasion assay, single lab","pmids":["24413082"],"is_preprint":false},{"year":2013,"finding":"ID4 forms a physical complex with mutant p53 and the acetyltransferase CBP/p300, resulting in acetylation of mutant p53 at K373; this acetylation restores mutant p53 DNA binding and transcriptional activity (BAX, PUMA, p21 expression and apoptosis). Loss of ID4 in LNCaP cells abrogates wild-type p53 DNA binding.","method":"Co-immunoprecipitation of ID4-p53-CBP/p300 complex, p53 acetylation Western blot, EMSA for p53 DNA binding, luciferase reporter for p53 transcriptional activity, apoptosis assay","journal":"Molecular cancer","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods: Co-IP for complex, acetylation analysis, DNA-binding assay, reporter assay, functional apoptosis readout in single rigorous study","pmids":["24330748"],"is_preprint":false},{"year":2014,"finding":"ID4 is a transcriptional target of gain-of-function p53 mutants (R175H, R273H, R280K); the mutant p53-E2F1 protein complex assembles on the ID4 promoter and positively controls ID4 expression. (Note: this finding was established in 2009; the 2014 review paper [PMID:25512197] elaborates on this but the original data is from PMID:19783986.)","method":"ChIP, reporter assay, siRNA depletion of mutant p53","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — ChIP for direct promoter occupancy, functional reporter, depletion experiments; original data from PMID:19783986","pmids":["19783986"],"is_preprint":false},{"year":2014,"finding":"EZH2-dependent H3K27me3 initiates ID4 promoter silencing in prostate cancer; EZH2 and H3K27me3 are enriched at the ID4 promoter along with DNMT1 in ID4-negative cell lines; EZH2 knockdown in DU145 leads to re-expression of ID4 with concomitant loss of EZH2, H3K27me3, and DNMT1 at the promoter.","method":"Chromatin immunoprecipitation (ChIP) for EZH2, H3K27me3, DNMT1 on ID4 promoter; siRNA knockdown of EZH2; RT-PCR for ID4 re-expression","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — ChIP for multiple histone marks and enzymes, knockdown rescue experiment, single lab with multiple orthogonal methods","pmids":["25115397"],"is_preprint":false},{"year":2014,"finding":"ID4 heterodimerizes with ID1, ID2, and ID3 via its HLH domain, acting as an inhibitor of inhibitors; ID4 promotes E47 (bHLH) DNA binding by sequestering ID1 (demonstrated by EMSA); the N-terminal alanine-stretch unique to ID4's HLH domain is required for optimal ID-ID interaction; ID4 and ID1 co-localize in prostate cancer cells.","method":"Co-immunoprecipitation, EMSA (electrophoretic mobility shift assay) for E47 DNA binding competition, co-localization by immunofluorescence, domain deletion mutagenesis","journal":"Biochimie","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct biochemical interaction by Co-IP, EMSA competition assay, mutagenesis of functional domain, single lab with multiple orthogonal methods","pmids":["25778840"],"is_preprint":false},{"year":2014,"finding":"Id4 acts as a downstream target of Notch2 signaling and maintains hippocampal neural stem cell (NSC) quiescence; Id4 knockdown rescues Notch2-induced inhibition of NSC proliferation; Id4 deletion activates NSC proliferation in the dentate gyrus without evoking neurogenesis; Id4 overexpression increases NSC maintenance and promotes astrogliogenesis at the expense of neurogenesis.","method":"Conditional Id4 knockout, lentiviral shRNA knockdown, Notch2 gain-of-function, BrdU proliferation assay, immunofluorescence, neurosphere assay","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO, knockdown epistasis with Notch2, gain-of-function, multiple cellular phenotype readouts establishing pathway position","pmids":["31390563"],"is_preprint":false},{"year":2014,"finding":"Id4 chromatin immunoprecipitation confirmed Id4 binding to the Esr1 and Foxa1 promoter regions in mammary cells; Id4 deficiency leads to striking increases in ERα (Esr1), PR, and FoxA1 expression in both basal and luminal mammary subsets, indicating that Id4 is a negative transcriptional regulator of the ERα signaling axis.","method":"Conditional MMTV-Cre/K14-Cre Id4 knockout mouse model, ChIP for Id4 on Esr1/Foxa1 promoters, RT-PCR/Western blot for target gene expression","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic loss-of-function plus direct ChIP evidence of promoter binding, multiple orthogonal methods, single focused study","pmids":["25038044"],"is_preprint":false},{"year":2014,"finding":"Loss of ID4 in LNCaP prostate cancer cells (L-ID4) promotes castration-resistant prostate cancer (CRPC) through constitutive AR activation and de novo steroidogenesis; RNA-seq revealed a gene expression signature mimicking constitutively active AR in L-ID4 cells; PSA expression persisted in castrated conditions in L-ID4 but not control cells.","method":"Stable shRNA silencing of ID4 in LNCaP, RNA-seq, xenograft in castrated vs. non-castrated mice, PSA expression assay","journal":"Molecular endocrinology","confidence":"High","confidence_rationale":"Tier 2 / Strong — stable knockdown, RNA-seq, and in vivo castration model with mechanistic pathway identification","pmids":["24921661"],"is_preprint":false},{"year":2015,"finding":"ID4 controls mammary stem cell self-renewal by suppressing key factors required for luminal differentiation; in an ID4-GFP knock-in reporter mouse, ID4 marks a stem cell-enriched basal mammary cell subset; single-cell transcriptomics revealed ID4-high cells have a distinct stem cell transcriptional signature.","method":"ID4-GFP knock-in reporter mouse, single-cell transcriptomics, FACS, mammary reconstitution assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — knock-in reporter, single-cell transcriptomics, and functional reconstitution assay in single comprehensive study","pmids":["25813983"],"is_preprint":false},{"year":2016,"finding":"ID4 directly interacts with FKBP52 (FK506-binding protein 52) in vitro (but not directly with AR); loss of ID4 increases FKBP52-dependent AR transcriptional activity and promotes CRPC; pharmacological inhibition of FKBP52-AR signaling with MJC13 attenuates tumor growth in L-ID4 xenografts.","method":"Proteomic analysis (proteomics of L-ID4 vs. L+ns), co-immunoprecipitation/protein interaction studies for ID4-FKBP52, in vitro binding assay, AR reporter assay, MJC13 pharmacological inhibition in xenograft","journal":"Molecular oncology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — direct in vitro interaction, proteomic identification, functional AR reporter, and in vivo pharmacological rescue; single lab with multiple orthogonal methods","pmids":["28252832"],"is_preprint":false},{"year":2017,"finding":"Mutant p53 and ID4 delocalize lncRNA MALAT1 from nuclear speckles and favor its association with chromatin by recruiting MALAT1 via splicing factor SRSF1; this enables aberrant recruitment of MALAT1 on VEGFA pre-mRNA and modulation of VEGFA isoform expression in breast cancer cells.","method":"RNA immunoprecipitation (RIP), co-immunoprecipitation, RNA FISH for MALAT1 localization, VEGFA isoform analysis by RT-PCR, shRNA knockdown","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — RIP, Co-IP, RNA localization by FISH, functional isoform analysis; multiple orthogonal methods in single study","pmids":["28652379"],"is_preprint":false},{"year":2017,"finding":"ID4 level dictates spermatogonial stem cell (SSC) vs. progenitor state: the ID4-EGFPBright population functions as SSCs, whereas ID4-EGFPDim cells are transitioning to progenitors; conditional overexpression of Id4 blocks transition from SSC to progenitor state.","method":"Id4-eGFP reporter mouse, conditional Id4 overexpression mouse model, RNA-seq, FACS, transplantation assay","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — reporter mouse, conditional overexpression, RNA-seq, and functional transplantation; multiple orthogonal methods across genetic models","pmids":["28087628"],"is_preprint":false},{"year":2017,"finding":"ID4 regulates wild-type and mutant p53 transcriptional activity by promoting acetylation at K373 (and to a lesser extent K320) via CBP/p300 interaction; acetylation-mimic (K373Q) and non-acetylation mimic (K373R) p53 mutants confirmed K373 as the critical residue mediating ID4-dependent p53 activation and downstream cell cycle arrest/apoptosis.","method":"Ectopic expression in p53-null prostate cancer cells, acetylation-mimic/non-mimic p53 mutants, p53 DNA-binding assay, target gene expression (RT-PCR, Western blot), apoptosis/cell cycle assay","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1 / Moderate — site-specific mutagenesis of p53 acetylation sites, functional DNA-binding assay, and downstream target validation; single lab but multiple rigorous methods","pmids":["27911860"],"is_preprint":false},{"year":2019,"finding":"Id4 promotes elimination of the pro-activation bHLH factor Ascl1 protein in hippocampal neural stem cells (NSCs) by sequestering Ascl1's heterodimerization partner E47, thereby promoting Ascl1 protein degradation and maintaining NSC quiescence; Id4 deletion causes abnormal Ascl1 protein accumulation and premature stem cell activation.","method":"Id4 conditional knockout in adult hippocampus, Id4 overexpression in NSC cultures, Ascl1 protein level analysis by Western blot, E47-Ascl1-Id4 interaction studies by Co-IP, BrdU proliferation assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout, protein interaction (Co-IP), gain-of-function, mechanistic link between Id4/E47/Ascl1 established with multiple orthogonal approaches","pmids":["31552825"],"is_preprint":false},{"year":2020,"finding":"ID4 interacts with mediator of DNA damage checkpoint protein 1 (MDC1) and, through MDC1, also with DNA repair proteins γH2AX and BRCA1 at fragile chromatin sites; ID4 binds to chromatin following DNA damage but does not affect transcription at these sites, suggesting a role in DNA damage response rather than transcription per se.","method":"RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins), ChIP-seq, RNA-seq, immunoprecipitation","journal":"Breast cancer research : BCR","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — RIME proteomics for endogenous interactions, ChIP-seq for genome-wide binding, RNA-seq for transcriptional impact; multiple orthogonal methods in single study","pmids":["32527287"],"is_preprint":false},{"year":2021,"finding":"ID4 interacts with the bHLH protein HEB (detected by mass spectrometry); ID4 antagonizes HEB transcriptional activity, preventing HEB from binding E-box motifs in regulatory elements of myoepithelial developmental genes (extracellular matrix, contractile cytoskeleton); loss of ID4 in mammary organoids/in vivo allows HEB-dependent myoepithelial differentiation.","method":"Mass spectrometry (proteomics) for ID4 interactome, ChIP-seq for HEB genomic binding, mammary organoid culture, conditional Id4 knockout mouse, RNA-seq","journal":"iScience","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — mass spectrometry for protein interaction, ChIP-seq for genomic targets, genetic KO with functional cellular phenotype; multiple orthogonal methods","pmids":["33554073"],"is_preprint":false},{"year":2021,"finding":"CRISPR/dCas9-DNMT3a-targeted methylation of the Id2 and Id4 promoters drives oligodendrocyte precursor cell (OPC) differentiation, confirming that DNA methylation-mediated suppression of ID4 (and ID2) is a direct epigenetic mechanism controlling OPC differentiation.","method":"Epigenetic editing with CRISPR/dCas9-DNMT3a for targeted methylation of Id4 promoter, OPC differentiation assay","journal":"Cellular and molecular life sciences : CMLS","confidence":"High","confidence_rationale":"Tier 1 / Moderate — targeted epigenetic editing (CRISPR-based) with direct functional readout; single lab but rigorous method","pmids":["34482420"],"is_preprint":false},{"year":2024,"finding":"ID4 expression in breast cancer cells promotes VEGFA secretion, which stimulates VEGFR2-integrin β3 interaction in a paracrine fashion; this interaction activates the focal adhesion pathway, promotes migration/invasion/stress fiber formation, and drives nuclear translocation and activation of YAP and TAZ.","method":"Overexpression and knockdown of ID4, conditioned media paracrine experiments, co-immunoprecipitation of VEGFR2-integrin β3, VEGFA ELISA, YAP/TAZ nuclear localization assay, invasion/migration assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — paracrine experiment, receptor interaction Co-IP, nuclear localization assay, single lab with multiple orthogonal methods","pmids":["38321003"],"is_preprint":false}],"current_model":"ID4 is a dominant-negative HLH protein that lacks a DNA-binding basic domain and functions by heterodimerizing with bHLH transcription factors (E47, MyoD, Twist1, HEB, Neurogenin, NeuroD) and with other ID proteins (ID1-3) to inhibit their transcriptional activity, thereby regulating cell proliferation, differentiation timing (in neural progenitors, oligodendrocytes, spermatogonial stem cells, mammary stem cells, osteoblasts, and adipocytes), and stem cell quiescence; ID4 is transcriptionally controlled by mutant p53-E2F1 complexes and Notch2 signaling, and is epigenetically silenced by EZH2/H3K27me3-initiated promoter methylation in multiple cancers; at the protein level, ID4 forms a complex with CBP/p300 and p53 to promote p53 acetylation at K373, restoring p53 transcriptional activity, and interacts directly with FKBP52 to suppress AR signaling; in the context of mRNA regulation, ID4 binds and stabilizes IL8 and GRO-alpha mRNAs (downstream of mutant p53) to promote angiogenesis, and recruits MALAT1 via SRSF1 to modulate VEGFA isoform splicing; in mammary stem cells, ID4 interacts with HEB to prevent myoepithelial differentiation, and promotes VEGFA secretion that activates VEGFR2-integrin β3-YAP/TAZ signaling."},"narrative":{"mechanistic_narrative":"ID4 is a helix-loop-helix (HLH) protein that lacks the DNA-binding basic region and acts as a dominant-negative regulator of differentiation timing, proliferation, and stem cell state across multiple lineages [PMID:8139914, PMID:15469968]. Its core biochemical activity is sequestration: through its HLH domain it heterodimerizes with bHLH transcription factors (E47, MyoD, Neurogenin, NeuroD, Twist1, HEB) to block their DNA binding and trans-activation [PMID:8139914, PMID:7665172, PMID:14651922, PMID:24413082, PMID:33554073], and it also dimerizes with other ID proteins (ID1-3), acting as an \"inhibitor of inhibitors\" that can paradoxically free E47 by titrating ID1 [PMID:25778840]. This sequestration logic underlies its developmental roles: ID4 sustains progenitor proliferation and prevents premature differentiation in cortical neural progenitors, oligodendrocyte precursors, mammary stem cells, and spermatogonial stem cells, with loss causing precocious differentiation in each [PMID:15469968, PMID:16862533, PMID:15882580, PMID:25813983, PMID:28087628]. In adult hippocampal neural stem cells ID4 enforces quiescence downstream of Notch2, in part by sequestering E47 to promote degradation of the pro-activation bHLH factor Ascl1 [PMID:31390563, PMID:31552825]. ID4 directs lineage choice through specific partners: it antagonizes HEB to prevent myoepithelial differentiation in mammary cells [PMID:33554073], releases Hes1 from Hes1-Hey2 complexes to stabilize Runx2 and promote osteoblast over adipocyte fate [PMID:20628571], and is required for adipocyte differentiation via C/EBPalpha and PPARgamma [PMID:20460371]. ID4 is itself tightly regulated transcriptionally — by USF1/Sp1-Sp3 promoter elements, cAMP/PKA signaling, MeCP2 repression, and a mutant p53-E2F1 complex — and is epigenetically silenced in cancers by EZH2/H3K27me3-initiated, DNMT-dependent promoter methylation [PMID:9516472, PMID:10066362, PMID:16682435, PMID:19783986, PMID:25115397, PMID:34482420]. Beyond transcription factor sequestration, ID4 has acquired direct protein- and RNA-level functions in cancer: it forms a complex with CBP/p300 and p53 to drive p53 acetylation at K373 and restore p53 transcriptional activity [PMID:24330748, PMID:27911860], binds and stabilizes pro-angiogenic IL8 and GRO-alpha mRNAs downstream of mutant p53 [PMID:19783986], recruits the lncRNA MALAT1 via SRSF1 to reshape VEGFA splicing [PMID:28652379], and interacts with FKBP52 to restrain androgen receptor signaling, with ID4 loss promoting castration-resistant prostate cancer [PMID:24921661, PMID:28252832]. ID4 is also a negative transcriptional regulator of the ERalpha axis (Esr1, FoxA1) and BRCA1, and associates with MDC1 at damaged chromatin, implicating it in the DNA damage response [PMID:11136250, PMID:25038044, PMID:32527287].","teleology":[{"year":1994,"claim":"Established ID4's foundational biochemical identity: a basic-region-less HLH protein that inhibits bHLH factors, defining its mechanism of action.","evidence":"In vitro EMSA with recombinant ID4 against E47 homodimers and E47/MyoD heterodimers","pmids":["8139914"],"confidence":"High","gaps":["In vitro binding does not establish which bHLH partners are physiologically relevant in vivo","No cellular phenotype demonstrated"]},{"year":1995,"claim":"Confirmed the dominant-negative activity in cells, showing ID4 represses MyoD-driven transcription at an E-box enhancer.","evidence":"MCK-luciferase reporter assay in transfected mammalian cells","pmids":["7665172"],"confidence":"Medium","gaps":["Single reporter system","Endogenous muscle differentiation not tested"]},{"year":1998,"claim":"Mapped the cis-regulatory architecture controlling ID4's own transcription, revealing USF1 activation and Sp1/Sp3 repression plus autoregulatory feedback.","evidence":"EMSA and reporter assays including heterologous Drosophila SL2 cells","pmids":["9516472"],"confidence":"High","gaps":["Promoter context studied in transfection, not endogenous chromatin","Physiological signals driving USF1/Sp1 occupancy not defined"]},{"year":1999,"claim":"Linked ID4 expression to cAMP signaling and revealed a pro-apoptotic consequence of overexpression in astrocytes.","evidence":"Pharmacological cAMP modulation of primary astrocytes and overexpression with apoptosis readout","pmids":["10066362"],"confidence":"Medium","gaps":["Mechanism connecting cAMP to Id4 promoter not resolved","Apoptosis phenotype may be overexpression artifact"]},{"year":2000,"claim":"Defined ID4 as a differentiation-timing switch in oligodendrocyte precursors, where its downregulation permits differentiation.","evidence":"Retroviral overexpression in OPCs with mitogen-withdrawal and thyroid hormone differentiation assays","pmids":["10790366"],"confidence":"High","gaps":["bHLH partners mediating the block not identified","Loss-of-function not yet tested"]},{"year":2001,"claim":"Distinguished ID4 from other ID family members through isoform-specific hormonal regulation (FSH/cAMP-induced) and dispensability for transferrin promoter activation.","evidence":"RT-PCR, Northern blot, antisense oligonucleotide plus reporter assay in Sertoli cells","pmids":["11316735"],"confidence":"Medium","gaps":["Functional role of FSH-induced Id4 in Sertoli cells unclear","No direct target identified"]},{"year":2001,"claim":"Identified ID4 as a negative regulator of the BRCA1 promoter and linked it to anchorage-independent growth.","evidence":"Ribozyme library screen with BRCA1-promoter EGFP reporter and expression modulation in ovarian cancer cells","pmids":["11136250"],"confidence":"Medium","gaps":["Mechanism of BRCA1 promoter repression not defined","Direct vs indirect regulation unresolved"]},{"year":2003,"claim":"Revealed epigenetic silencing of ID4 by promoter hypermethylation in gastric cancer, establishing ID4 as a methylation-regulated locus.","evidence":"Bisulfite sequencing, MSP, and pharmacological demethylation with RT-PCR","pmids":["14534543"],"confidence":"Medium","gaps":["Causality of silencing for tumor phenotype not tested","Enzymes initiating methylation not identified"]},{"year":2003,"claim":"Extended ID4's proliferation/differentiation switch and oncogenic potential to mammary epithelium.","evidence":"Stable/transient Id4 transfection in HC11 cells with proliferation, beta-casein reporter, and soft agar assays","pmids":["14633621"],"confidence":"Medium","gaps":["bHLH targets in mammary cells not identified","In vivo relevance not addressed"]},{"year":2003,"claim":"Demonstrated differential bHLH substrate specificity among ID proteins, with ID4 uniquely inhibiting neurogenin, neuroD, and MyoD.","evidence":"Xenopus animal cap and in vivo overexpression assays comparing Id2/3/4","pmids":["14651922"],"confidence":"Medium","gaps":["Structural basis of specificity not defined","Ortholog data may not fully translate to mammalian ID4"]},{"year":2004,"claim":"Provided genetic proof that ID4 controls neural progenitor G1-S transition and prevents premature neuronal differentiation in vivo.","evidence":"Id4-knockout mice with BrdU, IHC, and neurosphere assays","pmids":["15469968"],"confidence":"High","gaps":["Molecular bHLH targets in cortical progenitors not pinpointed","Cell-cycle machinery link not detailed"]},{"year":2005,"claim":"Genetically confirmed ID4 as a brake on oligodendrocyte and astrocyte differentiation, controlling neocortical neuron output and glial timing.","evidence":"Id4-knockout mice plus retroviral overexpression with myelin-gene reporters and BMP2 differentiation assays","pmids":["16862533","15882580"],"confidence":"High","gaps":["Direct transcriptional effectors of myelin gene regulation not resolved","Interplay with BMP signaling mechanistically incomplete"]},{"year":2006,"claim":"Placed ID4 within MeCP2-dependent neuronal gene regulation, with ID4 derepressed in Mecp2-deficient brain.","evidence":"ChIP, qRT-PCR, and immunofluorescence in Mecp2-deficient mouse and Rett syndrome brain","pmids":["16682435"],"confidence":"Medium","gaps":["Direct MeCP2 binding shown for ID1-3 but ID4 inferred from expression change","Functional consequence of ID4 derepression not tested"]},{"year":2009,"claim":"Connected ID4 to oncogenic gain-of-function p53, showing a mutant p53-E2F1 complex drives ID4 transcription and ID4 then stabilizes pro-angiogenic IL8/GRO-alpha mRNAs.","evidence":"ChIP on ID4 promoter, RIP and mRNA stability assays, mutant p53 knockdown, angiogenesis assays","pmids":["19783986"],"confidence":"High","gaps":["RNA-binding domain/mode of ID4 not structurally defined","Generality across mutant p53 alleles addressed only later"]},{"year":2009,"claim":"Implicated ID4 in prostate cancer cell cycle/apoptosis control and linked its loss to AR pathway changes, with promoter hypermethylation in DU145.","evidence":"Ectopic expression in DU145 with cell cycle, apoptosis, marker expression, and MSP","pmids":["19500415"],"confidence":"Medium","gaps":["Mechanism linking ID4 to AR/p21/p27/p53 not yet defined here","Correlative marker changes only"]},{"year":2010,"claim":"Defined ID4 as a determinant of mesenchymal lineage choice — required for adipogenesis and, via an Id4-Hes1-Hey2-Runx2 axis, for osteoblast over adipocyte fate.","evidence":"Id4-knockout mice, ST2 knockdown, expression profiling, Co-IP for Hes1-Hey2, Runx2 activity assays","pmids":["20460371","20628571"],"confidence":"High","gaps":["Direct ID4-Hes1 binding vs displacement mechanism not fully resolved","Reconciliation of pro-adipogenic and anti-adipogenic roles across tissues incomplete"]},{"year":2011,"claim":"Revealed non-canonical ID4 functions: a miR-9*/SOX2/ABC-transporter axis driving glioma chemoresistance and p38MAPK suppression controlling mammary ductal expansion.","evidence":"shRNA/miRNA modulation with 3'-UTR reporters and drug assays; Id4-KO/siRNA with p38MAPK phosphorylation and inhibitor rescue","pmids":["21531766","22069192"],"confidence":"High","gaps":["How ID4 suppresses miR-9* or p38MAPK mechanistically not defined","Direct vs indirect effects unresolved"]},{"year":2013,"claim":"Identified direct ID4-Twist1 interaction as a brake on glioblastoma invasiveness through MMP2 repression.","evidence":"Co-IP, MMP2 reporter, invasion assay, siRNA, and TCGA correlation","pmids":["24413082"],"confidence":"Medium","gaps":["Single Co-IP without reciprocal/structural validation of the interaction","In vivo invasion not tested"]},{"year":2013,"claim":"Established a protein-level ID4 function distinct from bHLH sequestration: ID4-CBP/p300-p53 complex formation drives p53 K373 acetylation to restore p53 DNA binding and transcriptional/apoptotic activity.","evidence":"Co-IP of ID4-p53-CBP/p300, acetylation Western, EMSA, reporter, and apoptosis assays","pmids":["24330748"],"confidence":"High","gaps":["Whether ID4 directly contacts p300 or p53 to position acetylation not dissected","Structural basis unknown"]},{"year":2017,"claim":"Pinpointed K373 as the critical ID4-dependent p53 acetylation site using acetylation-mimic mutants, solidifying the ID4-p53 activation mechanism.","evidence":"K373Q/K373R p53 mutants with DNA-binding, target expression, and cell cycle/apoptosis assays in p53-null cells","pmids":["27911860"],"confidence":"High","gaps":["Mechanism by which ID4 enhances CBP/p300 catalysis not resolved","In vivo relevance of K373 axis not tested"]},{"year":2014,"claim":"Defined ID4 as a downstream effector and quiescence factor of Notch2 in adult hippocampal neural stem cells, with loss activating NSC proliferation.","evidence":"Conditional Id4 knockout, shRNA, Notch2 gain-of-function, BrdU and neurosphere assays","pmids":["31390563"],"confidence":"High","gaps":["Direct Notch2-to-Id4 transcriptional link not mapped here","Effector bHLH targets defined only in later work"]},{"year":2014,"claim":"Established ID4's role in mammary stem cell biology as a negative regulator of the ERalpha signaling axis through direct promoter binding.","evidence":"Conditional Id4 knockout mice with ChIP on Esr1/Foxa1 promoters and target expression analysis","pmids":["25038044"],"confidence":"High","gaps":["How a basic-region-less protein binds these promoters (direct vs co-factor) not resolved","bHLH partner mediating repression not identified here"]},{"year":2014,"claim":"Defined the epigenetic silencing mechanism of ID4 in cancer: EZH2/H3K27me3 initiates and DNMT1 maintains promoter methylation.","evidence":"ChIP for EZH2/H3K27me3/DNMT1 on ID4 promoter and EZH2 knockdown reactivation in prostate cancer cells","pmids":["25115397"],"confidence":"High","gaps":["Upstream signals recruiting EZH2 to ID4 not defined","Tissue generality of this mechanism beyond prostate not established here"]},{"year":2014,"claim":"Refined the inter-ID interaction logic, showing ID4 dimerizes with ID1-3 and can free E47 by sequestering ID1, acting as an inhibitor of inhibitors.","evidence":"Co-IP, EMSA competition for E47 binding, immunofluorescence colocalization, and domain-deletion mutagenesis","pmids":["25778840"],"confidence":"High","gaps":["Physiological contexts where the inhibitor-of-inhibitors mode dominates not defined","Quantitative dimer preferences not measured"]},{"year":2014,"claim":"Connected ID4 loss to castration-resistant prostate cancer through constitutive AR activation and de novo steroidogenesis.","evidence":"Stable shRNA silencing in LNCaP, RNA-seq, and castration xenograft model","pmids":["24921661"],"confidence":"High","gaps":["Molecular link from ID4 to AR activation defined in subsequent FKBP52 work","Steroidogenic enzyme regulation mechanism incomplete"]},{"year":2015,"claim":"Identified ID4 as a marker and driver of mammary stem cell self-renewal that suppresses luminal differentiation factors.","evidence":"ID4-GFP knock-in reporter mouse, single-cell transcriptomics, FACS, and mammary reconstitution","pmids":["25813983"],"confidence":"High","gaps":["bHLH effectors of the self-renewal program defined only in later HEB work","Direct target genes from this study limited"]},{"year":2016,"claim":"Provided the molecular basis for ID4-mediated AR restraint: direct ID4-FKBP52 interaction (not direct AR binding) that limits FKBP52-dependent AR activity.","evidence":"Proteomics, in vitro binding/Co-IP, AR reporter, and MJC13 pharmacological rescue in xenografts","pmids":["28252832"],"confidence":"High","gaps":["Structural mode of ID4-FKBP52 binding not resolved","Whether HLH domain mediates this interaction not established"]},{"year":2017,"claim":"Extended ID4's RNA-level oncogenic functions, showing ID4 with mutant p53 recruits MALAT1 via SRSF1 to chromatin to reshape VEGFA splicing.","evidence":"RIP, Co-IP, RNA-FISH for MALAT1 localization, and VEGFA isoform analysis with shRNA","pmids":["28652379"],"confidence":"High","gaps":["Direct vs SRSF1-bridged ID4-MALAT1 association not dissected","Functional output of altered VEGFA isoforms only partly characterized"]},{"year":2017,"claim":"Established ID4 as a dosage-sensitive determinant of spermatogonial stem cell vs progenitor identity, with high ID4 maintaining the stem state.","evidence":"Id4-eGFP reporter and conditional overexpression mice, RNA-seq, FACS, and transplantation","pmids":["28087628"],"confidence":"High","gaps":["bHLH partners enforcing SSC state not identified","Transcriptional targets in SSCs not mapped"]},{"year":2019,"claim":"Resolved a mechanism for ID4-enforced NSC quiescence: ID4 sequesters E47 to drive degradation of the pro-activation factor Ascl1.","evidence":"Conditional Id4 knockout, overexpression in NSCs, Ascl1 protein analysis, and E47-Ascl1-Id4 Co-IP","pmids":["31552825"],"confidence":"High","gaps":["Degradation machinery acting on freed Ascl1 not identified","Generalizability to other quiescent niches untested"]},{"year":2020,"claim":"Revealed a transcription-independent chromatin role for ID4 in the DNA damage response via MDC1, gammaH2AX, and BRCA1 at fragile sites.","evidence":"RIME endogenous interactome, ChIP-seq, RNA-seq, and immunoprecipitation in breast cancer cells","pmids":["32527287"],"confidence":"High","gaps":["Functional consequence of ID4 at damage sites for repair efficiency not established","Whether ID4 recruits or stabilizes MDC1 unclear"]},{"year":2021,"claim":"Identified HEB as the key bHLH partner through which ID4 blocks mammary myoepithelial differentiation, linking sequestration to lineage gene control.","evidence":"Mass spectrometry interactome, HEB ChIP-seq, conditional Id4 knockout organoids/mice, and RNA-seq","pmids":["33554073"],"confidence":"High","gaps":["Stoichiometry of ID4-HEB control at individual loci not quantified","Interplay with the ERalpha-suppressing role not integrated"]},{"year":2021,"claim":"Provided causal epigenetic proof that DNA methylation of the Id4 promoter is sufficient to drive OPC differentiation.","evidence":"CRISPR/dCas9-DNMT3a targeted methylation of the Id4 promoter with OPC differentiation readout","pmids":["34482420"],"confidence":"High","gaps":["Endogenous signals triggering this methylation in vivo not defined","Contribution of co-targeted Id2 not fully separated"]},{"year":2024,"claim":"Described a paracrine ID4 oncogenic circuit: ID4-driven VEGFA secretion activates VEGFR2-integrin beta3-focal adhesion signaling and YAP/TAZ in breast cancer.","evidence":"ID4 overexpression/knockdown, conditioned-media paracrine assays, VEGFR2-integrin beta3 Co-IP, VEGFA ELISA, YAP/TAZ localization, invasion assays","pmids":["38321003"],"confidence":"Medium","gaps":["How ID4 transcriptionally/post-transcriptionally raises VEGFA secretion not pinned down","In vivo significance of the YAP/TAZ axis not tested"]},{"year":null,"claim":"How a protein lacking a DNA-binding basic region achieves direct promoter occupancy (e.g., on Esr1/Foxa1/ID4-target genes) and integrates its many partner-specific functions—bHLH sequestration, p53 acetylation, RNA stabilization/splicing, FKBP52-AR restraint, and DNA-damage chromatin association—into context-dependent outputs remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No structural model of ID4 in any of its protein/RNA complexes","Mechanism of ID4 chromatin recruitment without a basic domain unknown","Rules governing which partner dominates in a given cell type undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,9,24,26]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,17,24,33]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[24,33]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[14,30]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[21,30,32]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[21,24,26,34]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[34,35]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,26,35]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[10,11,12,16,17,25,28,31]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[14,23,27,30,37]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[14,30]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[34]}],"complexes":["ID4-CBP/p300-p53 complex"],"partners":["E47","MYOD","TWIST1","HEB","FKBP52","MDC1","SRSF1","ID1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P47928","full_name":"DNA-binding protein inhibitor ID-4","aliases":["Class B basic helix-loop-helix protein 27","bHLHb27","Inhibitor of DNA binding 4","Inhibitor of differentiation 4"],"length_aa":161,"mass_kda":16.6,"function":"Transcriptional regulator (lacking a basic DNA binding domain) which negatively regulates the basic helix-loop-helix (bHLH) transcription factors by forming heterodimers and inhibiting their DNA binding and transcriptional activity. Implicated in regulating a variety of cellular processes, including cellular growth, senescence, differentiation, apoptosis, angiogenesis, and neoplastic transformation (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P47928/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ID4","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ID4","total_profiled":1310},"omim":[{"mim_id":"620305","title":"NEUROOCULORENAL SYNDROME; NORS","url":"https://www.omim.org/entry/620305"},{"mim_id":"605274","title":"MESOMELIC DYSPLASIA, SAVARIRAYAN TYPE","url":"https://www.omim.org/entry/605274"},{"mim_id":"602430","title":"ROUNDABOUT GUIDANCE RECEPTOR 1; ROBO1","url":"https://www.omim.org/entry/602430"},{"mim_id":"600581","title":"INHIBITOR OF DNA BINDING 4; ID4","url":"https://www.omim.org/entry/600581"},{"mim_id":"600386","title":"INHIBITOR OF DNA BINDING 2; ID2","url":"https://www.omim.org/entry/600386"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"thyroid 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proteins\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct in vitro reconstitution of inhibitory activity on E47 and MyoD heterodimers, foundational biochemical characterization replicated across multiple papers\",\n      \"pmids\": [\"8139914\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"Enforced expression of ID4 (Id-4H) inhibits trans-activation of the muscle creatine kinase E-box enhancer by MyoD in transfection experiments, confirming dominant-negative function.\",\n      \"method\": \"Transient transfection reporter assay (E-box/MCK enhancer-luciferase) in mammalian cells\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — single lab, single method (reporter assay), but direct functional demonstration\",\n      \"pmids\": [\"7665172\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The human ID4 promoter is regulated by two functional elements: (1) an E-box bound by the bHLH-zip protein USF1 that stimulates transcription (enforced USF1 increases E-box-mediated promoter activity), and ID4 itself blocks this stimulation via heterodimerization with bHLH factors; (2) a downstream GA motif recognized by Sp1 and Sp3 that represses ID4 transcription ~20-fold.\",\n      \"method\": \"Transient transfection reporter assay, gel mobility shift assay (EMSA), transfection into Drosophila SL2 cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple orthogonal methods (EMSA, reporter assay, heterologous cell system), single lab, direct cis-element and trans-factor identification\",\n      \"pmids\": [\"9516472\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The cAMP-dependent signaling pathway (activated by dibutyryl-cAMP, forskolin, or pentoxifylline) selectively and dramatically decreases Id4 gene expression in primary astrocytes; overexpression of Id4 in an astrocyte-derived cell line induces apoptosis (cells round up and die).\",\n      \"method\": \"Pharmacological treatment of primary astrocyte cultures, Northern blot, overexpression by transfection with morphological/apoptosis readout\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal approaches (pharmacological regulation and overexpression phenotype), single lab\",\n      \"pmids\": [\"10066362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Enforced expression of Id4 in oligodendrocyte precursor cells (OPCs) stimulates proliferation and blocks differentiation induced by mitogen withdrawal or thyroid hormone; Id4 mRNA and protein decrease together under pro-differentiation conditions, indicating transcriptional control as part of a differentiation-timing mechanism.\",\n      \"method\": \"Retroviral overexpression in OPC cultures, differentiation induction by mitogen withdrawal and thyroid hormone treatment, Northern and Western blot for Id4\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal conditions (mitogen withdrawal, thyroid hormone), gain-of-function with specific cellular phenotypes, replicated across conditions\",\n      \"pmids\": [\"10790366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Id4 expression in Sertoli cells is induced by FSH or cAMP (mimicking FSH via PKA pathway), whereas Id1 is downregulated by FSH; antisense to Id4 had no effect on FSH-mediated transferrin promoter activation in Sertoli cells, unlike Id1 and Id2.\",\n      \"method\": \"Immunoprecipitation with isoform-specific antibodies, RT-PCR, Northern blot, antisense oligonucleotide transfection with transferrin promoter reporter assay\",\n      \"journal\": \"Endocrinology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal approach (antisense + reporter) and protein detection methods, single lab, two orthogonal methods\",\n      \"pmids\": [\"11316735\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Ribozyme-mediated knockdown of ID4 upregulates BRCA1 expression in human ovarian cancer cells (identified by inverse genomics), and increased ID4 expression correlates with anchorage-independent growth; ID4 acts as a negative regulator of BRCA1 promoter activity.\",\n      \"method\": \"Randomized ribozyme gene library screen with EGFP reporter under BRCA1 promoter, target gene identification and modulation experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional genomic screen plus direct expression modulation experiments, single lab\",\n      \"pmids\": [\"11136250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"ID4 promoter hypermethylation silences ID4 expression in gastric cancer; treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (with or without trichostatin A) restores ID4 expression and is accompanied by promoter demethylation. ID4 promoter methylation significantly associates with hMLH1 methylation and microsatellite instability.\",\n      \"method\": \"Bisulfite genomic sequencing, methylation-specific PCR, RT-PCR, pharmacological demethylation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (bisulfite sequencing, MSP, pharmacological reactivation), single lab\",\n      \"pmids\": [\"14534543\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Id4 overexpression in HC11 mammary epithelial cells increases cell proliferation, inhibits lactogenic hormone-mediated differentiation (reduces beta-casein promoter activity and expression), and induces anchorage-independent growth in soft agar.\",\n      \"method\": \"Transient and stable transfection of Id4, proliferation assay, beta-casein promoter reporter assay, soft agar colony assay\",\n      \"journal\": \"The American journal of pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal readouts (proliferation, differentiation marker, soft agar), single lab\",\n      \"pmids\": [\"14633621\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Xenopus Id4 can inhibit the neurogenic activity of neurogenin and neuroD and the myogenic activity of MyoD in animal cap and in vivo overexpression assays, whereas Id2 inhibits only MyoD and neuroD, and Id3 blocks only neuroD—demonstrating differential substrate specificity among Id proteins.\",\n      \"method\": \"Animal cap explant assay, in vivo overexpression in Xenopus embryos, reporter assay\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple bHLH substrates tested, ortholog study with direct functional comparison, single lab\",\n      \"pmids\": [\"14651922\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Id4 is required for normal G1-S transition in early cortical progenitors; Id4-null mice show reduced proliferation of ventricular zone stem cells and premature neuronal differentiation, demonstrating that Id4 regulates neural progenitor proliferation and differentiation in vivo.\",\n      \"method\": \"Id4-knockout mouse analysis, BrdU incorporation, immunohistochemistry, in vitro neurosphere cultures\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function (knockout mouse) with multiple orthogonal readouts (BrdU, IHC, neurosphere), replicated in vitro and in vivo\",\n      \"pmids\": [\"15469968\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Id4 deficiency in mice leads to precocious oligodendrocyte differentiation both in vitro and in vivo; overexpression of Id4 in OPC progenitors prevents differentiation and decreases myelin gene expression (MBP, CGT but not PLP promoters), and Id4 differentially regulates myelin protein expression and subcellular distribution.\",\n      \"method\": \"Id4-knockout mouse analysis, overexpression via retroviral transduction, luciferase reporter assay for CGT/MBP/PLP promoters, immunohistochemistry\",\n      \"journal\": \"Glia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function mouse plus gain-of-function with multiple reporter assays and in vivo/in vitro concordance\",\n      \"pmids\": [\"16862533\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Id4 deficiency causes premature neuronal differentiation resulting in a thicker neocortex with more early-born neurons; adult Id4-/- brains are smaller with depleted late-born cortical neurons and astrocytes, and neurosphere proliferation is impaired; BMP2-mediated astrocyte differentiation is accelerated in Id4-/- NPCs.\",\n      \"method\": \"Id4-knockout mouse analysis, immunohistochemistry, neurosphere culture, BMP2 treatment assay\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with multiple quantitative readouts in vivo and in vitro, replicated in neurosphere system\",\n      \"pmids\": [\"15882580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"All four ID genes (ID1-4) are neuronal targets repressed by MeCP2; chromatin immunoprecipitation confirmed MeCP2 binding near/within ID1, ID2, ID3 promoters; ID4 mRNA and protein are increased in Mecp2-deficient mouse brain.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), quantitative RT-PCR, immunofluorescence, laser scanning cytometry in Mecp2-deficient mouse and human RTT brain\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus expression validation in multiple systems (mouse and human tissue), single lab\",\n      \"pmids\": [\"16682435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The protein complex mutant p53-E2F1 assembles on specific regions of the ID4 promoter and positively controls ID4 transcription; ID4 protein then binds to and stabilizes mRNAs encoding pro-angiogenic factors IL8 and GRO-alpha, increasing angiogenic potential in mutant p53-expressing cancer cells.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) of mutant p53-E2F1 on ID4 promoter; RNA immunoprecipitation (RIP) and mRNA stability assays for IL8/GRO-alpha; siRNA knockdown of mutant p53; angiogenesis assays\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ChIP for promoter occupancy, RNA pulldown for mRNA binding, functional angiogenesis assay; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"19783986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Ectopic Id4 expression in DU145 prostate cancer cells increases apoptosis and decreases proliferation (S-phase arrest), associated with increased androgen receptor (AR), p21, p27, and p53 expression; Id4 promoter is hypermethylated in DU145 cells.\",\n      \"method\": \"Ectopic expression by transfection, cell cycle analysis (3H-thymidine, FACS), RT-PCR, real-time PCR, Western blot, immunocytochemistry, MSP\",\n      \"journal\": \"BMC cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal readouts (cell cycle, apoptosis, molecular markers), single lab\",\n      \"pmids\": [\"19500415\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"ID4 is required for adipocyte differentiation: Id4-/- mice have reduced body fat; MEFs from Id4-/- mice have reduced adipogenic potential with decreased C/EBPα and PPARγ expression; ID4 is induced during 3T3-L1 adipocyte differentiation.\",\n      \"method\": \"Id4-knockout mouse analysis, MEF isolation and adipogenic differentiation assay, Western blot for C/EBPα and PPARγ\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout plus MEF differentiation assay with molecular markers, in vitro and in vivo concordance\",\n      \"pmids\": [\"20460371\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Id4 promotes osteoblast differentiation via an Id4-Hes1-Hey2 mechanism: Id4 releases Hes1 from Hes1-Hey2 repressor complexes, and free Hes1 increases stability and transcriptional activity of Runx2, a key osteoblast differentiation factor. Loss of Id4 (Id4-/- mice) drastically reduces osteoblast differentiation and drives MSC differentiation toward adipocytes instead.\",\n      \"method\": \"Id4-knockout mouse analysis, ST2 cell line Id4 knockdown, genome-wide expression analysis, co-immunoprecipitation for Hes1-Hey2 interaction, Runx2 activity assays\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, knockdown, and biochemical interaction studies with mechanistic pathway placement (Id4-Hes1-Hey2-Runx2 axis)\",\n      \"pmids\": [\"20628571\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ID4 enhances SOX2 protein expression by suppressing miR-9* (which targets SOX2 3'-UTR), leading to SOX2-mediated induction of ABC transporters ABCC3 and ABCC6 and chemoresistance in glioma stem-like cells.\",\n      \"method\": \"shRNA knockdown, microRNA overexpression, luciferase 3'-UTR reporter assay, RT-PCR, Western blot, drug resistance assay\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — 3'-UTR reporter validation, shRNA knockdown of pathway components with rescue experiments, multiple orthogonal methods in single study\",\n      \"pmids\": [\"21531766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"ID4 regulates mammary gland ductal expansion by suppressing p38MAPK activity; p38MAPK is activated in Id4-null mammary cells and after siRNA-mediated knockdown; this p38MAPK activation is required for the reduced proliferation and increased apoptosis in Id4-ablated mammary glands.\",\n      \"method\": \"Id4-knockout mouse mammary analysis, siRNA knockdown in transformed mammary cells, p38MAPK phosphorylation Western blot, p38MAPK inhibitor treatment\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO, siRNA KD, and pharmacological inhibitor rescue for pathway placement, multiple orthogonal approaches\",\n      \"pmids\": [\"22069192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Id4 interacts directly with Twist1 (a bHLH transcription factor) to suppress Twist1-mediated MMP2 expression, thereby inhibiting invasiveness of glioblastoma cells; Id4 expression inversely correlates with MMP2 in GBM patient data.\",\n      \"method\": \"Co-immunoprecipitation of Id4 with Twist1, MMP2 reporter assay, invasion assay, siRNA knockdown, TCGA bioinformatic correlation\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction by Co-IP plus functional invasion assay, single lab\",\n      \"pmids\": [\"24413082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ID4 forms a physical complex with mutant p53 and the acetyltransferase CBP/p300, resulting in acetylation of mutant p53 at K373; this acetylation restores mutant p53 DNA binding and transcriptional activity (BAX, PUMA, p21 expression and apoptosis). Loss of ID4 in LNCaP cells abrogates wild-type p53 DNA binding.\",\n      \"method\": \"Co-immunoprecipitation of ID4-p53-CBP/p300 complex, p53 acetylation Western blot, EMSA for p53 DNA binding, luciferase reporter for p53 transcriptional activity, apoptosis assay\",\n      \"journal\": \"Molecular cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods: Co-IP for complex, acetylation analysis, DNA-binding assay, reporter assay, functional apoptosis readout in single rigorous study\",\n      \"pmids\": [\"24330748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ID4 is a transcriptional target of gain-of-function p53 mutants (R175H, R273H, R280K); the mutant p53-E2F1 protein complex assembles on the ID4 promoter and positively controls ID4 expression. (Note: this finding was established in 2009; the 2014 review paper [PMID:25512197] elaborates on this but the original data is from PMID:19783986.)\",\n      \"method\": \"ChIP, reporter assay, siRNA depletion of mutant p53\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — ChIP for direct promoter occupancy, functional reporter, depletion experiments; original data from PMID:19783986\",\n      \"pmids\": [\"19783986\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"EZH2-dependent H3K27me3 initiates ID4 promoter silencing in prostate cancer; EZH2 and H3K27me3 are enriched at the ID4 promoter along with DNMT1 in ID4-negative cell lines; EZH2 knockdown in DU145 leads to re-expression of ID4 with concomitant loss of EZH2, H3K27me3, and DNMT1 at the promoter.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) for EZH2, H3K27me3, DNMT1 on ID4 promoter; siRNA knockdown of EZH2; RT-PCR for ID4 re-expression\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — ChIP for multiple histone marks and enzymes, knockdown rescue experiment, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25115397\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"ID4 heterodimerizes with ID1, ID2, and ID3 via its HLH domain, acting as an inhibitor of inhibitors; ID4 promotes E47 (bHLH) DNA binding by sequestering ID1 (demonstrated by EMSA); the N-terminal alanine-stretch unique to ID4's HLH domain is required for optimal ID-ID interaction; ID4 and ID1 co-localize in prostate cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, EMSA (electrophoretic mobility shift assay) for E47 DNA binding competition, co-localization by immunofluorescence, domain deletion mutagenesis\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct biochemical interaction by Co-IP, EMSA competition assay, mutagenesis of functional domain, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25778840\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Id4 acts as a downstream target of Notch2 signaling and maintains hippocampal neural stem cell (NSC) quiescence; Id4 knockdown rescues Notch2-induced inhibition of NSC proliferation; Id4 deletion activates NSC proliferation in the dentate gyrus without evoking neurogenesis; Id4 overexpression increases NSC maintenance and promotes astrogliogenesis at the expense of neurogenesis.\",\n      \"method\": \"Conditional Id4 knockout, lentiviral shRNA knockdown, Notch2 gain-of-function, BrdU proliferation assay, immunofluorescence, neurosphere assay\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO, knockdown epistasis with Notch2, gain-of-function, multiple cellular phenotype readouts establishing pathway position\",\n      \"pmids\": [\"31390563\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Id4 chromatin immunoprecipitation confirmed Id4 binding to the Esr1 and Foxa1 promoter regions in mammary cells; Id4 deficiency leads to striking increases in ERα (Esr1), PR, and FoxA1 expression in both basal and luminal mammary subsets, indicating that Id4 is a negative transcriptional regulator of the ERα signaling axis.\",\n      \"method\": \"Conditional MMTV-Cre/K14-Cre Id4 knockout mouse model, ChIP for Id4 on Esr1/Foxa1 promoters, RT-PCR/Western blot for target gene expression\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic loss-of-function plus direct ChIP evidence of promoter binding, multiple orthogonal methods, single focused study\",\n      \"pmids\": [\"25038044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Loss of ID4 in LNCaP prostate cancer cells (L-ID4) promotes castration-resistant prostate cancer (CRPC) through constitutive AR activation and de novo steroidogenesis; RNA-seq revealed a gene expression signature mimicking constitutively active AR in L-ID4 cells; PSA expression persisted in castrated conditions in L-ID4 but not control cells.\",\n      \"method\": \"Stable shRNA silencing of ID4 in LNCaP, RNA-seq, xenograft in castrated vs. non-castrated mice, PSA expression assay\",\n      \"journal\": \"Molecular endocrinology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — stable knockdown, RNA-seq, and in vivo castration model with mechanistic pathway identification\",\n      \"pmids\": [\"24921661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"ID4 controls mammary stem cell self-renewal by suppressing key factors required for luminal differentiation; in an ID4-GFP knock-in reporter mouse, ID4 marks a stem cell-enriched basal mammary cell subset; single-cell transcriptomics revealed ID4-high cells have a distinct stem cell transcriptional signature.\",\n      \"method\": \"ID4-GFP knock-in reporter mouse, single-cell transcriptomics, FACS, mammary reconstitution assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knock-in reporter, single-cell transcriptomics, and functional reconstitution assay in single comprehensive study\",\n      \"pmids\": [\"25813983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ID4 directly interacts with FKBP52 (FK506-binding protein 52) in vitro (but not directly with AR); loss of ID4 increases FKBP52-dependent AR transcriptional activity and promotes CRPC; pharmacological inhibition of FKBP52-AR signaling with MJC13 attenuates tumor growth in L-ID4 xenografts.\",\n      \"method\": \"Proteomic analysis (proteomics of L-ID4 vs. L+ns), co-immunoprecipitation/protein interaction studies for ID4-FKBP52, in vitro binding assay, AR reporter assay, MJC13 pharmacological inhibition in xenograft\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — direct in vitro interaction, proteomic identification, functional AR reporter, and in vivo pharmacological rescue; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"28252832\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Mutant p53 and ID4 delocalize lncRNA MALAT1 from nuclear speckles and favor its association with chromatin by recruiting MALAT1 via splicing factor SRSF1; this enables aberrant recruitment of MALAT1 on VEGFA pre-mRNA and modulation of VEGFA isoform expression in breast cancer cells.\",\n      \"method\": \"RNA immunoprecipitation (RIP), co-immunoprecipitation, RNA FISH for MALAT1 localization, VEGFA isoform analysis by RT-PCR, shRNA knockdown\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — RIP, Co-IP, RNA localization by FISH, functional isoform analysis; multiple orthogonal methods in single study\",\n      \"pmids\": [\"28652379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ID4 level dictates spermatogonial stem cell (SSC) vs. progenitor state: the ID4-EGFPBright population functions as SSCs, whereas ID4-EGFPDim cells are transitioning to progenitors; conditional overexpression of Id4 blocks transition from SSC to progenitor state.\",\n      \"method\": \"Id4-eGFP reporter mouse, conditional Id4 overexpression mouse model, RNA-seq, FACS, transplantation assay\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reporter mouse, conditional overexpression, RNA-seq, and functional transplantation; multiple orthogonal methods across genetic models\",\n      \"pmids\": [\"28087628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"ID4 regulates wild-type and mutant p53 transcriptional activity by promoting acetylation at K373 (and to a lesser extent K320) via CBP/p300 interaction; acetylation-mimic (K373Q) and non-acetylation mimic (K373R) p53 mutants confirmed K373 as the critical residue mediating ID4-dependent p53 activation and downstream cell cycle arrest/apoptosis.\",\n      \"method\": \"Ectopic expression in p53-null prostate cancer cells, acetylation-mimic/non-mimic p53 mutants, p53 DNA-binding assay, target gene expression (RT-PCR, Western blot), apoptosis/cell cycle assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — site-specific mutagenesis of p53 acetylation sites, functional DNA-binding assay, and downstream target validation; single lab but multiple rigorous methods\",\n      \"pmids\": [\"27911860\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Id4 promotes elimination of the pro-activation bHLH factor Ascl1 protein in hippocampal neural stem cells (NSCs) by sequestering Ascl1's heterodimerization partner E47, thereby promoting Ascl1 protein degradation and maintaining NSC quiescence; Id4 deletion causes abnormal Ascl1 protein accumulation and premature stem cell activation.\",\n      \"method\": \"Id4 conditional knockout in adult hippocampus, Id4 overexpression in NSC cultures, Ascl1 protein level analysis by Western blot, E47-Ascl1-Id4 interaction studies by Co-IP, BrdU proliferation assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout, protein interaction (Co-IP), gain-of-function, mechanistic link between Id4/E47/Ascl1 established with multiple orthogonal approaches\",\n      \"pmids\": [\"31552825\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"ID4 interacts with mediator of DNA damage checkpoint protein 1 (MDC1) and, through MDC1, also with DNA repair proteins γH2AX and BRCA1 at fragile chromatin sites; ID4 binds to chromatin following DNA damage but does not affect transcription at these sites, suggesting a role in DNA damage response rather than transcription per se.\",\n      \"method\": \"RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins), ChIP-seq, RNA-seq, immunoprecipitation\",\n      \"journal\": \"Breast cancer research : BCR\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — RIME proteomics for endogenous interactions, ChIP-seq for genome-wide binding, RNA-seq for transcriptional impact; multiple orthogonal methods in single study\",\n      \"pmids\": [\"32527287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"ID4 interacts with the bHLH protein HEB (detected by mass spectrometry); ID4 antagonizes HEB transcriptional activity, preventing HEB from binding E-box motifs in regulatory elements of myoepithelial developmental genes (extracellular matrix, contractile cytoskeleton); loss of ID4 in mammary organoids/in vivo allows HEB-dependent myoepithelial differentiation.\",\n      \"method\": \"Mass spectrometry (proteomics) for ID4 interactome, ChIP-seq for HEB genomic binding, mammary organoid culture, conditional Id4 knockout mouse, RNA-seq\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — mass spectrometry for protein interaction, ChIP-seq for genomic targets, genetic KO with functional cellular phenotype; multiple orthogonal methods\",\n      \"pmids\": [\"33554073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"CRISPR/dCas9-DNMT3a-targeted methylation of the Id2 and Id4 promoters drives oligodendrocyte precursor cell (OPC) differentiation, confirming that DNA methylation-mediated suppression of ID4 (and ID2) is a direct epigenetic mechanism controlling OPC differentiation.\",\n      \"method\": \"Epigenetic editing with CRISPR/dCas9-DNMT3a for targeted methylation of Id4 promoter, OPC differentiation assay\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — targeted epigenetic editing (CRISPR-based) with direct functional readout; single lab but rigorous method\",\n      \"pmids\": [\"34482420\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ID4 expression in breast cancer cells promotes VEGFA secretion, which stimulates VEGFR2-integrin β3 interaction in a paracrine fashion; this interaction activates the focal adhesion pathway, promotes migration/invasion/stress fiber formation, and drives nuclear translocation and activation of YAP and TAZ.\",\n      \"method\": \"Overexpression and knockdown of ID4, conditioned media paracrine experiments, co-immunoprecipitation of VEGFR2-integrin β3, VEGFA ELISA, YAP/TAZ nuclear localization assay, invasion/migration assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — paracrine experiment, receptor interaction Co-IP, nuclear localization assay, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"38321003\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ID4 is a dominant-negative HLH protein that lacks a DNA-binding basic domain and functions by heterodimerizing with bHLH transcription factors (E47, MyoD, Twist1, HEB, Neurogenin, NeuroD) and with other ID proteins (ID1-3) to inhibit their transcriptional activity, thereby regulating cell proliferation, differentiation timing (in neural progenitors, oligodendrocytes, spermatogonial stem cells, mammary stem cells, osteoblasts, and adipocytes), and stem cell quiescence; ID4 is transcriptionally controlled by mutant p53-E2F1 complexes and Notch2 signaling, and is epigenetically silenced by EZH2/H3K27me3-initiated promoter methylation in multiple cancers; at the protein level, ID4 forms a complex with CBP/p300 and p53 to promote p53 acetylation at K373, restoring p53 transcriptional activity, and interacts directly with FKBP52 to suppress AR signaling; in the context of mRNA regulation, ID4 binds and stabilizes IL8 and GRO-alpha mRNAs (downstream of mutant p53) to promote angiogenesis, and recruits MALAT1 via SRSF1 to modulate VEGFA isoform splicing; in mammary stem cells, ID4 interacts with HEB to prevent myoepithelial differentiation, and promotes VEGFA secretion that activates VEGFR2-integrin β3-YAP/TAZ signaling.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ID4 is a helix-loop-helix (HLH) protein that lacks the DNA-binding basic region and acts as a dominant-negative regulator of differentiation timing, proliferation, and stem cell state across multiple lineages [#0, #10]. Its core biochemical activity is sequestration: through its HLH domain it heterodimerizes with bHLH transcription factors (E47, MyoD, Neurogenin, NeuroD, Twist1, HEB) to block their DNA binding and trans-activation [#0, #1, #9, #20, #35], and it also dimerizes with other ID proteins (ID1-3), acting as an \\\"inhibitor of inhibitors\\\" that can paradoxically free E47 by titrating ID1 [#24]. This sequestration logic underlies its developmental roles: ID4 sustains progenitor proliferation and prevents premature differentiation in cortical neural progenitors, oligodendrocyte precursors, mammary stem cells, and spermatogonial stem cells, with loss causing precocious differentiation in each [#10, #11, #12, #28, #31]. In adult hippocampal neural stem cells ID4 enforces quiescence downstream of Notch2, in part by sequestering E47 to promote degradation of the pro-activation bHLH factor Ascl1 [#25, #33]. ID4 directs lineage choice through specific partners: it antagonizes HEB to prevent myoepithelial differentiation in mammary cells [#35], releases Hes1 from Hes1-Hey2 complexes to stabilize Runx2 and promote osteoblast over adipocyte fate [#17], and is required for adipocyte differentiation via C/EBPalpha and PPARgamma [#16]. ID4 is itself tightly regulated transcriptionally — by USF1/Sp1-Sp3 promoter elements, cAMP/PKA signaling, MeCP2 repression, and a mutant p53-E2F1 complex — and is epigenetically silenced in cancers by EZH2/H3K27me3-initiated, DNMT-dependent promoter methylation [#2, #3, #13, #14, #23, #36]. Beyond transcription factor sequestration, ID4 has acquired direct protein- and RNA-level functions in cancer: it forms a complex with CBP/p300 and p53 to drive p53 acetylation at K373 and restore p53 transcriptional activity [#21, #32], binds and stabilizes pro-angiogenic IL8 and GRO-alpha mRNAs downstream of mutant p53 [#14], recruits the lncRNA MALAT1 via SRSF1 to reshape VEGFA splicing [#30], and interacts with FKBP52 to restrain androgen receptor signaling, with ID4 loss promoting castration-resistant prostate cancer [#27, #29]. ID4 is also a negative transcriptional regulator of the ERalpha axis (Esr1, FoxA1) and BRCA1, and associates with MDC1 at damaged chromatin, implicating it in the DNA damage response [#6, #26, #34].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Established ID4's foundational biochemical identity: a basic-region-less HLH protein that inhibits bHLH factors, defining its mechanism of action.\",\n      \"evidence\": \"In vitro EMSA with recombinant ID4 against E47 homodimers and E47/MyoD heterodimers\",\n      \"pmids\": [\"8139914\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro binding does not establish which bHLH partners are physiologically relevant in vivo\", \"No cellular phenotype demonstrated\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Confirmed the dominant-negative activity in cells, showing ID4 represses MyoD-driven transcription at an E-box enhancer.\",\n      \"evidence\": \"MCK-luciferase reporter assay in transfected mammalian cells\",\n      \"pmids\": [\"7665172\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single reporter system\", \"Endogenous muscle differentiation not tested\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Mapped the cis-regulatory architecture controlling ID4's own transcription, revealing USF1 activation and Sp1/Sp3 repression plus autoregulatory feedback.\",\n      \"evidence\": \"EMSA and reporter assays including heterologous Drosophila SL2 cells\",\n      \"pmids\": [\"9516472\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Promoter context studied in transfection, not endogenous chromatin\", \"Physiological signals driving USF1/Sp1 occupancy not defined\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Linked ID4 expression to cAMP signaling and revealed a pro-apoptotic consequence of overexpression in astrocytes.\",\n      \"evidence\": \"Pharmacological cAMP modulation of primary astrocytes and overexpression with apoptosis readout\",\n      \"pmids\": [\"10066362\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism connecting cAMP to Id4 promoter not resolved\", \"Apoptosis phenotype may be overexpression artifact\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined ID4 as a differentiation-timing switch in oligodendrocyte precursors, where its downregulation permits differentiation.\",\n      \"evidence\": \"Retroviral overexpression in OPCs with mitogen-withdrawal and thyroid hormone differentiation assays\",\n      \"pmids\": [\"10790366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"bHLH partners mediating the block not identified\", \"Loss-of-function not yet tested\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Distinguished ID4 from other ID family members through isoform-specific hormonal regulation (FSH/cAMP-induced) and dispensability for transferrin promoter activation.\",\n      \"evidence\": \"RT-PCR, Northern blot, antisense oligonucleotide plus reporter assay in Sertoli cells\",\n      \"pmids\": [\"11316735\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional role of FSH-induced Id4 in Sertoli cells unclear\", \"No direct target identified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified ID4 as a negative regulator of the BRCA1 promoter and linked it to anchorage-independent growth.\",\n      \"evidence\": \"Ribozyme library screen with BRCA1-promoter EGFP reporter and expression modulation in ovarian cancer cells\",\n      \"pmids\": [\"11136250\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of BRCA1 promoter repression not defined\", \"Direct vs indirect regulation unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Revealed epigenetic silencing of ID4 by promoter hypermethylation in gastric cancer, establishing ID4 as a methylation-regulated locus.\",\n      \"evidence\": \"Bisulfite sequencing, MSP, and pharmacological demethylation with RT-PCR\",\n      \"pmids\": [\"14534543\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Causality of silencing for tumor phenotype not tested\", \"Enzymes initiating methylation not identified\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Extended ID4's proliferation/differentiation switch and oncogenic potential to mammary epithelium.\",\n      \"evidence\": \"Stable/transient Id4 transfection in HC11 cells with proliferation, beta-casein reporter, and soft agar assays\",\n      \"pmids\": [\"14633621\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"bHLH targets in mammary cells not identified\", \"In vivo relevance not addressed\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Demonstrated differential bHLH substrate specificity among ID proteins, with ID4 uniquely inhibiting neurogenin, neuroD, and MyoD.\",\n      \"evidence\": \"Xenopus animal cap and in vivo overexpression assays comparing Id2/3/4\",\n      \"pmids\": [\"14651922\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of specificity not defined\", \"Ortholog data may not fully translate to mammalian ID4\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Provided genetic proof that ID4 controls neural progenitor G1-S transition and prevents premature neuronal differentiation in vivo.\",\n      \"evidence\": \"Id4-knockout mice with BrdU, IHC, and neurosphere assays\",\n      \"pmids\": [\"15469968\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular bHLH targets in cortical progenitors not pinpointed\", \"Cell-cycle machinery link not detailed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Genetically confirmed ID4 as a brake on oligodendrocyte and astrocyte differentiation, controlling neocortical neuron output and glial timing.\",\n      \"evidence\": \"Id4-knockout mice plus retroviral overexpression with myelin-gene reporters and BMP2 differentiation assays\",\n      \"pmids\": [\"16862533\", \"15882580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional effectors of myelin gene regulation not resolved\", \"Interplay with BMP signaling mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed ID4 within MeCP2-dependent neuronal gene regulation, with ID4 derepressed in Mecp2-deficient brain.\",\n      \"evidence\": \"ChIP, qRT-PCR, and immunofluorescence in Mecp2-deficient mouse and Rett syndrome brain\",\n      \"pmids\": [\"16682435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct MeCP2 binding shown for ID1-3 but ID4 inferred from expression change\", \"Functional consequence of ID4 derepression not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Connected ID4 to oncogenic gain-of-function p53, showing a mutant p53-E2F1 complex drives ID4 transcription and ID4 then stabilizes pro-angiogenic IL8/GRO-alpha mRNAs.\",\n      \"evidence\": \"ChIP on ID4 promoter, RIP and mRNA stability assays, mutant p53 knockdown, angiogenesis assays\",\n      \"pmids\": [\"19783986\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"RNA-binding domain/mode of ID4 not structurally defined\", \"Generality across mutant p53 alleles addressed only later\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Implicated ID4 in prostate cancer cell cycle/apoptosis control and linked its loss to AR pathway changes, with promoter hypermethylation in DU145.\",\n      \"evidence\": \"Ectopic expression in DU145 with cell cycle, apoptosis, marker expression, and MSP\",\n      \"pmids\": [\"19500415\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking ID4 to AR/p21/p27/p53 not yet defined here\", \"Correlative marker changes only\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Defined ID4 as a determinant of mesenchymal lineage choice — required for adipogenesis and, via an Id4-Hes1-Hey2-Runx2 axis, for osteoblast over adipocyte fate.\",\n      \"evidence\": \"Id4-knockout mice, ST2 knockdown, expression profiling, Co-IP for Hes1-Hey2, Runx2 activity assays\",\n      \"pmids\": [\"20460371\", \"20628571\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct ID4-Hes1 binding vs displacement mechanism not fully resolved\", \"Reconciliation of pro-adipogenic and anti-adipogenic roles across tissues incomplete\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed non-canonical ID4 functions: a miR-9*/SOX2/ABC-transporter axis driving glioma chemoresistance and p38MAPK suppression controlling mammary ductal expansion.\",\n      \"evidence\": \"shRNA/miRNA modulation with 3'-UTR reporters and drug assays; Id4-KO/siRNA with p38MAPK phosphorylation and inhibitor rescue\",\n      \"pmids\": [\"21531766\", \"22069192\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How ID4 suppresses miR-9* or p38MAPK mechanistically not defined\", \"Direct vs indirect effects unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified direct ID4-Twist1 interaction as a brake on glioblastoma invasiveness through MMP2 repression.\",\n      \"evidence\": \"Co-IP, MMP2 reporter, invasion assay, siRNA, and TCGA correlation\",\n      \"pmids\": [\"24413082\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single Co-IP without reciprocal/structural validation of the interaction\", \"In vivo invasion not tested\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Established a protein-level ID4 function distinct from bHLH sequestration: ID4-CBP/p300-p53 complex formation drives p53 K373 acetylation to restore p53 DNA binding and transcriptional/apoptotic activity.\",\n      \"evidence\": \"Co-IP of ID4-p53-CBP/p300, acetylation Western, EMSA, reporter, and apoptosis assays\",\n      \"pmids\": [\"24330748\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether ID4 directly contacts p300 or p53 to position acetylation not dissected\", \"Structural basis unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Pinpointed K373 as the critical ID4-dependent p53 acetylation site using acetylation-mimic mutants, solidifying the ID4-p53 activation mechanism.\",\n      \"evidence\": \"K373Q/K373R p53 mutants with DNA-binding, target expression, and cell cycle/apoptosis assays in p53-null cells\",\n      \"pmids\": [\"27911860\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which ID4 enhances CBP/p300 catalysis not resolved\", \"In vivo relevance of K373 axis not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined ID4 as a downstream effector and quiescence factor of Notch2 in adult hippocampal neural stem cells, with loss activating NSC proliferation.\",\n      \"evidence\": \"Conditional Id4 knockout, shRNA, Notch2 gain-of-function, BrdU and neurosphere assays\",\n      \"pmids\": [\"31390563\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Notch2-to-Id4 transcriptional link not mapped here\", \"Effector bHLH targets defined only in later work\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established ID4's role in mammary stem cell biology as a negative regulator of the ERalpha signaling axis through direct promoter binding.\",\n      \"evidence\": \"Conditional Id4 knockout mice with ChIP on Esr1/Foxa1 promoters and target expression analysis\",\n      \"pmids\": [\"25038044\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a basic-region-less protein binds these promoters (direct vs co-factor) not resolved\", \"bHLH partner mediating repression not identified here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Defined the epigenetic silencing mechanism of ID4 in cancer: EZH2/H3K27me3 initiates and DNMT1 maintains promoter methylation.\",\n      \"evidence\": \"ChIP for EZH2/H3K27me3/DNMT1 on ID4 promoter and EZH2 knockdown reactivation in prostate cancer cells\",\n      \"pmids\": [\"25115397\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Upstream signals recruiting EZH2 to ID4 not defined\", \"Tissue generality of this mechanism beyond prostate not established here\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Refined the inter-ID interaction logic, showing ID4 dimerizes with ID1-3 and can free E47 by sequestering ID1, acting as an inhibitor of inhibitors.\",\n      \"evidence\": \"Co-IP, EMSA competition for E47 binding, immunofluorescence colocalization, and domain-deletion mutagenesis\",\n      \"pmids\": [\"25778840\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological contexts where the inhibitor-of-inhibitors mode dominates not defined\", \"Quantitative dimer preferences not measured\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Connected ID4 loss to castration-resistant prostate cancer through constitutive AR activation and de novo steroidogenesis.\",\n      \"evidence\": \"Stable shRNA silencing in LNCaP, RNA-seq, and castration xenograft model\",\n      \"pmids\": [\"24921661\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular link from ID4 to AR activation defined in subsequent FKBP52 work\", \"Steroidogenic enzyme regulation mechanism incomplete\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified ID4 as a marker and driver of mammary stem cell self-renewal that suppresses luminal differentiation factors.\",\n      \"evidence\": \"ID4-GFP knock-in reporter mouse, single-cell transcriptomics, FACS, and mammary reconstitution\",\n      \"pmids\": [\"25813983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"bHLH effectors of the self-renewal program defined only in later HEB work\", \"Direct target genes from this study limited\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Provided the molecular basis for ID4-mediated AR restraint: direct ID4-FKBP52 interaction (not direct AR binding) that limits FKBP52-dependent AR activity.\",\n      \"evidence\": \"Proteomics, in vitro binding/Co-IP, AR reporter, and MJC13 pharmacological rescue in xenografts\",\n      \"pmids\": [\"28252832\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mode of ID4-FKBP52 binding not resolved\", \"Whether HLH domain mediates this interaction not established\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Extended ID4's RNA-level oncogenic functions, showing ID4 with mutant p53 recruits MALAT1 via SRSF1 to chromatin to reshape VEGFA splicing.\",\n      \"evidence\": \"RIP, Co-IP, RNA-FISH for MALAT1 localization, and VEGFA isoform analysis with shRNA\",\n      \"pmids\": [\"28652379\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs SRSF1-bridged ID4-MALAT1 association not dissected\", \"Functional output of altered VEGFA isoforms only partly characterized\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Established ID4 as a dosage-sensitive determinant of spermatogonial stem cell vs progenitor identity, with high ID4 maintaining the stem state.\",\n      \"evidence\": \"Id4-eGFP reporter and conditional overexpression mice, RNA-seq, FACS, and transplantation\",\n      \"pmids\": [\"28087628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"bHLH partners enforcing SSC state not identified\", \"Transcriptional targets in SSCs not mapped\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolved a mechanism for ID4-enforced NSC quiescence: ID4 sequesters E47 to drive degradation of the pro-activation factor Ascl1.\",\n      \"evidence\": \"Conditional Id4 knockout, overexpression in NSCs, Ascl1 protein analysis, and E47-Ascl1-Id4 Co-IP\",\n      \"pmids\": [\"31552825\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Degradation machinery acting on freed Ascl1 not identified\", \"Generalizability to other quiescent niches untested\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Revealed a transcription-independent chromatin role for ID4 in the DNA damage response via MDC1, gammaH2AX, and BRCA1 at fragile sites.\",\n      \"evidence\": \"RIME endogenous interactome, ChIP-seq, RNA-seq, and immunoprecipitation in breast cancer cells\",\n      \"pmids\": [\"32527287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of ID4 at damage sites for repair efficiency not established\", \"Whether ID4 recruits or stabilizes MDC1 unclear\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Identified HEB as the key bHLH partner through which ID4 blocks mammary myoepithelial differentiation, linking sequestration to lineage gene control.\",\n      \"evidence\": \"Mass spectrometry interactome, HEB ChIP-seq, conditional Id4 knockout organoids/mice, and RNA-seq\",\n      \"pmids\": [\"33554073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of ID4-HEB control at individual loci not quantified\", \"Interplay with the ERalpha-suppressing role not integrated\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Provided causal epigenetic proof that DNA methylation of the Id4 promoter is sufficient to drive OPC differentiation.\",\n      \"evidence\": \"CRISPR/dCas9-DNMT3a targeted methylation of the Id4 promoter with OPC differentiation readout\",\n      \"pmids\": [\"34482420\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous signals triggering this methylation in vivo not defined\", \"Contribution of co-targeted Id2 not fully separated\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Described a paracrine ID4 oncogenic circuit: ID4-driven VEGFA secretion activates VEGFR2-integrin beta3-focal adhesion signaling and YAP/TAZ in breast cancer.\",\n      \"evidence\": \"ID4 overexpression/knockdown, conditioned-media paracrine assays, VEGFR2-integrin beta3 Co-IP, VEGFA ELISA, YAP/TAZ localization, invasion assays\",\n      \"pmids\": [\"38321003\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How ID4 transcriptionally/post-transcriptionally raises VEGFA secretion not pinned down\", \"In vivo significance of the YAP/TAZ axis not tested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a protein lacking a DNA-binding basic region achieves direct promoter occupancy (e.g., on Esr1/Foxa1/ID4-target genes) and integrates its many partner-specific functions—bHLH sequestration, p53 acetylation, RNA stabilization/splicing, FKBP52-AR restraint, and DNA-damage chromatin association—into context-dependent outputs remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural model of ID4 in any of its protein/RNA complexes\", \"Mechanism of ID4 chromatin recruitment without a basic domain unknown\", \"Rules governing which partner dominates in a given cell type undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 9, 24, 26]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 17, 24, 33]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [24, 33]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [14, 30]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [21, 30, 32]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [21, 24, 26, 34]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [34, 35]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 26, 35]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [10, 11, 12, 16, 17, 25, 28, 31]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [14, 23, 27, 30, 37]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [14, 30]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [34]}\n    ],\n    \"complexes\": [\"ID4-CBP/p300-p53 complex\"],\n    \"partners\": [\"E47\", \"MyoD\", \"Twist1\", \"HEB\", \"FKBP52\", \"MDC1\", \"SRSF1\", \"ID1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}