{"gene":"E2F6","run_date":"2026-06-09T23:54:42","timeline":{"discoveries":[{"year":1998,"finding":"E2F6 lacks transactivation and retinoblastoma protein (pRB/p107/p130) binding domains, forms heterodimers with DP-1 and DP-2 proteins in vitro and in vivo, binds E2F recognition sites (preferring TTTCCCGC), and represses transcription of E2F-responsive promoters by a pRB-independent mechanism; its inhibitory effect depends on DNA binding activity and DP heterodimerization.","method":"Co-immunoprecipitation, in vitro binding assays, reporter gene assays, dominant-negative overexpression, DNA binding assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (in vitro binding, Co-IP, reporter assays, mutagenesis of DNA-binding domain); independently replicated across three labs in 1998 (PMID:9704927, PMID:9501179, PMID:9689056)","pmids":["9704927","9501179","9689056"],"is_preprint":false},{"year":1998,"finding":"E2F6 overexpression in NIH 3T3 cells inhibited entry into S phase of serum-stimulated quiescent cells but had no specific effect on asynchronously cycling cells, indicating it can regulate a subset of E2F-dependent genes required for cell cycle entry from G0.","method":"Cell cycle analysis by flow cytometry, serum-stimulation of G0-arrested NIH 3T3 cells with E2F6 overexpression","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean overexpression with defined cellular phenotype (G0 re-entry), single lab, two cell lines tested","pmids":["9689056"],"is_preprint":false},{"year":2001,"finding":"E2F6 interacts with RYBP (a mammalian polycomb complex member) through the E2F6 repression domain, and endogenous E2F6 associates with polycomb group proteins RYBP, Ring1, MEL-18, mph1, and Bmi1 in a multiprotein complex.","method":"Yeast two-hybrid screen to identify RYBP; co-immunoprecipitation of endogenous proteins; domain mapping","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP of endogenous proteins, yeast two-hybrid, domain mapping; replicated across multiple studies","pmids":["11171983"],"is_preprint":false},{"year":2002,"finding":"E2f6 knockout mice are viable but display homeotic transformations of the axial skeleton similar to polycomb mutant mice; E2f6−/− mouse embryonic fibroblasts proliferate normally, demonstrating that E2F6 is required for developmental patterning but not normal cell cycle progression.","method":"Targeted gene deletion in mice; skeletal analysis; MEF proliferation assays","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean knockout mouse model with specific skeletal phenotype and negative proliferation result; independently replicated by Courel et al. 2008","pmids":["12101104"],"is_preprint":false},{"year":2004,"finding":"E2F6 associates specifically with promoters of G1/S-regulated E2F target genes during S phase but not with G2/M-regulated E2F target genes; loss of E2F6 allows E2F4 to substitute at G1/S promoters, and combined inhibition of E2F4 and E2F6 causes specific derepression of G1/S genes during S phase.","method":"Chromatin immunoprecipitation (ChIP) during synchronized cell cycle; E2F6 knockdown and E2F4/6 double inhibition with specific derepression assays","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP across cell cycle combined with genetic epistasis (double loss-of-function), rigorous controls, single lab but multiple orthogonal methods","pmids":["15574595"],"is_preprint":false},{"year":2004,"finding":"E2F6 forms a stable core complex with DP1 and the polycomb group protein EPC1 in vitro and in vivo; this complex further associates with EZH2 and Sin3B specifically in proliferating cells (not quiescent cells), defining a novel proliferation-specific E2F6-PcG complex.","method":"Yeast two-hybrid screen; in vitro and in vivo co-immunoprecipitation; affinity purification from proliferating vs. quiescent cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — affinity purification, reciprocal Co-IP, in vitro binding, cell-state comparison; single lab but multiple orthogonal methods","pmids":["15536069"],"is_preprint":false},{"year":2003,"finding":"E2F6 represses endogenous target promoters including BRCA1, CTIP, ART27, HP1alpha, and RBAP48 in human cells; depletion of E2F6 by RNA interference results in recruitment of E2F1 to these promoters; repression does not require histone H3 methylation at lysine 9.","method":"ChIP combined with genomic microarrays (ChIP-chip) to identify E2F6-bound promoters; RNAi knockdown; ChIP before and after knockdown","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Moderate — ChIP-chip for genome-wide binding, RNAi loss-of-function with re-ChIP showing E2F1 replacement; multiple orthogonal methods, single lab","pmids":["12909625"],"is_preprint":false},{"year":2005,"finding":"E2F6 is required to silence meiosis-specific genes SMC1beta and STAG3 in somatic cells; E2F6 binds their promoters in vivo through a conserved binding site; transcriptional repression involves histone H3 methylation at both lysine 9 and lysine 27.","method":"cDNA microarray comparing wild-type and E2f6−/− MEFs; ChIP assays; re-expression rescue experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — knockout MEF transcriptomics + ChIP + rescue re-expression; multiple orthogonal methods; consistent with in vivo E2f6−/− phenotype","pmids":["16236716"],"is_preprint":false},{"year":2005,"finding":"E2F1 activating E2Fs bind the human E2F6 promoter at two functional E2F binding sites in vivo and transcriptionally upregulate E2F6 expression, establishing a regulatory feedback loop during G1/S transition.","method":"Promoter deletion/mutation reporter assays; EMSA; ChIP; E2F1 overexpression; synchronized cell cycle analysis","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP in vivo binding, EMSA, promoter reporter with mutagenesis; single lab, multiple orthogonal methods","pmids":["16107498"],"is_preprint":false},{"year":2006,"finding":"PHC3 (a hPRC-H polycomb complex component) associates with E2F6 and colocalizes with E2F6 in nuclear punctate bodies in differentiating/confluent (G0) cells but not in proliferating cells, suggesting a G0-specific E2F6-polycomb silencing complex.","method":"Co-immunoprecipitation; immunofluorescence colocalization in differentiating vs. proliferating cells","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP and colocalization; single lab, two orthogonal methods but no functional rescue","pmids":["17001316"],"is_preprint":false},{"year":2008,"finding":"The HPV E7 oncoprotein (low- and high-risk types), as well as SV40 T antigen and adenovirus E1A, associate with E2F6 and inactivate its transcriptional repression activity; E7-expressing cells show decreased E2F6/polycomb complex staining in a manner dependent on E2F6 association.","method":"Co-immunoprecipitation; transcriptional repression reporter assays; immunofluorescence staining of E2F6/polycomb complexes","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, reporter assay, immunofluorescence; single lab, multiple orthogonal methods","pmids":["18579589"],"is_preprint":false},{"year":2008,"finding":"A conserved TCCCGC E2F6-binding element is present in promoters of 79% of meiosis-specific genes; E2F6 binds in vivo to meiotic gene promoters (including Slc25a31/Ant4) and represses their expression in somatic cells; E2F6 overexpression reduces meiotic gene transcription.","method":"Bioinformatic promoter analysis; ChIP assays; E2f6 knockout MEF gene expression analysis; E2F6 overexpression","journal":"Biology of reproduction","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, knockout MEF analysis, overexpression rescue; single lab, multiple methods","pmids":["18667754"],"is_preprint":false},{"year":2008,"finding":"E2f6 and Bmi1 genetically cooperate in axial skeleton development and Hox gene repression in vivo (double mutant mice show enhanced skeletal transformations), but E2F6 does not cooperate with Bmi1 in repression of the Ink4a-Arf locus.","method":"Mouse genetic epistasis using E2f6−/− and Bmi1−/− compound mutants; skeletal analysis; gene expression","journal":"Developmental dynamics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in vivo with compound mutant mice; clean phenotypic readout for two distinct loci","pmids":["18366140"],"is_preprint":false},{"year":2010,"finding":"Dnmt3b is recruited to promoters of germline/meiotic genes in somatic tissues in an E2F6-dependent manner; E2F6 binding is a common feature of promoters upregulated when Dnmt3b activity is impaired; E2F6 binding is required for CpG methylation-based silencing of these genes.","method":"Dnmt3b hypomorphic mutant mice; DNA microarray; ChIP assays for E2F6 binding at target promoters; bisulfite sequencing for DNA methylation","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo mouse model, ChIP, bisulfite sequencing, microarray; multiple orthogonal methods, single lab","pmids":["20439742"],"is_preprint":false},{"year":2013,"finding":"Chk1 phosphorylates E2F6 in response to DNA replication stress, causing E2F6 to dissociate from G1/S target promoters; this releases repression and maintains high E2F-dependent transcription, which is required to prevent DNA damage and cell death during checkpoint activation.","method":"In vitro kinase assay (Chk1 phosphorylating E2F6); ChIP showing E2F6 dissociation from promoters after replication stress; siRNA knockdown of Chk1; cell viability and DNA damage assays","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1–2 / Moderate — in vitro kinase assay establishing direct phosphorylation, ChIP for chromatin binding, epistasis via Chk1 knockdown; single lab, multiple orthogonal methods","pmids":["23954429"],"is_preprint":false},{"year":2013,"finding":"E2f6-mediated repression of meiotic genes Stag3 and Smc1β during embryonic development requires EZH2 enzymatic activity (Polycomb repressive complex 2) but not Dnmt3b; EZH2 SET-domain deletion impairs repression during embryoid body differentiation.","method":"ESC-to-EpiSC transition analysis; EZH2 SET-domain deletion; Dnmt3b knockout; bisulfite sequencing; ChIP for E2f6 binding; gene expression analysis","journal":"Epigenetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic dissection with EZH2 and Dnmt3b knockouts, ChIP, bisulfite sequencing; single lab","pmids":["23880518"],"is_preprint":false},{"year":2012,"finding":"E2F6 interacts with BRG1 (ATPase subunit of SWI/SNF chromatin-remodeling complex) and BAF155; BRG1 co-occupies G1/S gene promoters coincident with E2F6 during S phase, suggesting E2F6 recruits SWI/SNF for transcriptional repression.","method":"Co-immunoprecipitation; ChIP showing co-occupancy of BRG1 and E2F6 at G1/S promoters during S phase","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ChIP co-occupancy; single lab, two orthogonal methods","pmids":["23082233"],"is_preprint":false},{"year":2018,"finding":"Within the PRC1.6 complex, MGA, L3MBTL2, and E2F6 each mediate recruitment to distinct sets of genomic loci; CRISPR/Cas-mediated ablation of MGA causes complete loss of PRC1.6 binding genome-wide, while depletion of L3MBTL2 or E2F6 (but not PCGF6) causes locus-specific loss; E2F6 thus mediates PRC1.6 loading to a distinct subset of promoters.","method":"ChIP-seq genome-wide; CRISPR/Cas ablation of individual subunits; rescue experiments with DNA-binding mutants","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide ChIP-seq, CRISPR knockouts of multiple subunits, rescue experiments; rigorous and comprehensive in single study","pmids":["29381691"],"is_preprint":false},{"year":2021,"finding":"E2F6 is required to initiate (but not maintain) CpG island DNA methylation at germline genes during peri-implantation development; E2F6 cooperates with MGA to silence germline genes in ESCs through the PRC1.6 complex; the E2F6 marked box domain is critical for this function.","method":"E2f6 knockout mice; genome-wide ChIP-seq in embryonic cells; WGBS/bisulfite sequencing for DNA methylation; domain mutagenesis; CRISPR in ESCs; RNA-seq","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vivo knockout, genome-wide ChIP-seq, bisulfite sequencing, domain mutagenesis, multiple developmental stages; single lab but extensive orthogonal methods","pmids":["34117224"],"is_preprint":false},{"year":2021,"finding":"USP22 deubiquitinase interacts with and stabilizes E2F6 protein; USP22-mediated stabilization of E2F6 leads to transcriptional repression of phosphatase DUSP1, which in turn strengthens AKT activation in hepatocellular carcinoma cells.","method":"Co-immunoprecipitation; ubiquitination assays; ChIP showing E2F6 binding at DUSP1 promoter; siRNA knockdown epistasis; Western blot","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, ChIP, epistasis knockdown; single lab, multiple methods","pmids":["34339800"],"is_preprint":false},{"year":2022,"finding":"CENPU physically interacts with E2F6 and promotes its ubiquitin-mediated proteasomal degradation; loss of E2F6 protein de-represses E2F1 transcription, and E2F1 in turn directly binds the CENPU promoter to upregulate CENPU expression, forming a positive feedback loop.","method":"Co-immunoprecipitation; ubiquitination assays; ChIP showing E2F1 binding at CENPU promoter; luciferase reporter assay; knockdown epistasis","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, ChIP, reporter assay; single lab, multiple orthogonal methods","pmids":["35844791"],"is_preprint":false},{"year":2022,"finding":"E2F6 interacts with KDM5C histone demethylase, recruits it to the SF3A3 promoter, and promotes demethylation of H3K4me2 at the CpG island, leading to increased SF3A3 expression in bladder cancer.","method":"Co-immunoprecipitation; luciferase reporter assay; ChIP for E2F6 and KDM5C co-occupancy at SF3A3 promoter; histone modification ChIP","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, reporter assay; single lab, multiple methods; unexpected activating rather than repressive role","pmids":["35248043"],"is_preprint":false},{"year":2006,"finding":"E2F6 represses BRCA1 transcription and negatively regulates UV-induced apoptosis; E2F6 overexpression inhibits UV-induced BRCA1 upregulation and cleavage; this effect depends on E2F6 association with the BRCA1 C-terminus in a UV-triggered manner and on E2F6 transcriptional repression of the BRCA1 promoter.","method":"Overexpression and siRNA knockdown of E2F6; ChIP at BRCA1 promoter; Co-immunoprecipitation of E2F6 with BRCA1; apoptosis assays; domain deletion mutants","journal":"Cell death and differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ChIP, rescue domain mutants, apoptosis assays; single lab","pmids":["17096023"],"is_preprint":false},{"year":2008,"finding":"E2F6 represses E2F1 promoter activity directly (shown by ChIP) and competes with E2F1 for DNA binding at target sites to inhibit hypoxia-induced apoptosis; the inhibitory effects require E2F6 DNA binding activity (abrogated by E2F6.E68 DNA-binding mutant) and are independent of physical E2F6-E2F1 protein-protein association.","method":"ChIP assay for E2F6 at E2F1 promoter; reporter assays; overexpression and siRNA knockdown; E2F6 domain mutants (C-terminal deletion and DNA-binding mutant); apoptosis assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, reporter assays, domain mutagenesis, loss- and gain-of-function; single lab","pmids":["18562691"],"is_preprint":false},{"year":2022,"finding":"E2F6 suppresses miR-193a expression by recruiting EZH2 to the miR-193a locus, resulting in repressive chromatin and DNA methylation at the miR-193a promoter; this E2F6-EZH2-miR-193a axis promotes ovarian cancer stemness.","method":"ChIP-PCR for E2F6 and EZH2 at miR-193a promoter; bisulfite pyrosequencing for DNA methylation; EZH2 depletion; luciferase reporter assay; cell stemness assays","journal":"Cancer science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, bisulfite sequencing, EZH2 knockdown epistasis; single lab, multiple methods","pmids":["30582655"],"is_preprint":false},{"year":2019,"finding":"E2F6 expression is controlled by the EGFRvIII/AKT/NF-κB signaling pathway in glioblastoma cells, and E2F6 drives temozolomide resistance downstream of this pathway.","method":"Genome-wide CRISPR-Cas9 library screen; pathway inhibition experiments; xenograft models","journal":"Advanced science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CRISPR screen validated by pathway inhibition and in vivo xenograft; single lab","pmids":["31508283"],"is_preprint":false},{"year":2025,"finding":"KDM4C histone demethylase directly binds the E2F6 promoter and epigenetically upregulates E2F6 expression in temozolomide-resistant GBM cells; pharmacological or genetic inhibition of KDM4C reduces E2F6 expression and restores TMZ sensitivity.","method":"ChIP for KDM4C at E2F6 promoter; KDM4C inhibitor (SD70); genetic knockdown; combination treatment assays","journal":"Archives of pharmacal research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP, pharmacological inhibition, genetic knockdown with defined phenotype; single lab","pmids":["41217736"],"is_preprint":false},{"year":2025,"finding":"LRRFIP1 binds the E2F6 promoter and suppresses E2F6 transcription; reduced E2F6 downregulates C/EBPα to inhibit white adipocyte differentiation; re-expression of WDR77 relieves GATA3-mediated growth inhibition, placing E2F6 in the LRRFIP1/E2F6/C/EBPα regulatory axis.","method":"Luciferase reporter assay; ChIP for LRRFIP1 at E2F6 promoter; overexpression/knockdown of LRRFIP1; Western blot; adipogenic differentiation assays","journal":"Diabetes & metabolism journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP and reporter assay, single lab, limited mechanistic validation of E2F6-specific function","pmids":["41224206"],"is_preprint":false},{"year":2025,"finding":"GATA3 and E2F6 directly bind the WDR77 gene promoter in vitro and in vivo and repress WDR77 promoter activity; re-expression of GATA3 and E2F6 in prostate cancer cells reduces WDR77 expression and cell growth.","method":"ChIP for E2F6 at WDR77 promoter; luciferase reporter assay; overexpression rescue experiments","journal":"Transcription","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP and reporter assay, single lab, single publication","pmids":["40071854"],"is_preprint":false},{"year":2022,"finding":"SUZ12 directly interacts with E2F6 (selectively among E2F family members) and forms a complex with EZH2; biochemical purification from HEK293 cells identified an E2F6-SUZ12-EZH2 complex; ChIP identified target genes of this complex, whose promoters are not tri-methylated at H3K27, and whose expression is paradoxically downregulated by EZH2 depletion, suggesting a non-canonical activating role.","method":"Co-immunoprecipitation; biochemical purification (Flag-SUZ12 stable cell line); ChIP; EZH2 knockdown","journal":"The Kurume medical journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP/pulldown, limited ChIP validation; single lab, single publication in low-impact journal","pmids":["36464274"],"is_preprint":false},{"year":2019,"finding":"E2F6 binds to the LINC01436 promoter and represses its transcription under normoxia; in a hypoxic microenvironment, this inhibitory effect is relieved, suggesting oxygen-sensitive regulation of E2F6 transcriptional repressor activity.","method":"ChIP assay for E2F6 at LINC01436 promoter; E2F6 knockdown/overexpression with gene expression analysis under normoxia and hypoxia","journal":"Molecular oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single ChIP experiment, single lab; lncRNA study with E2F6 as upstream regulator","pmids":["30614188"],"is_preprint":false},{"year":2016,"finding":"EBV nuclear antigen EBNA3C physically interacts with E2F6 at both its amino and carboxy terminal domains, stabilizes E2F6 protein, and colocalizes with E2F6 in the nucleus; EBNA3C recruits E2F6 to the E2F1 promoter to repress E2F1 transcription.","method":"Co-immunoprecipitation; domain mapping; nuclear colocalization by immunofluorescence; ChIP for E2F6 at E2F1 promoter; reporter assays","journal":"PLoS pathogens","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, ChIP, reporter assay, colocalization; single lab, multiple orthogonal methods","pmids":["27548379"],"is_preprint":false}],"current_model":"E2F6 is a pRB-independent transcriptional repressor that heterodimerizes with DP proteins to bind E2F recognition sites (preferring TTTCCCGC) and silence G1/S-phase target genes during S phase; it is a component of multiple Polycomb repressive complexes (including PRC1.6, with Bmi1/RYBP/Ring1/MEL-18/mph1, and EPC1/EZH2/Sin3B complexes) and recruits PcG machinery—as well as the SWI/SNF subunit BRG1—to target promoters; E2F6 directs CpG island DNA methylation at germline/meiotic genes in early embryogenesis in cooperation with MGA and Dnmt3b, enforcing their long-term somatic silencing through histone H3K9 and H3K27 methylation; during replication stress, Chk1 phosphorylates E2F6 to displace it from promoters and maintain survival-promoting transcription; E2F6 protein stability is regulated by USP22-mediated deubiquitination and CENPU-mediated ubiquitin-dependent degradation; E2F6 expression is transcriptionally activated by E2F1 (feedback loop), and its promoter is regulated by NRF-1/alpha-PAL, CREB, and KDM4C."},"narrative":{"mechanistic_narrative":"E2F6 is a pRB-independent transcriptional repressor of the E2F family that lacks transactivation and pocket-protein-binding domains, heterodimerizes with DP-1/DP-2, and binds E2F recognition sites (preferring TTTCCCGC) to silence target promoters in a manner dependent on its DNA-binding and DP-heterodimerization activities [PMID:9704927, PMID:9501179, PMID:9689056]. Rather than working through retinoblastoma proteins, E2F6 functions as a recruiting platform for Polycomb group machinery: it associates with RYBP, Ring1, MEL-18, mph1, and Bmi1 [PMID:11171983], forms proliferation-specific complexes with DP1/EPC1/EZH2/Sin3B [PMID:15536069], and within PRC1.6 mediates loading of the complex to a distinct subset of promoters defined by its DNA-binding specificity [PMID:29381691]. Consistent with this Polycomb partnership, E2f6-null mice are viable and proliferate normally but show homeotic axial-skeleton transformations and genetically cooperate with Bmi1 in Hox repression, establishing a developmental rather than cell-cycle-essential role [PMID:12101104, PMID:18366140]. During the cell cycle, E2F6 occupies G1/S (but not G2/M) target promoters specifically during S phase, where its loss permits E2F4 substitution and combined E2F4/E2F6 inhibition derepresses G1/S genes [PMID:15574595]; it can also recruit the SWI/SNF ATPase BRG1 to these promoters [PMID:23082233]. A major physiological function is the long-term silencing of germline and meiosis-specific genes (e.g. SMC1β, STAG3, Slc25a31) in somatic cells through H3K9/H3K27 methylation [PMID:16236716, PMID:18667754], and E2F6 is required to initiate CpG-island DNA methylation at germline genes during peri-implantation development by cooperating with MGA and directing Dnmt3b recruitment, an activity that depends on its marked-box domain [PMID:20439742, PMID:34117224]. Its repressive output is dynamically regulated: Chk1 phosphorylates E2F6 during replication stress to displace it from G1/S promoters and sustain survival-promoting transcription [PMID:23954429], and E2F6 abundance is set by USP22-mediated deubiquitination and CENPU-driven proteasomal degradation [PMID:34339800, PMID:35844791], while its own promoter is activated by E2F1 in a feedback loop [PMID:16107498].","teleology":[{"year":1998,"claim":"Established that E2F6 is a mechanistically distinct E2F member that represses without using the canonical pocket-protein route, answering how a transactivation-deficient E2F could regulate transcription.","evidence":"In vitro binding, Co-IP, reporter assays, and DNA-binding mutagenesis across three independent labs","pmids":["9704927","9501179","9689056"],"confidence":"High","gaps":["The identity of the corepressor machinery was not yet defined","Endogenous physiological targets were unknown"]},{"year":1998,"claim":"Showed E2F6 has a cell-context-restricted function, blocking S-phase entry from quiescence but not in cycling cells, framing it as a regulator of G0 exit rather than a general cell-cycle brake.","evidence":"E2F6 overexpression with flow-cytometric cell-cycle analysis in serum-stimulated NIH 3T3 cells","pmids":["9689056"],"confidence":"Medium","gaps":["Overexpression-based; endogenous requirement not tested","Specific target genes not identified"]},{"year":2001,"claim":"Identified E2F6's corepressor identity by linking it to Polycomb group proteins, explaining the molecular basis of its pRB-independent repression.","evidence":"Yeast two-hybrid (RYBP), endogenous reciprocal Co-IP, and domain mapping","pmids":["11171983"],"confidence":"High","gaps":["Functional consequence of the complex at promoters not yet shown","Whether complex composition varies by cell state unknown"]},{"year":2002,"claim":"Defined E2F6's in vivo role through knockout mice, separating a developmental Polycomb-like function from cell-cycle control.","evidence":"Targeted E2f6 deletion in mice with skeletal analysis and MEF proliferation assays","pmids":["12101104"],"confidence":"High","gaps":["Molecular targets driving homeotic phenotype not identified","Relationship to specific PcG complexes unresolved"]},{"year":2003,"claim":"Mapped genome-wide E2F6 targets and showed it acts by occluding activating E2F1, with repression not requiring H3K9 methylation at these loci.","evidence":"ChIP-chip, RNAi knockdown, and re-ChIP showing E2F1 recruitment after E2F6 loss","pmids":["12909625"],"confidence":"High","gaps":["Mechanism of repression at H3K9-independent promoters left open","Did not address meiotic/germline gene targets"]},{"year":2004,"claim":"Resolved E2F6's cell-cycle-specific chromatin behavior and functional redundancy, showing S-phase-restricted occupancy of G1/S promoters with E2F4 backup.","evidence":"ChIP across synchronized cell cycle plus E2F4/E2F6 double loss-of-function derepression assays","pmids":["15574595"],"confidence":"High","gaps":["What restricts E2F6 to G1/S vs G2/M promoters unknown","Recruited remodeling activity not yet defined"]},{"year":2004,"claim":"Demonstrated cell-state-specific complex assembly, defining a proliferation-specific E2F6-EPC1-EZH2-Sin3B complex.","evidence":"Yeast two-hybrid, affinity purification, and Co-IP comparing proliferating vs quiescent cells","pmids":["15536069"],"confidence":"High","gaps":["Genome-wide targets of this specific complex not mapped","Functional necessity of each subunit untested"]},{"year":2005,"claim":"Identified silencing of meiosis-specific genes in somatic cells as a core E2F6 function, linking it to H3K9/H3K27 methylation.","evidence":"Knockout MEF transcriptomics, ChIP at SMC1β/STAG3 promoters, and rescue re-expression","pmids":["16236716"],"confidence":"High","gaps":["Methyltransferase responsible not identified here","Whether DNA methylation is involved unaddressed"]},{"year":2005,"claim":"Established a feedback architecture in which E2F1 activates E2F6 expression at G1/S, providing a self-limiting circuit.","evidence":"Promoter reporter, EMSA, ChIP, and E2F1 overexpression in synchronized cells","pmids":["16107498"],"confidence":"Medium","gaps":["Quantitative impact of the loop on target gene timing unknown","Single-lab promoter analysis"]},{"year":2008,"claim":"Generalized the meiotic-silencing role across most meiosis-specific genes via a conserved E2F6 element.","evidence":"Bioinformatic promoter analysis, ChIP, knockout MEF expression, and overexpression","pmids":["18667754"],"confidence":"Medium","gaps":["Single-lab correlation between motif and direct binding","Mechanism of long-term maintenance not addressed"]},{"year":2008,"claim":"Provided in vivo genetic evidence that E2F6 and Bmi1 act in the same Polycomb pathway for Hox repression but diverge at the Ink4a-Arf locus.","evidence":"Compound E2f6/Bmi1 mutant mice with skeletal and gene-expression analysis","pmids":["18366140"],"confidence":"High","gaps":["Molecular basis for locus-specific cooperation unknown"]},{"year":2010,"claim":"Connected E2F6 to DNA-methylation-based silencing by showing it directs Dnmt3b recruitment to germline genes in somatic tissue.","evidence":"Dnmt3b hypomorph mice, ChIP for E2F6, microarray, and bisulfite sequencing","pmids":["20439742"],"confidence":"High","gaps":["Whether E2F6 directly contacts Dnmt3b unresolved","Temporal order of binding vs methylation not established"]},{"year":2012,"claim":"Added SWI/SNF chromatin remodeling to E2F6's repressive toolkit at G1/S promoters.","evidence":"Reciprocal Co-IP with BRG1/BAF155 and ChIP co-occupancy during S phase","pmids":["23082233"],"confidence":"Medium","gaps":["Functional requirement of BRG1 for E2F6 repression not tested","Single-lab Co-IP/ChIP"]},{"year":2013,"claim":"Defined a stress-responsive switch, showing Chk1 phosphorylation displaces E2F6 to sustain transcription and survival during replication stress.","evidence":"In vitro kinase assay, ChIP for promoter dissociation, Chk1 knockdown, and viability/DNA-damage assays","pmids":["23954429"],"confidence":"High","gaps":["Phosphosite mapping and phospho-mutant validation not detailed","Whether complex disassembly accompanies displacement unknown"]},{"year":2013,"claim":"Dissected the chromatin requirements for meiotic gene silencing, assigning EZH2 (PRC2) rather than Dnmt3b as the critical enzyme in development.","evidence":"ESC-to-EpiSC transition with EZH2 SET-domain and Dnmt3b deletions, ChIP, and bisulfite sequencing","pmids":["23880518"],"confidence":"Medium","gaps":["Reconciliation with Dnmt3b-dependent silencing in other contexts incomplete","Single-lab analysis"]},{"year":2018,"claim":"Defined E2F6's role within PRC1.6 as a DNA-binding-dependent recruiter for a distinct subset of loci, in a division of labor with MGA and L3MBTL2.","evidence":"Genome-wide ChIP-seq with CRISPR ablation of individual subunits and DNA-binding-mutant rescue","pmids":["29381691"],"confidence":"High","gaps":["What determines locus specificity between subunits unknown","Downstream chromatin output at E2F6-specific loci not detailed"]},{"year":2021,"claim":"Established E2F6 as the initiator of CpG-island DNA methylation at germline genes in early development, cooperating with MGA via its marked-box domain.","evidence":"E2f6 knockout mice, embryonic ChIP-seq, WGBS, domain mutagenesis, and ESC CRISPR/RNA-seq","pmids":["34117224"],"confidence":"High","gaps":["Mechanism separating initiation from maintenance unresolved","Direct enzyme-recruitment contacts of the marked box not defined"]},{"year":2021,"claim":"Revealed post-translational control of E2F6 abundance through USP22 deubiquitination, linking it to oncogenic AKT signaling via DUSP1 repression.","evidence":"Co-IP, ubiquitination assays, ChIP at DUSP1, and knockdown epistasis in hepatocellular carcinoma cells","pmids":["34339800"],"confidence":"Medium","gaps":["E3 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metastasis of hepatocellular carcinoma.","date":"2024","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/38166853","citation_count":8,"is_preprint":false},{"pmid":"34588094","id":"PMC_34588094","title":"Long Noncoding RNA LAMTOR5-AS1 Interference Affects MicroRNA-506-3p/E2F6-Mediated Behavior of Non-Small Cell Lung Cancer Cells.","date":"2021","source":"Oncology research","url":"https://pubmed.ncbi.nlm.nih.gov/34588094","citation_count":7,"is_preprint":false},{"pmid":"31768102","id":"PMC_31768102","title":"E2F6 is essential for cell viability in breast cancer cells during replication stress.","date":"2019","source":"Turkish journal of biology = Turk biyoloji dergisi","url":"https://pubmed.ncbi.nlm.nih.gov/31768102","citation_count":7,"is_preprint":false},{"pmid":"37178508","id":"PMC_37178508","title":"MicroRNA-31 regulates TNF-α and IL-17A co-induced-endothelial cell apoptosis by repressing E2F6.","date":"2023","source":"Biochemical and biophysical research 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heterodimerization.\",\n      \"method\": \"Co-immunoprecipitation, in vitro binding assays, reporter gene assays, dominant-negative overexpression, DNA binding assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (in vitro binding, Co-IP, reporter assays, mutagenesis of DNA-binding domain); independently replicated across three labs in 1998 (PMID:9704927, PMID:9501179, PMID:9689056)\",\n      \"pmids\": [\"9704927\", \"9501179\", \"9689056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"E2F6 overexpression in NIH 3T3 cells inhibited entry into S phase of serum-stimulated quiescent cells but had no specific effect on asynchronously cycling cells, indicating it can regulate a subset of E2F-dependent genes required for cell cycle entry from G0.\",\n      \"method\": \"Cell cycle analysis by flow cytometry, serum-stimulation of G0-arrested NIH 3T3 cells with E2F6 overexpression\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean overexpression with defined cellular phenotype (G0 re-entry), single lab, two cell lines tested\",\n      \"pmids\": [\"9689056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"E2F6 interacts with RYBP (a mammalian polycomb complex member) through the E2F6 repression domain, and endogenous E2F6 associates with polycomb group proteins RYBP, Ring1, MEL-18, mph1, and Bmi1 in a multiprotein complex.\",\n      \"method\": \"Yeast two-hybrid screen to identify RYBP; co-immunoprecipitation of endogenous proteins; domain mapping\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP of endogenous proteins, yeast two-hybrid, domain mapping; replicated across multiple studies\",\n      \"pmids\": [\"11171983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"E2f6 knockout mice are viable but display homeotic transformations of the axial skeleton similar to polycomb mutant mice; E2f6−/− mouse embryonic fibroblasts proliferate normally, demonstrating that E2F6 is required for developmental patterning but not normal cell cycle progression.\",\n      \"method\": \"Targeted gene deletion in mice; skeletal analysis; MEF proliferation assays\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean knockout mouse model with specific skeletal phenotype and negative proliferation result; independently replicated by Courel et al. 2008\",\n      \"pmids\": [\"12101104\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"E2F6 associates specifically with promoters of G1/S-regulated E2F target genes during S phase but not with G2/M-regulated E2F target genes; loss of E2F6 allows E2F4 to substitute at G1/S promoters, and combined inhibition of E2F4 and E2F6 causes specific derepression of G1/S genes during S phase.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) during synchronized cell cycle; E2F6 knockdown and E2F4/6 double inhibition with specific derepression assays\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP across cell cycle combined with genetic epistasis (double loss-of-function), rigorous controls, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15574595\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"E2F6 forms a stable core complex with DP1 and the polycomb group protein EPC1 in vitro and in vivo; this complex further associates with EZH2 and Sin3B specifically in proliferating cells (not quiescent cells), defining a novel proliferation-specific E2F6-PcG complex.\",\n      \"method\": \"Yeast two-hybrid screen; in vitro and in vivo co-immunoprecipitation; affinity purification from proliferating vs. quiescent cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — affinity purification, reciprocal Co-IP, in vitro binding, cell-state comparison; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15536069\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"E2F6 represses endogenous target promoters including BRCA1, CTIP, ART27, HP1alpha, and RBAP48 in human cells; depletion of E2F6 by RNA interference results in recruitment of E2F1 to these promoters; repression does not require histone H3 methylation at lysine 9.\",\n      \"method\": \"ChIP combined with genomic microarrays (ChIP-chip) to identify E2F6-bound promoters; RNAi knockdown; ChIP before and after knockdown\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP-chip for genome-wide binding, RNAi loss-of-function with re-ChIP showing E2F1 replacement; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"12909625\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"E2F6 is required to silence meiosis-specific genes SMC1beta and STAG3 in somatic cells; E2F6 binds their promoters in vivo through a conserved binding site; transcriptional repression involves histone H3 methylation at both lysine 9 and lysine 27.\",\n      \"method\": \"cDNA microarray comparing wild-type and E2f6−/− MEFs; ChIP assays; re-expression rescue experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — knockout MEF transcriptomics + ChIP + rescue re-expression; multiple orthogonal methods; consistent with in vivo E2f6−/− phenotype\",\n      \"pmids\": [\"16236716\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"E2F1 activating E2Fs bind the human E2F6 promoter at two functional E2F binding sites in vivo and transcriptionally upregulate E2F6 expression, establishing a regulatory feedback loop during G1/S transition.\",\n      \"method\": \"Promoter deletion/mutation reporter assays; EMSA; ChIP; E2F1 overexpression; synchronized cell cycle analysis\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP in vivo binding, EMSA, promoter reporter with mutagenesis; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"16107498\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PHC3 (a hPRC-H polycomb complex component) associates with E2F6 and colocalizes with E2F6 in nuclear punctate bodies in differentiating/confluent (G0) cells but not in proliferating cells, suggesting a G0-specific E2F6-polycomb silencing complex.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence colocalization in differentiating vs. proliferating cells\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP and colocalization; single lab, two orthogonal methods but no functional rescue\",\n      \"pmids\": [\"17001316\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The HPV E7 oncoprotein (low- and high-risk types), as well as SV40 T antigen and adenovirus E1A, associate with E2F6 and inactivate its transcriptional repression activity; E7-expressing cells show decreased E2F6/polycomb complex staining in a manner dependent on E2F6 association.\",\n      \"method\": \"Co-immunoprecipitation; transcriptional repression reporter assays; immunofluorescence staining of E2F6/polycomb complexes\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, reporter assay, immunofluorescence; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"18579589\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"A conserved TCCCGC E2F6-binding element is present in promoters of 79% of meiosis-specific genes; E2F6 binds in vivo to meiotic gene promoters (including Slc25a31/Ant4) and represses their expression in somatic cells; E2F6 overexpression reduces meiotic gene transcription.\",\n      \"method\": \"Bioinformatic promoter analysis; ChIP assays; E2f6 knockout MEF gene expression analysis; E2F6 overexpression\",\n      \"journal\": \"Biology of reproduction\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, knockout MEF analysis, overexpression rescue; single lab, multiple methods\",\n      \"pmids\": [\"18667754\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"E2f6 and Bmi1 genetically cooperate in axial skeleton development and Hox gene repression in vivo (double mutant mice show enhanced skeletal transformations), but E2F6 does not cooperate with Bmi1 in repression of the Ink4a-Arf locus.\",\n      \"method\": \"Mouse genetic epistasis using E2f6−/− and Bmi1−/− compound mutants; skeletal analysis; gene expression\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in vivo with compound mutant mice; clean phenotypic readout for two distinct loci\",\n      \"pmids\": [\"18366140\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Dnmt3b is recruited to promoters of germline/meiotic genes in somatic tissues in an E2F6-dependent manner; E2F6 binding is a common feature of promoters upregulated when Dnmt3b activity is impaired; E2F6 binding is required for CpG methylation-based silencing of these genes.\",\n      \"method\": \"Dnmt3b hypomorphic mutant mice; DNA microarray; ChIP assays for E2F6 binding at target promoters; bisulfite sequencing for DNA methylation\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo mouse model, ChIP, bisulfite sequencing, microarray; multiple orthogonal methods, single lab\",\n      \"pmids\": [\"20439742\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Chk1 phosphorylates E2F6 in response to DNA replication stress, causing E2F6 to dissociate from G1/S target promoters; this releases repression and maintains high E2F-dependent transcription, which is required to prevent DNA damage and cell death during checkpoint activation.\",\n      \"method\": \"In vitro kinase assay (Chk1 phosphorylating E2F6); ChIP showing E2F6 dissociation from promoters after replication stress; siRNA knockdown of Chk1; cell viability and DNA damage assays\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro kinase assay establishing direct phosphorylation, ChIP for chromatin binding, epistasis via Chk1 knockdown; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"23954429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"E2f6-mediated repression of meiotic genes Stag3 and Smc1β during embryonic development requires EZH2 enzymatic activity (Polycomb repressive complex 2) but not Dnmt3b; EZH2 SET-domain deletion impairs repression during embryoid body differentiation.\",\n      \"method\": \"ESC-to-EpiSC transition analysis; EZH2 SET-domain deletion; Dnmt3b knockout; bisulfite sequencing; ChIP for E2f6 binding; gene expression analysis\",\n      \"journal\": \"Epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic dissection with EZH2 and Dnmt3b knockouts, ChIP, bisulfite sequencing; single lab\",\n      \"pmids\": [\"23880518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"E2F6 interacts with BRG1 (ATPase subunit of SWI/SNF chromatin-remodeling complex) and BAF155; BRG1 co-occupies G1/S gene promoters coincident with E2F6 during S phase, suggesting E2F6 recruits SWI/SNF for transcriptional repression.\",\n      \"method\": \"Co-immunoprecipitation; ChIP showing co-occupancy of BRG1 and E2F6 at G1/S promoters during S phase\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ChIP co-occupancy; single lab, two orthogonal methods\",\n      \"pmids\": [\"23082233\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Within the PRC1.6 complex, MGA, L3MBTL2, and E2F6 each mediate recruitment to distinct sets of genomic loci; CRISPR/Cas-mediated ablation of MGA causes complete loss of PRC1.6 binding genome-wide, while depletion of L3MBTL2 or E2F6 (but not PCGF6) causes locus-specific loss; E2F6 thus mediates PRC1.6 loading to a distinct subset of promoters.\",\n      \"method\": \"ChIP-seq genome-wide; CRISPR/Cas ablation of individual subunits; rescue experiments with DNA-binding mutants\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide ChIP-seq, CRISPR knockouts of multiple subunits, rescue experiments; rigorous and comprehensive in single study\",\n      \"pmids\": [\"29381691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"E2F6 is required to initiate (but not maintain) CpG island DNA methylation at germline genes during peri-implantation development; E2F6 cooperates with MGA to silence germline genes in ESCs through the PRC1.6 complex; the E2F6 marked box domain is critical for this function.\",\n      \"method\": \"E2f6 knockout mice; genome-wide ChIP-seq in embryonic cells; WGBS/bisulfite sequencing for DNA methylation; domain mutagenesis; CRISPR in ESCs; RNA-seq\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vivo knockout, genome-wide ChIP-seq, bisulfite sequencing, domain mutagenesis, multiple developmental stages; single lab but extensive orthogonal methods\",\n      \"pmids\": [\"34117224\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"USP22 deubiquitinase interacts with and stabilizes E2F6 protein; USP22-mediated stabilization of E2F6 leads to transcriptional repression of phosphatase DUSP1, which in turn strengthens AKT activation in hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; ChIP showing E2F6 binding at DUSP1 promoter; siRNA knockdown epistasis; Western blot\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, ChIP, epistasis knockdown; single lab, multiple methods\",\n      \"pmids\": [\"34339800\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"CENPU physically interacts with E2F6 and promotes its ubiquitin-mediated proteasomal degradation; loss of E2F6 protein de-represses E2F1 transcription, and E2F1 in turn directly binds the CENPU promoter to upregulate CENPU expression, forming a positive feedback loop.\",\n      \"method\": \"Co-immunoprecipitation; ubiquitination assays; ChIP showing E2F1 binding at CENPU promoter; luciferase reporter assay; knockdown epistasis\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, ChIP, reporter assay; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"35844791\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"E2F6 interacts with KDM5C histone demethylase, recruits it to the SF3A3 promoter, and promotes demethylation of H3K4me2 at the CpG island, leading to increased SF3A3 expression in bladder cancer.\",\n      \"method\": \"Co-immunoprecipitation; luciferase reporter assay; ChIP for E2F6 and KDM5C co-occupancy at SF3A3 promoter; histone modification ChIP\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, reporter assay; single lab, multiple methods; unexpected activating rather than repressive role\",\n      \"pmids\": [\"35248043\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"E2F6 represses BRCA1 transcription and negatively regulates UV-induced apoptosis; E2F6 overexpression inhibits UV-induced BRCA1 upregulation and cleavage; this effect depends on E2F6 association with the BRCA1 C-terminus in a UV-triggered manner and on E2F6 transcriptional repression of the BRCA1 promoter.\",\n      \"method\": \"Overexpression and siRNA knockdown of E2F6; ChIP at BRCA1 promoter; Co-immunoprecipitation of E2F6 with BRCA1; apoptosis assays; domain deletion mutants\",\n      \"journal\": \"Cell death and differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ChIP, rescue domain mutants, apoptosis assays; single lab\",\n      \"pmids\": [\"17096023\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"E2F6 represses E2F1 promoter activity directly (shown by ChIP) and competes with E2F1 for DNA binding at target sites to inhibit hypoxia-induced apoptosis; the inhibitory effects require E2F6 DNA binding activity (abrogated by E2F6.E68 DNA-binding mutant) and are independent of physical E2F6-E2F1 protein-protein association.\",\n      \"method\": \"ChIP assay for E2F6 at E2F1 promoter; reporter assays; overexpression and siRNA knockdown; E2F6 domain mutants (C-terminal deletion and DNA-binding mutant); apoptosis assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, reporter assays, domain mutagenesis, loss- and gain-of-function; single lab\",\n      \"pmids\": [\"18562691\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"E2F6 suppresses miR-193a expression by recruiting EZH2 to the miR-193a locus, resulting in repressive chromatin and DNA methylation at the miR-193a promoter; this E2F6-EZH2-miR-193a axis promotes ovarian cancer stemness.\",\n      \"method\": \"ChIP-PCR for E2F6 and EZH2 at miR-193a promoter; bisulfite pyrosequencing for DNA methylation; EZH2 depletion; luciferase reporter assay; cell stemness assays\",\n      \"journal\": \"Cancer science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, bisulfite sequencing, EZH2 knockdown epistasis; single lab, multiple methods\",\n      \"pmids\": [\"30582655\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"E2F6 expression is controlled by the EGFRvIII/AKT/NF-κB signaling pathway in glioblastoma cells, and E2F6 drives temozolomide resistance downstream of this pathway.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 library screen; pathway inhibition experiments; xenograft models\",\n      \"journal\": \"Advanced science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CRISPR screen validated by pathway inhibition and in vivo xenograft; single lab\",\n      \"pmids\": [\"31508283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"KDM4C histone demethylase directly binds the E2F6 promoter and epigenetically upregulates E2F6 expression in temozolomide-resistant GBM cells; pharmacological or genetic inhibition of KDM4C reduces E2F6 expression and restores TMZ sensitivity.\",\n      \"method\": \"ChIP for KDM4C at E2F6 promoter; KDM4C inhibitor (SD70); genetic knockdown; combination treatment assays\",\n      \"journal\": \"Archives of pharmacal research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP, pharmacological inhibition, genetic knockdown with defined phenotype; single lab\",\n      \"pmids\": [\"41217736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"LRRFIP1 binds the E2F6 promoter and suppresses E2F6 transcription; reduced E2F6 downregulates C/EBPα to inhibit white adipocyte differentiation; re-expression of WDR77 relieves GATA3-mediated growth inhibition, placing E2F6 in the LRRFIP1/E2F6/C/EBPα regulatory axis.\",\n      \"method\": \"Luciferase reporter assay; ChIP for LRRFIP1 at E2F6 promoter; overexpression/knockdown of LRRFIP1; Western blot; adipogenic differentiation assays\",\n      \"journal\": \"Diabetes & metabolism journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP and reporter assay, single lab, limited mechanistic validation of E2F6-specific function\",\n      \"pmids\": [\"41224206\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GATA3 and E2F6 directly bind the WDR77 gene promoter in vitro and in vivo and repress WDR77 promoter activity; re-expression of GATA3 and E2F6 in prostate cancer cells reduces WDR77 expression and cell growth.\",\n      \"method\": \"ChIP for E2F6 at WDR77 promoter; luciferase reporter assay; overexpression rescue experiments\",\n      \"journal\": \"Transcription\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP and reporter assay, single lab, single publication\",\n      \"pmids\": [\"40071854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SUZ12 directly interacts with E2F6 (selectively among E2F family members) and forms a complex with EZH2; biochemical purification from HEK293 cells identified an E2F6-SUZ12-EZH2 complex; ChIP identified target genes of this complex, whose promoters are not tri-methylated at H3K27, and whose expression is paradoxically downregulated by EZH2 depletion, suggesting a non-canonical activating role.\",\n      \"method\": \"Co-immunoprecipitation; biochemical purification (Flag-SUZ12 stable cell line); ChIP; EZH2 knockdown\",\n      \"journal\": \"The Kurume medical journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP/pulldown, limited ChIP validation; single lab, single publication in low-impact journal\",\n      \"pmids\": [\"36464274\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"E2F6 binds to the LINC01436 promoter and represses its transcription under normoxia; in a hypoxic microenvironment, this inhibitory effect is relieved, suggesting oxygen-sensitive regulation of E2F6 transcriptional repressor activity.\",\n      \"method\": \"ChIP assay for E2F6 at LINC01436 promoter; E2F6 knockdown/overexpression with gene expression analysis under normoxia and hypoxia\",\n      \"journal\": \"Molecular oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single ChIP experiment, single lab; lncRNA study with E2F6 as upstream regulator\",\n      \"pmids\": [\"30614188\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"EBV nuclear antigen EBNA3C physically interacts with E2F6 at both its amino and carboxy terminal domains, stabilizes E2F6 protein, and colocalizes with E2F6 in the nucleus; EBNA3C recruits E2F6 to the E2F1 promoter to repress E2F1 transcription.\",\n      \"method\": \"Co-immunoprecipitation; domain mapping; nuclear colocalization by immunofluorescence; ChIP for E2F6 at E2F1 promoter; reporter assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, ChIP, reporter assay, colocalization; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"27548379\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"E2F6 is a pRB-independent transcriptional repressor that heterodimerizes with DP proteins to bind E2F recognition sites (preferring TTTCCCGC) and silence G1/S-phase target genes during S phase; it is a component of multiple Polycomb repressive complexes (including PRC1.6, with Bmi1/RYBP/Ring1/MEL-18/mph1, and EPC1/EZH2/Sin3B complexes) and recruits PcG machinery—as well as the SWI/SNF subunit BRG1—to target promoters; E2F6 directs CpG island DNA methylation at germline/meiotic genes in early embryogenesis in cooperation with MGA and Dnmt3b, enforcing their long-term somatic silencing through histone H3K9 and H3K27 methylation; during replication stress, Chk1 phosphorylates E2F6 to displace it from promoters and maintain survival-promoting transcription; E2F6 protein stability is regulated by USP22-mediated deubiquitination and CENPU-mediated ubiquitin-dependent degradation; E2F6 expression is transcriptionally activated by E2F1 (feedback loop), and its promoter is regulated by NRF-1/alpha-PAL, CREB, and KDM4C.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"E2F6 is a pRB-independent transcriptional repressor of the E2F family that lacks transactivation and pocket-protein-binding domains, heterodimerizes with DP-1/DP-2, and binds E2F recognition sites (preferring TTTCCCGC) to silence target promoters in a manner dependent on its DNA-binding and DP-heterodimerization activities [#0]. Rather than working through retinoblastoma proteins, E2F6 functions as a recruiting platform for Polycomb group machinery: it associates with RYBP, Ring1, MEL-18, mph1, and Bmi1 [#2], forms proliferation-specific complexes with DP1/EPC1/EZH2/Sin3B [#5], and within PRC1.6 mediates loading of the complex to a distinct subset of promoters defined by its DNA-binding specificity [#17]. Consistent with this Polycomb partnership, E2f6-null mice are viable and proliferate normally but show homeotic axial-skeleton transformations and genetically cooperate with Bmi1 in Hox repression, establishing a developmental rather than cell-cycle-essential role [#3, #12]. During the cell cycle, E2F6 occupies G1/S (but not G2/M) target promoters specifically during S phase, where its loss permits E2F4 substitution and combined E2F4/E2F6 inhibition derepresses G1/S genes [#4]; it can also recruit the SWI/SNF ATPase BRG1 to these promoters [#16]. A major physiological function is the long-term silencing of germline and meiosis-specific genes (e.g. SMC1\\u03b2, STAG3, Slc25a31) in somatic cells through H3K9/H3K27 methylation [#7, #11], and E2F6 is required to initiate CpG-island DNA methylation at germline genes during peri-implantation development by cooperating with MGA and directing Dnmt3b recruitment, an activity that depends on its marked-box domain [#13, #18]. Its repressive output is dynamically regulated: Chk1 phosphorylates E2F6 during replication stress to displace it from G1/S promoters and sustain survival-promoting transcription [#14], and E2F6 abundance is set by USP22-mediated deubiquitination and CENPU-driven proteasomal degradation [#19, #20], while its own promoter is activated by E2F1 in a feedback loop [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established that E2F6 is a mechanistically distinct E2F member that represses without using the canonical pocket-protein route, answering how a transactivation-deficient E2F could regulate transcription.\",\n      \"evidence\": \"In vitro binding, Co-IP, reporter assays, and DNA-binding mutagenesis across three independent labs\",\n      \"pmids\": [\"9704927\", \"9501179\", \"9689056\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The identity of the corepressor machinery was not yet defined\", \"Endogenous physiological targets were unknown\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Showed E2F6 has a cell-context-restricted function, blocking S-phase entry from quiescence but not in cycling cells, framing it as a regulator of G0 exit rather than a general cell-cycle brake.\",\n      \"evidence\": \"E2F6 overexpression with flow-cytometric cell-cycle analysis in serum-stimulated NIH 3T3 cells\",\n      \"pmids\": [\"9689056\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Overexpression-based; endogenous requirement not tested\", \"Specific target genes not identified\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identified E2F6's corepressor identity by linking it to Polycomb group proteins, explaining the molecular basis of its pRB-independent repression.\",\n      \"evidence\": \"Yeast two-hybrid (RYBP), endogenous reciprocal Co-IP, and domain mapping\",\n      \"pmids\": [\"11171983\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of the complex at promoters not yet shown\", \"Whether complex composition varies by cell state unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined E2F6's in vivo role through knockout mice, separating a developmental Polycomb-like function from cell-cycle control.\",\n      \"evidence\": \"Targeted E2f6 deletion in mice with skeletal analysis and MEF proliferation assays\",\n      \"pmids\": [\"12101104\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular targets driving homeotic phenotype not identified\", \"Relationship to specific PcG complexes unresolved\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Mapped genome-wide E2F6 targets and showed it acts by occluding activating E2F1, with repression not requiring H3K9 methylation at these loci.\",\n      \"evidence\": \"ChIP-chip, RNAi knockdown, and re-ChIP showing E2F1 recruitment after E2F6 loss\",\n      \"pmids\": [\"12909625\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of repression at H3K9-independent promoters left open\", \"Did not address meiotic/germline gene targets\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Resolved E2F6's cell-cycle-specific chromatin behavior and functional redundancy, showing S-phase-restricted occupancy of G1/S promoters with E2F4 backup.\",\n      \"evidence\": \"ChIP across synchronized cell cycle plus E2F4/E2F6 double loss-of-function derepression assays\",\n      \"pmids\": [\"15574595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What restricts E2F6 to G1/S vs G2/M promoters unknown\", \"Recruited remodeling activity not yet defined\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated cell-state-specific complex assembly, defining a proliferation-specific E2F6-EPC1-EZH2-Sin3B complex.\",\n      \"evidence\": \"Yeast two-hybrid, affinity purification, and Co-IP comparing proliferating vs quiescent cells\",\n      \"pmids\": [\"15536069\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide targets of this specific complex not mapped\", \"Functional necessity of each subunit untested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identified silencing of meiosis-specific genes in somatic cells as a core E2F6 function, linking it to H3K9/H3K27 methylation.\",\n      \"evidence\": \"Knockout MEF transcriptomics, ChIP at SMC1\\u03b2/STAG3 promoters, and rescue re-expression\",\n      \"pmids\": [\"16236716\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Methyltransferase responsible not identified here\", \"Whether DNA methylation is involved unaddressed\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Established a feedback architecture in which E2F1 activates E2F6 expression at G1/S, providing a self-limiting circuit.\",\n      \"evidence\": \"Promoter reporter, EMSA, ChIP, and E2F1 overexpression in synchronized cells\",\n      \"pmids\": [\"16107498\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Quantitative impact of the loop on target gene timing unknown\", \"Single-lab promoter analysis\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Generalized the meiotic-silencing role across most meiosis-specific genes via a conserved E2F6 element.\",\n      \"evidence\": \"Bioinformatic promoter analysis, ChIP, knockout MEF expression, and overexpression\",\n      \"pmids\": [\"18667754\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab correlation between motif and direct binding\", \"Mechanism of long-term maintenance not addressed\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Provided in vivo genetic evidence that E2F6 and Bmi1 act in the same Polycomb pathway for Hox repression but diverge at the Ink4a-Arf locus.\",\n      \"evidence\": \"Compound E2f6/Bmi1 mutant mice with skeletal and gene-expression analysis\",\n      \"pmids\": [\"18366140\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis for locus-specific cooperation unknown\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Connected E2F6 to DNA-methylation-based silencing by showing it directs Dnmt3b recruitment to germline genes in somatic tissue.\",\n      \"evidence\": \"Dnmt3b hypomorph mice, ChIP for E2F6, microarray, and bisulfite sequencing\",\n      \"pmids\": [\"20439742\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether E2F6 directly contacts Dnmt3b unresolved\", \"Temporal order of binding vs methylation not established\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Added SWI/SNF chromatin remodeling to E2F6's repressive toolkit at G1/S promoters.\",\n      \"evidence\": \"Reciprocal Co-IP with BRG1/BAF155 and ChIP co-occupancy during S phase\",\n      \"pmids\": [\"23082233\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional requirement of BRG1 for E2F6 repression not tested\", \"Single-lab Co-IP/ChIP\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined a stress-responsive switch, showing Chk1 phosphorylation displaces E2F6 to sustain transcription and survival during replication stress.\",\n      \"evidence\": \"In vitro kinase assay, ChIP for promoter dissociation, Chk1 knockdown, and viability/DNA-damage assays\",\n      \"pmids\": [\"23954429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosite mapping and phospho-mutant validation not detailed\", \"Whether complex disassembly accompanies displacement unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Dissected the chromatin requirements for meiotic gene silencing, assigning EZH2 (PRC2) rather than Dnmt3b as the critical enzyme in development.\",\n      \"evidence\": \"ESC-to-EpiSC transition with EZH2 SET-domain and Dnmt3b deletions, ChIP, and bisulfite sequencing\",\n      \"pmids\": [\"23880518\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation with Dnmt3b-dependent silencing in other contexts incomplete\", \"Single-lab analysis\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined E2F6's role within PRC1.6 as a DNA-binding-dependent recruiter for a distinct subset of loci, in a division of labor with MGA and L3MBTL2.\",\n      \"evidence\": \"Genome-wide ChIP-seq with CRISPR ablation of individual subunits and DNA-binding-mutant rescue\",\n      \"pmids\": [\"29381691\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"What determines locus specificity between subunits unknown\", \"Downstream chromatin output at E2F6-specific loci not detailed\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Established E2F6 as the initiator of CpG-island DNA methylation at germline genes in early development, cooperating with MGA via its marked-box domain.\",\n      \"evidence\": \"E2f6 knockout mice, embryonic ChIP-seq, WGBS, domain mutagenesis, and ESC CRISPR/RNA-seq\",\n      \"pmids\": [\"34117224\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism separating initiation from maintenance unresolved\", \"Direct enzyme-recruitment contacts of the marked box not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Revealed post-translational control of E2F6 abundance through USP22 deubiquitination, linking it to oncogenic AKT signaling via DUSP1 repression.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, ChIP at DUSP1, and knockdown epistasis in hepatocellular carcinoma cells\",\n      \"pmids\": [\"34339800\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"E3 ligase opposing USP22 not identified here\", \"Generality beyond HCC untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified CENPU-driven degradation of E2F6 embedded in a feedback loop with E2F1, providing a turnover mechanism that derepresses E2F1 targets.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, ChIP for E2F1 at CENPU, reporter assays, and knockdown epistasis\",\n      \"pmids\": [\"35844791\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether CENPU is the direct E3 ligase unresolved\", \"Single-context (single-lab) finding\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Documented non-canonical, context-dependent activating roles for E2F6 in cancer, recruiting KDM5C to demethylate H3K4me2 and increase SF3A3 expression.\",\n      \"evidence\": \"Co-IP, ChIP for E2F6/KDM5C co-occupancy, histone-modification ChIP, and reporter assays in bladder cancer\",\n      \"pmids\": [\"35248043\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How E2F6 switches from repressor to activator unexplained\", \"Single-lab, single-context\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed viral hijacking of E2F6 by EBNA3C to repress E2F1, illustrating exploitation of E2F6 repression by a pathogen.\",\n      \"evidence\": \"Co-IP with domain mapping, nuclear colocalization, ChIP at E2F1 promoter, and reporter assays\",\n      \"pmids\": [\"27548379\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological relevance to viral latency not fully established\", \"Single-lab\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrated that DNA tumor virus oncoproteins (HPV E7, SV40 T, adenovirus E1A) inactivate E2F6 repression, broadening the theme of viral subversion.\",\n      \"evidence\": \"Co-IP, repression reporter assays, and immunofluorescence of E2F6/PcG complexes\",\n      \"pmids\": [\"18579589\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of complex disruption not resolved\", \"Functional consequences on endogenous targets not mapped\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How E2F6's intrinsic repressive activity is switched to context-dependent transcriptional activation, and the rules governing which corepressor/coactivator complex it recruits at a given locus, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying model reconciles repressive PRC1.6/PRC2 roles with activating KDM5C/SUZ12 reports\", \"Locus-specificity determinants for complex choice unknown\", \"Structural basis of marked-box/repression-domain interactions undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 4, 6, 7, 17]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 17, 23]},\n      {\"term_id\": \"GO:0060089\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [2, 9, 31]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [4, 6, 17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 4, 6]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [2, 5, 17, 18]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 4]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [3, 12, 18]}\n    ],\n    \"complexes\": [\n      \"PRC1.6\",\n      \"E2F6-DP1-EPC1-EZH2-Sin3B complex\",\n      \"E2F6/RYBP-Ring1-MEL18-mph1-Bmi1 Polycomb complex\"\n    ],\n    \"partners\": [\n      \"DP1\",\n      \"RYBP\",\n      \"Bmi1\",\n      \"EPC1\",\n      \"EZH2\",\n      \"MGA\",\n      \"BRG1\",\n      \"Chk1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}