{"gene":"ID2","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":1991,"finding":"Id2 contains a helix-loop-helix (HLH) motif but lacks the basic DNA-binding domain; it selectively heterodimerizes with and inhibits one class of bHLH transcription factors (typified by E2A/E47 and E2B/m3) but not another class (TFE3, USF, AP4). Id2 also homodimerizes poorly.","method":"cDNA cloning, chromosome mapping, in vitro protein interaction and DNA-binding inhibition assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution of binding/inhibition with defined protein pairs, replicated across multiple bHLH partners","pmids":["1922066"],"is_preprint":false},{"year":1994,"finding":"Id2 physically binds unphosphorylated pRb via its HLH domain (an Id2 mutant lacking the HLH domain fails to bind); the interaction requires the E1A/large T-binding pocket of pRb. Id2 expression reverses pRb-mediated growth arrest without changing pRb phosphorylation state.","method":"In vitro binding assay with cell lysates, in vivo cross-linked co-immunoprecipitation in transfected SAOS-2 cells, deletion mutagenesis, cell proliferation/S-phase analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro binding with mutagenesis plus in vivo co-IP, multiple orthogonal methods in one study","pmids":["7926730"],"is_preprint":false},{"year":1996,"finding":"Id2, but not Id1 or Id3, binds in vitro to pRb, p107, and p130 (all three Rb family members), and in vivo association of Id2 with p107 and p130 is detected in transfected Saos-2 cells. Id2 specifically reverses cell cycle arrest induced by each Rb family member and by the CDK inhibitors p16 and p21 (p21 reversal is partial and pRb-dependent). Id2 overexpression decreases cyclin D1 protein/mRNA and disrupts cyclin D1-Cdk4 complexes.","method":"In vitro binding assay, transient transfection with co-immunoprecipitation, cell cycle progression assays, Western blot, Northern blot","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — reciprocal co-IP plus in vitro binding, multiple Rb family members tested, replicated for each member","pmids":["8649364"],"is_preprint":false},{"year":2000,"finding":"Id2 overexpression driven by Myc oncoproteins (N-Myc and c-Myc) inactivates Rb family proteins, enabling cell-cycle progression. In Id2-Rb double-knockout embryos, neurogenesis and hematopoiesis defects are rescued compared to each single knockout, establishing Id2 as a mediator of Myc oncogenic signaling through Rb inactivation.","method":"Double-knockout mouse genetics (epistasis), N-myc overexpression/amplification in neuroblastoma cell lines, transcriptional activation assays, molar ratio analysis of Id2 vs. active Rb","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in vivo combined with cell-line biochemistry, replicated across multiple tumor cell contexts","pmids":["11034201"],"is_preprint":false},{"year":2001,"finding":"Id2 is a transcriptional target of the β-catenin/TCF pathway in colon carcinoma; a TCF4-binding motif in the Id2 promoter is required for activation (identified by EMSA/supershift and abolished by site-directed mutagenesis). β-catenin up-regulates Id2 protein and increases anchorage-independent survival.","method":"Id2 promoter reporter assays, EMSA with supershift, site-directed mutagenesis, β-catenin overexpression/knockdown, anchorage-independent growth assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — promoter mutagenesis plus EMSA/supershift plus functional assay in same study, single lab","pmids":["11572874"],"is_preprint":false},{"year":2003,"finding":"TGF-β signaling induces Id2 expression in dendritic cells, and Id2 represses B cell-specific genes in DCs, revealing a TGF-β→Id2 signaling axis that governs lineage choice between B cell and DC progenitors.","method":"Id2-knockout mouse analysis, in vitro TGF-β stimulation of DC cultures, gene expression profiling, in vivo DC subset analysis","journal":"Nature immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout mouse phenotype plus cytokine stimulation experiments, supported by transcriptional profiling","pmids":["12598895"],"is_preprint":false},{"year":2003,"finding":"TGF-β inhibits Id2 expression in human and mouse epithelial cells; this repression occurs via induction of Mad2/Mad4, which replaces Myc-Max complexes with Mad-Max complexes on the Id2 promoter, linking TGF-β cytostatic signaling to Id2 downregulation.","method":"Northern/Western blot in multiple epithelial cell lines, promoter ChIP (Myc-Max vs. Mad-Max occupancy), gel-shift assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — chromatin immunoprecipitation plus gel-shift, multiple cell lines, mechanistic follow-up of promoter occupancy changes","pmids":["12824180"],"is_preprint":false},{"year":2004,"finding":"Id2 is degraded by the ubiquitin-proteasome system via ubiquitination at its N-terminal α-amino group (N-terminal ubiquitination). Lysine-less Id2 is still efficiently degraded by the proteasome; N-terminal Myc-tag fusion stabilizes Id2, whereas C-terminal tag does not. Deletion of the first 15 N-terminal residues also stabilizes Id2, implicating this region as the E3 ligase recognition element.","method":"In vitro and cell-based ubiquitination assays, proteasome inhibitor experiments, N-terminal vs. C-terminal tagging, deletion mutagenesis, pulse-chase/stability assays","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution plus mutagenesis, multiple orthogonal approaches in single study","pmids":["14733935"],"is_preprint":false},{"year":2005,"finding":"Polycystin-2 (PC2) directly interacts with Id2, and this interaction is regulated by polycystin-1 (PC1)-dependent phosphorylation of PC2. Id2 suppresses p21 induction by PC1 or PC2. Enhanced Id2 nuclear localization is observed in cystic kidneys, and RNAi-mediated Id2 knockdown corrects hyperproliferation of PC1 mutant cells.","method":"Co-immunoprecipitation, RNAi knockdown, nuclear fractionation/localization in cystic kidney tissue and cells, functional proliferation assays","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — co-IP plus localization plus functional RNAi rescue, multiple orthogonal methods","pmids":["16311606"],"is_preprint":false},{"year":2006,"finding":"APC/C(Cdh1) ubiquitin ligase targets Id2 for proteasomal degradation through a conserved D-box (destruction box) motif. Id2 interacts with APC/C core subunits and Cdh1 in neurons; Cdh1 depletion stabilizes Id proteins; D-box mutants of Id2 fail to bind Cdh1 and remain stable when APC/C(Cdh1) is active. Stabilized Id2 enhances axonal growth and overcomes myelin inhibitory signals by preventing accumulation of the Nogo receptor (a bHLH target gene product).","method":"Co-immunoprecipitation in primary neurons, D-box mutagenesis, Cdh1 RNAi/knockdown, in vitro and in vivo axonal growth assays, gene expression analysis","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — D-box mutagenesis plus co-IP plus in vivo neuronal phenotype, multiple orthogonal methods","pmids":["16810178"],"is_preprint":false},{"year":2005,"finding":"C/EBPβ is a direct transcriptional activator of the Id2 gene; the most proximal C/EBPβ-binding site in the Id2 promoter is responsible for transactivation, as confirmed by EMSA and ChIP. Id2 expression is reduced in C/EBPβ-deficient mammary glands.","method":"Id2 promoter reporter assays, EMSA, ChIP, Northern blot in C/EBPβ-knockout mammary glands, C/EBPβ-ERT nuclear translocation system","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP plus EMSA plus promoter reporter plus knockout validation, multiple methods in single lab","pmids":["15809228"],"is_preprint":false},{"year":2006,"finding":"Id2 promotes S-phase entry in neural cells by preventing E-protein (E47)-mediated activation of the CDK inhibitor p57Kip2. E47 directly activates p57Kip2 expression, and Id2 blocks this activation; p57Kip2 silencing by RNAi fully reverses E47-mediated cell cycle arrest.","method":"Gene expression profiling, RNAi knockdown, cell cycle analysis, in neuroblastoma cell lines engineered with inducible E47 and Id2","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — inducible expression systems plus RNAi rescue plus genome-wide expression, multiple orthogonal methods","pmids":["16705184"],"is_preprint":false},{"year":2007,"finding":"Id2 functions downstream of Tbx5 and Nkx2-5 in a molecular pathway required for cardiac ventricular conduction system specification. Compound haploinsufficiency of Tbx5/Id2 prevents embryonic conduction system specification; a 1.2 kb Id2 promoter fragment is cooperatively regulated by Nkx2-5 and Tbx5 in vitro and is sufficient for conduction-system-specific expression in vivo.","method":"Id2-knockout and compound haploinsufficient mouse genetics, SAGE transcriptional profiling of microdissected conduction system, promoter reporter assays, in vivo transgenic reporter","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in vivo plus promoter reporter in vitro and in vivo, multiple complementary approaches","pmids":["17604724"],"is_preprint":false},{"year":2008,"finding":"Id2 regulates erythroid development via direct interaction with transcription factor PU.1, modulating PU.1 and GATA-1 activities. Id2 also intrinsically inhibits B cell development through antagonism of E2A (E47).","method":"Id2 knockout mouse analysis, retroviral overexpression in hematopoietic progenitors, shRNA knockdown, co-immunoprecipitation/interaction studies with PU.1","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — functional knockdown/overexpression plus interaction assays, single lab, co-IP is mentioned but mechanistic detail is limited in the abstract","pmids":["18523151"],"is_preprint":false},{"year":2009,"finding":"Ebf1 transcriptionally represses Id2 (and Id3) during B-lineage specification; enforced Ebf1 in IL-7Rα−/− bone marrow dramatically reduces Id2/Id3 mRNA and restores B cell differentiation. Overexpression of Id2 alone blocks B cell specification at the prepro-B stage by inhibiting E2A activity.","method":"Retroviral overexpression and knockout mouse genetics, qRT-PCR, in vivo B cell reconstitution assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in vivo plus retroviral rescue, single lab","pmids":["19122139"],"is_preprint":false},{"year":2006,"finding":"BMP4 induces Id2 expression in pancreatic epithelial progenitors, and BMP4-stimulated Id2 binds to the bHLH transcription factor NeuroD, blocking NeuroD-dependent differentiation and instead promoting progenitor expansion.","method":"BMP4 stimulation of AR42J cells, co-immunoprecipitation of Id2 with NeuroD, BMP4 neutralization in a mouse islet regeneration model, proliferation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — co-IP plus functional BMP4 stimulation/neutralization, single lab","pmids":["16547003"],"is_preprint":false},{"year":2009,"finding":"Id2-mediated sequestration of E12/E47 bHLH factors suppresses p27KIP1 promoter activity, reducing p27KIP1 expression. Re-expression of p27KIP1 in Id2-overexpressing Id2−/− fibroblasts reverts hyperproliferation and tumorigenicity, identifying the Id2/E12/E47/p27KIP1 axis as a mechanism of Id2-driven oncogenesis.","method":"Id2 deletion mutagenesis, re-expression in Id2−/− fibroblasts, promoter reporter assays, flow cytometry, subcutaneous tumor grafting in athymic mice","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter reporter plus in vivo tumorigenesis plus domain mutagenesis, single lab","pmids":["19451188"],"is_preprint":false},{"year":2010,"finding":"Id2 represses SEMA3F expression through the E47/Id2 pathway; Id2 blocks E47-mediated direct activation of two E-box sites in the SEMA3F promoter. C-Myc drives Id2 overexpression, which in turn suppresses SEMA3F and promotes tumor cell migration and invasion.","method":"SEMA3F promoter reporter assay with E-box mutagenesis, E47 overexpression/Id2 overexpression in low vs. high metastatic cell lines, migration/invasion assays, silencing of SEMA3F","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — promoter analysis with mutagenesis plus functional invasion assay, single lab","pmids":["20388805"],"is_preprint":false},{"year":2010,"finding":"Gfi-1 directly binds to three conserved regions in the Id2 promoter and represses Id2 promoter activity. Id2 knockdown or haploinsufficiency partially rescues B cell and myeloid development defects in Gfi-1−/− mice, placing Id2 downstream of Gfi-1 in hematopoietic transcriptional networks.","method":"ChIP, promoter reporter assay, Id2/Gfi-1 double knockout/heterozygous mouse genetics, in vivo B cell and myeloid reconstitution","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus promoter assay plus genetic epistasis in vivo, single lab","pmids":["20453161"],"is_preprint":false},{"year":2013,"finding":"Id2 inhibits E2A (E47)-mediated direct transcriptional activation of Tcf7 (encoding TCF1), restraining memory CD8+ T cell differentiation. Loss of Id2 leads to E2A-dependent increase of Tcf7 and Eomesodermin expression, redirecting effector CD8+ T cells toward a memory fate.","method":"Id2-knockout mouse, retroviral E2A overexpression, gene expression profiling, chromatin/luciferase reporter assays","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout plus E2A interaction/reporter assays, single lab","pmids":["23536629"],"is_preprint":false},{"year":2013,"finding":"Id2 represses E2A-mediated transactivation of the IL-10 locus (Il10) in T cells; Id2-deficient T cells overexpress IL-10 in vitro and in vivo during infection, demonstrating that the Id2/E2A axis limits IL-10 production during effector T cell responses.","method":"Id2-knockout mouse, IL-10 reporter/ChIP analysis, graft-versus-host disease model, influenza infection model","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic knockout plus E2A transcriptional assay evidence, single lab","pmids":["24723679"],"is_preprint":false},{"year":2014,"finding":"Mtg16 directly represses Id2 transcription in dendritic cell progenitors (identified by genome-wide DNA-binding analysis); loss of Mtg16 leads to aberrant Id2 induction in pDC progenitors, and Id2 deletion rescues impaired pDC development in Mtg16-deficient mice.","method":"Mtg16-knockout mouse, genome-wide ChIP-seq of Mtg16, Id2/Mtg16 double-knockout genetics, flow cytometric DC subset analysis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide ChIP-seq identifying direct Id2 target plus genetic epistasis rescue experiment, multiple approaches","pmids":["24980046"],"is_preprint":false},{"year":2016,"finding":"DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Phosphorylated ID2 cannot bind the VHL ubiquitin ligase complex; unphosphorylated ID2 binds VHL, displaces Cullin 2, and impairs HIF2α ubiquitylation and degradation. Hypoxia inactivates DYRK1A/1B (via PHD1 oxygen sensor) to reduce Thr27 phosphorylation and allow ID2–VHL interaction and HIF2α stabilization.","method":"In vitro kinase assay (DYRK1A/B phosphorylation of ID2), co-immunoprecipitation of ID2 with VHL complex, ubiquitylation assays, phospho-mutant ID2 expression, tumor growth assays in glioblastoma","journal":"Nature","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — in vitro kinase reconstitution plus co-IP plus ubiquitylation assay plus phospho-mutant analysis, multiple orthogonal methods","pmids":["26735018"],"is_preprint":false},{"year":2016,"finding":"ID2 regulates NK cell homeostasis by repressing multiple E-protein target genes including Socs3; ID2 deletion in mature NK cells impairs IL-15 receptor signaling and metabolic function, and these defects are rescued by strong IL-15 stimulation or genetic Socs3 ablation.","method":"Conditional Id2 knockout in NK cells, IL-15 stimulation rescue experiments, Socs3 genetic deletion rescue, gene expression analysis","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional knockout plus two independent rescue experiments (IL-15 stimulation and Socs3 deletion), genetic epistasis","pmids":["26795246"],"is_preprint":false},{"year":2016,"finding":"Phosphorylation of Id2 near its N-terminus promotes its proteasomal degradation; Id2 mutants lacking these phosphorylation sites have longer half-lives and reduced proteasome-mediated degradation, and neural precursor cells expressing de-phosphorylated Id2 proliferate more rapidly. PP2A phosphatase expression stabilizes Id2 levels.","method":"Mass spectrometry identification of phosphorylation sites, stable expression of phospho-mutant Id2 in Id2−/− neural precursor cells, pulse-chase/half-life assays, proteasome inhibition, PP2A manipulation","journal":"Stem cells","confidence":"High","confidence_rationale":"Tier 1 / Moderate — MS-identified sites, mutagenesis, half-life assays, functional proliferation readout, single lab","pmids":["26756672"],"is_preprint":false},{"year":2017,"finding":"Id2 specifies embryonic Lgr5+ intestinal stem cell progenitors by restricting Wnt signaling activity; in the absence of Id2, Wnt target genes are prematurely activated and intestinal epithelial cells differentiate into Lgr5+ cells as early as E9.5. Id2-deficient embryonic epithelial cells strongly activate Wnt targets ex vivo.","method":"Id2-knockout mice, cell fate mapping, Wnt target gene expression analysis, ex vivo intestinal epithelial organoid cultures","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — knockout plus cell fate mapping plus ex vivo culture, single lab","pmids":["28077488"],"is_preprint":false},{"year":2018,"finding":"Pro-inflammatory cytokines enhance Id2 expression in Treg cells; Id2 sequesters E2A transcription activator, reducing Foxp3 expression and inducing TH17-related cytokines, thereby driving Treg-to-TH17 plasticity. Treg-specific ectopic Id2 expression in mice reduces the Treg compartment and exacerbates autoimmunity.","method":"In vitro iTreg differentiation with Id2 overexpression, Id2 Treg-specific transgenic mice, cellular fate-mapping, EAE model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic Treg-specific overexpression plus in vitro mechanistic E2A sequestration evidence, single lab","pmids":["30413714"],"is_preprint":false},{"year":2020,"finding":"Monoubiquitination of FANCD2 (within the FANCD2/FANCI = ID2 complex) promotes a large-scale conformational change that increases the complex's affinity for double-stranded DNA, causing it to encircle dsDNA. Monoubiquitination of FANCI protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being critical for this protection.","method":"Cryo-EM structural analysis, in vitro ubiquitination assays, DNA-binding affinity measurements, deubiquitination protection assays with USP1-UAF1","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus in vitro reconstitution of ubiquitination and DNA binding plus deubiquitination protection assay","pmids":["32510829"],"is_preprint":false},{"year":2021,"finding":"Id2 supports NK cell effector maturation by setting a threshold for TCF1 (encoded by Tcf7); ID2-deficient NK cells show increased TCF1 expression that arrests their maturation, reduces surface receptor expression, impairs IFN-γ production, and reduces ability to clear metastatic melanoma.","method":"Conditional Id2-knockout mouse, gene expression profiling, flow cytometry of NK cell maturation markers, cytokine stimulation assays, in vivo melanoma clearance assay","journal":"The Journal of experimental medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout with defined in vivo functional readout, single lab","pmids":["33857289"],"is_preprint":false},{"year":2021,"finding":"TGF-β signaling decelerates airway basal cell proliferation during development by inhibiting Id2 expression; in adult tissue regeneration, reduced TGF-β signaling restores Id2 expression to initiate regeneration. Persistent Id2 expression drives basal cell hyperplasia resembling a pre-cancerous state.","method":"Temporal single-cell transcriptomics, Id2 overexpression and Tgfbr2 conditional knockout in airway epithelium, genetic validation experiments","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — conditional knockout and overexpression with defined cellular phenotypes plus scRNA-seq, single lab","pmids":["34129836"],"is_preprint":false},{"year":2024,"finding":"Id2, via its HLH domain, binds and disrupts assembly of the Tcf3-Tal1 transcriptional regulatory complex, preventing Tcf3 from recruiting the histone demethylase LSD1 to the Slamf6 promoter. This preserves the permissive H3K4me2 mark on Tcf3-occupied E-boxes in the Slamf6 promoter, increasing chromatin accessibility and promoting generation of Slamf6+ progenitor exhausted CD8+ T cells.","method":"Id2-knockout mouse, ChIP-seq for H3K4me2 and chromatin accessibility (ATAC-seq), co-immunoprecipitation of Id2-HLH with Tcf3-Tal1 complex, LSD1 inhibitor rescue experiments","journal":"Cellular & molecular immunology","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — co-IP of protein complex plus ChIP-seq/ATAC-seq plus pharmacological rescue, multiple orthogonal methods, single lab","pmids":["38287103"],"is_preprint":false},{"year":2002,"finding":"Id2 inhibits BETA2/NeuroD binding to its E-box target sequence; coexpression of Id2 blocks BETA2/NeuroD-mediated E-box-driven transcription in a dose-dependent manner in neuroendocrine HIT cells.","method":"Yeast two-hybrid, EMSA, co-expression reporter assays in HIT and HeLa cells","journal":"Experimental & molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — EMSA plus reporter assay in multiple cell lines, single lab","pmids":["12526101"],"is_preprint":false},{"year":2007,"finding":"All-trans retinoic acid (atRA) suppresses Id2 expression in HaCaT keratinocytes by recruiting LSD1 histone demethylase to the Wnt-response element (TCF/LEF-binding site) in the Id2 promoter, reducing histone H3/H4 acetylation and H3K4 methylation specifically at this site. siRNA silencing of Id2 phenocopies atRA's antiproliferative effect.","method":"ChIP for histone modifications and LSD1 at Id2 promoter WRE, comparison with c-Myc and cyclin D1 WREs, siRNA, cell proliferation assays","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP-based epigenetic analysis plus siRNA functional readout, single lab","pmids":["17310985"],"is_preprint":false},{"year":2004,"finding":"FSH and cAMP (via protein kinase A) induce Id2 gene transcription in Sertoli cells; induction requires sequences between −122 and −82 of the Id2 promoter containing ATF/CRE-like and GATA motifs that bind 40–45 kDa proteins. FSH-induced Id2 expression correlates with decreased E-box-binding protein activity and E-box-mediated transcription.","method":"Northern blot, Id2 promoter reporter assays, EMSA, FSH and forskolin stimulation, protein kinase A overexpression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — promoter reporter with deletion mapping plus EMSA, single lab","pmids":["14761970"],"is_preprint":false},{"year":2023,"finding":"METTL3-mediated m6A modification stabilizes ID2 mRNA in pancreatic cancer cells; METTL3 knockdown reduces m6A modification and decreases ID2 mRNA stability. The m6A reader YTHDF2 is required for METTL3-mediated ID2 mRNA stabilization.","method":"m6A-seq/MeRIP, METTL3 knockdown, mRNA stability assay, YTHDF2 knockdown, Western blot/qRT-PCR","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MeRIP-seq plus mRNA stability assay plus YTHDF2 rescue, single lab","pmids":["37196908"],"is_preprint":false},{"year":2023,"finding":"BMPR1A activation induces physical interaction between ID2 and ZEB1, sequestering ZEB1 and attenuating transcription of Tgfbr2; this lowers TGFβ responsiveness and prevents excessive EndoMT. In Bmpr1a endothelial-conditional knockout mice, ZEB1-driven Tgfbr2 upregulation causes PAH-like disease.","method":"Co-immunoprecipitation of ID2 and ZEB1, conditional Bmpr1a endothelial knockout mouse, lineage tracing, siRNA-lipid nanoparticle therapeutic rescue, reporter assays","journal":"Cardiovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP plus conditional knockout plus in vivo therapeutic rescue, single lab","pmids":["36166408"],"is_preprint":false}],"current_model":"ID2 is a HLH-domain protein (lacking a DNA-binding basic region) that functions primarily as a dominant-negative inhibitor of bHLH E-protein transcription factors (e.g., E47/E2A) through heterodimerization, and also directly binds and inactivates all three Rb family tumor suppressors (pRb, p107, p130) via its HLH domain; it is degraded by both N-terminal ubiquitination and APC/C(Cdh1)-mediated D-box-dependent proteasomal degradation, and its stability is further regulated by DYRK1A/B-mediated phosphorylation on Thr27 (which blocks ID2 interaction with the VHL ubiquitin ligase complex to control HIF2α levels), while upstream signals including Myc oncoproteins, β-catenin/TCF, BMP4/TGF-β, C/EBPβ, Gfi-1, and cAMP/FSH directly control ID2 transcription; mechanistically, ID2 controls cell proliferation vs. differentiation across diverse lineages (neural, hematopoietic, epithelial, immune) by titrating E-protein activity to regulate downstream targets including p57Kip2, p27KIP1, Tcf7, IL-10, and SEMA3F, and epigenetically modulates chromatin by disrupting the Tcf3-LSD1 complex at target gene promoters."},"narrative":{"mechanistic_narrative":"ID2 is a helix-loop-helix (HLH) protein that lacks a basic DNA-binding region and acts as a dominant-negative regulator of the proliferation-versus-differentiation decision across neural, hematopoietic, epithelial, and immune lineages [PMID:1922066, PMID:11034201]. Its core biochemical activity is heterodimerization with and sequestration of class I bHLH E-proteins (E2A/E47, E12) and tissue-specific bHLH factors such as NeuroD/BETA2, thereby preventing their binding to E-box target promoters [PMID:1922066, PMID:12526101, PMID:16547003]. Through this E-protein titration ID2 derepresses or represses defined target genes to drive cell-cycle entry and lineage choice: it blocks E47-driven activation of the CDK inhibitors p57Kip2 and p27KIP1 to promote S-phase entry and tumorigenesis [PMID:16705184, PMID:19451188], suppresses SEMA3F to enhance tumor invasion [PMID:20388805], and restrains E2A-dependent Tcf7/TCF1, IL-10, and Socs3 induction to control CD8+ T cell, NK cell, and Treg fate [PMID:23536629, PMID:24723679, PMID:26795246, PMID:33857289, PMID:30413714]. Beyond E-proteins, ID2 directly binds the unphosphorylated forms of all three Rb-family pocket proteins (pRb, p107, p130) via its HLH domain, neutralizing their growth-suppressive activity and serving as a mediator of Myc oncogenic signaling [PMID:7926730, PMID:8649364, PMID:11034201]. ID2 also acts epigenetically, disrupting the Tcf3-Tal1 complex to block recruitment of the histone demethylase LSD1 and preserve permissive chromatin at target promoters [PMID:38287103]. ID2 protein levels are tightly controlled by proteasomal degradation through N-terminal ubiquitination, APC/C(Cdh1)-mediated D-box recognition, and phosphorylation-dependent turnover, while DYRK1A/B phosphorylation of Thr27 gates its interaction with the VHL ligase to control HIF2α stability [PMID:14733935, PMID:16810178, PMID:26756672, PMID:26735018]. ID2 transcription is directly driven by Myc, β-catenin/TCF, C/EBPβ, BMP4, and FSH/cAMP and repressed by TGF-β effectors, Gfi-1, Ebf1, and Mtg16, integrating diverse upstream signals into developmental and oncogenic programs [PMID:11572874, PMID:12824180, PMID:15809228, PMID:16547003, PMID:14761970, PMID:20453161, PMID:19122139, PMID:24980046]. Note that discovery [PMID:32510829] describes the FANCD2-FANCI \"ID2\" DNA-repair complex, an unrelated entity sharing the abbreviation, not the ID2 HLH protein.","teleology":[{"year":1991,"claim":"Established the founding biochemical activity of ID2: how a protein without a DNA-binding domain regulates transcription, by showing it selectively dimerizes with and inhibits a defined class of bHLH factors.","evidence":"cDNA cloning and in vitro protein interaction/DNA-binding inhibition assays with defined bHLH partners","pmids":["1922066"],"confidence":"High","gaps":["Did not establish in vivo targets or physiological lineages affected","Selectivity basis for class I vs class II bHLH partners left structurally undefined"]},{"year":1996,"claim":"Resolved whether ID2's growth control acts beyond E-proteins by demonstrating direct HLH-mediated binding to all three Rb-family proteins and reversal of Rb/CDK-inhibitor-induced arrest, defining a second mechanistic arm.","evidence":"In vitro binding, reciprocal co-IP in Saos-2 cells, deletion mutagenesis, and cell-cycle assays (builds on the 1994 pRb finding)","pmids":["8649364","7926730"],"confidence":"High","gaps":["Stoichiometry and competition between Rb-binding and E-protein-binding roles unresolved","Did not address physiological contexts where Rb binding dominates"]},{"year":2000,"claim":"Placed ID2 in an oncogenic signaling hierarchy by showing Myc drives ID2 to inactivate Rb, with genetic epistasis rescuing developmental defects, establishing ID2 as a Myc effector.","evidence":"Id2-Rb double-knockout mouse epistasis plus N-myc amplified neuroblastoma cell biochemistry","pmids":["11034201"],"confidence":"High","gaps":["Relative contribution of Rb inactivation vs E-protein sequestration to the rescue not separated"]},{"year":2003,"claim":"Defined ID2 as a transcriptionally controlled node where upstream signals converge, showing context-dependent induction (TGF-β in DCs) versus repression (TGF-β in epithelia via Mad-Max promoter switching).","evidence":"Id2-knockout mice, cytokine stimulation, promoter ChIP, and gel-shift across cell types; promoter direct activation also shown for β-catenin/TCF and C/EBPβ","pmids":["12598895","12824180","11572874","15809228"],"confidence":"High","gaps":["Mechanistic basis for opposite TGF-β outcomes across lineages not unified","Promoter element interplay between competing inputs not mapped"]},{"year":2006,"claim":"Mechanistically connected E-protein inhibition to specific cell-cycle and developmental outputs by identifying p57Kip2 as an E47 target blocked by ID2, and demonstrating bHLH-target control in neurons, pancreas, and heart.","evidence":"Inducible E47/Id2 systems with RNAi rescue, BMP4 stimulation with NeuroD co-IP, and Tbx5/Nkx2-5 promoter and genetic studies","pmids":["16705184","16547003","17604724"],"confidence":"High","gaps":["Tissue-specific E-protein partner identity not always defined","Quantitative threshold of E-protein titration needed for fate switches unknown"]},{"year":2006,"claim":"Resolved how ID2 protein abundance is set, identifying N-terminal ubiquitination and APC/C(Cdh1) D-box-dependent degradation, with stabilized ID2 driving axonal growth via suppression of bHLH targets.","evidence":"In vitro and cell ubiquitination assays, N-/C-terminal tagging, D-box mutagenesis, Cdh1 RNAi, and neuronal growth assays","pmids":["14733935","16810178"],"confidence":"High","gaps":["Identity of the N-terminal ubiquitination E3 ligase not established","Interplay between the two degradation routes not quantified"]},{"year":2013,"claim":"Extended the E-protein-titration model to adaptive and innate immunity, defining ID2 targets (Tcf7, Il10, Socs3) that govern effector-versus-memory CD8+ fate, NK homeostasis, and Treg plasticity.","evidence":"Conditional and germline Id2 knockouts with E2A reporter/ChIP analysis plus genetic rescue (Socs3 deletion, IL-15)","pmids":["23536629","24723679","26795246","30413714","33857289"],"confidence":"Medium","gaps":["Each axis characterized in a single lineage; cross-lineage generality of individual targets unconfirmed","Direct vs indirect status of some targets relies on reporter assays"]},{"year":2016,"claim":"Uncovered a phosphorylation-gated, oxygen-sensitive function: DYRK1A/B phosphorylation of Thr27 controls ID2's binding to the VHL complex, coupling ID2 to HIF2α stabilization, while N-terminal phosphorylation governs proteasomal turnover.","evidence":"In vitro kinase, co-IP, ubiquitylation, phospho-mutant analysis (VHL/HIF2α); MS-mapped sites with half-life and PP2A assays (turnover)","pmids":["26735018","26756672"],"confidence":"High","gaps":["Whether phospho-regulation of stability and of VHL binding act on the same pool unclear","In vivo relevance of HIF2α axis outside glioblastoma not broadly tested"]},{"year":2024,"claim":"Established a chromatin-level mechanism beyond simple E-protein sequestration: ID2's HLH domain disrupts the Tcf3-Tal1 complex to block LSD1 recruitment, preserving permissive H3K4me2 and shaping CD8+ T cell exhaustion programs.","evidence":"Id2-knockout mice, co-IP of Id2-HLH with Tcf3-Tal1, ChIP-seq/ATAC-seq, and LSD1 inhibitor rescue","pmids":["38287103"],"confidence":"High","gaps":["Generality of LSD1-blocking mechanism at other ID2-regulated promoters untested","Whether this acts in concert with or independently of E-protein heterodimerization unresolved"]},{"year":null,"claim":"It remains unresolved how ID2 partitions its activity among E-protein sequestration, Rb-family binding, VHL/HIF2α control, and chromatin remodeling within a single cell, and what determines which mechanism dominates in a given lineage.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model distinguishing Rb-binding from E-protein-binding HLH interfaces in vivo","No quantitative framework relating ID2 dose to choice of effector mechanism","Integrated regulation by the multiple degradation/phosphorylation routes not reconstituted"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,11,17,19]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1,2,31]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[16,26,35]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[8]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,3,11,16]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,30]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[5,19,20,23,26,28]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[12,25,29]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[7,9,24,22]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[4,6,15,35]}],"complexes":[],"partners":["E2A/E47","RB1","RBL1","RBL2","NEUROD1","VHL","TCF3","ZEB1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q02363","full_name":"DNA-binding protein inhibitor ID-2","aliases":["Class B basic helix-loop-helix protein 26","bHLHb26","Inhibitor of DNA binding 2","Inhibitor of differentiation 2"],"length_aa":134,"mass_kda":14.9,"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. 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Inhibits Bladder Cancer Progression and Metastasis via PI3K/AKT Signaling Pathway.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34746132","citation_count":25,"is_preprint":false},{"pmid":"8632670","id":"PMC_8632670","title":"Expression of Id2 and Id3 mRNA in human lymphocytes.","date":"1995","source":"Leukemia research","url":"https://pubmed.ncbi.nlm.nih.gov/8632670","citation_count":25,"is_preprint":false},{"pmid":"17310985","id":"PMC_17310985","title":"Id2 gene-targeted crosstalk between Wnt and retinoid signaling regulates proliferation in human keratinocytes.","date":"2007","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/17310985","citation_count":25,"is_preprint":false},{"pmid":"37230155","id":"PMC_37230155","title":"Quercetin prevents the ferroptosis of OPCs by inhibiting the Id2/transferrin pathway.","date":"2023","source":"Chemico-biological 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biophysics","url":"https://pubmed.ncbi.nlm.nih.gov/29753724","citation_count":21,"is_preprint":false},{"pmid":"26756672","id":"PMC_26756672","title":"Phosphorylation Regulates Id2 Degradation and Mediates the Proliferation of Neural Precursor Cells.","date":"2016","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/26756672","citation_count":21,"is_preprint":false},{"pmid":"27864336","id":"PMC_27864336","title":"BMP4 promotes mouse iPS cell differentiation to male germ cells via Smad1/5, Gata4, Id1 and Id2.","date":"2016","source":"Reproduction (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/27864336","citation_count":21,"is_preprint":false},{"pmid":"34920145","id":"PMC_34920145","title":"TOB1 Blocks Intestinal Mucosal Inflammation Through Inducing ID2-Mediated Suppression of Th1/Th17 Cell Immune Responses in IBD.","date":"2021","source":"Cellular and molecular gastroenterology and hepatology","url":"https://pubmed.ncbi.nlm.nih.gov/34920145","citation_count":20,"is_preprint":false},{"pmid":"35705978","id":"PMC_35705978","title":"GDF-11 promotes human trophoblast cell invasion by increasing ID2-mediated MMP2 expression.","date":"2022","source":"Cell communication and signaling : CCS","url":"https://pubmed.ncbi.nlm.nih.gov/35705978","citation_count":19,"is_preprint":false},{"pmid":"16252281","id":"PMC_16252281","title":"Zebrafish id2 developmental expression pattern contains evolutionary conserved and species-specific characteristics.","date":"2005","source":"Developmental dynamics : an official publication of the American Association of Anatomists","url":"https://pubmed.ncbi.nlm.nih.gov/16252281","citation_count":19,"is_preprint":false},{"pmid":"23390122","id":"PMC_23390122","title":"Elevated Id2 expression results in precocious neural stem cell depletion and abnormal brain development.","date":"2013","source":"Stem cells (Dayton, Ohio)","url":"https://pubmed.ncbi.nlm.nih.gov/23390122","citation_count":19,"is_preprint":false},{"pmid":"19451188","id":"PMC_19451188","title":"Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis.","date":"2009","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/19451188","citation_count":19,"is_preprint":false},{"pmid":"14966287","id":"PMC_14966287","title":"Id2 is dispensable for Myc-induced epidermal neoplasia.","date":"2004","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/14966287","citation_count":19,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47931,"output_tokens":9079,"usd":0.139989,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":19206,"output_tokens":4187,"usd":0.100352,"stage2_stop_reason":"end_turn"},"total_usd":0.240341,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"Id2 contains a helix-loop-helix (HLH) motif but lacks the basic DNA-binding domain; it selectively heterodimerizes with and inhibits one class of bHLH transcription factors (typified by E2A/E47 and E2B/m3) but not another class (TFE3, USF, AP4). Id2 also homodimerizes poorly.\",\n      \"method\": \"cDNA cloning, chromosome mapping, in vitro protein interaction and DNA-binding inhibition assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution of binding/inhibition with defined protein pairs, replicated across multiple bHLH partners\",\n      \"pmids\": [\"1922066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"Id2 physically binds unphosphorylated pRb via its HLH domain (an Id2 mutant lacking the HLH domain fails to bind); the interaction requires the E1A/large T-binding pocket of pRb. Id2 expression reverses pRb-mediated growth arrest without changing pRb phosphorylation state.\",\n      \"method\": \"In vitro binding assay with cell lysates, in vivo cross-linked co-immunoprecipitation in transfected SAOS-2 cells, deletion mutagenesis, cell proliferation/S-phase analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro binding with mutagenesis plus in vivo co-IP, multiple orthogonal methods in one study\",\n      \"pmids\": [\"7926730\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"Id2, but not Id1 or Id3, binds in vitro to pRb, p107, and p130 (all three Rb family members), and in vivo association of Id2 with p107 and p130 is detected in transfected Saos-2 cells. Id2 specifically reverses cell cycle arrest induced by each Rb family member and by the CDK inhibitors p16 and p21 (p21 reversal is partial and pRb-dependent). Id2 overexpression decreases cyclin D1 protein/mRNA and disrupts cyclin D1-Cdk4 complexes.\",\n      \"method\": \"In vitro binding assay, transient transfection with co-immunoprecipitation, cell cycle progression assays, Western blot, Northern blot\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — reciprocal co-IP plus in vitro binding, multiple Rb family members tested, replicated for each member\",\n      \"pmids\": [\"8649364\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Id2 overexpression driven by Myc oncoproteins (N-Myc and c-Myc) inactivates Rb family proteins, enabling cell-cycle progression. In Id2-Rb double-knockout embryos, neurogenesis and hematopoiesis defects are rescued compared to each single knockout, establishing Id2 as a mediator of Myc oncogenic signaling through Rb inactivation.\",\n      \"method\": \"Double-knockout mouse genetics (epistasis), N-myc overexpression/amplification in neuroblastoma cell lines, transcriptional activation assays, molar ratio analysis of Id2 vs. active Rb\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in vivo combined with cell-line biochemistry, replicated across multiple tumor cell contexts\",\n      \"pmids\": [\"11034201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Id2 is a transcriptional target of the β-catenin/TCF pathway in colon carcinoma; a TCF4-binding motif in the Id2 promoter is required for activation (identified by EMSA/supershift and abolished by site-directed mutagenesis). β-catenin up-regulates Id2 protein and increases anchorage-independent survival.\",\n      \"method\": \"Id2 promoter reporter assays, EMSA with supershift, site-directed mutagenesis, β-catenin overexpression/knockdown, anchorage-independent growth assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — promoter mutagenesis plus EMSA/supershift plus functional assay in same study, single lab\",\n      \"pmids\": [\"11572874\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TGF-β signaling induces Id2 expression in dendritic cells, and Id2 represses B cell-specific genes in DCs, revealing a TGF-β→Id2 signaling axis that governs lineage choice between B cell and DC progenitors.\",\n      \"method\": \"Id2-knockout mouse analysis, in vitro TGF-β stimulation of DC cultures, gene expression profiling, in vivo DC subset analysis\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout mouse phenotype plus cytokine stimulation experiments, supported by transcriptional profiling\",\n      \"pmids\": [\"12598895\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TGF-β inhibits Id2 expression in human and mouse epithelial cells; this repression occurs via induction of Mad2/Mad4, which replaces Myc-Max complexes with Mad-Max complexes on the Id2 promoter, linking TGF-β cytostatic signaling to Id2 downregulation.\",\n      \"method\": \"Northern/Western blot in multiple epithelial cell lines, promoter ChIP (Myc-Max vs. Mad-Max occupancy), gel-shift assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — chromatin immunoprecipitation plus gel-shift, multiple cell lines, mechanistic follow-up of promoter occupancy changes\",\n      \"pmids\": [\"12824180\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Id2 is degraded by the ubiquitin-proteasome system via ubiquitination at its N-terminal α-amino group (N-terminal ubiquitination). Lysine-less Id2 is still efficiently degraded by the proteasome; N-terminal Myc-tag fusion stabilizes Id2, whereas C-terminal tag does not. Deletion of the first 15 N-terminal residues also stabilizes Id2, implicating this region as the E3 ligase recognition element.\",\n      \"method\": \"In vitro and cell-based ubiquitination assays, proteasome inhibitor experiments, N-terminal vs. C-terminal tagging, deletion mutagenesis, pulse-chase/stability assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution plus mutagenesis, multiple orthogonal approaches in single study\",\n      \"pmids\": [\"14733935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Polycystin-2 (PC2) directly interacts with Id2, and this interaction is regulated by polycystin-1 (PC1)-dependent phosphorylation of PC2. Id2 suppresses p21 induction by PC1 or PC2. Enhanced Id2 nuclear localization is observed in cystic kidneys, and RNAi-mediated Id2 knockdown corrects hyperproliferation of PC1 mutant cells.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, nuclear fractionation/localization in cystic kidney tissue and cells, functional proliferation assays\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus localization plus functional RNAi rescue, multiple orthogonal methods\",\n      \"pmids\": [\"16311606\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"APC/C(Cdh1) ubiquitin ligase targets Id2 for proteasomal degradation through a conserved D-box (destruction box) motif. Id2 interacts with APC/C core subunits and Cdh1 in neurons; Cdh1 depletion stabilizes Id proteins; D-box mutants of Id2 fail to bind Cdh1 and remain stable when APC/C(Cdh1) is active. Stabilized Id2 enhances axonal growth and overcomes myelin inhibitory signals by preventing accumulation of the Nogo receptor (a bHLH target gene product).\",\n      \"method\": \"Co-immunoprecipitation in primary neurons, D-box mutagenesis, Cdh1 RNAi/knockdown, in vitro and in vivo axonal growth assays, gene expression analysis\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — D-box mutagenesis plus co-IP plus in vivo neuronal phenotype, multiple orthogonal methods\",\n      \"pmids\": [\"16810178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"C/EBPβ is a direct transcriptional activator of the Id2 gene; the most proximal C/EBPβ-binding site in the Id2 promoter is responsible for transactivation, as confirmed by EMSA and ChIP. Id2 expression is reduced in C/EBPβ-deficient mammary glands.\",\n      \"method\": \"Id2 promoter reporter assays, EMSA, ChIP, Northern blot in C/EBPβ-knockout mammary glands, C/EBPβ-ERT nuclear translocation system\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus EMSA plus promoter reporter plus knockout validation, multiple methods in single lab\",\n      \"pmids\": [\"15809228\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Id2 promotes S-phase entry in neural cells by preventing E-protein (E47)-mediated activation of the CDK inhibitor p57Kip2. E47 directly activates p57Kip2 expression, and Id2 blocks this activation; p57Kip2 silencing by RNAi fully reverses E47-mediated cell cycle arrest.\",\n      \"method\": \"Gene expression profiling, RNAi knockdown, cell cycle analysis, in neuroblastoma cell lines engineered with inducible E47 and Id2\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — inducible expression systems plus RNAi rescue plus genome-wide expression, multiple orthogonal methods\",\n      \"pmids\": [\"16705184\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Id2 functions downstream of Tbx5 and Nkx2-5 in a molecular pathway required for cardiac ventricular conduction system specification. Compound haploinsufficiency of Tbx5/Id2 prevents embryonic conduction system specification; a 1.2 kb Id2 promoter fragment is cooperatively regulated by Nkx2-5 and Tbx5 in vitro and is sufficient for conduction-system-specific expression in vivo.\",\n      \"method\": \"Id2-knockout and compound haploinsufficient mouse genetics, SAGE transcriptional profiling of microdissected conduction system, promoter reporter assays, in vivo transgenic reporter\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in vivo plus promoter reporter in vitro and in vivo, multiple complementary approaches\",\n      \"pmids\": [\"17604724\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Id2 regulates erythroid development via direct interaction with transcription factor PU.1, modulating PU.1 and GATA-1 activities. Id2 also intrinsically inhibits B cell development through antagonism of E2A (E47).\",\n      \"method\": \"Id2 knockout mouse analysis, retroviral overexpression in hematopoietic progenitors, shRNA knockdown, co-immunoprecipitation/interaction studies with PU.1\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — functional knockdown/overexpression plus interaction assays, single lab, co-IP is mentioned but mechanistic detail is limited in the abstract\",\n      \"pmids\": [\"18523151\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Ebf1 transcriptionally represses Id2 (and Id3) during B-lineage specification; enforced Ebf1 in IL-7Rα−/− bone marrow dramatically reduces Id2/Id3 mRNA and restores B cell differentiation. Overexpression of Id2 alone blocks B cell specification at the prepro-B stage by inhibiting E2A activity.\",\n      \"method\": \"Retroviral overexpression and knockout mouse genetics, qRT-PCR, in vivo B cell reconstitution assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in vivo plus retroviral rescue, single lab\",\n      \"pmids\": [\"19122139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"BMP4 induces Id2 expression in pancreatic epithelial progenitors, and BMP4-stimulated Id2 binds to the bHLH transcription factor NeuroD, blocking NeuroD-dependent differentiation and instead promoting progenitor expansion.\",\n      \"method\": \"BMP4 stimulation of AR42J cells, co-immunoprecipitation of Id2 with NeuroD, BMP4 neutralization in a mouse islet regeneration model, proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — co-IP plus functional BMP4 stimulation/neutralization, single lab\",\n      \"pmids\": [\"16547003\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Id2-mediated sequestration of E12/E47 bHLH factors suppresses p27KIP1 promoter activity, reducing p27KIP1 expression. Re-expression of p27KIP1 in Id2-overexpressing Id2−/− fibroblasts reverts hyperproliferation and tumorigenicity, identifying the Id2/E12/E47/p27KIP1 axis as a mechanism of Id2-driven oncogenesis.\",\n      \"method\": \"Id2 deletion mutagenesis, re-expression in Id2−/− fibroblasts, promoter reporter assays, flow cytometry, subcutaneous tumor grafting in athymic mice\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter reporter plus in vivo tumorigenesis plus domain mutagenesis, single lab\",\n      \"pmids\": [\"19451188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Id2 represses SEMA3F expression through the E47/Id2 pathway; Id2 blocks E47-mediated direct activation of two E-box sites in the SEMA3F promoter. C-Myc drives Id2 overexpression, which in turn suppresses SEMA3F and promotes tumor cell migration and invasion.\",\n      \"method\": \"SEMA3F promoter reporter assay with E-box mutagenesis, E47 overexpression/Id2 overexpression in low vs. high metastatic cell lines, migration/invasion assays, silencing of SEMA3F\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — promoter analysis with mutagenesis plus functional invasion assay, single lab\",\n      \"pmids\": [\"20388805\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Gfi-1 directly binds to three conserved regions in the Id2 promoter and represses Id2 promoter activity. Id2 knockdown or haploinsufficiency partially rescues B cell and myeloid development defects in Gfi-1−/− mice, placing Id2 downstream of Gfi-1 in hematopoietic transcriptional networks.\",\n      \"method\": \"ChIP, promoter reporter assay, Id2/Gfi-1 double knockout/heterozygous mouse genetics, in vivo B cell and myeloid reconstitution\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus promoter assay plus genetic epistasis in vivo, single lab\",\n      \"pmids\": [\"20453161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Id2 inhibits E2A (E47)-mediated direct transcriptional activation of Tcf7 (encoding TCF1), restraining memory CD8+ T cell differentiation. Loss of Id2 leads to E2A-dependent increase of Tcf7 and Eomesodermin expression, redirecting effector CD8+ T cells toward a memory fate.\",\n      \"method\": \"Id2-knockout mouse, retroviral E2A overexpression, gene expression profiling, chromatin/luciferase reporter assays\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout plus E2A interaction/reporter assays, single lab\",\n      \"pmids\": [\"23536629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Id2 represses E2A-mediated transactivation of the IL-10 locus (Il10) in T cells; Id2-deficient T cells overexpress IL-10 in vitro and in vivo during infection, demonstrating that the Id2/E2A axis limits IL-10 production during effector T cell responses.\",\n      \"method\": \"Id2-knockout mouse, IL-10 reporter/ChIP analysis, graft-versus-host disease model, influenza infection model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic knockout plus E2A transcriptional assay evidence, single lab\",\n      \"pmids\": [\"24723679\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Mtg16 directly represses Id2 transcription in dendritic cell progenitors (identified by genome-wide DNA-binding analysis); loss of Mtg16 leads to aberrant Id2 induction in pDC progenitors, and Id2 deletion rescues impaired pDC development in Mtg16-deficient mice.\",\n      \"method\": \"Mtg16-knockout mouse, genome-wide ChIP-seq of Mtg16, Id2/Mtg16 double-knockout genetics, flow cytometric DC subset analysis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide ChIP-seq identifying direct Id2 target plus genetic epistasis rescue experiment, multiple approaches\",\n      \"pmids\": [\"24980046\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Phosphorylated ID2 cannot bind the VHL ubiquitin ligase complex; unphosphorylated ID2 binds VHL, displaces Cullin 2, and impairs HIF2α ubiquitylation and degradation. Hypoxia inactivates DYRK1A/1B (via PHD1 oxygen sensor) to reduce Thr27 phosphorylation and allow ID2–VHL interaction and HIF2α stabilization.\",\n      \"method\": \"In vitro kinase assay (DYRK1A/B phosphorylation of ID2), co-immunoprecipitation of ID2 with VHL complex, ubiquitylation assays, phospho-mutant ID2 expression, tumor growth assays in glioblastoma\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — in vitro kinase reconstitution plus co-IP plus ubiquitylation assay plus phospho-mutant analysis, multiple orthogonal methods\",\n      \"pmids\": [\"26735018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"ID2 regulates NK cell homeostasis by repressing multiple E-protein target genes including Socs3; ID2 deletion in mature NK cells impairs IL-15 receptor signaling and metabolic function, and these defects are rescued by strong IL-15 stimulation or genetic Socs3 ablation.\",\n      \"method\": \"Conditional Id2 knockout in NK cells, IL-15 stimulation rescue experiments, Socs3 genetic deletion rescue, gene expression analysis\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional knockout plus two independent rescue experiments (IL-15 stimulation and Socs3 deletion), genetic epistasis\",\n      \"pmids\": [\"26795246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Phosphorylation of Id2 near its N-terminus promotes its proteasomal degradation; Id2 mutants lacking these phosphorylation sites have longer half-lives and reduced proteasome-mediated degradation, and neural precursor cells expressing de-phosphorylated Id2 proliferate more rapidly. PP2A phosphatase expression stabilizes Id2 levels.\",\n      \"method\": \"Mass spectrometry identification of phosphorylation sites, stable expression of phospho-mutant Id2 in Id2−/− neural precursor cells, pulse-chase/half-life assays, proteasome inhibition, PP2A manipulation\",\n      \"journal\": \"Stem cells\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — MS-identified sites, mutagenesis, half-life assays, functional proliferation readout, single lab\",\n      \"pmids\": [\"26756672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Id2 specifies embryonic Lgr5+ intestinal stem cell progenitors by restricting Wnt signaling activity; in the absence of Id2, Wnt target genes are prematurely activated and intestinal epithelial cells differentiate into Lgr5+ cells as early as E9.5. Id2-deficient embryonic epithelial cells strongly activate Wnt targets ex vivo.\",\n      \"method\": \"Id2-knockout mice, cell fate mapping, Wnt target gene expression analysis, ex vivo intestinal epithelial organoid cultures\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — knockout plus cell fate mapping plus ex vivo culture, single lab\",\n      \"pmids\": [\"28077488\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Pro-inflammatory cytokines enhance Id2 expression in Treg cells; Id2 sequesters E2A transcription activator, reducing Foxp3 expression and inducing TH17-related cytokines, thereby driving Treg-to-TH17 plasticity. Treg-specific ectopic Id2 expression in mice reduces the Treg compartment and exacerbates autoimmunity.\",\n      \"method\": \"In vitro iTreg differentiation with Id2 overexpression, Id2 Treg-specific transgenic mice, cellular fate-mapping, EAE model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic Treg-specific overexpression plus in vitro mechanistic E2A sequestration evidence, single lab\",\n      \"pmids\": [\"30413714\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Monoubiquitination of FANCD2 (within the FANCD2/FANCI = ID2 complex) promotes a large-scale conformational change that increases the complex's affinity for double-stranded DNA, causing it to encircle dsDNA. Monoubiquitination of FANCI protects the ubiquitin on FANCD2 from USP1-UAF1 deubiquitination, with key hydrophobic residues of FANCI's ubiquitin being critical for this protection.\",\n      \"method\": \"Cryo-EM structural analysis, in vitro ubiquitination assays, DNA-binding affinity measurements, deubiquitination protection assays with USP1-UAF1\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus in vitro reconstitution of ubiquitination and DNA binding plus deubiquitination protection assay\",\n      \"pmids\": [\"32510829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Id2 supports NK cell effector maturation by setting a threshold for TCF1 (encoded by Tcf7); ID2-deficient NK cells show increased TCF1 expression that arrests their maturation, reduces surface receptor expression, impairs IFN-γ production, and reduces ability to clear metastatic melanoma.\",\n      \"method\": \"Conditional Id2-knockout mouse, gene expression profiling, flow cytometry of NK cell maturation markers, cytokine stimulation assays, in vivo melanoma clearance assay\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout with defined in vivo functional readout, single lab\",\n      \"pmids\": [\"33857289\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TGF-β signaling decelerates airway basal cell proliferation during development by inhibiting Id2 expression; in adult tissue regeneration, reduced TGF-β signaling restores Id2 expression to initiate regeneration. Persistent Id2 expression drives basal cell hyperplasia resembling a pre-cancerous state.\",\n      \"method\": \"Temporal single-cell transcriptomics, Id2 overexpression and Tgfbr2 conditional knockout in airway epithelium, genetic validation experiments\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — conditional knockout and overexpression with defined cellular phenotypes plus scRNA-seq, single lab\",\n      \"pmids\": [\"34129836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Id2, via its HLH domain, binds and disrupts assembly of the Tcf3-Tal1 transcriptional regulatory complex, preventing Tcf3 from recruiting the histone demethylase LSD1 to the Slamf6 promoter. This preserves the permissive H3K4me2 mark on Tcf3-occupied E-boxes in the Slamf6 promoter, increasing chromatin accessibility and promoting generation of Slamf6+ progenitor exhausted CD8+ T cells.\",\n      \"method\": \"Id2-knockout mouse, ChIP-seq for H3K4me2 and chromatin accessibility (ATAC-seq), co-immunoprecipitation of Id2-HLH with Tcf3-Tal1 complex, LSD1 inhibitor rescue experiments\",\n      \"journal\": \"Cellular & molecular immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — co-IP of protein complex plus ChIP-seq/ATAC-seq plus pharmacological rescue, multiple orthogonal methods, single lab\",\n      \"pmids\": [\"38287103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Id2 inhibits BETA2/NeuroD binding to its E-box target sequence; coexpression of Id2 blocks BETA2/NeuroD-mediated E-box-driven transcription in a dose-dependent manner in neuroendocrine HIT cells.\",\n      \"method\": \"Yeast two-hybrid, EMSA, co-expression reporter assays in HIT and HeLa cells\",\n      \"journal\": \"Experimental & molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — EMSA plus reporter assay in multiple cell lines, single lab\",\n      \"pmids\": [\"12526101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"All-trans retinoic acid (atRA) suppresses Id2 expression in HaCaT keratinocytes by recruiting LSD1 histone demethylase to the Wnt-response element (TCF/LEF-binding site) in the Id2 promoter, reducing histone H3/H4 acetylation and H3K4 methylation specifically at this site. siRNA silencing of Id2 phenocopies atRA's antiproliferative effect.\",\n      \"method\": \"ChIP for histone modifications and LSD1 at Id2 promoter WRE, comparison with c-Myc and cyclin D1 WREs, siRNA, cell proliferation assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-based epigenetic analysis plus siRNA functional readout, single lab\",\n      \"pmids\": [\"17310985\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"FSH and cAMP (via protein kinase A) induce Id2 gene transcription in Sertoli cells; induction requires sequences between −122 and −82 of the Id2 promoter containing ATF/CRE-like and GATA motifs that bind 40–45 kDa proteins. FSH-induced Id2 expression correlates with decreased E-box-binding protein activity and E-box-mediated transcription.\",\n      \"method\": \"Northern blot, Id2 promoter reporter assays, EMSA, FSH and forskolin stimulation, protein kinase A overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — promoter reporter with deletion mapping plus EMSA, single lab\",\n      \"pmids\": [\"14761970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"METTL3-mediated m6A modification stabilizes ID2 mRNA in pancreatic cancer cells; METTL3 knockdown reduces m6A modification and decreases ID2 mRNA stability. The m6A reader YTHDF2 is required for METTL3-mediated ID2 mRNA stabilization.\",\n      \"method\": \"m6A-seq/MeRIP, METTL3 knockdown, mRNA stability assay, YTHDF2 knockdown, Western blot/qRT-PCR\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MeRIP-seq plus mRNA stability assay plus YTHDF2 rescue, single lab\",\n      \"pmids\": [\"37196908\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"BMPR1A activation induces physical interaction between ID2 and ZEB1, sequestering ZEB1 and attenuating transcription of Tgfbr2; this lowers TGFβ responsiveness and prevents excessive EndoMT. In Bmpr1a endothelial-conditional knockout mice, ZEB1-driven Tgfbr2 upregulation causes PAH-like disease.\",\n      \"method\": \"Co-immunoprecipitation of ID2 and ZEB1, conditional Bmpr1a endothelial knockout mouse, lineage tracing, siRNA-lipid nanoparticle therapeutic rescue, reporter assays\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP plus conditional knockout plus in vivo therapeutic rescue, single lab\",\n      \"pmids\": [\"36166408\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ID2 is a HLH-domain protein (lacking a DNA-binding basic region) that functions primarily as a dominant-negative inhibitor of bHLH E-protein transcription factors (e.g., E47/E2A) through heterodimerization, and also directly binds and inactivates all three Rb family tumor suppressors (pRb, p107, p130) via its HLH domain; it is degraded by both N-terminal ubiquitination and APC/C(Cdh1)-mediated D-box-dependent proteasomal degradation, and its stability is further regulated by DYRK1A/B-mediated phosphorylation on Thr27 (which blocks ID2 interaction with the VHL ubiquitin ligase complex to control HIF2α levels), while upstream signals including Myc oncoproteins, β-catenin/TCF, BMP4/TGF-β, C/EBPβ, Gfi-1, and cAMP/FSH directly control ID2 transcription; mechanistically, ID2 controls cell proliferation vs. differentiation across diverse lineages (neural, hematopoietic, epithelial, immune) by titrating E-protein activity to regulate downstream targets including p57Kip2, p27KIP1, Tcf7, IL-10, and SEMA3F, and epigenetically modulates chromatin by disrupting the Tcf3-LSD1 complex at target gene promoters.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ID2 is a helix-loop-helix (HLH) protein that lacks a basic DNA-binding region and acts as a dominant-negative regulator of the proliferation-versus-differentiation decision across neural, hematopoietic, epithelial, and immune lineages [#0, #3]. Its core biochemical activity is heterodimerization with and sequestration of class I bHLH E-proteins (E2A/E47, E12) and tissue-specific bHLH factors such as NeuroD/BETA2, thereby preventing their binding to E-box target promoters [#0, #31, #15]. Through this E-protein titration ID2 derepresses or represses defined target genes to drive cell-cycle entry and lineage choice: it blocks E47-driven activation of the CDK inhibitors p57Kip2 and p27KIP1 to promote S-phase entry and tumorigenesis [#11, #16], suppresses SEMA3F to enhance tumor invasion [#17], and restrains E2A-dependent Tcf7/TCF1, IL-10, and Socs3 induction to control CD8+ T cell, NK cell, and Treg fate [#19, #20, #23, #28, #26]. Beyond E-proteins, ID2 directly binds the unphosphorylated forms of all three Rb-family pocket proteins (pRb, p107, p130) via its HLH domain, neutralizing their growth-suppressive activity and serving as a mediator of Myc oncogenic signaling [#1, #2, #3]. ID2 also acts epigenetically, disrupting the Tcf3-Tal1 complex to block recruitment of the histone demethylase LSD1 and preserve permissive chromatin at target promoters [#30]. ID2 protein levels are tightly controlled by proteasomal degradation through N-terminal ubiquitination, APC/C(Cdh1)-mediated D-box recognition, and phosphorylation-dependent turnover, while DYRK1A/B phosphorylation of Thr27 gates its interaction with the VHL ligase to control HIF2\\u03b1 stability [#7, #9, #24, #22]. ID2 transcription is directly driven by Myc, \\u03b2-catenin/TCF, C/EBP\\u03b2, BMP4, and FSH/cAMP and repressed by TGF-\\u03b2 effectors, Gfi-1, Ebf1, and Mtg16, integrating diverse upstream signals into developmental and oncogenic programs [#4, #6, #10, #15, #33, #18, #14, #21]. Note that discovery [#27] describes the FANCD2-FANCI \\\"ID2\\\" DNA-repair complex, an unrelated entity sharing the abbreviation, not the ID2 HLH protein.\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Established the founding biochemical activity of ID2: how a protein without a DNA-binding domain regulates transcription, by showing it selectively dimerizes with and inhibits a defined class of bHLH factors.\",\n      \"evidence\": \"cDNA cloning and in vitro protein interaction/DNA-binding inhibition assays with defined bHLH partners\",\n      \"pmids\": [\"1922066\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish in vivo targets or physiological lineages affected\", \"Selectivity basis for class I vs class II bHLH partners left structurally undefined\"]\n    },\n    {\n      \"year\": 1996,\n      \"claim\": \"Resolved whether ID2's growth control acts beyond E-proteins by demonstrating direct HLH-mediated binding to all three Rb-family proteins and reversal of Rb/CDK-inhibitor-induced arrest, defining a second mechanistic arm.\",\n      \"evidence\": \"In vitro binding, reciprocal co-IP in Saos-2 cells, deletion mutagenesis, and cell-cycle assays (builds on the 1994 pRb finding)\",\n      \"pmids\": [\"8649364\", \"7926730\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and competition between Rb-binding and E-protein-binding roles unresolved\", \"Did not address physiological contexts where Rb binding dominates\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Placed ID2 in an oncogenic signaling hierarchy by showing Myc drives ID2 to inactivate Rb, with genetic epistasis rescuing developmental defects, establishing ID2 as a Myc effector.\",\n      \"evidence\": \"Id2-Rb double-knockout mouse epistasis plus N-myc amplified neuroblastoma cell biochemistry\",\n      \"pmids\": [\"11034201\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of Rb inactivation vs E-protein sequestration to the rescue not separated\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Defined ID2 as a transcriptionally controlled node where upstream signals converge, showing context-dependent induction (TGF-\\u03b2 in DCs) versus repression (TGF-\\u03b2 in epithelia via Mad-Max promoter switching).\",\n      \"evidence\": \"Id2-knockout mice, cytokine stimulation, promoter ChIP, and gel-shift across cell types; promoter direct activation also shown for \\u03b2-catenin/TCF and C/EBP\\u03b2\",\n      \"pmids\": [\"12598895\", \"12824180\", \"11572874\", \"15809228\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic basis for opposite TGF-\\u03b2 outcomes across lineages not unified\", \"Promoter element interplay between competing inputs not mapped\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mechanistically connected E-protein inhibition to specific cell-cycle and developmental outputs by identifying p57Kip2 as an E47 target blocked by ID2, and demonstrating bHLH-target control in neurons, pancreas, and heart.\",\n      \"evidence\": \"Inducible E47/Id2 systems with RNAi rescue, BMP4 stimulation with NeuroD co-IP, and Tbx5/Nkx2-5 promoter and genetic studies\",\n      \"pmids\": [\"16705184\", \"16547003\", \"17604724\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific E-protein partner identity not always defined\", \"Quantitative threshold of E-protein titration needed for fate switches unknown\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved how ID2 protein abundance is set, identifying N-terminal ubiquitination and APC/C(Cdh1) D-box-dependent degradation, with stabilized ID2 driving axonal growth via suppression of bHLH targets.\",\n      \"evidence\": \"In vitro and cell ubiquitination assays, N-/C-terminal tagging, D-box mutagenesis, Cdh1 RNAi, and neuronal growth assays\",\n      \"pmids\": [\"14733935\", \"16810178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the N-terminal ubiquitination E3 ligase not established\", \"Interplay between the two degradation routes not quantified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended the E-protein-titration model to adaptive and innate immunity, defining ID2 targets (Tcf7, Il10, Socs3) that govern effector-versus-memory CD8+ fate, NK homeostasis, and Treg plasticity.\",\n      \"evidence\": \"Conditional and germline Id2 knockouts with E2A reporter/ChIP analysis plus genetic rescue (Socs3 deletion, IL-15)\",\n      \"pmids\": [\"23536629\", \"24723679\", \"26795246\", \"30413714\", \"33857289\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Each axis characterized in a single lineage; cross-lineage generality of individual targets unconfirmed\", \"Direct vs indirect status of some targets relies on reporter assays\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Uncovered a phosphorylation-gated, oxygen-sensitive function: DYRK1A/B phosphorylation of Thr27 controls ID2's binding to the VHL complex, coupling ID2 to HIF2\\u03b1 stabilization, while N-terminal phosphorylation governs proteasomal turnover.\",\n      \"evidence\": \"In vitro kinase, co-IP, ubiquitylation, phospho-mutant analysis (VHL/HIF2\\u03b1); MS-mapped sites with half-life and PP2A assays (turnover)\",\n      \"pmids\": [\"26735018\", \"26756672\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phospho-regulation of stability and of VHL binding act on the same pool unclear\", \"In vivo relevance of HIF2\\u03b1 axis outside glioblastoma not broadly tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established a chromatin-level mechanism beyond simple E-protein sequestration: ID2's HLH domain disrupts the Tcf3-Tal1 complex to block LSD1 recruitment, preserving permissive H3K4me2 and shaping CD8+ T cell exhaustion programs.\",\n      \"evidence\": \"Id2-knockout mice, co-IP of Id2-HLH with Tcf3-Tal1, ChIP-seq/ATAC-seq, and LSD1 inhibitor rescue\",\n      \"pmids\": [\"38287103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of LSD1-blocking mechanism at other ID2-regulated promoters untested\", \"Whether this acts in concert with or independently of E-protein heterodimerization unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unresolved how ID2 partitions its activity among E-protein sequestration, Rb-family binding, VHL/HIF2\\u03b1 control, and chromatin remodeling within a single cell, and what determines which mechanism dominates in a given lineage.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model distinguishing Rb-binding from E-protein-binding HLH interfaces in vivo\", \"No quantitative framework relating ID2 dose to choice of effector mechanism\", \"Integrated regulation by the multiple degradation/phosphorylation routes not reconstituted\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 11, 17, 19]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1, 2, 31]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [16, 26, 35]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3, 11, 16]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 30]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [5, 19, 20, 23, 26, 28]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [12, 25, 29]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [7, 9, 24, 22]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [4, 6, 15, 35]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"E2A/E47\", \"RB1\", \"RBL1\", \"RBL2\", \"NEUROD1\", \"VHL\", \"TCF3\", \"ZEB1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}