{"gene":"AFF4","run_date":"2026-06-09T22:02:42","timeline":{"discoveries":[{"year":2010,"finding":"AFF4 is a core component of the Super Elongation Complex (SEC), which includes ELL, P-TEFb, and other factors. AFF4 is required for SEC stability and proper transcription by poised RNA polymerase II in metazoans. Knockdown of AFF4 in leukemic cells reduces MLL chimera target gene expression.","method":"Affinity purification of MLL chimeras followed by mass spectrometry identification of SEC components; siRNA knockdown with gene expression readout","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal purification, multiple MLL chimeras tested, functional knockdown with defined transcriptional phenotype; replicated across multiple chimeras in same study","pmids":["20159561"],"is_preprint":false},{"year":2010,"finding":"AFF4 bridges P-TEFb and ELL2 into a bifunctional elongation complex that greatly activates HIV-1 transcription. Through scaffolding functions of both Tat and AFF4, P-TEFb and ELL2 cooperate on the same RNA polymerase II. Without Tat, AFF4 can mediate the ELL2-P-TEFb interaction inefficiently; Tat overcomes this by bringing more ELL2 to P-TEFb and stabilizing ELL2 in a process requiring active P-TEFb.","method":"Sequential affinity-purification, Co-IP, in vitro binding assays, transcription reporter assays","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — sequential affinity purification plus functional transcription assays; multiple orthogonal methods in one rigorous study","pmids":["20471948"],"is_preprint":false},{"year":2002,"finding":"MCEF (AFF4) was identified as a binding partner of P-TEFb (CDK9/CyclinT1) by affinity purification from stably transfected cells expressing epitope-tagged CDK9; antisera against recombinant MCEF specifically immunoprecipitated P-TEFb.","method":"Affinity purification of P-TEFb complex followed by protein sequencing; co-immunoprecipitation","journal":"Journal of biomedical science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with direct sequencing identification, single lab, two complementary methods","pmids":["12065898"],"is_preprint":false},{"year":2012,"finding":"AFF4 acts as the central scaffold of the HIV-1 Tat elongation complex, recruiting ELL2, ENL/AF9, and P-TEFb through direct interactions with short hydrophobic regions along its structurally disordered axis. CycT1, ELL2, and ENL/AF9 act as bridging components linking the complex to P-TEFb and the PAF complex. Binding sites were mapped both in vitro and in vivo.","method":"In vitro binding mapping (pulldowns with truncation/mutation constructs), in vivo co-IP, structural analysis of binding sites","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — systematic in vitro binding site mapping combined with in vivo validation; multiple interaction partners mapped with orthogonal methods","pmids":["23251033"],"is_preprint":false},{"year":2013,"finding":"Crystal structure of AFF4 in complex with P-TEFb (CDK9/CycT1) revealed that AFF4 meanders over the surface of CycT1 but makes no stable contacts with CDK9. AFF4 is positioned to make direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. Interface mutations in AFF4 reduced CycT1 binding and AFF4-dependent transcription.","method":"X-ray crystal structure determination of tripartite complex; interface mutagenesis; transcription assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus mutagenesis plus functional transcription assay; multiple orthogonal methods in single rigorous study","pmids":["23471103"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of quaternary Tat-P-TEFb-AFF4 complex showed Tat and AFF4 fold on the CycT1 surface and interact directly. AFF4 binding partially orders the CycT1 Tat-TAR recognition motif (TRM) and increases the affinity of Tat-P-TEFb for TAR RNA 30-fold. Interface mutations in AFF1 reduced Tat-AFF1 affinity in vivo and Tat-dependent transcription from the HIV promoter. AFF4 acts as a two-step filter to increase selectivity of Tat and TAR for SECs over P-TEFb alone.","method":"X-ray crystal structure of quaternary complex; in vivo affinity assays with interface mutants; transcription reporter assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and functional transcription validation; multiple orthogonal methods","pmids":["24843025"],"is_preprint":false},{"year":2014,"finding":"Crystal structure of Tat·AFF4·P-TEFb complex revealed that Tat binding to AFF4·P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of CycT1 and the α-3(10) helix of AFF4. The Tat-TAR recognition motif (TRM) in CycT1 interacts with both Tat and AFF4, exposing arginine side chains for TAR RNA binding. Structural modeling suggests AFF1 and AFF4 are preferred over AFF2/3 for interaction with Tat·P-TEFb.","method":"X-ray crystallography of Tat·AFF4·P-TEFb complex","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure providing detailed atomic-level mechanistic insights; independent replication of Tat-AFF4-P-TEFb structural findings across labs","pmids":["24727379"],"is_preprint":false},{"year":2015,"finding":"Gain-of-function missense mutations in AFF4 cause CHOPS syndrome. Transcriptome and ChIP-seq analyses demonstrated altered genome-wide binding of AFF4, cohesin, and RNAP2 in CHOPS and Cornelia de Lange syndrome (CdLS). Direct molecular interaction between the SEC, cohesin, and RNAP2 was demonstrated, functionally linking the super elongation complex and cohesin.","method":"Exome sequencing; ChIP-seq; co-immunoprecipitation to demonstrate SEC-cohesin-RNAP2 interaction","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP-seq genome-wide binding analysis combined with direct Co-IP of SEC-cohesin-RNAP2, multiple orthogonal methods","pmids":["25730767"],"is_preprint":false},{"year":2016,"finding":"Cryo-EM/integrative structure of the HIV-1 TAR-Tat-AFF4-CDK9-CycT1 complex at 5.9 Å resolution showed TAR central loop contacts the CycT1 TRM and the second Tat Zn2+-binding loop. HDX showed AFF4 helix 2 is stabilized in the TAR complex despite not touching RNA, explaining how AFF4 enhances TAR binding to the SEC 50-fold. The Tat ARM enters the TAR major groove between the bulge and central loop.","method":"Cryo-EM structure; hydrogen-deuterium exchange (HDX); RNA SHAPE; SAXS; functional transcription assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — integrative structure (cryo-EM + SAXS + HDX) with functional assays; multiple orthogonal methods in single rigorous study","pmids":["27731797"],"is_preprint":false},{"year":2017,"finding":"Crystal structure (2.0 Å) of ELL2 C-terminal domain bound to its 50-residue binding site on AFF4 (ELLBow) revealed ELL2 has an arch-shaped fold similar to tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin (elbow joint) occupying the concave surface of ELL2. The AFF4-ELL2 interface surface is important for ELL2 promotion of HIV-1 Tat-mediated proviral transcription.","method":"X-ray crystallography at 2.0 Å; mutagenesis; transcription reporter assays","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with mutagenesis and functional validation in single study","pmids":["28134250"],"is_preprint":false},{"year":2017,"finding":"AFF4 depletion in MSCs inhibits osteogenic differentiation (decreased ALP activity, mineralization, osteogenic gene expression), while AFF4 overexpression enhances it. AFF4 is enriched at the promoter region of ID1, and AFF4 knockdown blunts BMP2-induced BRE luciferase activity and SP7/ALP expression.","method":"siRNA knockdown; lentiviral overexpression; ChIP at ID1 promoter; luciferase reporter assay; in vivo MSC-mediated bone formation","journal":"Bone research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase reporter assays combined with in vivo functional data; single lab","pmids":["28955517"],"is_preprint":false},{"year":2019,"finding":"X-ray crystal structure of AFF4 C-terminal homology domain (CHD) at 2.2 Å revealed a novel eight-helix domain distantly related to tetratricopeptide repeat motifs. AFF4-CHD mediates AFF4 homodimerization and AFF1-AFF4 heterodimerization. Fluorescence anisotropy experiments showed AFF4-CHD interacts with both RNA and DNA in vitro. A surface loop in AFF4-CHD was identified as a substrate for CDK9, which triggers release of Pol II from promoter-proximal pausing.","method":"X-ray crystallography at 2.2 Å; biochemical dimerization assays; fluorescence anisotropy; in vitro CDK9 kinase assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with multiple biochemical assays (dimerization, nucleic acid binding, kinase substrate identification) in single study","pmids":["31147444"],"is_preprint":false},{"year":2020,"finding":"Crystal structure of AFF4-THD (TPRL with Handle Region Dimerization Domain) at 2.4 Å revealed the α4, α5, and α6 helices of one AFF4-THD mediate dimer formation packing against equivalent regions of the second molecule. Single mutations F1014A or Y1096A of AFF4 impair dimer formation. AFF4 dimerization is essential for transactivation of HIV-1 provirus but mutations of AFF1/4 dimerization residues have no effect on interaction with other SEC subunits.","method":"X-ray crystallography at 2.4 Å; site-directed mutagenesis; co-IP for SEC subunit interactions; HIV-1 transactivation reporter assay","journal":"Cell discovery","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure with mutagenesis and functional transactivation assay; multiple orthogonal methods","pmids":["32128251"],"is_preprint":false},{"year":2007,"finding":"MCEF (AFF4) localizes exclusively to the nucleus. Three distinct protein sequences encoded by three separate exons mediate nuclear localization. Ectopic expression of MCEF represses HIV-1 LTR-directed RNA Pol II transcription at the level of Tat-transactivation.","method":"MCEF-EGFP fusion protein live cell imaging; 20 deletion mutant constructs; HIV-1 LTR reporter transcription assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — systematic deletion mapping with fluorescent protein fusions combined with functional HIV transcription assay; single lab","pmids":["17389929"],"is_preprint":false},{"year":2005,"finding":"AF5q31 (AFF4) knockout mice show male infertility with azoospermia due to arrest of spermiogenesis. AFF4 is preferentially expressed in Sertoli cells. Knockout mice display severely impaired expression of protamine 1, protamine 2, and transition protein 2 and increased apoptosis in seminiferous tubules, indicating AFF4 functions as a transcriptional regulator in testicular somatic cells essential for male germ cell differentiation.","method":"Knockout mouse model; histological analysis; gene expression analysis of spermatogenesis genes; cell-type-specific expression profiling","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout with specific phenotypic readout and molecular characterization of downstream gene expression; thorough histological and molecular analyses","pmids":["16024815"],"is_preprint":false},{"year":2012,"finding":"AFF4 expression in hypothalamic neurons is induced by ghrelin and fasting. AFF4 overexpression specifically induces AMPKα2 subunit expression and increases AMPKα2 promoter activity. AFF4 also increases phosphorylation of acetyl-CoA carboxylase α (ACCα) downstream of AMPK. Ghrelin-induced AMPKα2 expression and ACCα phosphorylation in the late phase of activation were attenuated by AFF4 siRNA knockdown.","method":"Overexpression and siRNA knockdown in hypothalamic neuronal GT1-7 cells; promoter luciferase assay; Western blot for phosphorylation","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — promoter luciferase plus knockdown with phosphorylation readout; single lab, multiple methods","pmids":["22528490"],"is_preprint":false},{"year":2019,"finding":"FUS (fused in sarcoma) physically interacts with AFF4 in cells and forms nuclear punctuated condensates with AFF4, which are disrupted by aliphatic alcohol treatment. FUS inhibits activation of HIV transcription by AFF4 and ELL2. FUS depletion elevates occupancy of AFF4 and Cdk9 on the viral promoter, genome-wide FUS knockdown leads to increased AFF4 and Cdk9 occupancy on gene promoters, and FUS knockout delays HIV entry into latency.","method":"Co-localization (live cell imaging); ChIP for AFF4/Cdk9 occupancy; siRNA/CRISPR knockout; HIV latency model; transcription assays","journal":"Retrovirology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live cell imaging of co-localization, ChIP occupancy, and functional HIV transcription assays; single lab, multiple orthogonal methods","pmids":["31238957"],"is_preprint":false},{"year":2019,"finding":"METTL3-mediated m6A modification directly targets AFF4 mRNA in bladder cancer cells. AFF4 binds to the MYC promoter and promotes MYC expression, operating as part of an AFF4/NF-κB/MYC signaling network downstream of METTL3-mediated m6A modification.","method":"m6A sequencing; m6A methylated RNA immunoprecipitation qRT-PCR; ChIP for AFF4 at MYC promoter; siRNA knockdown with gene expression readout","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — m6A-seq identifies AFF4 as direct METTL3 target, ChIP shows AFF4 at MYC promoter; single lab with two orthogonal methods","pmids":["30659266"],"is_preprint":false},{"year":2020,"finding":"AFF4 regulates m6A-dependent expression and promotes SOX2 and MYC transcription in bladder cancer stem cells. AFF4 binds to promoter regions of SOX2 and MYC to sustain their transcription; AFF4 knockdown phenocopies METTL3 ablation and diminishes tumor-initiating capability in vivo.","method":"siRNA knockdown; ChIP at SOX2 and MYC promoters; xenograft tumor-initiating assays; sphere-forming and ALDH activity assays","journal":"Stem cells international","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — ChIP at promoters combined with in vivo xenograft functional assay; single lab","pmids":["32676121"],"is_preprint":false},{"year":2022,"finding":"AFF4 regulates autophagy during adipogenesis by directly binding to autophagy-related proteins ATG5 and ATG16L1 and promoting their transcription. Adipose-specific Aff4 knockout mice have impaired adipocyte development and white fat depot formation. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overexpressing cells, while overexpression of ATG5/ATG16L1 rescues impaired adipogenesis in Aff4-knockout cells.","method":"Co-immunoprecipitation (AFF4-ATG5/ATG16L1 interaction); siRNA knockdown; lentiviral overexpression; Fabp4-cre Aff4 conditional knockout mice; rescue experiments","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP identifying direct binding partners, conditional knockout mouse model, epistasis rescue experiments across multiple cell types and in vivo","pmids":["36149892"],"is_preprint":false},{"year":2024,"finding":"AFF4 and AFF1 function antagonistically at transcription start sites: AFF4 is enriched downstream of the TSS while AFF1 binds upstream. AFF4 disruption causes slow elongation and early termination in a subset of AFF4-bound active genes; AFF1 deletion leads to fast elongation and transcriptional readthrough in the same gene subset. AFF1 knockdown increases AFF4 levels at chromatin and vice versa.","method":"ChIP-seq; PRO-seq; RNA-seq; CUT&Tag; siRNA knockdown of AFF1 and AFF4","journal":"Journal of molecular cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple genome-wide methods (ChIP-seq, PRO-seq, RNA-seq) with genetic knockdown of both paralogs; comprehensive mechanistic dissection","pmids":["37528066"],"is_preprint":false},{"year":2023,"finding":"AFF4 knockdown in HEL cells decreases cellular levels and global chromatin occupancy of CTD serine 2 phosphorylated Pol II. AFF4 promotes pause release likely by facilitating P-TEFb binding to Pol II. AFF4 loss increases promoter-proximal pause of Pol II on heat shock and thousands of non-heat shock genes.","method":"ChIP-seq (CTD Ser2-P Pol II); PRO-seq; CUT&Tag; RNA-seq; RNA interference knockdown","journal":"Yi chuan = Hereditas","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple genome-wide methods (ChIP-seq, PRO-seq) with consistent findings; single lab","pmids":["37609817"],"is_preprint":false},{"year":2025,"finding":"P70S6K phosphorylates AFF4 at S831 in an insulin-dependent manner, and this phosphorylation is attenuated in aged, insulin-resistant bone. Phosphorylation of S831 in AFF4 increases recruitment of chromatin remodelers ENL/AF9 to crotonylated histone via the YEATS domain, promoting gene-specific transcriptional elongation activation. In insulin-resistant osteoblasts, AFF4 S831 phosphorylation is defective and associated with reduced transcriptional elongation at discrete genomic locations.","method":"Phosphoproteomic analysis; in vitro kinase assay (P70S6K + AFF4); ChIP for ENL/AF9 at specific loci; zebrafish functional genomic screen; loss-of-function experiments in osteoblasts","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — phosphoproteomics identifying P70S6K as writer of AFF4 S831, in vitro kinase assay, ChIP showing downstream chromatin remodeler recruitment; multiple orthogonal methods","pmids":["41476161"],"is_preprint":false},{"year":2024,"finding":"MeCP2 directly binds AFF4 (the SEC scaffold) via the MeCP2 transcriptional repression domain. Loss of MeCP2 in mouse cortex reduces AFF4 binding at a subset of genes involved in synaptic function, which also show the strongest decrease in RNA Pol II genebody binding. MeCP2 physically interacts with the SEC in human cells and mouse brain.","method":"Co-immunoprecipitation (human cells and mouse brain); direct binding assay (MeCP2-AFF4); ChIP for AFF4 and Pol II; Drosophila genetic screen for SEC modifiers of MeCP2 phenotypes","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding demonstration and ChIP with in vivo mouse brain data; preprint, not yet peer-reviewed","pmids":[],"is_preprint":true},{"year":2027,"finding":"AFF4 regulates NFIC transcription during odontogenic differentiation in dental pulp cells. AFF4 depletion decreases ALP activity and odontogenic gene expression; overexpression of NFIC rescues restricted differentiation in AFF4-depleted cells.","method":"siRNA knockdown; lentiviral overexpression; ALP activity and mineralization assays; rescue experiment with NFIC overexpression","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — knockdown/overexpression with targeted rescue identifying NFIC as downstream transcriptional target; single lab","pmids":["32139123"],"is_preprint":false},{"year":2014,"finding":"A cyclin T1 mutant (V107E) that cannot bind Hexim1 or CDK9 and cannot assemble on HIV TAR or 7SK snRNA retains strong binding to AFF4, demonstrating that AFF4 binding to CycT1 is mediated by a distinct surface from CDK9 and Hexim1 binding sites. This mutant enforces HIV transcription repression, demonstrating the functional importance of the AFF4-CycT1 interaction for transcription activation.","method":"Site-directed mutagenesis of CycT1; co-immunoprecipitation/binding assays; HIV transcription reporter assays; T cell latency model","journal":"Retrovirology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic mutagenesis with binding and functional assays identifying distinct binding surfaces; single lab","pmids":["24985467"],"is_preprint":false},{"year":2023,"finding":"AFF4 promotes expression of HPRT1 and IMPDH2 (nucleotide metabolism enzymes) in pancreatic ductal carcinoma cells. CDK9 mediates AFF4 phosphorylation at S388, which is required for PAX2-mediated recruitment of AFF4 to drive HPRT1 and IMPDH2 expression. Xenograft studies confirmed HPRT1 and IMPDH2 function genetically downstream of AFF4.","method":"RNA-seq; CUT&Tag (AFF4 chromatin occupancy); xenograft rescue assays; phosphorylation assay","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — CUT&Tag and xenograft epistasis showing AFF4→HPRT1/IMPDH2 pathway; single lab, CDK9-AFF4 phosphorylation at S388 identified","pmids":["37063434"],"is_preprint":false}],"current_model":"AFF4 is the central intrinsically disordered scaffold protein of the Super Elongation Complex (SEC), directly binding P-TEFb (via CycT1), ELL2, ENL/AF9, and other elongation factors through distinct short interaction motifs to release promoter-proximally paused RNA Polymerase II; its CHD domain mediates AFF4 homodimerization and AFF1-AFF4 heterodimerization, its dimerization domain (THD) is required for HIV-1 proviral transactivation, it is phosphorylated by CDK9 and P70S6K at specific sites that regulate elongation complex assembly and chromatin remodeler recruitment, it directly interacts with cohesin and MeCP2, and loss-of-function studies demonstrate essential roles in spermatogenesis, osteogenic/adipogenic differentiation, and regulation of specific transcriptional programs including those driving leukemia and HIV transcription."},"narrative":{"mechanistic_narrative":"AFF4 is the central intrinsically disordered scaffold of the Super Elongation Complex (SEC), an assembly that releases promoter-proximally paused RNA Polymerase II to drive productive transcriptional elongation [PMID:20159561, PMID:37609817]. Along its disordered axis, AFF4 uses distinct short interaction motifs to directly recruit P-TEFb (through CycT1), ELL2, and ENL/AF9, integrating these elongation factors into a single complex acting on the same polymerase [PMID:20471948, PMID:23251033]. Crystallographic analysis shows AFF4 meanders over the CycT1 surface without contacting CDK9, binding through a surface distinct from the CDK9 and Hexim1 sites [PMID:23471103, PMID:24985467], while its C-terminal homology domain (CHD) and a dimerization domain (THD) mediate AFF4 homodimerization and AFF1–AFF4 heterodimerization, with dimerization being essential for HIV-1 proviral transactivation but dispensable for binding other SEC subunits [PMID:31147444, PMID:32128251]. AFF4 is a key cofactor for HIV-1 Tat: it bridges P-TEFb and ELL2 into a bifunctional elongation complex and, by partially ordering the CycT1 Tat-TAR recognition motif, increases the affinity of Tat-P-TEFb for TAR RNA, acting as a selectivity filter that favors SEC assembly over P-TEFb alone [PMID:20471948, PMID:24843025, PMID:27731797]. Its activity is tuned by phosphorylation—CDK9 modifies a CHD surface loop to trigger pause release and phosphorylates S388 for PAX2-directed recruitment, while P70S6K phosphorylates S831 in an insulin-dependent manner to enhance ENL/AF9 recruitment to crotonylated histones [PMID:31147444, PMID:41476161, PMID:37063434]. Through these activities AFF4 controls discrete transcriptional programs and physically engages cohesin and RNAP2; gain-of-function missense mutations in AFF4 cause CHOPS syndrome [PMID:25730767]. Functionally, AFF4 is essential for spermatogenesis via Sertoli-cell transcriptional control [PMID:16024815] and for osteogenic and adipogenic differentiation, the latter through direct transcriptional activation of autophagy genes ATG5 and ATG16L1 [PMID:28955517, PMID:36149892], and it sustains oncogenic transcription of MYC, SOX2 and nucleotide-metabolism genes in bladder and pancreatic cancers [PMID:30659266, PMID:32676121, PMID:37063434]. AFF4 acts antagonistically with its paralog AFF1 across the transcription start site to set elongation rate and termination [PMID:37528066].","teleology":[{"year":2002,"claim":"Establishing that AFF4 (MCEF) is a physical partner of P-TEFb placed it within the transcriptional elongation machinery, the first mechanistic anchor for its function.","evidence":"Affinity purification of epitope-tagged CDK9 with protein sequencing and reciprocal co-immunoprecipitation","pmids":["12065898"],"confidence":"Medium","gaps":["Did not define the binding interface or which P-TEFb subunit is contacted","No functional consequence of the interaction shown"]},{"year":2005,"claim":"A knockout established a physiological requirement for AFF4 in vivo, showing it is essential for spermatogenesis through somatic-cell transcriptional control.","evidence":"Aff4 knockout mouse with histology and spermatogenesis gene-expression profiling","pmids":["16024815"],"confidence":"High","gaps":["Did not connect the testicular phenotype to SEC scaffolding biochemistry","Direct transcriptional targets in Sertoli cells not mapped genome-wide"]},{"year":2007,"claim":"Mapping the determinants of AFF4 nuclear localization and its repressive effect on Tat-transactivation defined where and how it acts on HIV transcription.","evidence":"EGFP-fusion live-cell imaging with deletion mapping and HIV-1 LTR reporter assays","pmids":["17389929"],"confidence":"Medium","gaps":["Repression in this overexpression context contrasts with later activation models; context dependence unresolved","No interaction-partner mapping"]},{"year":2010,"claim":"Identifying AFF4 as a core SEC subunit required for complex stability and for MLL-chimera target gene expression defined its central scaffolding role and disease relevance in leukemia.","evidence":"Affinity purification of MLL chimeras with mass spectrometry and siRNA knockdown transcriptional readouts","pmids":["20159561","20471948"],"confidence":"High","gaps":["Atomic-level interaction architecture not yet defined","How AFF4 selects target genes not addressed"]},{"year":2012,"claim":"Systematic mapping of AFF4's short interaction motifs showed it recruits ELL2, ENL/AF9 and P-TEFb along a disordered axis, establishing the architecture of the Tat elongation complex.","evidence":"In vitro pulldowns with truncation/mutation constructs plus in vivo co-IP and binding-site analysis","pmids":["23251033"],"confidence":"High","gaps":["High-resolution structures of individual interfaces not yet available","Stoichiometry and dynamics of full assembly unresolved"]},{"year":2014,"claim":"A series of crystal and integrative structures of Tat·AFF4·P-TEFb and the TAR-bound complex revealed how AFF4 contacts CycT1 (but not CDK9), orders the Tat-TAR recognition motif, and acts as a selectivity filter that enhances TAR binding.","evidence":"X-ray crystallography, cryo-EM/SAXS/HDX integrative structures, interface mutagenesis and transcription assays","pmids":["23471103","24843025","24727379","27731797","24985467"],"confidence":"High","gaps":["Structures focus on the HIV Tat complex; cellular-target SEC architecture less defined","Conformational dynamics during pause release not fully captured"]},{"year":2015,"claim":"Gain-of-function AFF4 mutations causing CHOPS syndrome, with altered genome-wide AFF4/cohesin/RNAP2 binding, linked SEC dysregulation to human disease and connected the SEC physically to cohesin.","evidence":"Exome sequencing, ChIP-seq, and co-IP demonstrating SEC-cohesin-RNAP2 interaction","pmids":["25730767"],"confidence":"High","gaps":["Mechanism by which mutant AFF4 alters cohesin distribution not resolved","Causal target genes of the syndrome phenotype not pinpointed"]},{"year":2017,"claim":"High-resolution structure of the ELL2-AFF4 (ELLBow) interface and functional studies in mesenchymal stem cells extended AFF4 mechanism to both SEC assembly detail and tissue differentiation.","evidence":"2.0 Å crystallography with mutagenesis; siRNA/overexpression with ChIP at ID1 and BMP2 reporter assays in MSCs","pmids":["28134250","28955517"],"confidence":"High","gaps":["Whether ELLBow interface mutations affect non-HIV transcription not tested","Osteogenic role mechanistically separate from SEC scaffolding not fully established"]},{"year":2019,"claim":"Structural definition of the AFF4-CHD as a dimerization, nucleic-acid-binding and CDK9 substrate module, plus identification of FUS as a condensate-forming regulator, refined how AFF4 oligomerizes and is regulated.","evidence":"2.2 Å crystallography, dimerization and fluorescence anisotropy assays, in vitro CDK9 kinase assay; co-localization, ChIP and HIV latency models for FUS","pmids":["31147444","31238957"],"confidence":"High","gaps":["Physiological consequence of CHD nucleic-acid binding not defined","FUS data are single-lab and Medium confidence"]},{"year":2020,"claim":"Structure of the AFF4-THD dimerization domain and point mutants showed dimerization is essential for HIV-1 transactivation yet dispensable for other SEC-subunit contacts, separating two AFF4 functions.","evidence":"2.4 Å crystallography, site-directed mutagenesis, co-IP and HIV-1 transactivation reporter assays","pmids":["32128251","32139123"],"confidence":"High","gaps":["Why dimerization is selectively required for proviral transactivation not mechanistically explained","Role of dimerization at cellular target genes untested"]},{"year":2022,"claim":"Conditional knockout and direct binding to ATG5/ATG16L1 established AFF4 as a transcriptional driver of autophagy genes required for adipocyte development, broadening its differentiation roles.","evidence":"Co-IP, conditional Aff4 knockout mice, and epistasis rescue with ATG5/ATG16L1","pmids":["36149892"],"confidence":"High","gaps":["Whether ATG5/ATG16L1 activation occurs through canonical SEC elongation not directly shown","Tissue specificity of this program unexplained"]},{"year":2024,"claim":"Genome-wide dissection showed AFF4 and AFF1 act antagonistically across the TSS to control elongation rate and termination, and that AFF4 promotes Ser2-P Pol II and pause release, defining its quantitative role in elongation control.","evidence":"ChIP-seq, PRO-seq, RNA-seq and CUT&Tag with siRNA knockdown of both paralogs","pmids":["37528066","37609817"],"confidence":"High","gaps":["What distinguishes AFF1- versus AFF4-favored genes is unresolved","Mechanism of mutual chromatin-level antagonism unclear"]},{"year":2024,"claim":"MeCP2 was shown to directly bind AFF4 and recruit the SEC to synaptic genes, implicating AFF4-dependent elongation in MeCP2 (Rett-related) biology.","evidence":"Co-IP in human cells and mouse brain, direct binding assay, ChIP and Drosophila genetic screen (preprint)","pmids":[],"confidence":"Medium","gaps":["Preprint, not yet peer-reviewed","Whether MeCP2-AFF4 recruitment is direct or via additional factors needs further validation"]},{"year":2025,"claim":"Identification of P70S6K-mediated AFF4 S831 phosphorylation linking insulin signaling to ENL/AF9 recruitment onto crotonylated histones established a signaling-to-elongation axis relevant to insulin-resistant bone.","evidence":"Phosphoproteomics, in vitro kinase assay, ChIP for ENL/AF9, and zebrafish/osteoblast loss-of-function","pmids":["41476161"],"confidence":"High","gaps":["Full set of S831-dependent target genes not mapped","How phosphorylation alters the AFF4-ENL/AF9 interface structurally not defined"]},{"year":null,"claim":"How AFF4 selects its specific target gene sets across distinct tissues and disease states—and how its many phosphorylation and dimerization states are integrated to determine which elongation programs are activated—remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying model linking AFF4 post-translational states to target-gene specificity","Relationship between SEC scaffolding and tissue-specific differentiation programs incompletely defined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[14,17,18,24,19]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,3]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[8,11]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[11]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[4,5,25]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[13]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[7,20,21]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression 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In the SEC complex, AFF4 acts as a central scaffold that recruits other factors through direct interactions with ELL proteins (ELL, ELL2 or ELL3) and the P-TEFb complex. In case of infection by HIV-1 virus, the SEC complex is recruited by the viral Tat protein to stimulate viral gene expression","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q9UHB7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/AFF4","classification":"Not Classified","n_dependent_lines":7,"n_total_lines":1208,"dependency_fraction":0.005794701986754967},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/AFF4","total_profiled":1310},"omim":[{"mim_id":"619297","title":"KINSSHIP SYNDROME; KINS","url":"https://www.omim.org/entry/619297"},{"mim_id":"616368","title":"CHOPS SYNDROME; CHOPS","url":"https://www.omim.org/entry/616368"},{"mim_id":"611957","title":"MICRO RNA 378; MIRN378","url":"https://www.omim.org/entry/611957"},{"mim_id":"605043","title":"MEDIATOR COMPLEX SUBUNIT 26; MED26","url":"https://www.omim.org/entry/605043"},{"mim_id":"604417","title":"ALF TRANSCRIPTION ELONGATION FACTOR 4; AFF4","url":"https://www.omim.org/entry/604417"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli fibrillar center","reliability":"Additional"},{"location":"Nuclear bodies","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in 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AFF4 is required for SEC stability and proper transcription by poised RNA polymerase II in metazoans. Knockdown of AFF4 in leukemic cells reduces MLL chimera target gene expression.\",\n      \"method\": \"Affinity purification of MLL chimeras followed by mass spectrometry identification of SEC components; siRNA knockdown with gene expression readout\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal purification, multiple MLL chimeras tested, functional knockdown with defined transcriptional phenotype; replicated across multiple chimeras in same study\",\n      \"pmids\": [\"20159561\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"AFF4 bridges P-TEFb and ELL2 into a bifunctional elongation complex that greatly activates HIV-1 transcription. Through scaffolding functions of both Tat and AFF4, P-TEFb and ELL2 cooperate on the same RNA polymerase II. Without Tat, AFF4 can mediate the ELL2-P-TEFb interaction inefficiently; Tat overcomes this by bringing more ELL2 to P-TEFb and stabilizing ELL2 in a process requiring active P-TEFb.\",\n      \"method\": \"Sequential affinity-purification, Co-IP, in vitro binding assays, transcription reporter assays\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — sequential affinity purification plus functional transcription assays; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"20471948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"MCEF (AFF4) was identified as a binding partner of P-TEFb (CDK9/CyclinT1) by affinity purification from stably transfected cells expressing epitope-tagged CDK9; antisera against recombinant MCEF specifically immunoprecipitated P-TEFb.\",\n      \"method\": \"Affinity purification of P-TEFb complex followed by protein sequencing; co-immunoprecipitation\",\n      \"journal\": \"Journal of biomedical science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with direct sequencing identification, single lab, two complementary methods\",\n      \"pmids\": [\"12065898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AFF4 acts as the central scaffold of the HIV-1 Tat elongation complex, recruiting ELL2, ENL/AF9, and P-TEFb through direct interactions with short hydrophobic regions along its structurally disordered axis. CycT1, ELL2, and ENL/AF9 act as bridging components linking the complex to P-TEFb and the PAF complex. Binding sites were mapped both in vitro and in vivo.\",\n      \"method\": \"In vitro binding mapping (pulldowns with truncation/mutation constructs), in vivo co-IP, structural analysis of binding sites\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — systematic in vitro binding site mapping combined with in vivo validation; multiple interaction partners mapped with orthogonal methods\",\n      \"pmids\": [\"23251033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Crystal structure of AFF4 in complex with P-TEFb (CDK9/CycT1) revealed that AFF4 meanders over the surface of CycT1 but makes no stable contacts with CDK9. AFF4 is positioned to make direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. Interface mutations in AFF4 reduced CycT1 binding and AFF4-dependent transcription.\",\n      \"method\": \"X-ray crystal structure determination of tripartite complex; interface mutagenesis; transcription assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus mutagenesis plus functional transcription assay; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"23471103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of quaternary Tat-P-TEFb-AFF4 complex showed Tat and AFF4 fold on the CycT1 surface and interact directly. AFF4 binding partially orders the CycT1 Tat-TAR recognition motif (TRM) and increases the affinity of Tat-P-TEFb for TAR RNA 30-fold. Interface mutations in AFF1 reduced Tat-AFF1 affinity in vivo and Tat-dependent transcription from the HIV promoter. AFF4 acts as a two-step filter to increase selectivity of Tat and TAR for SECs over P-TEFb alone.\",\n      \"method\": \"X-ray crystal structure of quaternary complex; in vivo affinity assays with interface mutants; transcription reporter assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and functional transcription validation; multiple orthogonal methods\",\n      \"pmids\": [\"24843025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Crystal structure of Tat·AFF4·P-TEFb complex revealed that Tat binding to AFF4·P-TEFb causes concerted structural changes in AFF4 via a shift of helix H5' of CycT1 and the α-3(10) helix of AFF4. The Tat-TAR recognition motif (TRM) in CycT1 interacts with both Tat and AFF4, exposing arginine side chains for TAR RNA binding. Structural modeling suggests AFF1 and AFF4 are preferred over AFF2/3 for interaction with Tat·P-TEFb.\",\n      \"method\": \"X-ray crystallography of Tat·AFF4·P-TEFb complex\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure providing detailed atomic-level mechanistic insights; independent replication of Tat-AFF4-P-TEFb structural findings across labs\",\n      \"pmids\": [\"24727379\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Gain-of-function missense mutations in AFF4 cause CHOPS syndrome. Transcriptome and ChIP-seq analyses demonstrated altered genome-wide binding of AFF4, cohesin, and RNAP2 in CHOPS and Cornelia de Lange syndrome (CdLS). Direct molecular interaction between the SEC, cohesin, and RNAP2 was demonstrated, functionally linking the super elongation complex and cohesin.\",\n      \"method\": \"Exome sequencing; ChIP-seq; co-immunoprecipitation to demonstrate SEC-cohesin-RNAP2 interaction\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP-seq genome-wide binding analysis combined with direct Co-IP of SEC-cohesin-RNAP2, multiple orthogonal methods\",\n      \"pmids\": [\"25730767\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Cryo-EM/integrative structure of the HIV-1 TAR-Tat-AFF4-CDK9-CycT1 complex at 5.9 Å resolution showed TAR central loop contacts the CycT1 TRM and the second Tat Zn2+-binding loop. HDX showed AFF4 helix 2 is stabilized in the TAR complex despite not touching RNA, explaining how AFF4 enhances TAR binding to the SEC 50-fold. The Tat ARM enters the TAR major groove between the bulge and central loop.\",\n      \"method\": \"Cryo-EM structure; hydrogen-deuterium exchange (HDX); RNA SHAPE; SAXS; functional transcription assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — integrative structure (cryo-EM + SAXS + HDX) with functional assays; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"27731797\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Crystal structure (2.0 Å) of ELL2 C-terminal domain bound to its 50-residue binding site on AFF4 (ELLBow) revealed ELL2 has an arch-shaped fold similar to tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin (elbow joint) occupying the concave surface of ELL2. The AFF4-ELL2 interface surface is important for ELL2 promotion of HIV-1 Tat-mediated proviral transcription.\",\n      \"method\": \"X-ray crystallography at 2.0 Å; mutagenesis; transcription reporter assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with mutagenesis and functional validation in single study\",\n      \"pmids\": [\"28134250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"AFF4 depletion in MSCs inhibits osteogenic differentiation (decreased ALP activity, mineralization, osteogenic gene expression), while AFF4 overexpression enhances it. AFF4 is enriched at the promoter region of ID1, and AFF4 knockdown blunts BMP2-induced BRE luciferase activity and SP7/ALP expression.\",\n      \"method\": \"siRNA knockdown; lentiviral overexpression; ChIP at ID1 promoter; luciferase reporter assay; in vivo MSC-mediated bone formation\",\n      \"journal\": \"Bone research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase reporter assays combined with in vivo functional data; single lab\",\n      \"pmids\": [\"28955517\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"X-ray crystal structure of AFF4 C-terminal homology domain (CHD) at 2.2 Å revealed a novel eight-helix domain distantly related to tetratricopeptide repeat motifs. AFF4-CHD mediates AFF4 homodimerization and AFF1-AFF4 heterodimerization. Fluorescence anisotropy experiments showed AFF4-CHD interacts with both RNA and DNA in vitro. A surface loop in AFF4-CHD was identified as a substrate for CDK9, which triggers release of Pol II from promoter-proximal pausing.\",\n      \"method\": \"X-ray crystallography at 2.2 Å; biochemical dimerization assays; fluorescence anisotropy; in vitro CDK9 kinase assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with multiple biochemical assays (dimerization, nucleic acid binding, kinase substrate identification) in single study\",\n      \"pmids\": [\"31147444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Crystal structure of AFF4-THD (TPRL with Handle Region Dimerization Domain) at 2.4 Å revealed the α4, α5, and α6 helices of one AFF4-THD mediate dimer formation packing against equivalent regions of the second molecule. Single mutations F1014A or Y1096A of AFF4 impair dimer formation. AFF4 dimerization is essential for transactivation of HIV-1 provirus but mutations of AFF1/4 dimerization residues have no effect on interaction with other SEC subunits.\",\n      \"method\": \"X-ray crystallography at 2.4 Å; site-directed mutagenesis; co-IP for SEC subunit interactions; HIV-1 transactivation reporter assay\",\n      \"journal\": \"Cell discovery\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure with mutagenesis and functional transactivation assay; multiple orthogonal methods\",\n      \"pmids\": [\"32128251\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"MCEF (AFF4) localizes exclusively to the nucleus. Three distinct protein sequences encoded by three separate exons mediate nuclear localization. Ectopic expression of MCEF represses HIV-1 LTR-directed RNA Pol II transcription at the level of Tat-transactivation.\",\n      \"method\": \"MCEF-EGFP fusion protein live cell imaging; 20 deletion mutant constructs; HIV-1 LTR reporter transcription assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — systematic deletion mapping with fluorescent protein fusions combined with functional HIV transcription assay; single lab\",\n      \"pmids\": [\"17389929\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"AF5q31 (AFF4) knockout mice show male infertility with azoospermia due to arrest of spermiogenesis. AFF4 is preferentially expressed in Sertoli cells. Knockout mice display severely impaired expression of protamine 1, protamine 2, and transition protein 2 and increased apoptosis in seminiferous tubules, indicating AFF4 functions as a transcriptional regulator in testicular somatic cells essential for male germ cell differentiation.\",\n      \"method\": \"Knockout mouse model; histological analysis; gene expression analysis of spermatogenesis genes; cell-type-specific expression profiling\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout with specific phenotypic readout and molecular characterization of downstream gene expression; thorough histological and molecular analyses\",\n      \"pmids\": [\"16024815\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"AFF4 expression in hypothalamic neurons is induced by ghrelin and fasting. AFF4 overexpression specifically induces AMPKα2 subunit expression and increases AMPKα2 promoter activity. AFF4 also increases phosphorylation of acetyl-CoA carboxylase α (ACCα) downstream of AMPK. Ghrelin-induced AMPKα2 expression and ACCα phosphorylation in the late phase of activation were attenuated by AFF4 siRNA knockdown.\",\n      \"method\": \"Overexpression and siRNA knockdown in hypothalamic neuronal GT1-7 cells; promoter luciferase assay; Western blot for phosphorylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — promoter luciferase plus knockdown with phosphorylation readout; single lab, multiple methods\",\n      \"pmids\": [\"22528490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"FUS (fused in sarcoma) physically interacts with AFF4 in cells and forms nuclear punctuated condensates with AFF4, which are disrupted by aliphatic alcohol treatment. FUS inhibits activation of HIV transcription by AFF4 and ELL2. FUS depletion elevates occupancy of AFF4 and Cdk9 on the viral promoter, genome-wide FUS knockdown leads to increased AFF4 and Cdk9 occupancy on gene promoters, and FUS knockout delays HIV entry into latency.\",\n      \"method\": \"Co-localization (live cell imaging); ChIP for AFF4/Cdk9 occupancy; siRNA/CRISPR knockout; HIV latency model; transcription assays\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live cell imaging of co-localization, ChIP occupancy, and functional HIV transcription assays; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"31238957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"METTL3-mediated m6A modification directly targets AFF4 mRNA in bladder cancer cells. AFF4 binds to the MYC promoter and promotes MYC expression, operating as part of an AFF4/NF-κB/MYC signaling network downstream of METTL3-mediated m6A modification.\",\n      \"method\": \"m6A sequencing; m6A methylated RNA immunoprecipitation qRT-PCR; ChIP for AFF4 at MYC promoter; siRNA knockdown with gene expression readout\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — m6A-seq identifies AFF4 as direct METTL3 target, ChIP shows AFF4 at MYC promoter; single lab with two orthogonal methods\",\n      \"pmids\": [\"30659266\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AFF4 regulates m6A-dependent expression and promotes SOX2 and MYC transcription in bladder cancer stem cells. AFF4 binds to promoter regions of SOX2 and MYC to sustain their transcription; AFF4 knockdown phenocopies METTL3 ablation and diminishes tumor-initiating capability in vivo.\",\n      \"method\": \"siRNA knockdown; ChIP at SOX2 and MYC promoters; xenograft tumor-initiating assays; sphere-forming and ALDH activity assays\",\n      \"journal\": \"Stem cells international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — ChIP at promoters combined with in vivo xenograft functional assay; single lab\",\n      \"pmids\": [\"32676121\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"AFF4 regulates autophagy during adipogenesis by directly binding to autophagy-related proteins ATG5 and ATG16L1 and promoting their transcription. Adipose-specific Aff4 knockout mice have impaired adipocyte development and white fat depot formation. Depleting ATG5 or ATG16L1 abrogates adipogenesis in AFF4-overexpressing cells, while overexpression of ATG5/ATG16L1 rescues impaired adipogenesis in Aff4-knockout cells.\",\n      \"method\": \"Co-immunoprecipitation (AFF4-ATG5/ATG16L1 interaction); siRNA knockdown; lentiviral overexpression; Fabp4-cre Aff4 conditional knockout mice; rescue experiments\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP identifying direct binding partners, conditional knockout mouse model, epistasis rescue experiments across multiple cell types and in vivo\",\n      \"pmids\": [\"36149892\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"AFF4 and AFF1 function antagonistically at transcription start sites: AFF4 is enriched downstream of the TSS while AFF1 binds upstream. AFF4 disruption causes slow elongation and early termination in a subset of AFF4-bound active genes; AFF1 deletion leads to fast elongation and transcriptional readthrough in the same gene subset. AFF1 knockdown increases AFF4 levels at chromatin and vice versa.\",\n      \"method\": \"ChIP-seq; PRO-seq; RNA-seq; CUT&Tag; siRNA knockdown of AFF1 and AFF4\",\n      \"journal\": \"Journal of molecular cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple genome-wide methods (ChIP-seq, PRO-seq, RNA-seq) with genetic knockdown of both paralogs; comprehensive mechanistic dissection\",\n      \"pmids\": [\"37528066\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AFF4 knockdown in HEL cells decreases cellular levels and global chromatin occupancy of CTD serine 2 phosphorylated Pol II. AFF4 promotes pause release likely by facilitating P-TEFb binding to Pol II. AFF4 loss increases promoter-proximal pause of Pol II on heat shock and thousands of non-heat shock genes.\",\n      \"method\": \"ChIP-seq (CTD Ser2-P Pol II); PRO-seq; CUT&Tag; RNA-seq; RNA interference knockdown\",\n      \"journal\": \"Yi chuan = Hereditas\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple genome-wide methods (ChIP-seq, PRO-seq) with consistent findings; single lab\",\n      \"pmids\": [\"37609817\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"P70S6K phosphorylates AFF4 at S831 in an insulin-dependent manner, and this phosphorylation is attenuated in aged, insulin-resistant bone. Phosphorylation of S831 in AFF4 increases recruitment of chromatin remodelers ENL/AF9 to crotonylated histone via the YEATS domain, promoting gene-specific transcriptional elongation activation. In insulin-resistant osteoblasts, AFF4 S831 phosphorylation is defective and associated with reduced transcriptional elongation at discrete genomic locations.\",\n      \"method\": \"Phosphoproteomic analysis; in vitro kinase assay (P70S6K + AFF4); ChIP for ENL/AF9 at specific loci; zebrafish functional genomic screen; loss-of-function experiments in osteoblasts\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — phosphoproteomics identifying P70S6K as writer of AFF4 S831, in vitro kinase assay, ChIP showing downstream chromatin remodeler recruitment; multiple orthogonal methods\",\n      \"pmids\": [\"41476161\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MeCP2 directly binds AFF4 (the SEC scaffold) via the MeCP2 transcriptional repression domain. Loss of MeCP2 in mouse cortex reduces AFF4 binding at a subset of genes involved in synaptic function, which also show the strongest decrease in RNA Pol II genebody binding. MeCP2 physically interacts with the SEC in human cells and mouse brain.\",\n      \"method\": \"Co-immunoprecipitation (human cells and mouse brain); direct binding assay (MeCP2-AFF4); ChIP for AFF4 and Pol II; Drosophila genetic screen for SEC modifiers of MeCP2 phenotypes\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding demonstration and ChIP with in vivo mouse brain data; preprint, not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2027,\n      \"finding\": \"AFF4 regulates NFIC transcription during odontogenic differentiation in dental pulp cells. AFF4 depletion decreases ALP activity and odontogenic gene expression; overexpression of NFIC rescues restricted differentiation in AFF4-depleted cells.\",\n      \"method\": \"siRNA knockdown; lentiviral overexpression; ALP activity and mineralization assays; rescue experiment with NFIC overexpression\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — knockdown/overexpression with targeted rescue identifying NFIC as downstream transcriptional target; single lab\",\n      \"pmids\": [\"32139123\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A cyclin T1 mutant (V107E) that cannot bind Hexim1 or CDK9 and cannot assemble on HIV TAR or 7SK snRNA retains strong binding to AFF4, demonstrating that AFF4 binding to CycT1 is mediated by a distinct surface from CDK9 and Hexim1 binding sites. This mutant enforces HIV transcription repression, demonstrating the functional importance of the AFF4-CycT1 interaction for transcription activation.\",\n      \"method\": \"Site-directed mutagenesis of CycT1; co-immunoprecipitation/binding assays; HIV transcription reporter assays; T cell latency model\",\n      \"journal\": \"Retrovirology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic mutagenesis with binding and functional assays identifying distinct binding surfaces; single lab\",\n      \"pmids\": [\"24985467\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"AFF4 promotes expression of HPRT1 and IMPDH2 (nucleotide metabolism enzymes) in pancreatic ductal carcinoma cells. CDK9 mediates AFF4 phosphorylation at S388, which is required for PAX2-mediated recruitment of AFF4 to drive HPRT1 and IMPDH2 expression. Xenograft studies confirmed HPRT1 and IMPDH2 function genetically downstream of AFF4.\",\n      \"method\": \"RNA-seq; CUT&Tag (AFF4 chromatin occupancy); xenograft rescue assays; phosphorylation assay\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — CUT&Tag and xenograft epistasis showing AFF4→HPRT1/IMPDH2 pathway; single lab, CDK9-AFF4 phosphorylation at S388 identified\",\n      \"pmids\": [\"37063434\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"AFF4 is the central intrinsically disordered scaffold protein of the Super Elongation Complex (SEC), directly binding P-TEFb (via CycT1), ELL2, ENL/AF9, and other elongation factors through distinct short interaction motifs to release promoter-proximally paused RNA Polymerase II; its CHD domain mediates AFF4 homodimerization and AFF1-AFF4 heterodimerization, its dimerization domain (THD) is required for HIV-1 proviral transactivation, it is phosphorylated by CDK9 and P70S6K at specific sites that regulate elongation complex assembly and chromatin remodeler recruitment, it directly interacts with cohesin and MeCP2, and loss-of-function studies demonstrate essential roles in spermatogenesis, osteogenic/adipogenic differentiation, and regulation of specific transcriptional programs including those driving leukemia and HIV transcription.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"AFF4 is the central intrinsically disordered scaffold of the Super Elongation Complex (SEC), an assembly that releases promoter-proximally paused RNA Polymerase II to drive productive transcriptional elongation [#0, #21]. Along its disordered axis, AFF4 uses distinct short interaction motifs to directly recruit P-TEFb (through CycT1), ELL2, and ENL/AF9, integrating these elongation factors into a single complex acting on the same polymerase [#1, #3]. Crystallographic analysis shows AFF4 meanders over the CycT1 surface without contacting CDK9, binding through a surface distinct from the CDK9 and Hexim1 sites [#4, #25], while its C-terminal homology domain (CHD) and a dimerization domain (THD) mediate AFF4 homodimerization and AFF1–AFF4 heterodimerization, with dimerization being essential for HIV-1 proviral transactivation but dispensable for binding other SEC subunits [#11, #12]. AFF4 is a key cofactor for HIV-1 Tat: it bridges P-TEFb and ELL2 into a bifunctional elongation complex and, by partially ordering the CycT1 Tat-TAR recognition motif, increases the affinity of Tat-P-TEFb for TAR RNA, acting as a selectivity filter that favors SEC assembly over P-TEFb alone [#1, #5, #8]. Its activity is tuned by phosphorylation—CDK9 modifies a CHD surface loop to trigger pause release and phosphorylates S388 for PAX2-directed recruitment, while P70S6K phosphorylates S831 in an insulin-dependent manner to enhance ENL/AF9 recruitment to crotonylated histones [#11, #22, #26]. Through these activities AFF4 controls discrete transcriptional programs and physically engages cohesin and RNAP2; gain-of-function missense mutations in AFF4 cause CHOPS syndrome [#7]. Functionally, AFF4 is essential for spermatogenesis via Sertoli-cell transcriptional control [#14] and for osteogenic and adipogenic differentiation, the latter through direct transcriptional activation of autophagy genes ATG5 and ATG16L1 [#10, #19], and it sustains oncogenic transcription of MYC, SOX2 and nucleotide-metabolism genes in bladder and pancreatic cancers [#17, #18, #26]. AFF4 acts antagonistically with its paralog AFF1 across the transcription start site to set elongation rate and termination [#20].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Establishing that AFF4 (MCEF) is a physical partner of P-TEFb placed it within the transcriptional elongation machinery, the first mechanistic anchor for its function.\",\n      \"evidence\": \"Affinity purification of epitope-tagged CDK9 with protein sequencing and reciprocal co-immunoprecipitation\",\n      \"pmids\": [\"12065898\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not define the binding interface or which P-TEFb subunit is contacted\", \"No functional consequence of the interaction shown\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"A knockout established a physiological requirement for AFF4 in vivo, showing it is essential for spermatogenesis through somatic-cell transcriptional control.\",\n      \"evidence\": \"Aff4 knockout mouse with histology and spermatogenesis gene-expression profiling\",\n      \"pmids\": [\"16024815\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Did not connect the testicular phenotype to SEC scaffolding biochemistry\", \"Direct transcriptional targets in Sertoli cells not mapped genome-wide\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Mapping the determinants of AFF4 nuclear localization and its repressive effect on Tat-transactivation defined where and how it acts on HIV transcription.\",\n      \"evidence\": \"EGFP-fusion live-cell imaging with deletion mapping and HIV-1 LTR reporter assays\",\n      \"pmids\": [\"17389929\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Repression in this overexpression context contrasts with later activation models; context dependence unresolved\", \"No interaction-partner mapping\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Identifying AFF4 as a core SEC subunit required for complex stability and for MLL-chimera target gene expression defined its central scaffolding role and disease relevance in leukemia.\",\n      \"evidence\": \"Affinity purification of MLL chimeras with mass spectrometry and siRNA knockdown transcriptional readouts\",\n      \"pmids\": [\"20159561\", \"20471948\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Atomic-level interaction architecture not yet defined\", \"How AFF4 selects target genes not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Systematic mapping of AFF4's short interaction motifs showed it recruits ELL2, ENL/AF9 and P-TEFb along a disordered axis, establishing the architecture of the Tat elongation complex.\",\n      \"evidence\": \"In vitro pulldowns with truncation/mutation constructs plus in vivo co-IP and binding-site analysis\",\n      \"pmids\": [\"23251033\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"High-resolution structures of individual interfaces not yet available\", \"Stoichiometry and dynamics of full assembly unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"A series of crystal and integrative structures of Tat·AFF4·P-TEFb and the TAR-bound complex revealed how AFF4 contacts CycT1 (but not CDK9), orders the Tat-TAR recognition motif, and acts as a selectivity filter that enhances TAR binding.\",\n      \"evidence\": \"X-ray crystallography, cryo-EM/SAXS/HDX integrative structures, interface mutagenesis and transcription assays\",\n      \"pmids\": [\"23471103\", \"24843025\", \"24727379\", \"27731797\", \"24985467\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Structures focus on the HIV Tat complex; cellular-target SEC architecture less defined\", \"Conformational dynamics during pause release not fully captured\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Gain-of-function AFF4 mutations causing CHOPS syndrome, with altered genome-wide AFF4/cohesin/RNAP2 binding, linked SEC dysregulation to human disease and connected the SEC physically to cohesin.\",\n      \"evidence\": \"Exome sequencing, ChIP-seq, and co-IP demonstrating SEC-cohesin-RNAP2 interaction\",\n      \"pmids\": [\"25730767\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Mechanism by which mutant AFF4 alters cohesin distribution not resolved\", \"Causal target genes of the syndrome phenotype not pinpointed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"High-resolution structure of the ELL2-AFF4 (ELLBow) interface and functional studies in mesenchymal stem cells extended AFF4 mechanism to both SEC assembly detail and tissue differentiation.\",\n      \"evidence\": \"2.0 Å crystallography with mutagenesis; siRNA/overexpression with ChIP at ID1 and BMP2 reporter assays in MSCs\",\n      \"pmids\": [\"28134250\", \"28955517\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether ELLBow interface mutations affect non-HIV transcription not tested\", \"Osteogenic role mechanistically separate from SEC scaffolding not fully established\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Structural definition of the AFF4-CHD as a dimerization, nucleic-acid-binding and CDK9 substrate module, plus identification of FUS as a condensate-forming regulator, refined how AFF4 oligomerizes and is regulated.\",\n      \"evidence\": \"2.2 Å crystallography, dimerization and fluorescence anisotropy assays, in vitro CDK9 kinase assay; co-localization, ChIP and HIV latency models for FUS\",\n      \"pmids\": [\"31147444\", \"31238957\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Physiological consequence of CHD nucleic-acid binding not defined\", \"FUS data are single-lab and Medium confidence\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Structure of the AFF4-THD dimerization domain and point mutants showed dimerization is essential for HIV-1 transactivation yet dispensable for other SEC-subunit contacts, separating two AFF4 functions.\",\n      \"evidence\": \"2.4 Å crystallography, site-directed mutagenesis, co-IP and HIV-1 transactivation reporter assays\",\n      \"pmids\": [\"32128251\", \"32139123\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Why dimerization is selectively required for proviral transactivation not mechanistically explained\", \"Role of dimerization at cellular target genes untested\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Conditional knockout and direct binding to ATG5/ATG16L1 established AFF4 as a transcriptional driver of autophagy genes required for adipocyte development, broadening its differentiation roles.\",\n      \"evidence\": \"Co-IP, conditional Aff4 knockout mice, and epistasis rescue with ATG5/ATG16L1\",\n      \"pmids\": [\"36149892\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether ATG5/ATG16L1 activation occurs through canonical SEC elongation not directly shown\", \"Tissue specificity of this program unexplained\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Genome-wide dissection showed AFF4 and AFF1 act antagonistically across the TSS to control elongation rate and termination, and that AFF4 promotes Ser2-P Pol II and pause release, defining its quantitative role in elongation control.\",\n      \"evidence\": \"ChIP-seq, PRO-seq, RNA-seq and CUT&Tag with siRNA knockdown of both paralogs\",\n      \"pmids\": [\"37528066\", \"37609817\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"What distinguishes AFF1- versus AFF4-favored genes is unresolved\", \"Mechanism of mutual chromatin-level antagonism unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"MeCP2 was shown to directly bind AFF4 and recruit the SEC to synaptic genes, implicating AFF4-dependent elongation in MeCP2 (Rett-related) biology.\",\n      \"evidence\": \"Co-IP in human cells and mouse brain, direct binding assay, ChIP and Drosophila genetic screen (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Whether MeCP2-AFF4 recruitment is direct or via additional factors needs further validation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identification of P70S6K-mediated AFF4 S831 phosphorylation linking insulin signaling to ENL/AF9 recruitment onto crotonylated histones established a signaling-to-elongation axis relevant to insulin-resistant bone.\",\n      \"evidence\": \"Phosphoproteomics, in vitro kinase assay, ChIP for ENL/AF9, and zebrafish/osteoblast loss-of-function\",\n      \"pmids\": [\"41476161\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Full set of S831-dependent target genes not mapped\", \"How phosphorylation alters the AFF4-ENL/AF9 interface structurally not defined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AFF4 selects its specific target gene sets across distinct tissues and disease states—and how its many phosphorylation and dimerization states are integrated to determine which elongation programs are activated—remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No unifying model linking AFF4 post-translational states to target-gene specificity\", \"Relationship between SEC scaffolding and tissue-specific differentiation programs incompletely defined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [14, 17, 18, 24, 19]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [8, 11]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [11]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [4, 5, 25]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [13]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [7, 20, 21]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 3, 20, 21]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [1, 5, 7, 17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [14, 10, 19]}\n    ],\n    \"complexes\": [\"Super Elongation Complex (SEC)\", \"P-TEFb\", \"Tat-AFF4-P-TEFb-TAR complex\"],\n    \"partners\": [\"CCNT1\", \"ELL2\", \"MLLT3\", \"MLLT1\", \"AFF1\", \"FUS\", \"MECP2\", \"ATG5\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}