{"gene":"TRIM28","run_date":"2026-06-10T10:51:55","timeline":{"discoveries":[{"year":2005,"finding":"MDM2 interacts with KAP1/TRIM28 via the N-terminal coiled-coil domain of KAP1 and the central acidic domain of MDM2; this interaction stimulates formation of a p53-HDAC1 complex, inhibits p53 acetylation, and promotes p53 ubiquitination and degradation. ARF competes with KAP1 for MDM2 binding, reducing KAP1-MDM2 interaction.","method":"Co-immunoprecipitation, RNAi knockdown, ubiquitination assay, competition binding assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal functional assays (RNAi, ubiquitination, competition), mechanistic domain mapping","pmids":["16107876"],"is_preprint":false},{"year":2007,"finding":"ATM-dependent phosphorylation of KAP1 at Ser-824 in response to genotoxic stress reduces KAP1 sumoylation, leading to de-repression of p21WAF1/CIP1 and Gadd45alpha. Conversely, a phospho-mimetic S824D mutation decreases sumoylation and stimulates p21 transcription, while S824A increases sumoylation and maintains repression. SENP1 deSUMOylase is involved in regulating basal KAP1 Ser-824 phosphorylation.","method":"Site-directed mutagenesis (S824A, S824D), ATM kinase assay, sumoylation assay, reporter gene assay, ChIP","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis with multiple functional readouts, phosphorylation-sumoylation cross-talk mechanistically defined in single study","pmids":["17942393"],"is_preprint":false},{"year":2008,"finding":"The tandem PHD finger-bromodomain of KAP1 forms a unified structural scaffold in which the PHD finger and bromodomain cooperate as an intramolecular SUMO E3 ligase, facilitating lysine SUMOylation that is required for KAP1 co-repressor activity in gene silencing. Structure-function mutagenesis correlating UBC9 binding, SUMOylation, and transcriptional repression confirmed this.","method":"NMR solution structure determination, mutation-based structure-function analysis, SUMOylation assay, transcriptional repression assay, UBC9 binding assay","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure plus mutagenesis plus functional SUMOylation and transcription assays in one study","pmids":["18488044"],"is_preprint":false},{"year":2008,"finding":"Phosphorylation of TIF1beta/KAP1 at Ser473 by PKCdelta disrupts its interaction with HP1beta (at the PXVXL HP1-box), reducing co-repressor function and permitting expression of cell cycle genes (cyclin A2, Cdc2, Cdc25A). Unphosphorylated KAP1 allows HP1beta co-occupancy at target promoters and transcriptional repression; Ser473 phosphorylation coincides with S-phase.","method":"Site-directed mutagenesis (S473A, S473E), ChIP, co-immunoprecipitation, reporter assays, PKCdelta inhibitor treatment","journal":"BMC molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with ChIP and Co-IP in a single lab study with multiple orthogonal methods","pmids":["18590578"],"is_preprint":false},{"year":2011,"finding":"KAP1 genomic recruitment requires its RBCC domain for binding to 3' coding exons of zinc finger genes (via KRAB-ZNF interaction), but RBCC deletion does not abolish KAP1 binding to promoter regions, indicating a second, KRAB-ZNF-independent recruitment mechanism for promoter-bound KAP1.","method":"ChIP-seq, stable cell lines expressing tagged wild-type and RBCC-deletion mutant KAP1, KAP1 knockdown","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP-seq with deletion mutants and knockdown, two complementary approaches in one study","pmids":["21343339"],"is_preprint":false},{"year":2013,"finding":"De novo DNA methylation of endogenous retroviruses (ERVs) in embryonic stem cells requires a KRAB-ZFP that recognizes the specific ERV sequence, plus both KAP1 and ESET (SETDB1). KAP1 knockout in early embryos reduces methylation of ERVs. KAP1 thus acts downstream of KRAB-ZFPs and upstream of ESET to install stable DNA methylation marks at ERV loci.","method":"Genetic knockout (KAP1 KO in early embryos), reporter assay with introduced ERV sequences, bisulfite sequencing, ES cell differentiation model","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo KO plus reporter assay epistasis defining pathway order, replicated across multiple experimental systems","pmids":["23293284"],"is_preprint":false},{"year":2014,"finding":"TRIM28 stabilizes paused RNA Pol II at promoter-proximal regions of many genes and regulates Pol II pause release in a transcription-coupled phosphorylation-dependent manner. TRIM28 depletion triggers de novo expression of genes regulated by paused Pol II genome-wide.","method":"In vitro Pol II pausing assay, RNAi knockdown, genome-wide ChIP-seq of Pol II occupancy, gene expression analysis","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro pausing assay plus genome-wide ChIP-seq plus in vivo knockdown, multiple orthogonal methods","pmids":["25173174"],"is_preprint":false},{"year":2015,"finding":"The nuclear oncogene SET interacts with KAP1 and its overexpression causes sustained retention of KAP1 and HP1 on chromatin at double-strand breaks, leading to inhibition of DNA end resection and homologous recombination-mediated DNA repair.","method":"Co-immunoprecipitation, overexpression and depletion studies, chromatin fractionation, HR repair assay (I-SceI system)","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction plus functional HR assay, single lab","pmids":["25818296"],"is_preprint":false},{"year":2016,"finding":"URI (unconventional prefoldin RPB5 interactor) forms a nuclear complex with KAP1 and PP2A phosphatase; PP2A recruited by URI decreases KAP1 phosphorylation. This URI-KAP1-PP2A complex mediates retrotransposon (LINE-1, L1PA2) repression, functionally overlapping with the KAP1-SETDB1 silencing complex.","method":"Co-immunoprecipitation, mass spectrometry, URI knockdown, microarray of transposon expression, phosphorylation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus functional knockdown with transposon expression readout, single lab","pmids":["27780869"],"is_preprint":false},{"year":2017,"finding":"TRIM28 forms a complex with EZH2 and SWI/SNF (distinct from PRC2) to activate, rather than repress, a set of stem cell maintenance genes in breast cancer cells. TRIM28 depletion represses EZH2 recruitment to chromatin and reduces mammosphere formation; rescue by EZH2 requires intact TRIM28 interaction (pre-SET domain).","method":"Co-immunoprecipitation, mass spectrometry, ChIP, siRNA knockdown, mammosphere formation assay, EZH2 mutant rescue experiment","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with MS, ChIP, functional rescue with domain mutant, multiple orthogonal methods in single study","pmids":["28068325"],"is_preprint":false},{"year":2018,"finding":"TRIM28 acts as an E3 ubiquitin ligase for BCL2A1 at the mitochondria; endogenous TRIM28 and BCL2A1 physically interact and TRIM28 knockdown decreases BCL2A1 ubiquitination. TRIM17 antagonizes TRIM28 by blocking its binding to BCL2A1, stabilizing BCL2A1 and promoting chemoresistance.","method":"Co-immunoprecipitation, ubiquitination assay, TRIM28 knockdown, TRIM17 overexpression and knockout","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP, ubiquitination assay, genetic loss-of-function with mechanistic antagonist characterization in single study","pmids":["30042493"],"is_preprint":false},{"year":2018,"finding":"TRIM28 physically interacts with TRIM24 and prevents TRIM24 ubiquitination and SPOP-mediated degradation; TRIM28 also facilitates TRIM24 chromatin occupancy and augments AR signaling in prostate cancer.","method":"Co-immunoprecipitation, ubiquitination assay, ChIP, knockdown and overexpression in cell lines and xenograft models","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP with ubiquitination and ChIP assays plus in vivo xenograft, multiple orthogonal methods","pmids":["30479348"],"is_preprint":false},{"year":2018,"finding":"KAP1 associates with DNA replication factors PCNA, MCM3, and MCM6; these interactions are promoted by KAP1 phosphorylation at serine 473 during S phase. KAP1 forms a complex with PCNA and Suv39h1 histone methyltransferase to reinstate H3K9 methylation-dependent heterochromatin after DNA replication.","method":"Co-immunoprecipitation, phosphorylation-specific analysis (S473), ChIP, cell fractionation during S phase","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple Co-IPs identifying complex components, phosphorylation-regulated interaction, H3K9me3 reinstatement shown by ChIP","pmids":["29955894"],"is_preprint":false},{"year":2019,"finding":"TRIM28 promotes HIV-1 latency by SUMOylating CDK9 (catalytic subunit of P-TEFb) at lysines K44, K56, and K68 with SUMO4. This SUMOylation inhibits CDK9 kinase activity and/or prevents P-TEFb assembly by blocking CDK9-Cyclin T1 interaction, thereby suppressing HIV-1 transcriptional elongation.","method":"Global site-specific SUMO-MS, serial SUMOylation assays, site-directed mutagenesis of CDK9 residues, Co-immunoprecipitation, kinase activity assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — mass spectrometry-identified SUMOylation sites confirmed by mutagenesis and kinase assay, multiple orthogonal methods","pmids":["30652970"],"is_preprint":false},{"year":2019,"finding":"The KAP1 TRIM domain forms antiparallel dimers that further assemble into tetramers and higher-order oligomers. Crystal structure identifies the KRAB domain binding site in the coiled-coil domain near the dimer dyad; mutations at this site abolish KRAB binding and retrotransposon silencing activity. B-box 1 mutations that prevent higher-order oligomerization do not significantly impair silencing.","method":"Crystal structure determination, biophysical analysis (SAXS, SEC-MALS), site-directed mutagenesis, retrotransposon silencing assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus functional silencing assay in single study","pmids":["31289231"],"is_preprint":false},{"year":2019,"finding":"KAP1 is an elongated antiparallel dimer with functional asymmetry at the C-terminal domains. The RING domain contributes to KAP1 auto-SUMOylation. HP1 occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry.","method":"Solution scattering (SAXS), integrative modeling, single-molecule experiments, biochemical assays","journal":"Life science alliance","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural determination with multiple biophysical methods plus biochemical validation of auto-SUMOylation and HP1 stoichiometry","pmids":["31427381"],"is_preprint":false},{"year":2020,"finding":"KAP1/TRIM28 uses a chromatin reader cassette to bind hypo-acetylated histone H4 tails at promoters, then associates with RNA Pol II and recruits SMAD2 upon TGF-beta signaling to enable CDK9-dependent Pol II pause release. This couples chromatin reading to both Pol II pausing and pause release for transcriptional elongation.","method":"ChIP-seq, chromatin reader domain characterization, Co-immunoprecipitation, CDK9-dependent pause release assay, SMAD2 recruitment assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP-seq, Co-IP, functional elongation assay, identification of novel reader cassette with ligand-responsive mechanism in single study","pmids":["32402252"],"is_preprint":false},{"year":2021,"finding":"TRIM28 acts as an E3 SUMO ligase that binds NLRP3, promotes SUMO1/2/3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation, stabilizing NLRP3 protein levels to facilitate inflammasome assembly and activation. Trim28 deficiency attenuates NLRP3 inflammasome activation in vitro and in vivo.","method":"Co-immunoprecipitation, SUMOylation assay, ubiquitination assay, Trim28 conditional knockout, NLRP3 inflammasome activation assays (IL-1β secretion, caspase-1 cleavage)","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, SUMOylation and ubiquitination assays, in vivo KO, multiple readouts of inflammasome activity","pmids":["34373456"],"is_preprint":false},{"year":2021,"finding":"KAP1/TRIM28 represses HIV-1 expression in myeloid cells by interacting with and colocalizing with the viral transactivator Tat, promoting Tat's degradation via the proteasome. KAP1 is also bound to the latent HIV-1 promoter and cooperates with CTIP2, an epigenetic silencer; Tat and CTIP2 compete for KAP1 binding.","method":"Co-immunoprecipitation, colocalization (confocal microscopy), proteasome inhibitor treatment, KAP1 depletion, ChIP, myeloid HIV-1 latency model reactivation","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ChIP with functional knockdown, single lab","pmids":["33514850"],"is_preprint":false},{"year":2021,"finding":"UBE2S interacts with TRIM28 in the nucleus; together they enhance ubiquitination of p27 to facilitate its proteasomal degradation and promote G1/S cell cycle progression in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ubiquitination assay, cell cycle analysis, siRNA knockdown","journal":"Signal transduction and targeted therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ubiquitination assay, functional cell cycle readout, single lab","pmids":["33589597"],"is_preprint":false},{"year":2021,"finding":"The SETDB1-TRIM28 complex suppresses antitumor immunity; inhibition of this complex upregulates PD-L1 and activates the cGAS-STING innate immune pathway by promoting micronuclei formation in the cytoplasm, increasing CD8+ T cell infiltration.","method":"CRISPR-Cas9 epigenetic screen, flow cytometry, cGAS-STING pathway activation assay, syngeneic mouse tumor model, SETDB1 KO","journal":"Cancer immunology research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional CRISPR screen and in vivo mouse model, mechanistic pathway placement via cGAS-STING, single lab","pmids":["34848497"],"is_preprint":false},{"year":2021,"finding":"TRIM28 acts as a negative regulator of aggresome formation through direct interaction with CTIF; this interaction leads to inefficient aggresomal targeting of misfolded polypeptides. TRIM28 phosphorylation at S473 by double-stranded RNA-activated protein kinase (PKR) promotes TRIM28-CTIF association, inhibits aggresome formation, and suppresses viral proliferation.","method":"Co-immunoprecipitation, proximity ligation assay, phosphorylation analysis (S473), aggresome formation assay, influenza A virus infection model","journal":"Autophagy","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct interaction by Co-IP and PLA, phosphorylation-regulated mechanism with functional viral readout, multiple orthogonal methods","pmids":["33783327"],"is_preprint":false},{"year":2021,"finding":"TRIM28 is a transcriptional activator of the mutant TERT promoter. TRIM28 is recruited to the mutant hTERT promoter and interacts with TRIM24, which inhibits TRIM28 activity. Phosphorylation of TRIM28 via mTORC1 releases it from TRIM24 and induces hTERT transcription; mTORC1 inhibition suppresses TRIM28 phosphorylation and hTERT expression.","method":"CRISPR-Cas9 kinome knockout screen (FACS-based), ChIP, Co-immunoprecipitation, mTORC1 inhibition (rapamycin analog), phosphorylation assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen validated by ChIP, Co-IP, and pharmacological inhibition with mechanistic phosphorylation characterization","pmids":["34518220"],"is_preprint":false},{"year":2022,"finding":"Crystal structure of the KAP1 TRIM in complex with the KRAB domain of ZNF93 identifies the KAP1-KRAB binding interface. Structure-guided mutations in this interface abolish repressive activity in a transcriptional silencing assay and eliminate genome-wide H3K9me3 deposition at thousands of KAP1/KRAB-ZFP target loci.","method":"Crystal structure determination, site-directed mutagenesis, epigenetic transcriptional silencing assay, ChIP-seq (H3K9me3 genome-wide)","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus genome-wide ChIP-seq functional validation","pmids":["36341546"],"is_preprint":false},{"year":2022,"finding":"TRIM28-dependent SUMOylation is required to maintain the adult ovarian cell fate by repressing testicular-specific genes. TRIM28 is recruited to chromatin near FOXL2 binding sites, and its E3-SUMO ligase activity regulates the sex-specific SUMOylation profile of ovarian-specific genes. Loss of Trim28 in granulosa cells leads to their transdifferentiation into Sertoli cells.","method":"Conditional knockout (Trim28 deletion in granulosa cells), ChIP, SUMOylation profiling, histology/lineage tracing","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO with mechanistic SUMOylation profiling and ChIP, phenotype directly linked to SUMO ligase activity","pmids":["35906245"],"is_preprint":false},{"year":2022,"finding":"KAP1 and the KRAB domain residues forming the molecular interface were structurally mapped using AlphaFold2; leucine 301 on each chain of the TRIM28 coiled-coil acts as a 'pin' inserting into a hydrophobic pocket on the KRAB domain. Site-directed mutations at this interface abolish KRAB domain binding.","method":"AlphaFold2 modeling, site-directed mutagenesis, binding assays","journal":"Protein science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — computational structural model with mutagenesis-based binding validation, no X-ray or cryo-EM confirmation","pmids":["36173157"],"is_preprint":false},{"year":2023,"finding":"TRIM28 directly binds and stabilizes PD-L1 by inhibiting PD-L1 ubiquitination and promoting PD-L1 SUMOylation. Additionally, TRIM28 facilitates K63 polyubiquitination of TBK1, activating TBK1-IRF1 and TBK1-mTOR pathways to enhance PD-L1 transcription.","method":"CRISPR-Cas9 genome-wide screen, Co-immunoprecipitation, ubiquitination assay, SUMOylation assay, in vivo mouse tumor model, TBK1 pathway inhibitor studies","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR screen, multiple biochemical assays (Co-IP, ubiquitination, SUMOylation), in vivo validation with mechanistic pathway dissection","pmids":["37357254"],"is_preprint":false},{"year":2023,"finding":"TRIM28 promotes ubiquitination and proteasome-mediated degradation of TFE3 transcription factor in renal cancer cells, thereby suppressing TFE3-driven autophagic gene expression and cell proliferation. This defines a TRIM28-TFE3-KDM6A signaling axis in kidney cancer.","method":"Co-immunoprecipitation, ubiquitination assay, TRIM28 knockdown, ChIP, autophagy assays, cell proliferation assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, ChIP with functional readouts, single lab","pmids":["36935008"],"is_preprint":false},{"year":2023,"finding":"TRIM28 physically interacts with RIPK1 and promotes K63-linked ubiquitination of RIPK1, sustaining NF-κB pathway activation and upregulation of CXCL1, which recruits MDSCs. Mutagenesis of the TRIM28 RING/E3 ligase domain (C65, C68) abrogates NF-κB activation.","method":"Co-immunoprecipitation, K63-ubiquitination assay, RING domain mutagenesis, NF-κB reporter, CXCL1 ELISA, syngeneic mouse tumor models","journal":"Journal of experimental & clinical cancer research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mutagenesis validation, ubiquitination assay, and in vivo tumor models in single study","pmids":["37865804"],"is_preprint":false},{"year":2023,"finding":"TRIM28 acts as the E3 SUMO ligase responsible for SUMOylation of the SARS-CoV-2 nucleocapsid protein (SARS2-NP) at lysine K65, which mediates NP homo-oligomerization, RNA association, liquid-liquid phase separation, and suppression of innate antiviral immunity. An interfering peptide targeting the TRIM28-SARS2-NP interaction blocks NP SUMOylation and LLPS.","method":"SUMOylation assay, site-directed mutagenesis (K65), LLPS assay, Co-immunoprecipitation, antiviral immune assay, interfering peptide screen","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — mutagenesis of SUMOylation site plus biochemical reconstitution of LLPS plus functional immune assay in single study","pmids":["38172120"],"is_preprint":false},{"year":2023,"finding":"TRIM28 negatively regulates the RLR signaling pathway by targeting MAVS for K48-linked polyubiquitination and proteasome-mediated degradation. The RING domain of TRIM28 (specifically C65 and C68 residues) is critical for this activity; ubiquitin chains are transferred to K7, K10, K371, K420, and K500 of MAVS.","method":"Ubiquitination assay, proteasome inhibitor treatment, RING domain mutagenesis, Co-immunoprecipitation, type I interferon production assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — RING domain mutagenesis with site-specific ubiquitin mapping (K-residues identified) plus functional IFN assay, single lab but multiple orthogonal methods","pmids":["37119745"],"is_preprint":false},{"year":2023,"finding":"TIAM1 interacts with TRIM28 in the nucleus of NSCLC cells and the TIAM1-TRIM28 complex promotes H3K9me3-induced silencing of protocadherins and decreases E-cadherin expression, driving epithelial-to-mesenchymal transition and cell migration.","method":"Co-immunoprecipitation, ChIP (H3K9me3), siRNA knockdown, migration/invasion assay, rescue by protocadherin depletion","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ChIP plus functional rescue experiment, single lab","pmids":["37748077"],"is_preprint":false},{"year":2023,"finding":"TRIM28 binds ACSL4 and promotes SUMO3 modification at ACSL4 lysine K532, inhibiting K63-linked ACSL4 ubiquitination and thereby suppressing OPTN-dependent autophagic degradation of ACSL4, promoting neuronal ferroptosis. SENP3 acts as the opposing deSUMOylation enzyme.","method":"Co-immunoprecipitation, SUMOylation assay, ubiquitination assay, mutagenesis (K532), autophagic degradation assay, Trim28 genetic deletion, in vivo SCI mouse model","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP, site-specific mutagenesis, multiple PTM assays (SUMO, ubiquitin), in vivo knockout with behavioral readout, multiple orthogonal methods","pmids":["39875520"],"is_preprint":false},{"year":2024,"finding":"TRIM28 facilitates type I interferon activation by interacting with TBK1 and mediating K63-linked ubiquitination of TBK1, augmenting TBK1 signal transmission; TRIM28 KO cells display defective TBK1 phosphorylation and impaired complex assembly with IRF3.","method":"CRISPR-Cas9 TRIM28 knockout, Co-immunoprecipitation, K63-ubiquitination assay, TBK1 phosphorylation assay, virus infection susceptibility assay","journal":"Frontiers in immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR KO with Co-IP and ubiquitination assay, single lab","pmids":["38495890"],"is_preprint":false},{"year":2024,"finding":"TRIM28 acts as an E3 ubiquitin ligase for BRD7; the coiled-coil region of TRIM28 binds the N-terminal domain of BRD7 and mediates BRD7 ubiquitination at K21, promoting its proteasomal degradation. This promotes breast cancer malignant progression.","method":"Co-immunoprecipitation with mass spectrometry, ubiquitination assay, domain deletion/mutagenesis (K21), xenograft mouse model","journal":"Cellular oncology (Dordrecht, Netherlands)","confidence":"High","confidence_rationale":"Tier 2 / Moderate — Co-IP/MS for interaction, site-specific ubiquitin mutagenesis, in vivo xenograft, multiple orthogonal methods in single study","pmids":["39222175"],"is_preprint":false},{"year":2012,"finding":"KAP1 directly interacts with IRF5 via IRF5's N-terminus (DNA-binding domain and disordered region); KAP1 knockdown potentiates IRF5-mediated TNF and M1 macrophage marker expression. This inhibitory effect is linked to SETDB1 methyltransferase activity (H3K9me3 deposition) at the TNF locus.","method":"Affinity purification with mass spectrometry (IRF5 interactome), Co-immunoprecipitation, domain mapping, KAP1 siRNA knockdown, ChIP (H3K9me3)","journal":"Immunobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — AP-MS interaction confirmed by Co-IP and domain mapping, functional knockdown with ChIP, single lab","pmids":["22995936"],"is_preprint":false},{"year":2007,"finding":"KAP1 physically associates with endogenous STAT3 in cells; KAP1 knockdown enhances IL-6-induced STAT3-dependent transcription and causes marked accumulation of STAT3 phosphorylated on Ser727 in the nucleus, indicating KAP1 acts as a transcriptional repressor of the IL-6/STAT3 signaling pathway.","method":"Yeast two-hybrid screen, endogenous Co-immunoprecipitation, siRNA knockdown, reporter gene assay, phospho-STAT3 analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — endogenous Co-IP plus functional siRNA knockdown, single lab","pmids":["18037959"],"is_preprint":false},{"year":2020,"finding":"Kap1 interacts with Oct4 and inhibits Itch-mediated ubiquitination of Oct4 at lysine K133, thereby stabilizing Oct4 protein in embryonic stem cells and promoting self-renewal and cellular reprogramming.","method":"Affinity purification with mass spectrometry, Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K133R), Kap1 knockdown and overexpression, reprogramming assay","journal":"Cell death and differentiation","confidence":"High","confidence_rationale":"Tier 2 / Moderate — AP-MS identification confirmed by Co-IP, site-specific mutagenesis of ubiquitination site, functional reprogramming assay, multiple orthogonal methods","pmids":["32895487"],"is_preprint":false},{"year":2012,"finding":"KAP1-dependent H3K9me3 repressive chromatin is established at T-cell-specific cis-regulatory elements enriched in Ikaros/NuRD complexes; KAP1 directly controls FoxO1 expression and TCR/cytokine signaling genes. T-cell-specific Kap1 deletion causes expansion of immature thymocytes and altered CD4+/CD8+ ratios.","method":"T-cell-specific conditional Kap1 KO (Cre-lox), ChIP/ChIP-seq, transcriptome analysis","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo conditional KO with ChIP-seq, single lab","pmids":["22872677"],"is_preprint":false},{"year":2023,"finding":"TRIM28 modulates uterine function by complexing with estrogen receptor α (ERα) and progesterone receptor (PR); TRIM28 ablation suppresses PR and ERα chromatin binding in uterine epithelium and stroma, impairing implantation and early pregnancy.","method":"RIME (rapid immunoprecipitation mass spectrometry), Co-immunoprecipitation, conditional knockout, ChIP","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Moderate — RIME and Co-IP for interaction, conditional KO for function, ChIP for chromatin binding, multiple orthogonal methods in single study","pmids":["37528140"],"is_preprint":false},{"year":2012,"finding":"MAGE-C2 binds KAP1 and increases co-precipitation of KAP1 with ATM kinase; MAGE-C2 binding to KAP1 is required for enhanced ATM-dependent phosphorylation of KAP1-Ser824, which facilitates heterochromatin relaxation and DNA double-strand break repair.","method":"Co-immunoprecipitation, KAP1 S824 phosphorylation assay, I-SceI endonuclease DSB repair assay, MAGE-C2 knockdown/overexpression","journal":"The Journal of investigative dermatology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP interaction with functional phosphorylation and repair assays, single lab","pmids":["23096706"],"is_preprint":false},{"year":2015,"finding":"CCAR2/DBC1 is required for Chk2-dependent KAP1 phosphorylation at Ser824; loss of CCAR2 impairs Chk2 activation and reduces KAP1 S824 phosphorylation, leading to defective heterochromatin relaxation and impaired repair of heterochromatic DSBs.","method":"CCAR2 knockout, Chk2 activation assay, KAP1 S824 phosphorylation assay, DNA damage foci resolution (late time-point), HP1β depletion rescue","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO with phosphorylation assay and functional DNA repair epistasis, single lab","pmids":["26158765"],"is_preprint":false},{"year":2022,"finding":"At the onset of X chromosome inactivation, KAP1 is recruited to the Xist promoter allele that will upregulate Xist and is required for the increase in Tsix levels preceding cell fate choice. RIF1 and KAP1 show mutual exclusion at the Xist promoters, establishing an asymmetric self-sustaining loop for X chromosome choice.","method":"mESC differentiation, allele-specific ChIP, KAP1 and RIF1 depletion, Tsix/Xist RNA FISH","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — allele-specific ChIP and functional depletion, single lab","pmids":["34786738"],"is_preprint":false},{"year":2018,"finding":"KAP1 interacts with KAP1 regulates ERV (including HERV-T, HERV-S) and ZNF gene expression in adult differentiated human cells including peripheral blood mononuclear cells, requiring H3K9me3. KAP1 depletion leads to decreased H3K9me3 at ERVs and ZNF loci and activates an RNA-sensing response mediated through MAVS signaling.","method":"KAP1 knockout, RNA-seq, ChIP-seq (H3K9me3, KAP1), MAVS signaling assay, interferon response measurement","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with ChIP-seq and pathway activation assay, single lab","pmids":["30061100"],"is_preprint":false}],"current_model":"TRIM28/KAP1 is a multifunctional nuclear scaffold protein that acts as: (1) a co-repressor recruited by KRAB-ZFPs to silence endogenous retroviruses and transposable elements via assembly of an H3K9me3/SETDB1 heterochromatin complex, using its RBCC/TRIM domain to bind KRAB domains and its PHD finger-bromodomain as an intramolecular SUMO E3 ligase; (2) a regulator of RNA Pol II pausing and pause release through a chromatin reader cassette that binds hypo-acetylated histone H4 and recruits CDK9/P-TEFb; (3) an E3 ubiquitin and E3 SUMO ligase for diverse substrates including NLRP3, MAVS, BCL2A1, RIPK1, TBK1, ACSL4, and TFE3, with substrate specificity regulated by phosphorylation (ATM at S824 in DNA damage, mTORC1 and PKCdelta at S473); and (4) a signaling scaffold that controls p53 stability (via MDM2), pluripotency (via Oct4), and innate immune responses, with all activities tightly regulated by a phosphorylation-SUMOylation cross-talk switch."},"narrative":{"mechanistic_narrative":"TRIM28 (KAP1/TIF1beta) is a multifunctional nuclear scaffold that couples sequence-specific transcription factor recruitment to heterochromatin assembly, RNA Pol II control, and post-translational modification of diverse substrates [PMID:23293284, PMID:32402252, PMID:36341546]. Its RBCC/TRIM domain forms antiparallel dimers that present a coiled-coil interface—pinned by Leu301—for binding the KRAB domains of zinc-finger proteins, and structure-guided disruption of this interface abolishes transcriptional silencing and genome-wide H3K9me3 deposition at thousands of KAP1/KRAB-ZFP loci [PMID:31289231, PMID:36341546, PMID:36173157]. Recruited by KRAB-ZFPs, TRIM28 acts downstream of these factors and upstream of the SETDB1/ESET methyltransferase to install stable H3K9me3 and de novo DNA methylation that silence endogenous retroviruses and transposable elements [PMID:23293284, PMID:30061100], with HP1 binding contributing to repressive complex assembly [PMID:31427381]. Its tandem PHD finger–bromodomain functions as an intramolecular SUMO E3 ligase whose auto-SUMOylation is required for co-repressor activity [PMID:18488044, PMID:31427381]. Beyond silencing, a chromatin reader cassette binds hypo-acetylated histone H4 at promoters, stabilizes paused Pol II, and recruits CDK9/P-TEFb and SMAD2 to drive pause release upon TGF-beta signaling [PMID:25173174, PMID:32402252]. TRIM28 additionally serves as an E3 ubiquitin and SUMO ligase for an extensive substrate set—targeting MAVS, RIPK1, TBK1, NLRP3, BCL2A1, ACSL4, TFE3, PD-L1, BRD7, and p27 for ubiquitination or SUMOylation—thereby controlling innate immune signaling, inflammasome activation, ferroptosis, cell-cycle progression, and antitumor immunity [PMID:30042493, PMID:34373456, PMID:37357254, PMID:37865804, PMID:37119745, PMID:39875520, PMID:38495890]. TRIM28 also stabilizes p53-degrading MDM2 complexes and Oct4 to govern p53 turnover and pluripotency [PMID:16107876, PMID:32895487]. All of these activities are gated by a phosphorylation–SUMOylation cross-talk switch: ATM phosphorylation of Ser824 reduces SUMOylation to de-repress damage-response genes, while PKCdelta/mTORC1/PKR phosphorylation of Ser473 reconfigures partner interactions across the cell cycle, DNA replication, and stress responses [PMID:17942393, PMID:18590578, PMID:29955894, PMID:33783327, PMID:34518220].","teleology":[{"year":2005,"claim":"Established TRIM28 as a positive regulator of p53 turnover, linking it to oncogenic signaling before its silencing roles were fully appreciated.","evidence":"Reciprocal Co-IP, RNAi, ubiquitination and competition assays mapping the KAP1 coiled-coil to the MDM2 acidic domain","pmids":["16107876"],"confidence":"High","gaps":["Did not address whether SUMOylation or phosphorylation gates the MDM2 interaction","Physiological contexts where ARF competition dominates unresolved"]},{"year":2007,"claim":"Defined the central phosphorylation–SUMOylation switch by showing ATM phosphorylation of Ser824 antagonizes SUMOylation to de-repress damage-response genes.","evidence":"S824A/S824D mutagenesis, ATM kinase and SUMOylation assays, reporter assays, ChIP","pmids":["17942393"],"confidence":"High","gaps":["Which SUMO sites are coupled to S824 not pinpointed","Genome-wide scope of the switch not assessed"]},{"year":2008,"claim":"Resolved how TRIM28 generates its own activating modification and how a second phospho-site tunes HP1 binding, mechanizing the PTM-controlled co-repressor.","evidence":"NMR structure of the PHD–bromodomain SUMO ligase; S473 mutagenesis with ChIP and Co-IP showing PKCdelta disrupts HP1beta binding","pmids":["18488044","18590578"],"confidence":"High","gaps":["Intramolecular vs intermolecular SUMO transfer in vivo not fully delineated","S473 kinase repertoire beyond PKCdelta unknown at this stage"]},{"year":2011,"claim":"Showed KAP1 recruitment is bipartite—RBCC/KRAB-ZFP-dependent at zinc-finger gene bodies but KRAB-independent at promoters—revealing more than one targeting mode.","evidence":"ChIP-seq with RBCC-deletion mutants and knockdown","pmids":["21343339"],"confidence":"High","gaps":["Identity of the KRAB-independent promoter recruiter not determined"]},{"year":2013,"claim":"Placed KAP1 epistatically between KRAB-ZFPs and SETDB1 in establishing stable DNA methylation at ERVs, ordering the silencing pathway.","evidence":"KAP1 KO in early embryos, ERV reporter assays, bisulfite sequencing in ES cells","pmids":["23293284"],"confidence":"High","gaps":["Mechanism linking H3K9me3 to de novo DNA methyltransferase recruitment not resolved"]},{"year":2014,"claim":"Expanded TRIM28 function beyond silencing to direct control of Pol II promoter-proximal pausing genome-wide.","evidence":"In vitro pausing assay, Pol II ChIP-seq, RNAi knockdown","pmids":["25173174"],"confidence":"High","gaps":["Chromatin features directing TRIM28 to paused genes not yet defined"]},{"year":2015,"claim":"Identified TRIM28 partners that retune its DNA-repair and stem-cell roles, showing context-dependent regulation of chromatin retention.","evidence":"Co-IP and HR repair assays for SET; reciprocal Co-IP/MS, ChIP and mammosphere assays for an EZH2/SWI/SNF activating complex","pmids":["25818296","28068325"],"confidence":"Medium","gaps":["SET-KAP1 interaction shown by single-lab Co-IP without reciprocal structural mapping","How TRIM28 switches between PRC2-independent activation and repression unclear"]},{"year":2016,"claim":"Defined a URI-KAP1-PP2A complex that dephosphorylates KAP1 to sustain retrotransposon repression, mechanizing phosphatase control of the switch.","evidence":"Co-IP/MS, URI knockdown, transposon microarray, phosphorylation assay","pmids":["27780869"],"confidence":"Medium","gaps":["Single-lab; specific KAP1 phospho-sites targeted by PP2A not mapped"]},{"year":2018,"claim":"Catalogued TRIM28 as a substrate-specific E3 ligase and replication-coupled heterochromatin factor, broadening its enzymatic and cell-cycle roles.","evidence":"Co-IP and ubiquitination assays for BCL2A1 (TRIM17-antagonized) and TRIM24 (anti-SPOP); S473-promoted Co-IP with PCNA/MCM3/MCM6 and Suv39h1 during S phase","pmids":["30042493","30479348","29955894"],"confidence":"High","gaps":["Whether E3 activity requires RING dimerization not addressed here","Direct vs scaffolded ubiquitin transfer to each substrate not always distinguished"]},{"year":2019,"claim":"Provided high-resolution structural and biophysical definition of the TRIM28 dimer, KRAB-binding interface, and asymmetric HP1 stoichiometry.","evidence":"Crystal structure, SAXS/SEC-MALS, single-molecule and SUMOylation assays; CDK9 SUMOylation by SUMO-MS and mutagenesis for HIV latency","pmids":["31289231","31427381","30652970"],"confidence":"High","gaps":["Functional role of higher-order oligomerization beyond silencing unclear","How asymmetry is read out by partners not resolved"]},{"year":2020,"claim":"Mechanized the chromatin-reading-to-elongation coupling, showing TRIM28 reads hypo-acetylated H4 and recruits SMAD2/CDK9 for ligand-responsive pause release; also stabilizes Oct4.","evidence":"ChIP-seq, reader-domain characterization, Co-IP, CDK9-dependent pause-release assay; AP-MS/Co-IP and K133R mutagenesis showing KAP1 blocks Itch ubiquitination of Oct4","pmids":["32402252","32895487"],"confidence":"High","gaps":["How the same protein switches between Pol II stabilization and release at a given gene not fully defined"]},{"year":2021,"claim":"Established TRIM28 as a master post-translational regulator of innate immunity and inflammasome stability, with antitumor-immunity consequences.","evidence":"Co-IP, SUMOylation/ubiquitination assays and conditional KO for NLRP3 stabilization; CRISPR screen and cGAS-STING readouts for SETDB1-TRIM28 immune suppression; PKR-S473-driven CTIF interaction for aggresome/viral control; mTORC1-driven mutant TERT activation","pmids":["34373456","34848497","33783327","34518220","33514850","33589597"],"confidence":"High","gaps":["Direct vs indirect SUMO transfer to NLRP3 across cell types not fully resolved","How distinct phospho-sites partition immune versus chromatin functions unclear"]},{"year":2022,"claim":"Confirmed the KRAB-binding interface by crystallography and AlphaFold2, and extended SUMO-ligase control to cell-fate maintenance and X-inactivation choice.","evidence":"Crystal structure of TRIM/ZNF93-KRAB with mutagenesis and H3K9me3 ChIP-seq; AlphaFold2 Leu301 'pin' model; granulosa-cell conditional KO with SUMO profiling; allele-specific ChIP at Xist","pmids":["36341546","36173157","35906245","34786738"],"confidence":"High","gaps":["AlphaFold2 interface lacks independent X-ray/cryo-EM confirmation","How SUMOylation enforces lineage-specific gene sets mechanistically incomplete"]},{"year":2023,"claim":"Mapped TRIM28's substrate-specific ubiquitin/SUMO ligase activities onto immune checkpoint, RLR signaling, ferroptosis, EMT, and reproductive programs, with RING residues C65/C68 as the catalytic determinant.","evidence":"CRISPR screens, Co-IP, ubiquitination and SUMOylation assays, RING mutagenesis, in vivo tumor and SCI models, RIME for ERalpha/PR; substrates PD-L1, RIPK1, MAVS, ACSL4, TFE3, SARS2-NP","pmids":["37357254","37865804","37119745","39875520","36935008","37748077","38172120","37528140"],"confidence":"High","gaps":["Opposing ubiquitin- vs SUMO-promoting outcomes on shared substrates not unified into a single rule","How substrate selection is encoded in TRIM28 not defined"]},{"year":2024,"claim":"Extended the E3 ligase substrate repertoire (BRD7, TBK1) and clarified that TRIM28 can both activate and degrade innate signaling components depending on chain linkage.","evidence":"Co-IP/MS, K21 and chain-type-specific ubiquitination assays, CRISPR KO, xenograft and infection models","pmids":["39222175","38495890"],"confidence":"Medium","gaps":["Reconciliation of TRIM28 promoting (K63-TBK1) versus degrading (K48-MAVS) innate signaling not mechanistically integrated","TBK1 result single-lab"]},{"year":null,"claim":"How a single scaffold partitions its silencing, Pol II-elongation, and dozens of distinct ubiquitin/SUMO substrate activities—and whether the phospho-SUMO switch alone encodes this selectivity—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking specific phospho-states to specific substrate or chromatin outputs","Determinants of ubiquitin-chain-linkage choice (K48 vs K63) versus SUMO modification on substrates undefined","Stoichiometry/competition among the many named partners in cells not measured"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[10,17,26,28,30,32,33,34]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[2,13,17,24,29,32]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[6,16,22,23]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[16,12]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,16,0]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[4,23]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[16,19,31,36]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[5,12,23,42]},{"term_id":"GO:0005739","term_label":"mitochondrion","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[5,12,23,31,43]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[6,16,22]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[17,26,30,33,35,43]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,10,19,27,34,37]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[1,40,41]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[3,12,19]}],"complexes":["KAP1-SETDB1 heterochromatin complex","URI-KAP1-PP2A complex","TRIM28-EZH2-SWI/SNF complex"],"partners":["SETDB1","HP1","MDM2","TRIM24","CDK9","SMAD2","OCT4","EZH2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q13263","full_name":"Transcription intermediary factor 1-beta","aliases":["E3 SUMO-protein ligase TRIM28","KRAB-associated protein 1","KAP-1","KRAB-interacting protein 1","KRIP-1","Nuclear corepressor KAP-1","RING finger protein 96","RING-type E3 ubiquitin transferase TIF1-beta","Tripartite motif-containing protein 28"],"length_aa":835,"mass_kda":88.5,"function":"Nuclear corepressor for KRAB domain-containing zinc finger proteins (KRAB-ZFPs). Mediates gene silencing by recruiting CHD3, a subunit of the nucleosome remodeling and deacetylation (NuRD) complex, and SETDB1 (which specifically methylates histone H3 at 'Lys-9' (H3K9me)) to the promoter regions of KRAB target genes. Enhances transcriptional repression by coordinating the increase in H3K9me, the decrease in histone H3 'Lys-9 and 'Lys-14' acetylation (H3K9ac and H3K14ac, respectively) and the disposition of HP1 proteins to silence gene expression. Recruitment of SETDB1 induces heterochromatinization. May play a role as a coactivator for CEBPB and NR3C1 in the transcriptional activation of ORM1. Also a corepressor for ERBB4. Inhibits E2F1 activity by stimulating E2F1-HDAC1 complex formation and inhibiting E2F1 acetylation. May serve as a partial backup to prevent E2F1-mediated apoptosis in the absence of RB1. Important regulator of CDKN1A/p21(CIP1). Has E3 SUMO-protein ligase activity toward itself via its PHD-type zinc finger. Also specifically sumoylates IRF7, thereby inhibiting its transactivation activity. Ubiquitinates p53/TP53 leading to its proteasomal degradation; the function is enhanced by MAGEC2 and MAGEA2, and possibly MAGEA3 and MAGEA6. Mediates the nuclear localization of KOX1, ZNF268 and ZNF300 transcription factors. In association with isoform 2 of ZFP90, is required for the transcriptional repressor activity of FOXP3 and the suppressive function of regulatory T-cells (Treg) (PubMed:23543754). Probably forms a corepressor complex required for activated KRAS-mediated promoter hypermethylation and transcriptional silencing of tumor suppressor genes (TSGs) or other tumor-related genes in colorectal cancer (CRC) cells (PubMed:24623306). Required to maintain a transcriptionally repressive state of genes in undifferentiated embryonic stem cells (ESCs) (PubMed:24623306). In ESCs, in collaboration with SETDB1, is also required for H3K9me3 and silencing of endogenous and introduced retroviruses in a DNA-methylation independent-pathway (By similarity). Associates at promoter regions of tumor suppressor genes (TSGs) leading to their gene silencing (PubMed:24623306). The SETDB1-TRIM28-ZNF274 complex may play a role in recruiting ATRX to the 3'-exons of zinc-finger coding genes with atypical chromatin signatures to establish or maintain/protect H3K9me3 at these transcriptionally active regions (PubMed:27029610) (Microbial infection) Plays a critical role in the shutdown of lytic gene expression during the early stage of herpes virus 8 primary infection. This inhibition is mediated through interaction with herpes virus 8 protein LANA1","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q13263/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/TRIM28","classification":"Not Classified","n_dependent_lines":249,"n_total_lines":1208,"dependency_fraction":0.20612582781456953},"opencell":{"profiled":true,"resolved_as":"","ensg_id":"ENSG00000130726","cell_line_id":"CID001292","localizations":[{"compartment":"nucleoplasm","grade":3}],"interactors":[{"gene":"CBX1","stoichiometry":10.0},{"gene":"IPO7","stoichiometry":4.0},{"gene":"CBX3","stoichiometry":4.0},{"gene":"CAPZB","stoichiometry":0.2},{"gene":"CSNK2A1","stoichiometry":0.2},{"gene":"CSNK2A2","stoichiometry":0.2},{"gene":"IPO8","stoichiometry":0.2},{"gene":"PPM1G","stoichiometry":0.2},{"gene":"PSME3","stoichiometry":0.2},{"gene":"SMARCAD1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/target/CID001292","total_profiled":1310},"omim":[{"mim_id":"621332","title":"WILMS TUMOR 7; WT7","url":"https://www.omim.org/entry/621332"},{"mim_id":"619511","title":"ZINC FINGER PROTEIN 354C; ZNF354C","url":"https://www.omim.org/entry/619511"},{"mim_id":"618359","title":"ZINC FINGER PROTEIN 197; ZNF197","url":"https://www.omim.org/entry/618359"},{"mim_id":"618033","title":"ZINC FINGER PROTEIN 689; ZNF689","url":"https://www.omim.org/entry/618033"},{"mim_id":"617851","title":"SERTA DOMAIN-CONTAINING PROTEIN 2; SERTAD2","url":"https://www.omim.org/entry/617851"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/TRIM28"},"hgnc":{"alias_symbol":["TIF1B","KAP1","TF1B","RNF96","PPP1R157","KAP-1","TIF1-beta","TIF1beta"],"prev_symbol":[]},"alphafold":{"accession":"Q13263","domains":[{"cath_id":"3.30.40.10","chopping":"61-93_113-138","consensus_level":"medium","plddt":87.4402,"start":61,"end":138},{"cath_id":"-","chopping":"245-390","consensus_level":"medium","plddt":92.2862,"start":245,"end":390},{"cath_id":"3.30.40.10","chopping":"624-668","consensus_level":"medium","plddt":80.1691,"start":624,"end":668},{"cath_id":"1.20.920.10","chopping":"694-724_736-812","consensus_level":"medium","plddt":74.1745,"start":694,"end":812},{"cath_id":"3.30.160","chopping":"150-198","consensus_level":"medium","plddt":82.6588,"start":150,"end":198}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13263","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q13263-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q13263-F1-predicted_aligned_error_v6.png","plddt_mean":65.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=TRIM28","jax_strain_url":"https://www.jax.org/strain/search?query=TRIM28"},"sequence":{"accession":"Q13263","fasta_url":"https://rest.uniprot.org/uniprotkb/Q13263.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q13263/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q13263"}},"corpus_meta":[{"pmid":"21652716","id":"PMC_21652716","title":"KAP1 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ubiquitination and degradation. ARF competes with KAP1 for MDM2 binding, reducing KAP1-MDM2 interaction.\",\n      \"method\": \"Co-immunoprecipitation, RNAi knockdown, ubiquitination assay, competition binding assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, multiple orthogonal functional assays (RNAi, ubiquitination, competition), mechanistic domain mapping\",\n      \"pmids\": [\"16107876\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ATM-dependent phosphorylation of KAP1 at Ser-824 in response to genotoxic stress reduces KAP1 sumoylation, leading to de-repression of p21WAF1/CIP1 and Gadd45alpha. Conversely, a phospho-mimetic S824D mutation decreases sumoylation and stimulates p21 transcription, while S824A increases sumoylation and maintains repression. SENP1 deSUMOylase is involved in regulating basal KAP1 Ser-824 phosphorylation.\",\n      \"method\": \"Site-directed mutagenesis (S824A, S824D), ATM kinase assay, sumoylation assay, reporter gene assay, ChIP\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis with multiple functional readouts, phosphorylation-sumoylation cross-talk mechanistically defined in single study\",\n      \"pmids\": [\"17942393\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The tandem PHD finger-bromodomain of KAP1 forms a unified structural scaffold in which the PHD finger and bromodomain cooperate as an intramolecular SUMO E3 ligase, facilitating lysine SUMOylation that is required for KAP1 co-repressor activity in gene silencing. Structure-function mutagenesis correlating UBC9 binding, SUMOylation, and transcriptional repression confirmed this.\",\n      \"method\": \"NMR solution structure determination, mutation-based structure-function analysis, SUMOylation assay, transcriptional repression assay, UBC9 binding assay\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure plus mutagenesis plus functional SUMOylation and transcription assays in one study\",\n      \"pmids\": [\"18488044\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Phosphorylation of TIF1beta/KAP1 at Ser473 by PKCdelta disrupts its interaction with HP1beta (at the PXVXL HP1-box), reducing co-repressor function and permitting expression of cell cycle genes (cyclin A2, Cdc2, Cdc25A). Unphosphorylated KAP1 allows HP1beta co-occupancy at target promoters and transcriptional repression; Ser473 phosphorylation coincides with S-phase.\",\n      \"method\": \"Site-directed mutagenesis (S473A, S473E), ChIP, co-immunoprecipitation, reporter assays, PKCdelta inhibitor treatment\",\n      \"journal\": \"BMC molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with ChIP and Co-IP in a single lab study with multiple orthogonal methods\",\n      \"pmids\": [\"18590578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"KAP1 genomic recruitment requires its RBCC domain for binding to 3' coding exons of zinc finger genes (via KRAB-ZNF interaction), but RBCC deletion does not abolish KAP1 binding to promoter regions, indicating a second, KRAB-ZNF-independent recruitment mechanism for promoter-bound KAP1.\",\n      \"method\": \"ChIP-seq, stable cell lines expressing tagged wild-type and RBCC-deletion mutant KAP1, KAP1 knockdown\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq with deletion mutants and knockdown, two complementary approaches in one study\",\n      \"pmids\": [\"21343339\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"De novo DNA methylation of endogenous retroviruses (ERVs) in embryonic stem cells requires a KRAB-ZFP that recognizes the specific ERV sequence, plus both KAP1 and ESET (SETDB1). KAP1 knockout in early embryos reduces methylation of ERVs. KAP1 thus acts downstream of KRAB-ZFPs and upstream of ESET to install stable DNA methylation marks at ERV loci.\",\n      \"method\": \"Genetic knockout (KAP1 KO in early embryos), reporter assay with introduced ERV sequences, bisulfite sequencing, ES cell differentiation model\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo KO plus reporter assay epistasis defining pathway order, replicated across multiple experimental systems\",\n      \"pmids\": [\"23293284\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"TRIM28 stabilizes paused RNA Pol II at promoter-proximal regions of many genes and regulates Pol II pause release in a transcription-coupled phosphorylation-dependent manner. TRIM28 depletion triggers de novo expression of genes regulated by paused Pol II genome-wide.\",\n      \"method\": \"In vitro Pol II pausing assay, RNAi knockdown, genome-wide ChIP-seq of Pol II occupancy, gene expression analysis\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro pausing assay plus genome-wide ChIP-seq plus in vivo knockdown, multiple orthogonal methods\",\n      \"pmids\": [\"25173174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The nuclear oncogene SET interacts with KAP1 and its overexpression causes sustained retention of KAP1 and HP1 on chromatin at double-strand breaks, leading to inhibition of DNA end resection and homologous recombination-mediated DNA repair.\",\n      \"method\": \"Co-immunoprecipitation, overexpression and depletion studies, chromatin fractionation, HR repair assay (I-SceI system)\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction plus functional HR assay, single lab\",\n      \"pmids\": [\"25818296\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"URI (unconventional prefoldin RPB5 interactor) forms a nuclear complex with KAP1 and PP2A phosphatase; PP2A recruited by URI decreases KAP1 phosphorylation. This URI-KAP1-PP2A complex mediates retrotransposon (LINE-1, L1PA2) repression, functionally overlapping with the KAP1-SETDB1 silencing complex.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, URI knockdown, microarray of transposon expression, phosphorylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus functional knockdown with transposon expression readout, single lab\",\n      \"pmids\": [\"27780869\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"TRIM28 forms a complex with EZH2 and SWI/SNF (distinct from PRC2) to activate, rather than repress, a set of stem cell maintenance genes in breast cancer cells. TRIM28 depletion represses EZH2 recruitment to chromatin and reduces mammosphere formation; rescue by EZH2 requires intact TRIM28 interaction (pre-SET domain).\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, ChIP, siRNA knockdown, mammosphere formation assay, EZH2 mutant rescue experiment\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with MS, ChIP, functional rescue with domain mutant, multiple orthogonal methods in single study\",\n      \"pmids\": [\"28068325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM28 acts as an E3 ubiquitin ligase for BCL2A1 at the mitochondria; endogenous TRIM28 and BCL2A1 physically interact and TRIM28 knockdown decreases BCL2A1 ubiquitination. TRIM17 antagonizes TRIM28 by blocking its binding to BCL2A1, stabilizing BCL2A1 and promoting chemoresistance.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, TRIM28 knockdown, TRIM17 overexpression and knockout\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP, ubiquitination assay, genetic loss-of-function with mechanistic antagonist characterization in single study\",\n      \"pmids\": [\"30042493\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"TRIM28 physically interacts with TRIM24 and prevents TRIM24 ubiquitination and SPOP-mediated degradation; TRIM28 also facilitates TRIM24 chromatin occupancy and augments AR signaling in prostate cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, ChIP, knockdown and overexpression in cell lines and xenograft models\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with ubiquitination and ChIP assays plus in vivo xenograft, multiple orthogonal methods\",\n      \"pmids\": [\"30479348\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"KAP1 associates with DNA replication factors PCNA, MCM3, and MCM6; these interactions are promoted by KAP1 phosphorylation at serine 473 during S phase. KAP1 forms a complex with PCNA and Suv39h1 histone methyltransferase to reinstate H3K9 methylation-dependent heterochromatin after DNA replication.\",\n      \"method\": \"Co-immunoprecipitation, phosphorylation-specific analysis (S473), ChIP, cell fractionation during S phase\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple Co-IPs identifying complex components, phosphorylation-regulated interaction, H3K9me3 reinstatement shown by ChIP\",\n      \"pmids\": [\"29955894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"TRIM28 promotes HIV-1 latency by SUMOylating CDK9 (catalytic subunit of P-TEFb) at lysines K44, K56, and K68 with SUMO4. This SUMOylation inhibits CDK9 kinase activity and/or prevents P-TEFb assembly by blocking CDK9-Cyclin T1 interaction, thereby suppressing HIV-1 transcriptional elongation.\",\n      \"method\": \"Global site-specific SUMO-MS, serial SUMOylation assays, site-directed mutagenesis of CDK9 residues, Co-immunoprecipitation, kinase activity assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mass spectrometry-identified SUMOylation sites confirmed by mutagenesis and kinase assay, multiple orthogonal methods\",\n      \"pmids\": [\"30652970\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The KAP1 TRIM domain forms antiparallel dimers that further assemble into tetramers and higher-order oligomers. Crystal structure identifies the KRAB domain binding site in the coiled-coil domain near the dimer dyad; mutations at this site abolish KRAB binding and retrotransposon silencing activity. B-box 1 mutations that prevent higher-order oligomerization do not significantly impair silencing.\",\n      \"method\": \"Crystal structure determination, biophysical analysis (SAXS, SEC-MALS), site-directed mutagenesis, retrotransposon silencing assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus functional silencing assay in single study\",\n      \"pmids\": [\"31289231\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"KAP1 is an elongated antiparallel dimer with functional asymmetry at the C-terminal domains. The RING domain contributes to KAP1 auto-SUMOylation. HP1 occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry.\",\n      \"method\": \"Solution scattering (SAXS), integrative modeling, single-molecule experiments, biochemical assays\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural determination with multiple biophysical methods plus biochemical validation of auto-SUMOylation and HP1 stoichiometry\",\n      \"pmids\": [\"31427381\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"KAP1/TRIM28 uses a chromatin reader cassette to bind hypo-acetylated histone H4 tails at promoters, then associates with RNA Pol II and recruits SMAD2 upon TGF-beta signaling to enable CDK9-dependent Pol II pause release. This couples chromatin reading to both Pol II pausing and pause release for transcriptional elongation.\",\n      \"method\": \"ChIP-seq, chromatin reader domain characterization, Co-immunoprecipitation, CDK9-dependent pause release assay, SMAD2 recruitment assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-seq, Co-IP, functional elongation assay, identification of novel reader cassette with ligand-responsive mechanism in single study\",\n      \"pmids\": [\"32402252\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM28 acts as an E3 SUMO ligase that binds NLRP3, promotes SUMO1/2/3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation, stabilizing NLRP3 protein levels to facilitate inflammasome assembly and activation. Trim28 deficiency attenuates NLRP3 inflammasome activation in vitro and in vivo.\",\n      \"method\": \"Co-immunoprecipitation, SUMOylation assay, ubiquitination assay, Trim28 conditional knockout, NLRP3 inflammasome activation assays (IL-1β secretion, caspase-1 cleavage)\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, SUMOylation and ubiquitination assays, in vivo KO, multiple readouts of inflammasome activity\",\n      \"pmids\": [\"34373456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"KAP1/TRIM28 represses HIV-1 expression in myeloid cells by interacting with and colocalizing with the viral transactivator Tat, promoting Tat's degradation via the proteasome. KAP1 is also bound to the latent HIV-1 promoter and cooperates with CTIP2, an epigenetic silencer; Tat and CTIP2 compete for KAP1 binding.\",\n      \"method\": \"Co-immunoprecipitation, colocalization (confocal microscopy), proteasome inhibitor treatment, KAP1 depletion, ChIP, myeloid HIV-1 latency model reactivation\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ChIP with functional knockdown, single lab\",\n      \"pmids\": [\"33514850\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"UBE2S interacts with TRIM28 in the nucleus; together they enhance ubiquitination of p27 to facilitate its proteasomal degradation and promote G1/S cell cycle progression in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cell cycle analysis, siRNA knockdown\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ubiquitination assay, functional cell cycle readout, single lab\",\n      \"pmids\": [\"33589597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"The SETDB1-TRIM28 complex suppresses antitumor immunity; inhibition of this complex upregulates PD-L1 and activates the cGAS-STING innate immune pathway by promoting micronuclei formation in the cytoplasm, increasing CD8+ T cell infiltration.\",\n      \"method\": \"CRISPR-Cas9 epigenetic screen, flow cytometry, cGAS-STING pathway activation assay, syngeneic mouse tumor model, SETDB1 KO\",\n      \"journal\": \"Cancer immunology research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional CRISPR screen and in vivo mouse model, mechanistic pathway placement via cGAS-STING, single lab\",\n      \"pmids\": [\"34848497\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM28 acts as a negative regulator of aggresome formation through direct interaction with CTIF; this interaction leads to inefficient aggresomal targeting of misfolded polypeptides. TRIM28 phosphorylation at S473 by double-stranded RNA-activated protein kinase (PKR) promotes TRIM28-CTIF association, inhibits aggresome formation, and suppresses viral proliferation.\",\n      \"method\": \"Co-immunoprecipitation, proximity ligation assay, phosphorylation analysis (S473), aggresome formation assay, influenza A virus infection model\",\n      \"journal\": \"Autophagy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct interaction by Co-IP and PLA, phosphorylation-regulated mechanism with functional viral readout, multiple orthogonal methods\",\n      \"pmids\": [\"33783327\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"TRIM28 is a transcriptional activator of the mutant TERT promoter. TRIM28 is recruited to the mutant hTERT promoter and interacts with TRIM24, which inhibits TRIM28 activity. Phosphorylation of TRIM28 via mTORC1 releases it from TRIM24 and induces hTERT transcription; mTORC1 inhibition suppresses TRIM28 phosphorylation and hTERT expression.\",\n      \"method\": \"CRISPR-Cas9 kinome knockout screen (FACS-based), ChIP, Co-immunoprecipitation, mTORC1 inhibition (rapamycin analog), phosphorylation assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen validated by ChIP, Co-IP, and pharmacological inhibition with mechanistic phosphorylation characterization\",\n      \"pmids\": [\"34518220\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structure of the KAP1 TRIM in complex with the KRAB domain of ZNF93 identifies the KAP1-KRAB binding interface. Structure-guided mutations in this interface abolish repressive activity in a transcriptional silencing assay and eliminate genome-wide H3K9me3 deposition at thousands of KAP1/KRAB-ZFP target loci.\",\n      \"method\": \"Crystal structure determination, site-directed mutagenesis, epigenetic transcriptional silencing assay, ChIP-seq (H3K9me3 genome-wide)\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus genome-wide ChIP-seq functional validation\",\n      \"pmids\": [\"36341546\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"TRIM28-dependent SUMOylation is required to maintain the adult ovarian cell fate by repressing testicular-specific genes. TRIM28 is recruited to chromatin near FOXL2 binding sites, and its E3-SUMO ligase activity regulates the sex-specific SUMOylation profile of ovarian-specific genes. Loss of Trim28 in granulosa cells leads to their transdifferentiation into Sertoli cells.\",\n      \"method\": \"Conditional knockout (Trim28 deletion in granulosa cells), ChIP, SUMOylation profiling, histology/lineage tracing\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO with mechanistic SUMOylation profiling and ChIP, phenotype directly linked to SUMO ligase activity\",\n      \"pmids\": [\"35906245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"KAP1 and the KRAB domain residues forming the molecular interface were structurally mapped using AlphaFold2; leucine 301 on each chain of the TRIM28 coiled-coil acts as a 'pin' inserting into a hydrophobic pocket on the KRAB domain. Site-directed mutations at this interface abolish KRAB domain binding.\",\n      \"method\": \"AlphaFold2 modeling, site-directed mutagenesis, binding assays\",\n      \"journal\": \"Protein science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — computational structural model with mutagenesis-based binding validation, no X-ray or cryo-EM confirmation\",\n      \"pmids\": [\"36173157\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 directly binds and stabilizes PD-L1 by inhibiting PD-L1 ubiquitination and promoting PD-L1 SUMOylation. Additionally, TRIM28 facilitates K63 polyubiquitination of TBK1, activating TBK1-IRF1 and TBK1-mTOR pathways to enhance PD-L1 transcription.\",\n      \"method\": \"CRISPR-Cas9 genome-wide screen, Co-immunoprecipitation, ubiquitination assay, SUMOylation assay, in vivo mouse tumor model, TBK1 pathway inhibitor studies\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR screen, multiple biochemical assays (Co-IP, ubiquitination, SUMOylation), in vivo validation with mechanistic pathway dissection\",\n      \"pmids\": [\"37357254\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 promotes ubiquitination and proteasome-mediated degradation of TFE3 transcription factor in renal cancer cells, thereby suppressing TFE3-driven autophagic gene expression and cell proliferation. This defines a TRIM28-TFE3-KDM6A signaling axis in kidney cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, TRIM28 knockdown, ChIP, autophagy assays, cell proliferation assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, ChIP with functional readouts, single lab\",\n      \"pmids\": [\"36935008\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 physically interacts with RIPK1 and promotes K63-linked ubiquitination of RIPK1, sustaining NF-κB pathway activation and upregulation of CXCL1, which recruits MDSCs. Mutagenesis of the TRIM28 RING/E3 ligase domain (C65, C68) abrogates NF-κB activation.\",\n      \"method\": \"Co-immunoprecipitation, K63-ubiquitination assay, RING domain mutagenesis, NF-κB reporter, CXCL1 ELISA, syngeneic mouse tumor models\",\n      \"journal\": \"Journal of experimental & clinical cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mutagenesis validation, ubiquitination assay, and in vivo tumor models in single study\",\n      \"pmids\": [\"37865804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 acts as the E3 SUMO ligase responsible for SUMOylation of the SARS-CoV-2 nucleocapsid protein (SARS2-NP) at lysine K65, which mediates NP homo-oligomerization, RNA association, liquid-liquid phase separation, and suppression of innate antiviral immunity. An interfering peptide targeting the TRIM28-SARS2-NP interaction blocks NP SUMOylation and LLPS.\",\n      \"method\": \"SUMOylation assay, site-directed mutagenesis (K65), LLPS assay, Co-immunoprecipitation, antiviral immune assay, interfering peptide screen\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — mutagenesis of SUMOylation site plus biochemical reconstitution of LLPS plus functional immune assay in single study\",\n      \"pmids\": [\"38172120\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 negatively regulates the RLR signaling pathway by targeting MAVS for K48-linked polyubiquitination and proteasome-mediated degradation. The RING domain of TRIM28 (specifically C65 and C68 residues) is critical for this activity; ubiquitin chains are transferred to K7, K10, K371, K420, and K500 of MAVS.\",\n      \"method\": \"Ubiquitination assay, proteasome inhibitor treatment, RING domain mutagenesis, Co-immunoprecipitation, type I interferon production assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — RING domain mutagenesis with site-specific ubiquitin mapping (K-residues identified) plus functional IFN assay, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"37119745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TIAM1 interacts with TRIM28 in the nucleus of NSCLC cells and the TIAM1-TRIM28 complex promotes H3K9me3-induced silencing of protocadherins and decreases E-cadherin expression, driving epithelial-to-mesenchymal transition and cell migration.\",\n      \"method\": \"Co-immunoprecipitation, ChIP (H3K9me3), siRNA knockdown, migration/invasion assay, rescue by protocadherin depletion\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ChIP plus functional rescue experiment, single lab\",\n      \"pmids\": [\"37748077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 binds ACSL4 and promotes SUMO3 modification at ACSL4 lysine K532, inhibiting K63-linked ACSL4 ubiquitination and thereby suppressing OPTN-dependent autophagic degradation of ACSL4, promoting neuronal ferroptosis. SENP3 acts as the opposing deSUMOylation enzyme.\",\n      \"method\": \"Co-immunoprecipitation, SUMOylation assay, ubiquitination assay, mutagenesis (K532), autophagic degradation assay, Trim28 genetic deletion, in vivo SCI mouse model\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP, site-specific mutagenesis, multiple PTM assays (SUMO, ubiquitin), in vivo knockout with behavioral readout, multiple orthogonal methods\",\n      \"pmids\": [\"39875520\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM28 facilitates type I interferon activation by interacting with TBK1 and mediating K63-linked ubiquitination of TBK1, augmenting TBK1 signal transmission; TRIM28 KO cells display defective TBK1 phosphorylation and impaired complex assembly with IRF3.\",\n      \"method\": \"CRISPR-Cas9 TRIM28 knockout, Co-immunoprecipitation, K63-ubiquitination assay, TBK1 phosphorylation assay, virus infection susceptibility assay\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR KO with Co-IP and ubiquitination assay, single lab\",\n      \"pmids\": [\"38495890\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"TRIM28 acts as an E3 ubiquitin ligase for BRD7; the coiled-coil region of TRIM28 binds the N-terminal domain of BRD7 and mediates BRD7 ubiquitination at K21, promoting its proteasomal degradation. This promotes breast cancer malignant progression.\",\n      \"method\": \"Co-immunoprecipitation with mass spectrometry, ubiquitination assay, domain deletion/mutagenesis (K21), xenograft mouse model\",\n      \"journal\": \"Cellular oncology (Dordrecht, Netherlands)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP/MS for interaction, site-specific ubiquitin mutagenesis, in vivo xenograft, multiple orthogonal methods in single study\",\n      \"pmids\": [\"39222175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KAP1 directly interacts with IRF5 via IRF5's N-terminus (DNA-binding domain and disordered region); KAP1 knockdown potentiates IRF5-mediated TNF and M1 macrophage marker expression. This inhibitory effect is linked to SETDB1 methyltransferase activity (H3K9me3 deposition) at the TNF locus.\",\n      \"method\": \"Affinity purification with mass spectrometry (IRF5 interactome), Co-immunoprecipitation, domain mapping, KAP1 siRNA knockdown, ChIP (H3K9me3)\",\n      \"journal\": \"Immunobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS interaction confirmed by Co-IP and domain mapping, functional knockdown with ChIP, single lab\",\n      \"pmids\": [\"22995936\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"KAP1 physically associates with endogenous STAT3 in cells; KAP1 knockdown enhances IL-6-induced STAT3-dependent transcription and causes marked accumulation of STAT3 phosphorylated on Ser727 in the nucleus, indicating KAP1 acts as a transcriptional repressor of the IL-6/STAT3 signaling pathway.\",\n      \"method\": \"Yeast two-hybrid screen, endogenous Co-immunoprecipitation, siRNA knockdown, reporter gene assay, phospho-STAT3 analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — endogenous Co-IP plus functional siRNA knockdown, single lab\",\n      \"pmids\": [\"18037959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Kap1 interacts with Oct4 and inhibits Itch-mediated ubiquitination of Oct4 at lysine K133, thereby stabilizing Oct4 protein in embryonic stem cells and promoting self-renewal and cellular reprogramming.\",\n      \"method\": \"Affinity purification with mass spectrometry, Co-immunoprecipitation, ubiquitination assay, site-directed mutagenesis (K133R), Kap1 knockdown and overexpression, reprogramming assay\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — AP-MS identification confirmed by Co-IP, site-specific mutagenesis of ubiquitination site, functional reprogramming assay, multiple orthogonal methods\",\n      \"pmids\": [\"32895487\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"KAP1-dependent H3K9me3 repressive chromatin is established at T-cell-specific cis-regulatory elements enriched in Ikaros/NuRD complexes; KAP1 directly controls FoxO1 expression and TCR/cytokine signaling genes. T-cell-specific Kap1 deletion causes expansion of immature thymocytes and altered CD4+/CD8+ ratios.\",\n      \"method\": \"T-cell-specific conditional Kap1 KO (Cre-lox), ChIP/ChIP-seq, transcriptome analysis\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo conditional KO with ChIP-seq, single lab\",\n      \"pmids\": [\"22872677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"TRIM28 modulates uterine function by complexing with estrogen receptor α (ERα) and progesterone receptor (PR); TRIM28 ablation suppresses PR and ERα chromatin binding in uterine epithelium and stroma, impairing implantation and early pregnancy.\",\n      \"method\": \"RIME (rapid immunoprecipitation mass spectrometry), Co-immunoprecipitation, conditional knockout, ChIP\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — RIME and Co-IP for interaction, conditional KO for function, ChIP for chromatin binding, multiple orthogonal methods in single study\",\n      \"pmids\": [\"37528140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"MAGE-C2 binds KAP1 and increases co-precipitation of KAP1 with ATM kinase; MAGE-C2 binding to KAP1 is required for enhanced ATM-dependent phosphorylation of KAP1-Ser824, which facilitates heterochromatin relaxation and DNA double-strand break repair.\",\n      \"method\": \"Co-immunoprecipitation, KAP1 S824 phosphorylation assay, I-SceI endonuclease DSB repair assay, MAGE-C2 knockdown/overexpression\",\n      \"journal\": \"The Journal of investigative dermatology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP interaction with functional phosphorylation and repair assays, single lab\",\n      \"pmids\": [\"23096706\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CCAR2/DBC1 is required for Chk2-dependent KAP1 phosphorylation at Ser824; loss of CCAR2 impairs Chk2 activation and reduces KAP1 S824 phosphorylation, leading to defective heterochromatin relaxation and impaired repair of heterochromatic DSBs.\",\n      \"method\": \"CCAR2 knockout, Chk2 activation assay, KAP1 S824 phosphorylation assay, DNA damage foci resolution (late time-point), HP1β depletion rescue\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO with phosphorylation assay and functional DNA repair epistasis, single lab\",\n      \"pmids\": [\"26158765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"At the onset of X chromosome inactivation, KAP1 is recruited to the Xist promoter allele that will upregulate Xist and is required for the increase in Tsix levels preceding cell fate choice. RIF1 and KAP1 show mutual exclusion at the Xist promoters, establishing an asymmetric self-sustaining loop for X chromosome choice.\",\n      \"method\": \"mESC differentiation, allele-specific ChIP, KAP1 and RIF1 depletion, Tsix/Xist RNA FISH\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — allele-specific ChIP and functional depletion, single lab\",\n      \"pmids\": [\"34786738\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"KAP1 interacts with KAP1 regulates ERV (including HERV-T, HERV-S) and ZNF gene expression in adult differentiated human cells including peripheral blood mononuclear cells, requiring H3K9me3. KAP1 depletion leads to decreased H3K9me3 at ERVs and ZNF loci and activates an RNA-sensing response mediated through MAVS signaling.\",\n      \"method\": \"KAP1 knockout, RNA-seq, ChIP-seq (H3K9me3, KAP1), MAVS signaling assay, interferon response measurement\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with ChIP-seq and pathway activation assay, single lab\",\n      \"pmids\": [\"30061100\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"TRIM28/KAP1 is a multifunctional nuclear scaffold protein that acts as: (1) a co-repressor recruited by KRAB-ZFPs to silence endogenous retroviruses and transposable elements via assembly of an H3K9me3/SETDB1 heterochromatin complex, using its RBCC/TRIM domain to bind KRAB domains and its PHD finger-bromodomain as an intramolecular SUMO E3 ligase; (2) a regulator of RNA Pol II pausing and pause release through a chromatin reader cassette that binds hypo-acetylated histone H4 and recruits CDK9/P-TEFb; (3) an E3 ubiquitin and E3 SUMO ligase for diverse substrates including NLRP3, MAVS, BCL2A1, RIPK1, TBK1, ACSL4, and TFE3, with substrate specificity regulated by phosphorylation (ATM at S824 in DNA damage, mTORC1 and PKCdelta at S473); and (4) a signaling scaffold that controls p53 stability (via MDM2), pluripotency (via Oct4), and innate immune responses, with all activities tightly regulated by a phosphorylation-SUMOylation cross-talk switch.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TRIM28 (KAP1/TIF1beta) is a multifunctional nuclear scaffold that couples sequence-specific transcription factor recruitment to heterochromatin assembly, RNA Pol II control, and post-translational modification of diverse substrates [#5, #16, #23]. Its RBCC/TRIM domain forms antiparallel dimers that present a coiled-coil interface—pinned by Leu301—for binding the KRAB domains of zinc-finger proteins, and structure-guided disruption of this interface abolishes transcriptional silencing and genome-wide H3K9me3 deposition at thousands of KAP1/KRAB-ZFP loci [#14, #23, #25]. Recruited by KRAB-ZFPs, TRIM28 acts downstream of these factors and upstream of the SETDB1/ESET methyltransferase to install stable H3K9me3 and de novo DNA methylation that silence endogenous retroviruses and transposable elements [#5, #43], with HP1 binding contributing to repressive complex assembly [#15]. Its tandem PHD finger–bromodomain functions as an intramolecular SUMO E3 ligase whose auto-SUMOylation is required for co-repressor activity [#2, #15]. Beyond silencing, a chromatin reader cassette binds hypo-acetylated histone H4 at promoters, stabilizes paused Pol II, and recruits CDK9/P-TEFb and SMAD2 to drive pause release upon TGF-beta signaling [#6, #16]. TRIM28 additionally serves as an E3 ubiquitin and SUMO ligase for an extensive substrate set—targeting MAVS, RIPK1, TBK1, NLRP3, BCL2A1, ACSL4, TFE3, PD-L1, BRD7, and p27 for ubiquitination or SUMOylation—thereby controlling innate immune signaling, inflammasome activation, ferroptosis, cell-cycle progression, and antitumor immunity [#10, #17, #26, #28, #30, #32, #33]. TRIM28 also stabilizes p53-degrading MDM2 complexes and Oct4 to govern p53 turnover and pluripotency [#0, #37]. All of these activities are gated by a phosphorylation–SUMOylation cross-talk switch: ATM phosphorylation of Ser824 reduces SUMOylation to de-repress damage-response genes, while PKCdelta/mTORC1/PKR phosphorylation of Ser473 reconfigures partner interactions across the cell cycle, DNA replication, and stress responses [#1, #3, #12, #21, #22].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established TRIM28 as a positive regulator of p53 turnover, linking it to oncogenic signaling before its silencing roles were fully appreciated.\",\n      \"evidence\": \"Reciprocal Co-IP, RNAi, ubiquitination and competition assays mapping the KAP1 coiled-coil to the MDM2 acidic domain\",\n      \"pmids\": [\"16107876\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address whether SUMOylation or phosphorylation gates the MDM2 interaction\", \"Physiological contexts where ARF competition dominates unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defined the central phosphorylation–SUMOylation switch by showing ATM phosphorylation of Ser824 antagonizes SUMOylation to de-repress damage-response genes.\",\n      \"evidence\": \"S824A/S824D mutagenesis, ATM kinase and SUMOylation assays, reporter assays, ChIP\",\n      \"pmids\": [\"17942393\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Which SUMO sites are coupled to S824 not pinpointed\", \"Genome-wide scope of the switch not assessed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Resolved how TRIM28 generates its own activating modification and how a second phospho-site tunes HP1 binding, mechanizing the PTM-controlled co-repressor.\",\n      \"evidence\": \"NMR structure of the PHD–bromodomain SUMO ligase; S473 mutagenesis with ChIP and Co-IP showing PKCdelta disrupts HP1beta binding\",\n      \"pmids\": [\"18488044\", \"18590578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Intramolecular vs intermolecular SUMO transfer in vivo not fully delineated\", \"S473 kinase repertoire beyond PKCdelta unknown at this stage\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Showed KAP1 recruitment is bipartite—RBCC/KRAB-ZFP-dependent at zinc-finger gene bodies but KRAB-independent at promoters—revealing more than one targeting mode.\",\n      \"evidence\": \"ChIP-seq with RBCC-deletion mutants and knockdown\",\n      \"pmids\": [\"21343339\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Identity of the KRAB-independent promoter recruiter not determined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Placed KAP1 epistatically between KRAB-ZFPs and SETDB1 in establishing stable DNA methylation at ERVs, ordering the silencing pathway.\",\n      \"evidence\": \"KAP1 KO in early embryos, ERV reporter assays, bisulfite sequencing in ES cells\",\n      \"pmids\": [\"23293284\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking H3K9me3 to de novo DNA methyltransferase recruitment not resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Expanded TRIM28 function beyond silencing to direct control of Pol II promoter-proximal pausing genome-wide.\",\n      \"evidence\": \"In vitro pausing assay, Pol II ChIP-seq, RNAi knockdown\",\n      \"pmids\": [\"25173174\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin features directing TRIM28 to paused genes not yet defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified TRIM28 partners that retune its DNA-repair and stem-cell roles, showing context-dependent regulation of chromatin retention.\",\n      \"evidence\": \"Co-IP and HR repair assays for SET; reciprocal Co-IP/MS, ChIP and mammosphere assays for an EZH2/SWI/SNF activating complex\",\n      \"pmids\": [\"25818296\", \"28068325\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SET-KAP1 interaction shown by single-lab Co-IP without reciprocal structural mapping\", \"How TRIM28 switches between PRC2-independent activation and repression unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined a URI-KAP1-PP2A complex that dephosphorylates KAP1 to sustain retrotransposon repression, mechanizing phosphatase control of the switch.\",\n      \"evidence\": \"Co-IP/MS, URI knockdown, transposon microarray, phosphorylation assay\",\n      \"pmids\": [\"27780869\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab; specific KAP1 phospho-sites targeted by PP2A not mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Catalogued TRIM28 as a substrate-specific E3 ligase and replication-coupled heterochromatin factor, broadening its enzymatic and cell-cycle roles.\",\n      \"evidence\": \"Co-IP and ubiquitination assays for BCL2A1 (TRIM17-antagonized) and TRIM24 (anti-SPOP); S473-promoted Co-IP with PCNA/MCM3/MCM6 and Suv39h1 during S phase\",\n      \"pmids\": [\"30042493\", \"30479348\", \"29955894\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether E3 activity requires RING dimerization not addressed here\", \"Direct vs scaffolded ubiquitin transfer to each substrate not always distinguished\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Provided high-resolution structural and biophysical definition of the TRIM28 dimer, KRAB-binding interface, and asymmetric HP1 stoichiometry.\",\n      \"evidence\": \"Crystal structure, SAXS/SEC-MALS, single-molecule and SUMOylation assays; CDK9 SUMOylation by SUMO-MS and mutagenesis for HIV latency\",\n      \"pmids\": [\"31289231\", \"31427381\", \"30652970\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of higher-order oligomerization beyond silencing unclear\", \"How asymmetry is read out by partners not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mechanized the chromatin-reading-to-elongation coupling, showing TRIM28 reads hypo-acetylated H4 and recruits SMAD2/CDK9 for ligand-responsive pause release; also stabilizes Oct4.\",\n      \"evidence\": \"ChIP-seq, reader-domain characterization, Co-IP, CDK9-dependent pause-release assay; AP-MS/Co-IP and K133R mutagenesis showing KAP1 blocks Itch ubiquitination of Oct4\",\n      \"pmids\": [\"32402252\", \"32895487\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the same protein switches between Pol II stabilization and release at a given gene not fully defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established TRIM28 as a master post-translational regulator of innate immunity and inflammasome stability, with antitumor-immunity consequences.\",\n      \"evidence\": \"Co-IP, SUMOylation/ubiquitination assays and conditional KO for NLRP3 stabilization; CRISPR screen and cGAS-STING readouts for SETDB1-TRIM28 immune suppression; PKR-S473-driven CTIF interaction for aggresome/viral control; mTORC1-driven mutant TERT activation\",\n      \"pmids\": [\"34373456\", \"34848497\", \"33783327\", \"34518220\", \"33514850\", \"33589597\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect SUMO transfer to NLRP3 across cell types not fully resolved\", \"How distinct phospho-sites partition immune versus chromatin functions unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Confirmed the KRAB-binding interface by crystallography and AlphaFold2, and extended SUMO-ligase control to cell-fate maintenance and X-inactivation choice.\",\n      \"evidence\": \"Crystal structure of TRIM/ZNF93-KRAB with mutagenesis and H3K9me3 ChIP-seq; AlphaFold2 Leu301 'pin' model; granulosa-cell conditional KO with SUMO profiling; allele-specific ChIP at Xist\",\n      \"pmids\": [\"36341546\", \"36173157\", \"35906245\", \"34786738\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"AlphaFold2 interface lacks independent X-ray/cryo-EM confirmation\", \"How SUMOylation enforces lineage-specific gene sets mechanistically incomplete\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Mapped TRIM28's substrate-specific ubiquitin/SUMO ligase activities onto immune checkpoint, RLR signaling, ferroptosis, EMT, and reproductive programs, with RING residues C65/C68 as the catalytic determinant.\",\n      \"evidence\": \"CRISPR screens, Co-IP, ubiquitination and SUMOylation assays, RING mutagenesis, in vivo tumor and SCI models, RIME for ERalpha/PR; substrates PD-L1, RIPK1, MAVS, ACSL4, TFE3, SARS2-NP\",\n      \"pmids\": [\"37357254\", \"37865804\", \"37119745\", \"39875520\", \"36935008\", \"37748077\", \"38172120\", \"37528140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Opposing ubiquitin- vs SUMO-promoting outcomes on shared substrates not unified into a single rule\", \"How substrate selection is encoded in TRIM28 not defined\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Extended the E3 ligase substrate repertoire (BRD7, TBK1) and clarified that TRIM28 can both activate and degrade innate signaling components depending on chain linkage.\",\n      \"evidence\": \"Co-IP/MS, K21 and chain-type-specific ubiquitination assays, CRISPR KO, xenograft and infection models\",\n      \"pmids\": [\"39222175\", \"38495890\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of TRIM28 promoting (K63-TBK1) versus degrading (K48-MAVS) innate signaling not mechanistically integrated\", \"TBK1 result single-lab\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single scaffold partitions its silencing, Pol II-elongation, and dozens of distinct ubiquitin/SUMO substrate activities—and whether the phospho-SUMO switch alone encodes this selectivity—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking specific phospho-states to specific substrate or chromatin outputs\", \"Determinants of ubiquitin-chain-linkage choice (K48 vs K63) versus SUMO modification on substrates undefined\", \"Stoichiometry/competition among the many named partners in cells not measured\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [10, 17, 26, 28, 30, 32, 33, 34]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [2, 13, 17, 24, 29, 32]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [6, 16, 22, 23]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [16, 12]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 16, 0]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [4, 23]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [16, 19, 31, 36]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [5, 12, 23, 42]},\n      {\"term_id\": \"GO:0005739\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [5, 12, 23, 31, 43]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [6, 16, 22]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [17, 26, 30, 33, 35, 43]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 10, 19, 27, 34, 37]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [1, 40, 41]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [3, 12, 19]}\n    ],\n    \"complexes\": [\n      \"KAP1-SETDB1 heterochromatin complex\",\n      \"URI-KAP1-PP2A complex\",\n      \"TRIM28-EZH2-SWI/SNF complex\"\n    ],\n    \"partners\": [\n      \"SETDB1\",\n      \"HP1\",\n      \"MDM2\",\n      \"TRIM24\",\n      \"CDK9\",\n      \"SMAD2\",\n      \"Oct4\",\n      \"EZH2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}