{"gene":"TDG","run_date":"2026-06-10T10:51:54","timeline":{"discoveries":[{"year":2011,"finding":"TDG specifically recognizes and excises 5-carboxylcytosine (5caC) from DNA, a product of iterative TET dioxygenase-mediated oxidation of 5-methylcytosine (5mC). Depletion of TDG in mouse embryonic stem cells leads to accumulation of 5caC to detectable levels, establishing TDG as the glycosylase that removes 5caC in the TET-initiated active DNA demethylation pathway.","method":"In vitro glycosylase assay, cell-based depletion (siRNA/genetic) with LC-MS quantification of 5caC","journal":"Science","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro biochemical assay with defined substrates plus genetic depletion in ESCs, replicated across multiple labs subsequently","pmids":["21817016"],"is_preprint":false},{"year":2011,"finding":"TDG is essential for embryonic development (Tdg null mice die in utero). TDG maintains epigenetic stability by contributing to active and bivalent chromatin at developmental gene promoters, counteracting aberrant de novo methylation and facilitating proper assembly of chromatin-modifying complexes via base excision repair.","method":"Mouse genetic knockout, ChIP for histone modifications and CpG methylation at gene promoters in MEFs and ESCs","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout lethal phenotype combined with multiple orthogonal epigenomic assays (ChIP, methylation analysis)","pmids":["21278727"],"is_preprint":false},{"year":2013,"finding":"TDG excises 5-formylcytosine (5fC) and 5caC genome-wide, particularly at gene regulatory elements (proximal and distal). Genome-wide maps in Tdg-deficient mouse ESCs reveal marked accumulation of 5fC and 5caC at these sites, demonstrating that TET/TDG-dependent active demethylation occurs extensively at regulatory regions.","method":"Genome-wide modification-specific antibody profiling (ChIP-seq/immunoprecipitation) in wild-type and Tdg-deficient ESCs","journal":"Cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide approach with genetic controls (Tdg KO), multiple modification marks, replicated findings","pmids":["23602152"],"is_preprint":false},{"year":2016,"finding":"TET1 and TDG physically interact to form a functional complex; biochemical reconstitution demonstrates that the TET-TDG-BER system can achieve productive DNA demethylation of symmetrically methylated CpGs in a sequential manner, avoiding DNA double-strand break formation.","method":"Co-immunoprecipitation, biochemical reconstitution with purified TET1 and TDG, in vitro demethylation assay","journal":"Nature Communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — biochemical reconstitution with purified components and physical interaction validated, single lab but multiple orthogonal methods","pmids":["26932196"],"is_preprint":false},{"year":2016,"finding":"NEIL1 and NEIL2 DNA glycosylases stimulate TDG substrate turnover during active demethylation: TDG occupies the abasic site after base excision and is displaced by NEIL proteins, which process the baseless sugar as AP lyases, thereby accelerating TDG cycling on 5fC/5caC substrates.","method":"In vitro glycosylase/AP lyase assay with purified proteins, Xenopus embryo loss-of-function experiments","journal":"Nature Structural & Molecular Biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with purified components plus in vivo genetic validation in Xenopus, two orthogonal model systems","pmids":["26751644"],"is_preprint":false},{"year":2004,"finding":"TDG (TDGb isoform) binds SUMO-1 non-covalently via a specific region (residues within the N-terminus), and this non-covalent SUMO-1 binding is required for covalent SUMO-1 conjugation at an adjacent lysine residue. SUMO-1 modification of TDG promotes its interaction with PML and co-localization to PML nuclear bodies.","method":"Yeast two-hybrid screen, in vitro pulldown, co-immunoprecipitation, fluorescence co-localization in transfected cells, deletion and point mutagenesis","journal":"Journal of Biological Chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and localization with systematic mutagenesis dissecting binding domain, single lab but multiple orthogonal methods","pmids":["15569683"],"is_preprint":false},{"year":2007,"finding":"TDG is degraded by the ubiquitin-proteasome system at the onset of S-phase and is absent throughout S and G2 phases; this cell cycle regulation is strictly inverse to UNG2, functionally separating the two repair enzymes. Incomplete TDG degradation impedes S-phase progression and cell proliferation.","method":"Flow cytometry cell cycle analysis, proteasome inhibition, ectopic expression of non-degradable TDG, cell proliferation assays","journal":"Nucleic Acids Research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean loss-of-function and gain-of-function with defined proliferation phenotype, cell cycle fractionation, single lab with multiple orthogonal approaches","pmids":["17526518"],"is_preprint":false},{"year":2007,"finding":"TDG interacts with Rad9, Rad1, and Hus1 individually and as the 9-1-1 checkpoint complex. The Hus1-interacting domain is mapped to residues 67–110 of TDG (Val74 critical). 9-1-1 components significantly stimulate TDG glycosylase activity on U:G and T:G mispairs; TDG foci co-localize with Rad9 foci after DNA damage.","method":"Co-immunoprecipitation, GST pulldown, deletion/point mutagenesis, in vitro glycosylase stimulation assay, immunofluorescence co-localization after MNNG treatment","journal":"Nucleic Acids Research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction mapping with mutagenesis plus functional stimulation assay and cellular co-localization, single lab multiple orthogonal methods","pmids":["17855402"],"is_preprint":false},{"year":2015,"finding":"Gadd45a promotes active DNA demethylation through TDG: Gadd45a physically interacts with TDG and stimulates TDG-mediated removal of 5fC and 5caC from genomic and plasmid DNA. Knockout of both Gadd45a and Gadd45b in mouse ESCs causes hypermethylation at loci that are also TDG targets.","method":"Co-immunoprecipitation, in vitro 5fC/5caC excision assay, methylated reporter gene assay in HEK293T cells, double-KO mouse ESC methylation analysis","journal":"Nucleic Acids Research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — physical interaction plus stimulation assay plus genetic epistasis (double KO), single lab with multiple orthogonal methods","pmids":["25845601"],"is_preprint":false},{"year":2014,"finding":"TET-mediated oxidation of 5mC at a methylated Oct4 promoter reporter leads to gene reactivation in a TDG-dependent (but not MBD4-dependent) manner. NEIL1, NEIL2, and NEIL3 can partially rescue TDG loss for this gene reactivation. TDG co-immunoprecipitates with TET proteins and BER factors PARP1, XRCC1, and LIG3.","method":"In vitro methylated reporter gene reactivation assay, TDG/MBD4/NEIL KO cell lines with rescue, co-immunoprecipitation of TET-interacting factors","journal":"Nucleic Acids Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional reporter assay plus genetic KO plus Co-IP, single lab","pmids":["24948610"],"is_preprint":false},{"year":2005,"finding":"TDG interacts with the p160 coactivator SRC1 in vitro and in vivo via a novel Y-X-X-X-Y tyrosine repeat motif in TDG; site-directed mutagenesis of these tyrosines abolishes the interaction. TDG functions as a coactivator in transcriptional complexes at nuclear receptor target genes.","method":"In vitro GST pulldown, co-immunoprecipitation, site-directed mutagenesis of tyrosine motif","journal":"Nucleic Acids Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal interaction with mutagenesis defining the binding motif, single lab","pmids":["16282588"],"is_preprint":false},{"year":2014,"finding":"TDG is required for mesenchymal-to-epithelial transition (MET) during somatic cell reprogramming to iPSCs. The block in reprogramming in TDG-deficient MEFs is caused at least partly by defective activation of key miRNAs that depend on oxidative demethylation promoted by catalytically active TDG. Reintroduction of catalytically active TDG restores reprogramming.","method":"TDG-deficient MEF reprogramming assay (iPSC generation), miRNA expression analysis, rescue with catalytically active vs inactive TDG","journal":"Cell Stem Cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean KO with defined cellular phenotype plus catalytic-activity rescue experiment distinguishing enzymatic from structural function","pmids":["24529596"],"is_preprint":false},{"year":2003,"finding":"TDG can excise thymine glycol (a common oxidative DNA lesion) when it is mispaired with guanine but not when paired with adenine, demonstrating that TDG substrate recognition is mismatch-dependent and extends to oxidatively modified bases at CpG sites.","method":"In vitro glycosylase assay with oligonucleotides containing thymine glycol in different pairing contexts","journal":"Nucleic Acids Research","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with defined substrates, single lab","pmids":["12954776"],"is_preprint":false},{"year":2017,"finding":"Retinoic acid receptor (RAR) activation recruits a complex containing RAR/RXR, TDG, the acetyltransferase CBP, and TET1/2 to the Hic1 promoter, causing transient 5fC/5caC accumulation and TDG-dependent upregulation of Hic1. Conditional Tdg deletion in vivo results in Hic1 silencing and promoter hypermethylation.","method":"ChIP, gene expression analysis, conditional Tdg knockout mice, 5fC/5caC quantification at promoter","journal":"Cell Reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO with defined phenotype plus ChIP demonstrating complex recruitment, multiple orthogonal methods","pmids":["28538185"],"is_preprint":false},{"year":2014,"finding":"TDG forms a functional ternary complex with CBP (histone acetyltransferase) and activated RARα; a point mutation in TDG that does not affect overall structure or BER activity reduces ternary complex stability and deregulates RA-dependent target genes, demonstrating a structural scaffolding role of TDG independent of its catalytic activity.","method":"Co-immunoprecipitation, point mutagenesis, global transcriptome profiling, reporter assays","journal":"Genomics, Proteomics & Bioinformatics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus mutagenesis plus transcriptomics, single lab","pmids":["24394593"],"is_preprint":false},{"year":2012,"finding":"p53 directly binds two consensus response elements in the TDG promoter and transcriptionally activates TDG expression. DNA damage in p53-competent cells leads to TDG nuclear re-localization that does not occur in p53-deficient cells, indicating p53 activity influences TDG nuclear translocation.","method":"ChIP, luciferase reporter assay, isogenic cell lines with different p53 status, nuclear/cytoplasmic fractionation after DNA damage","journal":"Cell Cycle","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus reporter assay plus fractionation, single lab with multiple orthogonal methods","pmids":["23165212"],"is_preprint":false},{"year":2017,"finding":"Alpha-ketoglutarate (αKG) allosterically activates TDG by binding at Arg275, significantly increasing TDG activity on G:T mismatches and 5fC. In diabetic cardiac mesenchymal cells, reduced αKG synthesis compromises TDG and TET1 association and function; exogenous αKG restores TDG function, TET1 nuclear localization, and TET/TDG association.","method":"Molecular dynamics simulation, mutational analysis (Arg275), in vitro glycosylase activity assay, co-immunoprecipitation, TET1 localization by immunofluorescence, CRISPR/Cas9 TDG KO","journal":"Circulation Research","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — computational + mutagenesis + enzymatic activity assay + Co-IP, single lab","pmids":["29158345"],"is_preprint":false},{"year":2019,"finding":"Chromatin compaction and nucleosome positioning dramatically inhibit TDG-mediated excision of 5fC from chromatin substrates. The H2A.Z/H3.3 double-variant nucleosome and the pioneering transcription factor FOXA1 differentially regulate TDG activity on chromatin, providing direct evidence that higher-order chromatin structure regulates active DNA demethylation through TDG.","method":"In vitro glycosylase assay on chemically defined nucleosome arrays with site-specific 5fC, reconstituted chromatin compaction assays","journal":"Journal of the American Chemical Society","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted chromatin system with defined substrates and systematic variation of chromatin state, single lab with multiple conditions","pmids":["31460763"],"is_preprint":false},{"year":2017,"finding":"NEIL1 directly excises 5caC from dsDNA and both directly binds TDG and displaces it from abasic sites to process the 2'-deoxyribose as an AP lyase; NEIL1 also enhances TDG glycosylase activity specifically on 5fC and 5caC substrates but not on T:G mismatches.","method":"In vitro glycosylase assay with purified NEIL1 wild-type and catalytic mutants (P2T, E3Q), direct TDG-NEIL1 binding assay","journal":"Scientific Reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro assay with purified proteins and catalytic mutant controls, single lab","pmids":["28827588"],"is_preprint":false},{"year":2014,"finding":"TDG excises thymine mispaired with multiple exocyclic etheno-DNA adducts (εC, BεC, BεG, HεC, HεG) in vitro; TDG-knockdown human cells show higher resistance to cell death from etheno-adduct induction, lower repair of εC, and modest increase in εC-induced mutations, demonstrating TDG has repair activity toward lipid peroxidation-derived etheno adducts.","method":"In vitro DNA cleavage assay with oligonucleotides containing defined adducts, siRNA knockdown in human cells with cell viability, repair, and mutation frequency assays","journal":"Free Radical Biology & Medicine","confidence":"Medium","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic assay plus cellular KD with multiple functional readouts, single lab","pmids":["25151120"],"is_preprint":false},{"year":2014,"finding":"TDG N151A mutation inhibits 5caC excision activity while retaining activity on 5fC and T:G mismatches; N157D mutation creates a more 5caC-specific glycosylase; crystal structures show TDG recognizes G:5caC DNA from the minor groove similarly to mismatch DNA, providing structural basis for 5caC discrimination.","method":"Crystal structure determination, site-directed mutagenesis, in vitro glycosylase activity assay","journal":"Biophysics","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — crystal structure plus mutagenesis, single review/structural paper","pmids":["27493500"],"is_preprint":false},{"year":2014,"finding":"SUMO-modified TDG interacts with the SUMO-targeted ubiquitin E3 ligase RNF4; both SUMOylated and non-modified TDG fluctuate during the cell cycle. A SUMOylation-independent association between TDG and RNF4 also exists; both TDG forms are efficiently degraded in RNF4-depleted cells during S phase arrest.","method":"Co-immunoprecipitation (in vitro and in vivo), cell cycle synchronization with hydroxyurea/nocodazole, RNF4 siRNA depletion, western blot","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus cell cycle perturbation experiments, single lab","pmids":["24727457"],"is_preprint":false},{"year":2020,"finding":"TDG physically interacts with DNMT3A and promotes ubiquitination and degradation of DNMT3A. This leads to demethylation of the TIMP2 promoter and upregulation of TIMP2 expression, inhibiting colon cancer cell migration and invasion in vitro and in vivo.","method":"Co-immunoprecipitation, siRNA knockdown, ChIP, methylation-specific PCR, migration/invasion assays, xenograft mouse model","journal":"International Journal of Biological Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP demonstrating physical interaction plus functional pathway (DNMT3A ubiquitination/degradation) with in vivo validation, single lab","pmids":["35414793"],"is_preprint":false},{"year":2020,"finding":"TDG-mediated DNA demethylation is transactivated by c-Myc upon insulin treatment, leading to decreased 5caC abundance at the SREBP1 promoter and upregulation of lipogenic genes. AMPK activation by metformin increases DNMT3A activity to hypermethylate the TDG promoter, reducing TDG expression and reversing this demethylation.","method":"ChIP, bisulfite sequencing, siRNA knockdown, luciferase reporter assay, 5caC quantification by dot blot/sequencing","journal":"Molecular Therapy Oncolytics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP plus epigenomic quantification plus functional rescue, single lab","pmids":["32728616"],"is_preprint":false},{"year":2021,"finding":"TDG-mediated active demethylation (replication-independent) occurs at detectable levels in T cells (5fC/5caC accumulate in TDG-deleted T cells), but this process contributes negligibly to overall DNA demethylation during primary T cell differentiation, which occurs mainly through passive replication-dependent dilution of oxidized methylcytosines.","method":"Inducible TDG gene disruption in mice, pyridine borane sequencing (PB-seq) for 5fC/5caC at single-base resolution, analysis of differentiation markers","journal":"Genome Biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo conditional KO with single-base resolution sequencing, quantitative comparison of passive vs active pathways, well-controlled study","pmids":["34158086"],"is_preprint":false},{"year":2021,"finding":"TDG excision of 5fC and 5caC is only modestly dependent on CpG context, in contrast to its strong CpG context dependence for thymine excision. TET2 and TDG collaborative demethylation activity is only marginally reduced for CA versus CG contexts, indicating the TET-TDG pathway is not limited to CpG sites.","method":"In vitro TET2 oxidation assay and TDG excision assay with systematic CG and CH context substrates, quantitative kinetics","journal":"Journal of Molecular Biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic assay with purified proteins and systematic substrate variation, single lab","pmids":["33561435"],"is_preprint":false},{"year":2020,"finding":"TDG forms a multiprotein complex with DNMT3A at distinct allosteric sites from histone H3 tail binding; TDG plays a dominant role in modulating DNMT3A activity on nucleosome substrates, even overriding histone tail signals, and this regulation operates on DNA within single and adjacent nucleosomes.","method":"In vitro DNMT3A methylation assay on mononucleosomes and polynucleosomes, binding assays with synthetic histone tails, multi-component complex formation","journal":"Journal of Biological Chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstituted enzymatic assay with defined nucleosome substrates, single lab","pmids":["33172892"],"is_preprint":false},{"year":2025,"finding":"TDG binds tightly to R-loops in vitro and can excise 5fC and 5caC from DNA within DNA/RNA hybrid duplexes. R-loops guide the strand-specific activity of TDG at CpG sites, and 19F NMR provides mechanistic evidence for base excision on DNA/RNA hybrid substrates.","method":"In vitro R-loop binding assay, in vitro glycosylase assay on DNA/RNA hybrid substrates with defined 5fC/5caC, 19F NMR","journal":"bioRxiv (preprint)","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with structural NMR validation, preprint not yet peer-reviewed, single lab","pmids":["bio_10.1101_2025.08.05.668694"],"is_preprint":true},{"year":2025,"finding":"TDG is an RNA-binding protein that interacts with long non-coding RNAs including the paraspeckle-organizing lncRNA Neat1, and can excise oxidized 5-methylcytosine (5fC/5caC) in RNA:DNA hybrids (R-loops), suggesting TDG participates in active DNA demethylation through R-loop regulation. TDG proximity interactome also encompasses chromatin remodelers RUVBL2 and H3K4 methyltransferase complex tethering factor HCFC1.","method":"BioID2 proximity labeling proteomics in mESCs, RNA immunoprecipitation, in vitro glycosylase assay on RNA:DNA hybrid substrates","journal":"Cellular and Molecular Life Sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — proximity proteomics plus RNA-IP plus in vitro assay, single lab multiple orthogonal methods","pmids":["41291101"],"is_preprint":false},{"year":2026,"finding":"TDG occupies a majority of active promoters in pluripotent cells, co-occupying sites with the transcription factor ATF4. During retinoic acid-induced neural differentiation, TDG maintains ATF4-dependent gene expression and mTORC1 pathway activity in a manner that does not require TDG catalytic activity, linked instead to TDG-associated nucleosome positioning at promoters.","method":"Tdg KO epiblast stem-like cells, ChIP-seq for TDG and ATF4, transcriptomics, mTORC1 pathway analysis, rescue with catalytically inactive TDG","journal":"Nucleic Acids Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with catalytic mutant rescue distinguishing structural from enzymatic function, epigenomics + transcriptomics, single lab","pmids":["41773019"],"is_preprint":false}],"current_model":"TDG is a multifunctional DNA glycosylase that initiates base excision repair by excising uracil, thymine, thymine glycol, etheno-adducts, and oxidized 5-methylcytosine derivatives (5fC and 5caC) from G:X mismatches; in the TET-TDG active DNA demethylation pathway, TET dioxygenases iteratively oxidize 5mC to 5hmC, 5fC, and 5caC, TDG specifically excises 5fC and 5caC (stimulated by physical interactions with TET1, NEIL1/2, Gadd45a, and the 9-1-1 checkpoint complex), BER then restores unmodified cytosine, and this activity is regulated by allosteric activation by α-ketoglutarate (at Arg275), SUMO modification, cell-cycle-dependent proteasomal degradation during S-phase, chromatin compaction state, and transcriptional control by p53; additionally, TDG acts as a structural scaffold in transcriptional coactivator complexes (with CBP, SRC1, RAR/RXR) and can function on RNA:DNA hybrid substrates (R-loops), placing it at the intersection of DNA repair, epigenetic regulation, and transcriptional control."},"narrative":{"mechanistic_narrative":"TDG is a bifunctional DNA glycosylase that operates at the intersection of base excision repair, active DNA demethylation, and transcriptional control. Its canonical activity is mismatch-dependent excision of bases from G:X pairs, including thymine glycol [PMID:12954776] and lipid peroxidation-derived etheno adducts [PMID:25151120], with crystal structures showing it engages substrates from the DNA minor groove [PMID:27493500]. Its defining epigenetic role is as the terminal glycosylase of the TET-initiated demethylation pathway: TDG specifically excises the oxidized 5-methylcytosine derivatives 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated by TET dioxygenases, and its loss causes genome-wide accumulation of these marks at gene regulatory elements in mouse ESCs [PMID:21817016, PMID:23602152]. Biochemical reconstitution established that the TET-TDG-BER system completes demethylation of symmetrically methylated CpGs without generating double-strand breaks [PMID:26932196], and this excision is only modestly CpG-context-dependent, extending the pathway beyond CpG sites [PMID:33561435]. TDG glycosylase function is potentiated by physical and functional partnerships with TET1 [PMID:26932196], NEIL1/NEIL2 (which displace TDG from abasic sites and process them as AP lyases to accelerate turnover) [PMID:26751644, PMID:28827588], Gadd45a [PMID:25845601], and the 9-1-1 checkpoint complex (Rad9-Rad1-Hus1) [PMID:17855402], and is allosterically activated by alpha-ketoglutarate binding at Arg275 [PMID:29158345]. TDG abundance and access are tightly regulated: it is degraded by the ubiquitin-proteasome system at S-phase onset inverse to UNG2 [PMID:17526518] via the SUMO-targeted ligase RNF4 [PMID:24727457], its transcription is activated by p53 [PMID:23165212], and its activity on chromatin is inhibited by nucleosome compaction and modulated by histone variants and the pioneer factor FOXA1 [PMID:31460763]. Beyond catalysis, TDG functions as a structural scaffold in transcriptional coactivator complexes, binding SRC1 through a tyrosine-repeat motif [PMID:16282588] and forming a ternary complex with CBP and activated RARalpha that is required for retinoic acid target gene regulation independent of its catalytic activity [PMID:28538185, PMID:24394593]; it also restrains DNA methylation by binding DNMT3A and modulating its nucleosomal activity [PMID:35414793, PMID:33172892]. TDG is essential for embryonic development and epigenomic stability [PMID:21278727] and is required for catalysis-dependent reprogramming and lineage transitions [PMID:24529596], while its catalysis-independent occupancy of active promoters sustains ATF4-dependent gene expression during differentiation [PMID:41773019]. Emerging evidence places TDG on RNA:DNA hybrid (R-loop) substrates and identifies it as an RNA-binding protein associating with lncRNAs such as Neat1 [PMID:bio_10.1101_2025.08.05.668694, PMID:41291101].","teleology":[{"year":2003,"claim":"Established that TDG substrate recognition is governed by mispairing rather than the lesion alone, extending its repair scope to oxidatively modified bases.","evidence":"In vitro glycosylase assays with thymine glycol in defined pairing contexts","pmids":["12954776"],"confidence":"Medium","gaps":["Did not address cellular relevance of thymine glycol repair","No structural basis for mismatch discrimination at this point"]},{"year":2004,"claim":"Defined how SUMO modification controls TDG, linking non-covalent SUMO binding to covalent conjugation and subnuclear targeting.","evidence":"Yeast two-hybrid, pulldown, Co-IP, and co-localization with mutagenesis in transfected cells","pmids":["15569683"],"confidence":"High","gaps":["Functional consequence of PML body localization for repair unresolved","Did not connect SUMOylation to turnover during demethylation"]},{"year":2007,"claim":"Revealed cell-cycle control of TDG, showing it is eliminated during S/G2 inverse to UNG2, functionally partitioning the two enzymes, and that failure to degrade it impairs proliferation.","evidence":"Cell cycle flow cytometry, proteasome inhibition, non-degradable TDG expression, proliferation assays","pmids":["17526518"],"confidence":"High","gaps":["Did not identify the responsible E3 ligase","Mechanistic reason S-phase TDG impairs progression unclear"]},{"year":2007,"claim":"Connected TDG to the DNA damage checkpoint by mapping its interaction with the 9-1-1 complex and showing 9-1-1 stimulates its glycosylase activity.","evidence":"Co-IP, GST pulldown, domain mapping (residues 67-110, Val74), in vitro stimulation assay, damage-induced co-localization","pmids":["17855402"],"confidence":"High","gaps":["In vivo significance of 9-1-1 stimulation not tested genetically","Whether stimulation applies to 5fC/5caC substrates not addressed"]},{"year":2011,"claim":"Identified TDG as the glycosylase that removes 5caC, placing it as the terminal excision step of TET-driven active demethylation.","evidence":"In vitro glycosylase assay with defined substrates plus TDG depletion in mouse ESCs with LC-MS quantification of 5caC","pmids":["21817016"],"confidence":"High","gaps":["Genome-wide distribution of 5caC accumulation not yet mapped","Coupling to downstream BER steps not reconstituted"]},{"year":2011,"claim":"Demonstrated TDG is essential for development and epigenomic stability, establishing physiological importance beyond a repair enzyme.","evidence":"Tdg null mouse lethality, ChIP for histone marks and CpG methylation at developmental promoters","pmids":["21278727"],"confidence":"High","gaps":["Did not separate catalytic from scaffolding contributions to the phenotype","Specific developmental lethal mechanism not pinpointed"]},{"year":2013,"claim":"Mapped TDG-dependent demethylation genome-wide, showing 5fC and 5caC accumulate at regulatory elements when TDG is lost.","evidence":"Modification-specific genome-wide profiling in WT and Tdg-deficient ESCs","pmids":["23602152"],"confidence":"High","gaps":["Functional consequence at individual regulatory elements not dissected","Turnover kinetics in vivo not measured"]},{"year":2014,"claim":"Distinguished TDG's catalytic from structural roles by showing reprogramming requires its enzymatic activity while RAR/CBP target gene control depends on scaffolding.","evidence":"TDG-deficient MEF reprogramming with catalytic-vs-inactive rescue; ternary complex Co-IP with point mutant and transcriptomics; methylated reporter reactivation with NEIL rescue","pmids":["24529596","24394593","24948610"],"confidence":"Medium","gaps":["Molecular basis of scaffolding-dependent transcription incompletely defined","How miRNA loci are selected for TDG-dependent activation unknown"]},{"year":2015,"claim":"Identified Gadd45a as a stimulatory partner coupling TDG to active demethylation, supported by genetic epistasis at shared loci.","evidence":"Co-IP, in vitro 5fC/5caC excision stimulation, reporter assay, Gadd45a/b double-KO ESC methylation analysis","pmids":["25845601"],"confidence":"High","gaps":["Mechanism by which Gadd45a accelerates excision not defined","Overlap with NEIL stimulation pathways unresolved"]},{"year":2016,"claim":"Reconstituted productive, break-free demethylation of symmetric CpGs and showed NEIL glycosylases accelerate TDG turnover by displacing it from abasic sites.","evidence":"Purified TET1-TDG reconstitution and demethylation assay; in vitro glycosylase/AP lyase assays with Xenopus loss-of-function validation","pmids":["26932196","26751644"],"confidence":"High","gaps":["Stoichiometry and ordering of full multienzyme handoff in cells not established","Regulation of NEIL recruitment in vivo unknown"]},{"year":2017,"claim":"Showed how TDG activity is tuned by metabolism and chromatin: alpha-ketoglutarate allosterically activates it at Arg275, and nucleosome compaction inhibits its action on chromatin.","evidence":"Molecular dynamics, Arg275 mutagenesis, glycosylase assays, Co-IP in diabetic cells (alphaKG); defined nucleosome arrays with site-specific 5fC and FOXA1/histone variants (chromatin)","pmids":["29158345","31460763"],"confidence":"High","gaps":["In vivo contribution of alphaKG regulation to demethylation untested genome-wide","How FOXA1 mechanistically licenses TDG access unresolved"]},{"year":2017,"claim":"Refined the demethylation handoff by showing NEIL1 itself excises 5caC and substrate-specifically enhances TDG on 5fC/5caC but not T:G, and connected TDG to coactivator complex recruitment in vivo.","evidence":"In vitro assays with NEIL1 catalytic mutants and binding assays; ChIP and conditional Tdg KO at the Hic1 promoter with RAR/RXR/CBP/TET recruitment","pmids":["28827588","28538185"],"confidence":"High","gaps":["Generality of the RAR-recruited demethylation complex across target genes not established","Substrate-specificity basis of NEIL1 stimulation structurally undefined"]},{"year":2020,"claim":"Uncovered TDG as a regulator of DNA methyltransferase activity, binding DNMT3A to promote its degradation and dominantly modulate its nucleosomal activity, with disease relevance in cancer.","evidence":"Co-IP, ubiquitination and degradation assays, MSP, migration/invasion and xenografts (TIMP2); in vitro DNMT3A nucleosome methylation with allosteric binding mapping","pmids":["35414793","33172892"],"confidence":"Medium","gaps":["Whether DNMT3A regulation is catalysis-dependent unclear","Physiological scope beyond cancer models untested"]},{"year":2021,"claim":"Quantified the in vivo contribution of TDG-dependent active demethylation, showing it operates but is minor relative to passive dilution during T cell differentiation, and that 5fC/5caC excision is largely CpG-context-independent.","evidence":"Inducible Tdg disruption with single-base PB-seq in mice; in vitro TET2/TDG kinetics across CG and CH contexts","pmids":["34158086","33561435"],"confidence":"High","gaps":["Cell types where active demethylation dominates not delineated","Determinants directing TDG to specific loci in vivo unknown"]},{"year":2025,"claim":"Extended TDG's substrate range to RNA:DNA hybrids and identified it as an RNA-binding protein, implicating R-loops and lncRNAs in directing strand-specific demethylation.","evidence":"In vitro R-loop binding and glycosylase assays with 19F NMR (preprint); BioID2 proximity proteomics, RNA-IP, and hybrid-substrate assays in mESCs","pmids":["bio_10.1101_2025.08.05.668694","41291101"],"confidence":"Medium","gaps":["In vivo role of R-loop-directed TDG activity not established","Functional consequence of Neat1 and RUVBL2/HCFC1 associations undefined"]},{"year":2026,"claim":"Defined a catalysis-independent genomic function, showing TDG occupies active promoters with ATF4 and sustains ATF4-dependent expression and mTORC1 signaling via nucleosome positioning during differentiation.","evidence":"Tdg KO epiblast-like cells, TDG and ATF4 ChIP-seq, transcriptomics, mTORC1 analysis, catalytically inactive rescue","pmids":["41773019"],"confidence":"Medium","gaps":["Mechanism by which TDG positions nucleosomes at promoters unknown","Relationship between scaffolding and demethylation roles at the same loci unresolved"]},{"year":null,"claim":"How TDG is targeted to specific genomic loci, and how its catalytic, scaffolding, and RNA-associated functions are coordinated and partitioned across cell states, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model of locus selection for active demethylation versus structural occupancy","Interplay of SUMO/RNF4 turnover, p53 transcription, and chromatin state in directing function not integrated","In vivo significance of R-loop and RNA-binding activities undetermined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140097","term_label":"catalytic activity, acting on DNA","supporting_discovery_ids":[0,2,3,12,19,20,25,27]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,12,19]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[20,27]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[27,28]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[10,13,14]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[10,14,29]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2,15]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[2,17]}],"pathway":[{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[7,12,19]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,1,2,3,17]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[10,13,14,29]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,11,13,29]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[6,21]}],"complexes":["9-1-1 checkpoint complex (Rad9-Rad1-Hus1)","RAR/RXR-CBP-TDG coactivator complex"],"partners":["TET1","NEIL1","NEIL2","GADD45A","HUS1","CBP","SRC1","DNMT3A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13569","full_name":"G/T mismatch-specific thymine DNA glycosylase","aliases":["Thymine-DNA glycosylase","hTDG"],"length_aa":410,"mass_kda":46.1,"function":"DNA glycosylase that plays a key role in active DNA demethylation: specifically recognizes and binds 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in the context of CpG sites and mediates their excision through base-excision repair (BER) to install an unmethylated cytosine. Cannot remove 5-hydroxymethylcytosine (5hmC). According to an alternative model, involved in DNA demethylation by mediating DNA glycolase activity toward 5-hydroxymethyluracil (5hmU) produced by deamination of 5hmC. Also involved in DNA repair by acting as a thymine-DNA glycosylase that mediates correction of G/T mispairs to G/C pairs: in the DNA of higher eukaryotes, hydrolytic deamination of 5-methylcytosine to thymine leads to the formation of G/T mismatches. Its role in the repair of canonical base damage is however minor compared to its role in DNA demethylation. It is capable of hydrolyzing the carbon-nitrogen bond between the sugar-phosphate backbone of the DNA and a mispaired thymine. In addition to the G/T, it can remove thymine also from C/T and T/T mispairs in the order G/T >> C/T > T/T. It has no detectable activity on apyrimidinic sites and does not catalyze the removal of thymine from A/T pairs or from single-stranded DNA. 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Toxicology & pharmacology : CBP","url":"https://pubmed.ncbi.nlm.nih.gov/37004899","citation_count":1,"is_preprint":false},{"pmid":"41773019","id":"PMC_41773019","title":"TDG orchestrates ATF4-dependent gene transcription during retinoic acid-induced cell fate acquisition.","date":"2026","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/41773019","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.06.687040","title":"Integrating Longitudinal Metabolite Profiles Improves Trait Prediction in Pigs in a Trait- and Timepoint-Dependent Manner","date":"2025-11-08","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.06.687040","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.08.05.668694","title":"Thymine DNA Glycosylase Binds to R-Loops and Excises 5-Formyl and 5-Carboxyl Cytosine from DNA/RNA Hybrids","date":"2025-08-05","source":"bioRxiv","url":"https://doi.org/10.1101/2025.08.05.668694","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.06.18.660344","title":"Epigenetic genes are differentially methylated in the blood of persons with mild cognitive impairment and Alzheimer’s disease","date":"2025-06-24","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.18.660344","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.12.17.628571","title":"Mbd4 and MutSα protect cells from spontaneous deamination of 5-methylcytosine","date":"2024-12-18","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.17.628571","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":31610,"output_tokens":7476,"usd":0.103485,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16692,"output_tokens":5628,"usd":0.11208,"stage2_stop_reason":"end_turn"},"total_usd":0.215565,"stage1_batch_id":"msgbatch_017qqVz33Ga63SCDwzb35XPb","stage2_batch_id":"msgbatch_01Scr2Ei9x4HJMiRGxwrmvLK","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2011,\n      \"finding\": \"TDG specifically recognizes and excises 5-carboxylcytosine (5caC) from DNA, a product of iterative TET dioxygenase-mediated oxidation of 5-methylcytosine (5mC). Depletion of TDG in mouse embryonic stem cells leads to accumulation of 5caC to detectable levels, establishing TDG as the glycosylase that removes 5caC in the TET-initiated active DNA demethylation pathway.\",\n      \"method\": \"In vitro glycosylase assay, cell-based depletion (siRNA/genetic) with LC-MS quantification of 5caC\",\n      \"journal\": \"Science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro biochemical assay with defined substrates plus genetic depletion in ESCs, replicated across multiple labs subsequently\",\n      \"pmids\": [\"21817016\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"TDG is essential for embryonic development (Tdg null mice die in utero). TDG maintains epigenetic stability by contributing to active and bivalent chromatin at developmental gene promoters, counteracting aberrant de novo methylation and facilitating proper assembly of chromatin-modifying complexes via base excision repair.\",\n      \"method\": \"Mouse genetic knockout, ChIP for histone modifications and CpG methylation at gene promoters in MEFs and ESCs\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout lethal phenotype combined with multiple orthogonal epigenomic assays (ChIP, methylation analysis)\",\n      \"pmids\": [\"21278727\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"TDG excises 5-formylcytosine (5fC) and 5caC genome-wide, particularly at gene regulatory elements (proximal and distal). Genome-wide maps in Tdg-deficient mouse ESCs reveal marked accumulation of 5fC and 5caC at these sites, demonstrating that TET/TDG-dependent active demethylation occurs extensively at regulatory regions.\",\n      \"method\": \"Genome-wide modification-specific antibody profiling (ChIP-seq/immunoprecipitation) in wild-type and Tdg-deficient ESCs\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide approach with genetic controls (Tdg KO), multiple modification marks, replicated findings\",\n      \"pmids\": [\"23602152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"TET1 and TDG physically interact to form a functional complex; biochemical reconstitution demonstrates that the TET-TDG-BER system can achieve productive DNA demethylation of symmetrically methylated CpGs in a sequential manner, avoiding DNA double-strand break formation.\",\n      \"method\": \"Co-immunoprecipitation, biochemical reconstitution with purified TET1 and TDG, in vitro demethylation assay\",\n      \"journal\": \"Nature Communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — biochemical reconstitution with purified components and physical interaction validated, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"26932196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NEIL1 and NEIL2 DNA glycosylases stimulate TDG substrate turnover during active demethylation: TDG occupies the abasic site after base excision and is displaced by NEIL proteins, which process the baseless sugar as AP lyases, thereby accelerating TDG cycling on 5fC/5caC substrates.\",\n      \"method\": \"In vitro glycosylase/AP lyase assay with purified proteins, Xenopus embryo loss-of-function experiments\",\n      \"journal\": \"Nature Structural & Molecular Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with purified components plus in vivo genetic validation in Xenopus, two orthogonal model systems\",\n      \"pmids\": [\"26751644\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TDG (TDGb isoform) binds SUMO-1 non-covalently via a specific region (residues within the N-terminus), and this non-covalent SUMO-1 binding is required for covalent SUMO-1 conjugation at an adjacent lysine residue. SUMO-1 modification of TDG promotes its interaction with PML and co-localization to PML nuclear bodies.\",\n      \"method\": \"Yeast two-hybrid screen, in vitro pulldown, co-immunoprecipitation, fluorescence co-localization in transfected cells, deletion and point mutagenesis\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and localization with systematic mutagenesis dissecting binding domain, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15569683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TDG is degraded by the ubiquitin-proteasome system at the onset of S-phase and is absent throughout S and G2 phases; this cell cycle regulation is strictly inverse to UNG2, functionally separating the two repair enzymes. Incomplete TDG degradation impedes S-phase progression and cell proliferation.\",\n      \"method\": \"Flow cytometry cell cycle analysis, proteasome inhibition, ectopic expression of non-degradable TDG, cell proliferation assays\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean loss-of-function and gain-of-function with defined proliferation phenotype, cell cycle fractionation, single lab with multiple orthogonal approaches\",\n      \"pmids\": [\"17526518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"TDG interacts with Rad9, Rad1, and Hus1 individually and as the 9-1-1 checkpoint complex. The Hus1-interacting domain is mapped to residues 67–110 of TDG (Val74 critical). 9-1-1 components significantly stimulate TDG glycosylase activity on U:G and T:G mispairs; TDG foci co-localize with Rad9 foci after DNA damage.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, deletion/point mutagenesis, in vitro glycosylase stimulation assay, immunofluorescence co-localization after MNNG treatment\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction mapping with mutagenesis plus functional stimulation assay and cellular co-localization, single lab multiple orthogonal methods\",\n      \"pmids\": [\"17855402\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Gadd45a promotes active DNA demethylation through TDG: Gadd45a physically interacts with TDG and stimulates TDG-mediated removal of 5fC and 5caC from genomic and plasmid DNA. Knockout of both Gadd45a and Gadd45b in mouse ESCs causes hypermethylation at loci that are also TDG targets.\",\n      \"method\": \"Co-immunoprecipitation, in vitro 5fC/5caC excision assay, methylated reporter gene assay in HEK293T cells, double-KO mouse ESC methylation analysis\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — physical interaction plus stimulation assay plus genetic epistasis (double KO), single lab with multiple orthogonal methods\",\n      \"pmids\": [\"25845601\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TET-mediated oxidation of 5mC at a methylated Oct4 promoter reporter leads to gene reactivation in a TDG-dependent (but not MBD4-dependent) manner. NEIL1, NEIL2, and NEIL3 can partially rescue TDG loss for this gene reactivation. TDG co-immunoprecipitates with TET proteins and BER factors PARP1, XRCC1, and LIG3.\",\n      \"method\": \"In vitro methylated reporter gene reactivation assay, TDG/MBD4/NEIL KO cell lines with rescue, co-immunoprecipitation of TET-interacting factors\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional reporter assay plus genetic KO plus Co-IP, single lab\",\n      \"pmids\": [\"24948610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TDG interacts with the p160 coactivator SRC1 in vitro and in vivo via a novel Y-X-X-X-Y tyrosine repeat motif in TDG; site-directed mutagenesis of these tyrosines abolishes the interaction. TDG functions as a coactivator in transcriptional complexes at nuclear receptor target genes.\",\n      \"method\": \"In vitro GST pulldown, co-immunoprecipitation, site-directed mutagenesis of tyrosine motif\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal interaction with mutagenesis defining the binding motif, single lab\",\n      \"pmids\": [\"16282588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TDG is required for mesenchymal-to-epithelial transition (MET) during somatic cell reprogramming to iPSCs. The block in reprogramming in TDG-deficient MEFs is caused at least partly by defective activation of key miRNAs that depend on oxidative demethylation promoted by catalytically active TDG. Reintroduction of catalytically active TDG restores reprogramming.\",\n      \"method\": \"TDG-deficient MEF reprogramming assay (iPSC generation), miRNA expression analysis, rescue with catalytically active vs inactive TDG\",\n      \"journal\": \"Cell Stem Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO with defined cellular phenotype plus catalytic-activity rescue experiment distinguishing enzymatic from structural function\",\n      \"pmids\": [\"24529596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"TDG can excise thymine glycol (a common oxidative DNA lesion) when it is mispaired with guanine but not when paired with adenine, demonstrating that TDG substrate recognition is mismatch-dependent and extends to oxidatively modified bases at CpG sites.\",\n      \"method\": \"In vitro glycosylase assay with oligonucleotides containing thymine glycol in different pairing contexts\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with defined substrates, single lab\",\n      \"pmids\": [\"12954776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Retinoic acid receptor (RAR) activation recruits a complex containing RAR/RXR, TDG, the acetyltransferase CBP, and TET1/2 to the Hic1 promoter, causing transient 5fC/5caC accumulation and TDG-dependent upregulation of Hic1. Conditional Tdg deletion in vivo results in Hic1 silencing and promoter hypermethylation.\",\n      \"method\": \"ChIP, gene expression analysis, conditional Tdg knockout mice, 5fC/5caC quantification at promoter\",\n      \"journal\": \"Cell Reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO with defined phenotype plus ChIP demonstrating complex recruitment, multiple orthogonal methods\",\n      \"pmids\": [\"28538185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TDG forms a functional ternary complex with CBP (histone acetyltransferase) and activated RARα; a point mutation in TDG that does not affect overall structure or BER activity reduces ternary complex stability and deregulates RA-dependent target genes, demonstrating a structural scaffolding role of TDG independent of its catalytic activity.\",\n      \"method\": \"Co-immunoprecipitation, point mutagenesis, global transcriptome profiling, reporter assays\",\n      \"journal\": \"Genomics, Proteomics & Bioinformatics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus mutagenesis plus transcriptomics, single lab\",\n      \"pmids\": [\"24394593\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"p53 directly binds two consensus response elements in the TDG promoter and transcriptionally activates TDG expression. DNA damage in p53-competent cells leads to TDG nuclear re-localization that does not occur in p53-deficient cells, indicating p53 activity influences TDG nuclear translocation.\",\n      \"method\": \"ChIP, luciferase reporter assay, isogenic cell lines with different p53 status, nuclear/cytoplasmic fractionation after DNA damage\",\n      \"journal\": \"Cell Cycle\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus reporter assay plus fractionation, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"23165212\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Alpha-ketoglutarate (αKG) allosterically activates TDG by binding at Arg275, significantly increasing TDG activity on G:T mismatches and 5fC. In diabetic cardiac mesenchymal cells, reduced αKG synthesis compromises TDG and TET1 association and function; exogenous αKG restores TDG function, TET1 nuclear localization, and TET/TDG association.\",\n      \"method\": \"Molecular dynamics simulation, mutational analysis (Arg275), in vitro glycosylase activity assay, co-immunoprecipitation, TET1 localization by immunofluorescence, CRISPR/Cas9 TDG KO\",\n      \"journal\": \"Circulation Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — computational + mutagenesis + enzymatic activity assay + Co-IP, single lab\",\n      \"pmids\": [\"29158345\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Chromatin compaction and nucleosome positioning dramatically inhibit TDG-mediated excision of 5fC from chromatin substrates. The H2A.Z/H3.3 double-variant nucleosome and the pioneering transcription factor FOXA1 differentially regulate TDG activity on chromatin, providing direct evidence that higher-order chromatin structure regulates active DNA demethylation through TDG.\",\n      \"method\": \"In vitro glycosylase assay on chemically defined nucleosome arrays with site-specific 5fC, reconstituted chromatin compaction assays\",\n      \"journal\": \"Journal of the American Chemical Society\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted chromatin system with defined substrates and systematic variation of chromatin state, single lab with multiple conditions\",\n      \"pmids\": [\"31460763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEIL1 directly excises 5caC from dsDNA and both directly binds TDG and displaces it from abasic sites to process the 2'-deoxyribose as an AP lyase; NEIL1 also enhances TDG glycosylase activity specifically on 5fC and 5caC substrates but not on T:G mismatches.\",\n      \"method\": \"In vitro glycosylase assay with purified NEIL1 wild-type and catalytic mutants (P2T, E3Q), direct TDG-NEIL1 binding assay\",\n      \"journal\": \"Scientific Reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro assay with purified proteins and catalytic mutant controls, single lab\",\n      \"pmids\": [\"28827588\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TDG excises thymine mispaired with multiple exocyclic etheno-DNA adducts (εC, BεC, BεG, HεC, HεG) in vitro; TDG-knockdown human cells show higher resistance to cell death from etheno-adduct induction, lower repair of εC, and modest increase in εC-induced mutations, demonstrating TDG has repair activity toward lipid peroxidation-derived etheno adducts.\",\n      \"method\": \"In vitro DNA cleavage assay with oligonucleotides containing defined adducts, siRNA knockdown in human cells with cell viability, repair, and mutation frequency assays\",\n      \"journal\": \"Free Radical Biology & Medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic assay plus cellular KD with multiple functional readouts, single lab\",\n      \"pmids\": [\"25151120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TDG N151A mutation inhibits 5caC excision activity while retaining activity on 5fC and T:G mismatches; N157D mutation creates a more 5caC-specific glycosylase; crystal structures show TDG recognizes G:5caC DNA from the minor groove similarly to mismatch DNA, providing structural basis for 5caC discrimination.\",\n      \"method\": \"Crystal structure determination, site-directed mutagenesis, in vitro glycosylase activity assay\",\n      \"journal\": \"Biophysics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure plus mutagenesis, single review/structural paper\",\n      \"pmids\": [\"27493500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"SUMO-modified TDG interacts with the SUMO-targeted ubiquitin E3 ligase RNF4; both SUMOylated and non-modified TDG fluctuate during the cell cycle. A SUMOylation-independent association between TDG and RNF4 also exists; both TDG forms are efficiently degraded in RNF4-depleted cells during S phase arrest.\",\n      \"method\": \"Co-immunoprecipitation (in vitro and in vivo), cell cycle synchronization with hydroxyurea/nocodazole, RNF4 siRNA depletion, western blot\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus cell cycle perturbation experiments, single lab\",\n      \"pmids\": [\"24727457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TDG physically interacts with DNMT3A and promotes ubiquitination and degradation of DNMT3A. This leads to demethylation of the TIMP2 promoter and upregulation of TIMP2 expression, inhibiting colon cancer cell migration and invasion in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, ChIP, methylation-specific PCR, migration/invasion assays, xenograft mouse model\",\n      \"journal\": \"International Journal of Biological Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP demonstrating physical interaction plus functional pathway (DNMT3A ubiquitination/degradation) with in vivo validation, single lab\",\n      \"pmids\": [\"35414793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TDG-mediated DNA demethylation is transactivated by c-Myc upon insulin treatment, leading to decreased 5caC abundance at the SREBP1 promoter and upregulation of lipogenic genes. AMPK activation by metformin increases DNMT3A activity to hypermethylate the TDG promoter, reducing TDG expression and reversing this demethylation.\",\n      \"method\": \"ChIP, bisulfite sequencing, siRNA knockdown, luciferase reporter assay, 5caC quantification by dot blot/sequencing\",\n      \"journal\": \"Molecular Therapy Oncolytics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus epigenomic quantification plus functional rescue, single lab\",\n      \"pmids\": [\"32728616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TDG-mediated active demethylation (replication-independent) occurs at detectable levels in T cells (5fC/5caC accumulate in TDG-deleted T cells), but this process contributes negligibly to overall DNA demethylation during primary T cell differentiation, which occurs mainly through passive replication-dependent dilution of oxidized methylcytosines.\",\n      \"method\": \"Inducible TDG gene disruption in mice, pyridine borane sequencing (PB-seq) for 5fC/5caC at single-base resolution, analysis of differentiation markers\",\n      \"journal\": \"Genome Biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo conditional KO with single-base resolution sequencing, quantitative comparison of passive vs active pathways, well-controlled study\",\n      \"pmids\": [\"34158086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TDG excision of 5fC and 5caC is only modestly dependent on CpG context, in contrast to its strong CpG context dependence for thymine excision. TET2 and TDG collaborative demethylation activity is only marginally reduced for CA versus CG contexts, indicating the TET-TDG pathway is not limited to CpG sites.\",\n      \"method\": \"In vitro TET2 oxidation assay and TDG excision assay with systematic CG and CH context substrates, quantitative kinetics\",\n      \"journal\": \"Journal of Molecular Biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic assay with purified proteins and systematic substrate variation, single lab\",\n      \"pmids\": [\"33561435\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"TDG forms a multiprotein complex with DNMT3A at distinct allosteric sites from histone H3 tail binding; TDG plays a dominant role in modulating DNMT3A activity on nucleosome substrates, even overriding histone tail signals, and this regulation operates on DNA within single and adjacent nucleosomes.\",\n      \"method\": \"In vitro DNMT3A methylation assay on mononucleosomes and polynucleosomes, binding assays with synthetic histone tails, multi-component complex formation\",\n      \"journal\": \"Journal of Biological Chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstituted enzymatic assay with defined nucleosome substrates, single lab\",\n      \"pmids\": [\"33172892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TDG binds tightly to R-loops in vitro and can excise 5fC and 5caC from DNA within DNA/RNA hybrid duplexes. R-loops guide the strand-specific activity of TDG at CpG sites, and 19F NMR provides mechanistic evidence for base excision on DNA/RNA hybrid substrates.\",\n      \"method\": \"In vitro R-loop binding assay, in vitro glycosylase assay on DNA/RNA hybrid substrates with defined 5fC/5caC, 19F NMR\",\n      \"journal\": \"bioRxiv (preprint)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with structural NMR validation, preprint not yet peer-reviewed, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.08.05.668694\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"TDG is an RNA-binding protein that interacts with long non-coding RNAs including the paraspeckle-organizing lncRNA Neat1, and can excise oxidized 5-methylcytosine (5fC/5caC) in RNA:DNA hybrids (R-loops), suggesting TDG participates in active DNA demethylation through R-loop regulation. TDG proximity interactome also encompasses chromatin remodelers RUVBL2 and H3K4 methyltransferase complex tethering factor HCFC1.\",\n      \"method\": \"BioID2 proximity labeling proteomics in mESCs, RNA immunoprecipitation, in vitro glycosylase assay on RNA:DNA hybrid substrates\",\n      \"journal\": \"Cellular and Molecular Life Sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — proximity proteomics plus RNA-IP plus in vitro assay, single lab multiple orthogonal methods\",\n      \"pmids\": [\"41291101\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"TDG occupies a majority of active promoters in pluripotent cells, co-occupying sites with the transcription factor ATF4. During retinoic acid-induced neural differentiation, TDG maintains ATF4-dependent gene expression and mTORC1 pathway activity in a manner that does not require TDG catalytic activity, linked instead to TDG-associated nucleosome positioning at promoters.\",\n      \"method\": \"Tdg KO epiblast stem-like cells, ChIP-seq for TDG and ATF4, transcriptomics, mTORC1 pathway analysis, rescue with catalytically inactive TDG\",\n      \"journal\": \"Nucleic Acids Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with catalytic mutant rescue distinguishing structural from enzymatic function, epigenomics + transcriptomics, single lab\",\n      \"pmids\": [\"41773019\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TDG is a multifunctional DNA glycosylase that initiates base excision repair by excising uracil, thymine, thymine glycol, etheno-adducts, and oxidized 5-methylcytosine derivatives (5fC and 5caC) from G:X mismatches; in the TET-TDG active DNA demethylation pathway, TET dioxygenases iteratively oxidize 5mC to 5hmC, 5fC, and 5caC, TDG specifically excises 5fC and 5caC (stimulated by physical interactions with TET1, NEIL1/2, Gadd45a, and the 9-1-1 checkpoint complex), BER then restores unmodified cytosine, and this activity is regulated by allosteric activation by α-ketoglutarate (at Arg275), SUMO modification, cell-cycle-dependent proteasomal degradation during S-phase, chromatin compaction state, and transcriptional control by p53; additionally, TDG acts as a structural scaffold in transcriptional coactivator complexes (with CBP, SRC1, RAR/RXR) and can function on RNA:DNA hybrid substrates (R-loops), placing it at the intersection of DNA repair, epigenetic regulation, and transcriptional control.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TDG is a bifunctional DNA glycosylase that operates at the intersection of base excision repair, active DNA demethylation, and transcriptional control. Its canonical activity is mismatch-dependent excision of bases from G:X pairs, including thymine glycol [#12] and lipid peroxidation-derived etheno adducts [#19], with crystal structures showing it engages substrates from the DNA minor groove [#20]. Its defining epigenetic role is as the terminal glycosylase of the TET-initiated demethylation pathway: TDG specifically excises the oxidized 5-methylcytosine derivatives 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated by TET dioxygenases, and its loss causes genome-wide accumulation of these marks at gene regulatory elements in mouse ESCs [#0, #2]. Biochemical reconstitution established that the TET-TDG-BER system completes demethylation of symmetrically methylated CpGs without generating double-strand breaks [#3], and this excision is only modestly CpG-context-dependent, extending the pathway beyond CpG sites [#25]. TDG glycosylase function is potentiated by physical and functional partnerships with TET1 [#3], NEIL1/NEIL2 (which displace TDG from abasic sites and process them as AP lyases to accelerate turnover) [#4, #18], Gadd45a [#8], and the 9-1-1 checkpoint complex (Rad9-Rad1-Hus1) [#7], and is allosterically activated by alpha-ketoglutarate binding at Arg275 [#16]. TDG abundance and access are tightly regulated: it is degraded by the ubiquitin-proteasome system at S-phase onset inverse to UNG2 [#6] via the SUMO-targeted ligase RNF4 [#21], its transcription is activated by p53 [#15], and its activity on chromatin is inhibited by nucleosome compaction and modulated by histone variants and the pioneer factor FOXA1 [#17]. Beyond catalysis, TDG functions as a structural scaffold in transcriptional coactivator complexes, binding SRC1 through a tyrosine-repeat motif [#10] and forming a ternary complex with CBP and activated RARalpha that is required for retinoic acid target gene regulation independent of its catalytic activity [#13, #14]; it also restrains DNA methylation by binding DNMT3A and modulating its nucleosomal activity [#22, #26]. TDG is essential for embryonic development and epigenomic stability [#1] and is required for catalysis-dependent reprogramming and lineage transitions [#11], while its catalysis-independent occupancy of active promoters sustains ATF4-dependent gene expression during differentiation [#29]. Emerging evidence places TDG on RNA:DNA hybrid (R-loop) substrates and identifies it as an RNA-binding protein associating with lncRNAs such as Neat1 [#27, #28].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Established that TDG substrate recognition is governed by mispairing rather than the lesion alone, extending its repair scope to oxidatively modified bases.\",\n      \"evidence\": \"In vitro glycosylase assays with thymine glycol in defined pairing contexts\",\n      \"pmids\": [\"12954776\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not address cellular relevance of thymine glycol repair\", \"No structural basis for mismatch discrimination at this point\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined how SUMO modification controls TDG, linking non-covalent SUMO binding to covalent conjugation and subnuclear targeting.\",\n      \"evidence\": \"Yeast two-hybrid, pulldown, Co-IP, and co-localization with mutagenesis in transfected cells\",\n      \"pmids\": [\"15569683\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of PML body localization for repair unresolved\", \"Did not connect SUMOylation to turnover during demethylation\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Revealed cell-cycle control of TDG, showing it is eliminated during S/G2 inverse to UNG2, functionally partitioning the two enzymes, and that failure to degrade it impairs proliferation.\",\n      \"evidence\": \"Cell cycle flow cytometry, proteasome inhibition, non-degradable TDG expression, proliferation assays\",\n      \"pmids\": [\"17526518\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the responsible E3 ligase\", \"Mechanistic reason S-phase TDG impairs progression unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected TDG to the DNA damage checkpoint by mapping its interaction with the 9-1-1 complex and showing 9-1-1 stimulates its glycosylase activity.\",\n      \"evidence\": \"Co-IP, GST pulldown, domain mapping (residues 67-110, Val74), in vitro stimulation assay, damage-induced co-localization\",\n      \"pmids\": [\"17855402\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo significance of 9-1-1 stimulation not tested genetically\", \"Whether stimulation applies to 5fC/5caC substrates not addressed\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified TDG as the glycosylase that removes 5caC, placing it as the terminal excision step of TET-driven active demethylation.\",\n      \"evidence\": \"In vitro glycosylase assay with defined substrates plus TDG depletion in mouse ESCs with LC-MS quantification of 5caC\",\n      \"pmids\": [\"21817016\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide distribution of 5caC accumulation not yet mapped\", \"Coupling to downstream BER steps not reconstituted\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrated TDG is essential for development and epigenomic stability, establishing physiological importance beyond a repair enzyme.\",\n      \"evidence\": \"Tdg null mouse lethality, ChIP for histone marks and CpG methylation at developmental promoters\",\n      \"pmids\": [\"21278727\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate catalytic from scaffolding contributions to the phenotype\", \"Specific developmental lethal mechanism not pinpointed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Mapped TDG-dependent demethylation genome-wide, showing 5fC and 5caC accumulate at regulatory elements when TDG is lost.\",\n      \"evidence\": \"Modification-specific genome-wide profiling in WT and Tdg-deficient ESCs\",\n      \"pmids\": [\"23602152\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence at individual regulatory elements not dissected\", \"Turnover kinetics in vivo not measured\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Distinguished TDG's catalytic from structural roles by showing reprogramming requires its enzymatic activity while RAR/CBP target gene control depends on scaffolding.\",\n      \"evidence\": \"TDG-deficient MEF reprogramming with catalytic-vs-inactive rescue; ternary complex Co-IP with point mutant and transcriptomics; methylated reporter reactivation with NEIL rescue\",\n      \"pmids\": [\"24529596\", \"24394593\", \"24948610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular basis of scaffolding-dependent transcription incompletely defined\", \"How miRNA loci are selected for TDG-dependent activation unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified Gadd45a as a stimulatory partner coupling TDG to active demethylation, supported by genetic epistasis at shared loci.\",\n      \"evidence\": \"Co-IP, in vitro 5fC/5caC excision stimulation, reporter assay, Gadd45a/b double-KO ESC methylation analysis\",\n      \"pmids\": [\"25845601\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which Gadd45a accelerates excision not defined\", \"Overlap with NEIL stimulation pathways unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Reconstituted productive, break-free demethylation of symmetric CpGs and showed NEIL glycosylases accelerate TDG turnover by displacing it from abasic sites.\",\n      \"evidence\": \"Purified TET1-TDG reconstitution and demethylation assay; in vitro glycosylase/AP lyase assays with Xenopus loss-of-function validation\",\n      \"pmids\": [\"26932196\", \"26751644\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and ordering of full multienzyme handoff in cells not established\", \"Regulation of NEIL recruitment in vivo unknown\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed how TDG activity is tuned by metabolism and chromatin: alpha-ketoglutarate allosterically activates it at Arg275, and nucleosome compaction inhibits its action on chromatin.\",\n      \"evidence\": \"Molecular dynamics, Arg275 mutagenesis, glycosylase assays, Co-IP in diabetic cells (alphaKG); defined nucleosome arrays with site-specific 5fC and FOXA1/histone variants (chromatin)\",\n      \"pmids\": [\"29158345\", \"31460763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo contribution of alphaKG regulation to demethylation untested genome-wide\", \"How FOXA1 mechanistically licenses TDG access unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Refined the demethylation handoff by showing NEIL1 itself excises 5caC and substrate-specifically enhances TDG on 5fC/5caC but not T:G, and connected TDG to coactivator complex recruitment in vivo.\",\n      \"evidence\": \"In vitro assays with NEIL1 catalytic mutants and binding assays; ChIP and conditional Tdg KO at the Hic1 promoter with RAR/RXR/CBP/TET recruitment\",\n      \"pmids\": [\"28827588\", \"28538185\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Generality of the RAR-recruited demethylation complex across target genes not established\", \"Substrate-specificity basis of NEIL1 stimulation structurally undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Uncovered TDG as a regulator of DNA methyltransferase activity, binding DNMT3A to promote its degradation and dominantly modulate its nucleosomal activity, with disease relevance in cancer.\",\n      \"evidence\": \"Co-IP, ubiquitination and degradation assays, MSP, migration/invasion and xenografts (TIMP2); in vitro DNMT3A nucleosome methylation with allosteric binding mapping\",\n      \"pmids\": [\"35414793\", \"33172892\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether DNMT3A regulation is catalysis-dependent unclear\", \"Physiological scope beyond cancer models untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Quantified the in vivo contribution of TDG-dependent active demethylation, showing it operates but is minor relative to passive dilution during T cell differentiation, and that 5fC/5caC excision is largely CpG-context-independent.\",\n      \"evidence\": \"Inducible Tdg disruption with single-base PB-seq in mice; in vitro TET2/TDG kinetics across CG and CH contexts\",\n      \"pmids\": [\"34158086\", \"33561435\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cell types where active demethylation dominates not delineated\", \"Determinants directing TDG to specific loci in vivo unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Extended TDG's substrate range to RNA:DNA hybrids and identified it as an RNA-binding protein, implicating R-loops and lncRNAs in directing strand-specific demethylation.\",\n      \"evidence\": \"In vitro R-loop binding and glycosylase assays with 19F NMR (preprint); BioID2 proximity proteomics, RNA-IP, and hybrid-substrate assays in mESCs\",\n      \"pmids\": [\"bio_10.1101_2025.08.05.668694\", \"41291101\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo role of R-loop-directed TDG activity not established\", \"Functional consequence of Neat1 and RUVBL2/HCFC1 associations undefined\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Defined a catalysis-independent genomic function, showing TDG occupies active promoters with ATF4 and sustains ATF4-dependent expression and mTORC1 signaling via nucleosome positioning during differentiation.\",\n      \"evidence\": \"Tdg KO epiblast-like cells, TDG and ATF4 ChIP-seq, transcriptomics, mTORC1 analysis, catalytically inactive rescue\",\n      \"pmids\": [\"41773019\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which TDG positions nucleosomes at promoters unknown\", \"Relationship between scaffolding and demethylation roles at the same loci unresolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How TDG is targeted to specific genomic loci, and how its catalytic, scaffolding, and RNA-associated functions are coordinated and partitioned across cell states, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model of locus selection for active demethylation versus structural occupancy\", \"Interplay of SUMO/RNF4 turnover, p53 transcription, and chromatin state in directing function not integrated\", \"In vivo significance of R-loop and RNA-binding activities undetermined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": [0, 2, 3, 12, 19, 20, 25, 27]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 12, 19]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [20, 27]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [27, 28]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [10, 13, 14]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [10, 14, 29]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2, 15]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [2, 17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [7, 12, 19]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 1, 2, 3, 17]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [10, 13, 14, 29]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 11, 13, 29]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [6, 21]}\n    ],\n    \"complexes\": [\"9-1-1 checkpoint complex (Rad9-Rad1-Hus1)\", \"RAR/RXR-CBP-TDG coactivator complex\"],\n    \"partners\": [\"TET1\", \"NEIL1\", \"NEIL2\", \"GADD45A\", \"HUS1\", \"CBP\", \"SRC1\", \"DNMT3A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}