{"gene":"MLLT6","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1994,"finding":"AF17 (MLLT6) encodes a protein of 1093 amino acids containing a leucine-zipper dimerization motif and a cysteine-rich N-terminal domain arrangeable as three zinc fingers; the leucine-zipper motif is located 3' of the MLL fusion breakpoint, suggesting it contributes to leukemogenic function of MLL-AF17 chimeric proteins.","method":"cDNA cloning and sequence characterization of the AF17 gene fused to ALL-1/MLL in t(11;17)(q23;q21) acute leukemia","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — single lab, molecular cloning with domain analysis but no functional mutagenesis assay in this paper","pmids":["8058765"],"is_preprint":false},{"year":1995,"finding":"The N-terminal cysteine-rich domain of MLLT6 defines the LAP (leukemia-associated protein) zinc finger motif, a structurally distinct zinc-binding domain shared with AF10 and other proteins; the consensus sequence was characterized and proposed to be involved in DNA binding.","method":"Sequence homology analysis and characterization of the LAP finger consensus across 25 proteins including MLLT6 and its C. elegans homolog CEZF","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Low","confidence_rationale":"Tier 4 / Weak — computational/sequence-based prediction with no direct biochemical validation of zinc binding or DNA binding for MLLT6 specifically","pmids":["7568208"],"is_preprint":false},{"year":2005,"finding":"Both known forms of MLL-AF17 fusion transcripts retain the leucine zipper domain of AF17, indicating that the AF17 leucine-zipper dimerization domain is critical for leukemogenic activity of MLL-AF17.","method":"RT-PCR and cDNA panhandle PCR mapping of chromosomal breakpoints and fusion transcripts in a patient with t(11;17)(q23;q21) AML","journal":"International journal of hematology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single case, breakpoint mapping supports structural inference but no direct functional assay","pmids":["16105757"],"is_preprint":false},{"year":2001,"finding":"AF17 (MLLT6) is a transcriptional target of the beta-catenin/TCF pathway; its overexpression stimulates NIH3T3 cell growth and promotes cell-cycle progression primarily at the G2-M transition.","method":"cDNA microarray identification, beta-catenin accumulation assays, plasmid overexpression, and FACS cell-cycle analysis in NIH3T3 cells","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — two orthogonal methods (microarray + FACS cell-cycle), single lab","pmids":["11522623"],"is_preprint":false},{"year":2009,"finding":"AF17 and AF9 competitively bind to the same domain of Dot1a; AF17 overexpression redirects Dot1a from the nucleus to the cytoplasm (blocked by nuclear export inhibitor leptomycin B), reduces histone H3 K79 methylation, and relieves Dot1a·AF9-mediated repression of alpha-ENaC and other target genes, whereas AF17 RNAi causes nuclear enrichment of Dot1a and H3 K79 hypermethylation.","method":"Co-IP/pulldown competitive binding assays, luciferase reporter assays, RNAi knockdown, immunofluorescence localization, whole-cell patch clamping in HEK 293T cells","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (competitive binding, localization with chemical inhibitor validation, knockdown phenotype, electrophysiology), replicated in subsequent papers","pmids":["19864429"],"is_preprint":false},{"year":2011,"finding":"AF17 facilitates nuclear export of Dot1a in renal collecting duct (M-1) cells; AF17 overexpression impairs the Dot1a-AF9 interaction and H3 K79 methylation at the αENaC promoter (without affecting AF9 promoter binding), increases mRNA and protein expression of ENaC subunits (α, β, γ) and Sgk1, and elevates ENaC-mediated Na+ transport; AF17 knockdown causes opposite effects. These actions are not additive with aldosterone.","method":"Overexpression and siRNA knockdown in M-1 cells, immunoblot, real-time RT-qPCR, chromatin immunoprecipitation (ChIP), nuclear export inhibitor leptomycin B, single-cell fluorescence Na+ imaging, epithelial short-circuit current measurement","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ChIP, localization with inhibitor, electrophysiology, KD and OE reciprocal experiments), single lab but rigorous","pmids":["22087315"],"is_preprint":false},{"year":2011,"finding":"Af17-deficient mice show increased H3 K79 dimethylation, reduced ENaC mRNA and protein expression, fewer active channels, lower open probability, and decreased ENaC-mediated Na+ transport, resulting in lower blood pressure and increased sodium excretion; high aldosterone levels fully compensate for Af17 loss.","method":"Af17 knockout mouse generation, blood pressure measurement, urine sodium analysis, histone methylation assays, patch-clamp electrophysiology of collecting duct cells","journal":"Journal of the American Society of Nephrology : JASN","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout with multiple orthogonal phenotypic and molecular readouts, consistent with in vitro mechanistic data from other labs/papers","pmids":["21546577"],"is_preprint":false},{"year":2001,"finding":"The Drosophila ortholog of AF17/AF10 (Dalf) encodes a zinc finger/leucine zipper nuclear protein; loss-of-function mutation abolishes EVE expression maintenance specifically in RP2 motoneurons, and larval growth retardation is rescued by neuron-specific Dalf transgene expression, establishing a requirement for Dalf in the nervous system for gene expression maintenance.","method":"P-element mutagenesis, immunostaining, genetic rescue with neuron-specific GAL4-driven transgene in Drosophila","journal":"Mechanisms of development","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with transgenic rescue, single lab, Drosophila ortholog","pmids":["11165485"],"is_preprint":false},{"year":2020,"finding":"MLLT6 is required for efficient PD-L1 protein expression and cell-surface presentation in cancer cells; MLLT6 depletion impairs STAT1 signaling and renders cells insensitive to IFN-γ-induced expression of IDO1, GBP5, CD74, and MHC class II genes, thereby alleviating suppression of CD8+ T cell-mediated cytolysis.","method":"CRISPR/Cas9 screen followed by functional validation: MLLT6 CRISPR knockout, flow cytometry for PD-L1, T cell cytolysis co-culture assays, STAT1 signaling measurement, IFN-γ stimulation assays in human cancer cells","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide CRISPR screen plus targeted KO with multiple orthogonal functional readouts (PD-L1 surface expression, T cell killing, STAT1 signaling, gene expression), single lab but comprehensive","pmids":["33063451"],"is_preprint":false},{"year":2024,"finding":"MLLT6 forms a complex with transcription factor ATF2; this MLLT6/ATF2 axis induces DDIT3/4 expression by establishing an active chromatin structure at their promoters, promoting breast cancer cell apoptosis; AKT signaling inhibition reinforces this axis, and DNMT1-mediated methylation of the MLLT6 promoter under hypoxia downregulates MLLT6 expression.","method":"Co-IP identifying ATF2 as binding partner, chromatin assays (active chromatin structure at DDIT3/4 promoters), RNA profiling, stable MLLT6 knockdown and DDIT3/4 restoration experiments, colony formation and migration assays, in vivo colonization assays","journal":"Molecular cancer research : MCR","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus chromatin assays plus functional rescue experiments, single lab, mechanistic pathway placement","pmids":["38757913"],"is_preprint":false}],"current_model":"MLLT6 (AF17) is a nuclear zinc finger/leucine-zipper transcriptional regulator that competes with AF9 for binding to the histone H3 K79 methyltransferase Dot1a, facilitates Dot1a nuclear export to reduce H3 K79 methylation and relieve Dot1a·AF9-mediated gene repression (notably of ENaC subunits in renal collecting duct cells), partners with ATF2 to establish active chromatin and drive DDIT3/4-dependent apoptosis, supports STAT1 signaling and IFN-γ-induced PD-L1 expression in cancer cells, and when fused to MLL via t(11;17) contributes to acute leukemia through its retained leucine-zipper dimerization domain."},"narrative":{"mechanistic_narrative":"MLLT6 (AF17) is a nuclear zinc finger/leucine-zipper protein that acts as a chromatin-associated transcriptional regulator [PMID:8058765]. Its best-characterized mechanism is competition with AF9 for binding to the same domain of the H3 K79 methyltransferase Dot1a: MLLT6 redirects Dot1a from the nucleus to the cytoplasm in a leptomycin B-sensitive manner, lowers H3 K79 methylation, and relieves Dot1a·AF9-mediated gene repression [PMID:19864429]. In renal collecting duct cells this axis derepresses the ENaC subunits (α, β, γ) and Sgk1 to enhance Na+ transport, and Af17-deficient mice display elevated H3 K79 dimethylation, reduced ENaC activity, increased sodium excretion, and lower blood pressure, with these effects compensated by high aldosterone [PMID:22087315, PMID:21546577]. MLLT6 also functions in transcriptional activation through chromatin remodeling: it complexes with ATF2 to establish active chromatin at the DDIT3/4 promoters and drive breast cancer cell apoptosis [PMID:38757913], and it supports STAT1 signaling required for IFN-γ-induced PD-L1 expression and antigen-presentation gene induction, modulating CD8+ T cell-mediated cytolysis [PMID:33063451]. The N-terminal cysteine-rich domain is retained alongside the leucine-zipper dimerization motif 3' of the MLL breakpoint in t(11;17) acute leukemia fusions, and the dimerization domain is implicated in leukemogenic activity of MLL-AF17 [PMID:8058765, PMID:16105757]. Beyond these contexts, MLLT6 is a β-catenin/TCF transcriptional target that promotes cell growth and G2-M progression [PMID:11522623].","teleology":[{"year":1994,"claim":"Establishing the molecular identity of the t(11;17) MLL fusion partner defined MLLT6 as a leucine-zipper/zinc-finger protein and located the dimerization motif 3' of the breakpoint, framing a structural basis for its leukemogenic contribution.","evidence":"cDNA cloning and sequence/domain analysis of AF17 fused to MLL in t(11;17) AML","pmids":["8058765"],"confidence":"Medium","gaps":["No functional mutagenesis of the leucine zipper in this work","DNA-binding or partner activity of the zinc-finger domain not tested"]},{"year":1995,"claim":"Classifying the N-terminal cysteine-rich domain as a LAP zinc-finger motif shared with AF10 proposed a DNA-binding function for MLLT6.","evidence":"Sequence homology analysis of the LAP finger consensus across 25 proteins including MLLT6","pmids":["7568208"],"confidence":"Low","gaps":["Computational prediction only — no biochemical validation of zinc or DNA binding for MLLT6","No demonstration of a specific DNA target"]},{"year":2001,"claim":"Placing MLLT6 downstream of Wnt signaling connected it to proliferative control, showing it is a β-catenin/TCF target that drives G2-M progression.","evidence":"cDNA microarray, β-catenin accumulation assays, overexpression and FACS cell-cycle analysis in NIH3T3 cells","pmids":["11522623"],"confidence":"Medium","gaps":["Mechanism linking MLLT6 to G2-M transition not defined","No direct transcriptional targets identified in this context"]},{"year":2001,"claim":"Ortholog genetics established an in vivo requirement for the AF17/AF10 family in maintaining cell-type-specific gene expression.","evidence":"P-element mutagenesis, immunostaining, and neuron-specific transgenic rescue of the Drosophila ortholog Dalf","pmids":["11165485"],"confidence":"Medium","gaps":["Molecular partners of Dalf not identified","Relevance to mammalian MLLT6 mechanism inferred from orthology only"]},{"year":2009,"claim":"Defining MLLT6 as a competitive Dot1a-binding partner of AF9 revealed its core mechanism: controlling Dot1a nucleocytoplasmic localization to set H3 K79 methylation and relieve gene repression.","evidence":"Competitive Co-IP/pulldown, luciferase reporters, RNAi, leptomycin B localization, and patch clamp in HEK 293T cells","pmids":["19864429"],"confidence":"High","gaps":["Nuclear export signal/mechanism driving Dot1a relocalization not mapped","Scope of repressed targets beyond α-ENaC not delineated"]},{"year":2011,"claim":"Tissue-level and in vivo work tied the MLLT6–Dot1a axis to ENaC-dependent sodium handling and blood pressure, with aldosterone acting as a compensating input.","evidence":"Reciprocal OE/knockdown with ChIP and electrophysiology in M-1 cells, plus Af17-knockout mice with blood pressure, sodium, and channel assays","pmids":["22087315","21546577"],"confidence":"High","gaps":["Crosstalk between MLLT6 and aldosterone signaling at the molecular level unresolved","Whether the same axis operates in non-renal tissues not addressed"]},{"year":2020,"claim":"A CRISPR screen extended MLLT6 function into immune evasion, showing it supports STAT1 signaling and IFN-γ-induced PD-L1 and antigen-presentation gene expression in cancer cells.","evidence":"Genome-wide CRISPR screen, MLLT6 knockout, PD-L1 flow cytometry, STAT1 readouts, IFN-γ stimulation, and T cell cytolysis co-culture in human cancer cells","pmids":["33063451"],"confidence":"High","gaps":["Direct molecular link between MLLT6 and STAT1 not defined","Whether the Dot1a/H3 K79 mechanism underlies this immune phenotype unknown"]},{"year":2024,"claim":"Identifying the MLLT6/ATF2 complex showed MLLT6 can act as a transcriptional activator establishing active chromatin to drive a pro-apoptotic DDIT3/4 program.","evidence":"Co-IP, chromatin assays at DDIT3/4 promoters, RNA profiling, stable knockdown with restoration, and in vivo colonization assays in breast cancer","pmids":["38757913"],"confidence":"Medium","gaps":["Direct vs indirect nature of MLLT6 recruitment to DDIT3/4 promoters not resolved","Reciprocal validation of the ATF2 interaction limited to single-lab Co-IP"]},{"year":null,"claim":"How MLLT6's distinct activities — Dot1a relocalization/repression relief, ATF2-driven activation, and STAT1 support — are integrated through its zinc-finger and leucine-zipper domains remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of MLLT6 domain function in any complex","Direct DNA-binding activity of the LAP zinc finger never demonstrated","Unifying mechanism across renal, oncogenic, and immune contexts unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[4,5,9]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[4]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,4,7]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[4,9]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[8]},{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[5,6]}],"complexes":[],"partners":["DOT1L","AF9","ATF2","STAT1","KMT2A"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P55198","full_name":"Protein AF-17","aliases":["ALL1-fused gene from chromosome 17 protein"],"length_aa":1093,"mass_kda":112.0,"function":"","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P55198/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MLLT6","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MLLT6","total_profiled":1310},"omim":[{"mim_id":"604083","title":"ZINC FINGER PROTEIN 142; ZNF142","url":"https://www.omim.org/entry/604083"},{"mim_id":"602410","title":"BROMODOMAIN- AND PHD FINGER-CONTAINING PROTEIN; BRPF1","url":"https://www.omim.org/entry/602410"},{"mim_id":"600328","title":"MLLT6, PHD FINGER-CONTAINING; MLLT6","url":"https://www.omim.org/entry/600328"},{"mim_id":"116806","title":"CATENIN, BETA-1; CTNNB1","url":"https://www.omim.org/entry/116806"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MLLT6"},"hgnc":{"alias_symbol":["AF17","FLJ23480"],"prev_symbol":[]},"alphafold":{"accession":"P55198","domains":[{"cath_id":"3.30.40.10","chopping":"23-187","consensus_level":"medium","plddt":92.3864,"start":23,"end":187}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P55198","model_url":"https://alphafold.ebi.ac.uk/files/AF-P55198-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P55198-F1-predicted_aligned_error_v6.png","plddt_mean":49.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MLLT6","jax_strain_url":"https://www.jax.org/strain/search?query=MLLT6"},"sequence":{"accession":"P55198","fasta_url":"https://rest.uniprot.org/uniprotkb/P55198.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P55198/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P55198"}},"corpus_meta":[{"pmid":"8058765","id":"PMC_8058765","title":"Leucine-zipper dimerization motif encoded by the AF17 gene fused to ALL-1 (MLL) in acute leukemia.","date":"1994","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/8058765","citation_count":144,"is_preprint":false},{"pmid":"7568208","id":"PMC_7568208","title":"The leukemia-associated-protein (LAP) domain, a cysteine-rich motif, is present in a wide range of proteins, including MLL, AF10, and MLLT6 proteins.","date":"1995","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/7568208","citation_count":137,"is_preprint":false},{"pmid":"19864429","id":"PMC_19864429","title":"AF17 competes with AF9 for binding to Dot1a to up-regulate transcription of epithelial Na+ channel alpha.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19864429","citation_count":44,"is_preprint":false},{"pmid":"11522623","id":"PMC_11522623","title":"Identification of AF17 as a downstream gene of the beta-catenin/T-cell factor pathway and its involvement in colorectal carcinogenesis.","date":"2001","source":"Cancer research","url":"https://pubmed.ncbi.nlm.nih.gov/11522623","citation_count":43,"is_preprint":false},{"pmid":"21546577","id":"PMC_21546577","title":"Af17 deficiency increases sodium excretion and decreases blood pressure.","date":"2011","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/21546577","citation_count":27,"is_preprint":false},{"pmid":"33063451","id":"PMC_33063451","title":"MLLT6 maintains PD-L1 expression and mediates tumor immune resistance.","date":"2020","source":"EMBO reports","url":"https://pubmed.ncbi.nlm.nih.gov/33063451","citation_count":20,"is_preprint":false},{"pmid":"16105757","id":"PMC_16105757","title":"Identification of a chromosomal breakpoint and detection of a novel form of an MLL-AF17 fusion transcript in acute monocytic leukemia with t(11;17)(q23;q21).","date":"2005","source":"International journal of hematology","url":"https://pubmed.ncbi.nlm.nih.gov/16105757","citation_count":17,"is_preprint":false},{"pmid":"22087315","id":"PMC_22087315","title":"AF17 facilitates Dot1a nuclear export and upregulates ENaC-mediated Na+ transport in renal collecting duct cells.","date":"2011","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22087315","citation_count":16,"is_preprint":false},{"pmid":"11165485","id":"PMC_11165485","title":"The Drosophila homolog of human AF10/AF17 leukemia fusion genes (Dalf) encodes a zinc finger/leucine zipper nuclear protein required in the nervous system for maintaining EVE expression and normal growth.","date":"2001","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/11165485","citation_count":14,"is_preprint":false},{"pmid":"21170927","id":"PMC_21170927","title":"Widely expressed Af17 is likely not required for embryogenesis, hematopoiesis, and animal survival.","date":"2010","source":"Genesis (New York, N.Y. : 2000)","url":"https://pubmed.ncbi.nlm.nih.gov/21170927","citation_count":7,"is_preprint":false},{"pmid":"38757913","id":"PMC_38757913","title":"MLLT6/ATF2 Axis Restrains Breast Cancer Progression by Driving DDIT3/4 Expression.","date":"2024","source":"Molecular cancer research : MCR","url":"https://pubmed.ncbi.nlm.nih.gov/38757913","citation_count":5,"is_preprint":false},{"pmid":"8946209","id":"PMC_8946209","title":"Gene BR140, which is related to AF10 and AF17, maps to chromosome band 3p25.","date":"1996","source":"Genes, chromosomes & cancer","url":"https://pubmed.ncbi.nlm.nih.gov/8946209","citation_count":5,"is_preprint":false},{"pmid":"41094568","id":"PMC_41094568","title":"Development of a recurrence-related gene signature and functional role of MLLT6 in ovarian cancer progression and Paclitaxel resistance.","date":"2025","source":"Journal of ovarian research","url":"https://pubmed.ncbi.nlm.nih.gov/41094568","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8175,"output_tokens":2746,"usd":0.032857,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10066,"output_tokens":3117,"usd":0.064128,"stage2_stop_reason":"end_turn"},"total_usd":0.096985,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1994,\n      \"finding\": \"AF17 (MLLT6) encodes a protein of 1093 amino acids containing a leucine-zipper dimerization motif and a cysteine-rich N-terminal domain arrangeable as three zinc fingers; the leucine-zipper motif is located 3' of the MLL fusion breakpoint, suggesting it contributes to leukemogenic function of MLL-AF17 chimeric proteins.\",\n      \"method\": \"cDNA cloning and sequence characterization of the AF17 gene fused to ALL-1/MLL in t(11;17)(q23;q21) acute leukemia\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — single lab, molecular cloning with domain analysis but no functional mutagenesis assay in this paper\",\n      \"pmids\": [\"8058765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"The N-terminal cysteine-rich domain of MLLT6 defines the LAP (leukemia-associated protein) zinc finger motif, a structurally distinct zinc-binding domain shared with AF10 and other proteins; the consensus sequence was characterized and proposed to be involved in DNA binding.\",\n      \"method\": \"Sequence homology analysis and characterization of the LAP finger consensus across 25 proteins including MLLT6 and its C. elegans homolog CEZF\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 4 / Weak — computational/sequence-based prediction with no direct biochemical validation of zinc binding or DNA binding for MLLT6 specifically\",\n      \"pmids\": [\"7568208\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Both known forms of MLL-AF17 fusion transcripts retain the leucine zipper domain of AF17, indicating that the AF17 leucine-zipper dimerization domain is critical for leukemogenic activity of MLL-AF17.\",\n      \"method\": \"RT-PCR and cDNA panhandle PCR mapping of chromosomal breakpoints and fusion transcripts in a patient with t(11;17)(q23;q21) AML\",\n      \"journal\": \"International journal of hematology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single case, breakpoint mapping supports structural inference but no direct functional assay\",\n      \"pmids\": [\"16105757\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"AF17 (MLLT6) is a transcriptional target of the beta-catenin/TCF pathway; its overexpression stimulates NIH3T3 cell growth and promotes cell-cycle progression primarily at the G2-M transition.\",\n      \"method\": \"cDNA microarray identification, beta-catenin accumulation assays, plasmid overexpression, and FACS cell-cycle analysis in NIH3T3 cells\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — two orthogonal methods (microarray + FACS cell-cycle), single lab\",\n      \"pmids\": [\"11522623\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"AF17 and AF9 competitively bind to the same domain of Dot1a; AF17 overexpression redirects Dot1a from the nucleus to the cytoplasm (blocked by nuclear export inhibitor leptomycin B), reduces histone H3 K79 methylation, and relieves Dot1a·AF9-mediated repression of alpha-ENaC and other target genes, whereas AF17 RNAi causes nuclear enrichment of Dot1a and H3 K79 hypermethylation.\",\n      \"method\": \"Co-IP/pulldown competitive binding assays, luciferase reporter assays, RNAi knockdown, immunofluorescence localization, whole-cell patch clamping in HEK 293T cells\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (competitive binding, localization with chemical inhibitor validation, knockdown phenotype, electrophysiology), replicated in subsequent papers\",\n      \"pmids\": [\"19864429\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"AF17 facilitates nuclear export of Dot1a in renal collecting duct (M-1) cells; AF17 overexpression impairs the Dot1a-AF9 interaction and H3 K79 methylation at the αENaC promoter (without affecting AF9 promoter binding), increases mRNA and protein expression of ENaC subunits (α, β, γ) and Sgk1, and elevates ENaC-mediated Na+ transport; AF17 knockdown causes opposite effects. These actions are not additive with aldosterone.\",\n      \"method\": \"Overexpression and siRNA knockdown in M-1 cells, immunoblot, real-time RT-qPCR, chromatin immunoprecipitation (ChIP), nuclear export inhibitor leptomycin B, single-cell fluorescence Na+ imaging, epithelial short-circuit current measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ChIP, localization with inhibitor, electrophysiology, KD and OE reciprocal experiments), single lab but rigorous\",\n      \"pmids\": [\"22087315\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Af17-deficient mice show increased H3 K79 dimethylation, reduced ENaC mRNA and protein expression, fewer active channels, lower open probability, and decreased ENaC-mediated Na+ transport, resulting in lower blood pressure and increased sodium excretion; high aldosterone levels fully compensate for Af17 loss.\",\n      \"method\": \"Af17 knockout mouse generation, blood pressure measurement, urine sodium analysis, histone methylation assays, patch-clamp electrophysiology of collecting duct cells\",\n      \"journal\": \"Journal of the American Society of Nephrology : JASN\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout with multiple orthogonal phenotypic and molecular readouts, consistent with in vitro mechanistic data from other labs/papers\",\n      \"pmids\": [\"21546577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The Drosophila ortholog of AF17/AF10 (Dalf) encodes a zinc finger/leucine zipper nuclear protein; loss-of-function mutation abolishes EVE expression maintenance specifically in RP2 motoneurons, and larval growth retardation is rescued by neuron-specific Dalf transgene expression, establishing a requirement for Dalf in the nervous system for gene expression maintenance.\",\n      \"method\": \"P-element mutagenesis, immunostaining, genetic rescue with neuron-specific GAL4-driven transgene in Drosophila\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with transgenic rescue, single lab, Drosophila ortholog\",\n      \"pmids\": [\"11165485\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MLLT6 is required for efficient PD-L1 protein expression and cell-surface presentation in cancer cells; MLLT6 depletion impairs STAT1 signaling and renders cells insensitive to IFN-γ-induced expression of IDO1, GBP5, CD74, and MHC class II genes, thereby alleviating suppression of CD8+ T cell-mediated cytolysis.\",\n      \"method\": \"CRISPR/Cas9 screen followed by functional validation: MLLT6 CRISPR knockout, flow cytometry for PD-L1, T cell cytolysis co-culture assays, STAT1 signaling measurement, IFN-γ stimulation assays in human cancer cells\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide CRISPR screen plus targeted KO with multiple orthogonal functional readouts (PD-L1 surface expression, T cell killing, STAT1 signaling, gene expression), single lab but comprehensive\",\n      \"pmids\": [\"33063451\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MLLT6 forms a complex with transcription factor ATF2; this MLLT6/ATF2 axis induces DDIT3/4 expression by establishing an active chromatin structure at their promoters, promoting breast cancer cell apoptosis; AKT signaling inhibition reinforces this axis, and DNMT1-mediated methylation of the MLLT6 promoter under hypoxia downregulates MLLT6 expression.\",\n      \"method\": \"Co-IP identifying ATF2 as binding partner, chromatin assays (active chromatin structure at DDIT3/4 promoters), RNA profiling, stable MLLT6 knockdown and DDIT3/4 restoration experiments, colony formation and migration assays, in vivo colonization assays\",\n      \"journal\": \"Molecular cancer research : MCR\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus chromatin assays plus functional rescue experiments, single lab, mechanistic pathway placement\",\n      \"pmids\": [\"38757913\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"MLLT6 (AF17) is a nuclear zinc finger/leucine-zipper transcriptional regulator that competes with AF9 for binding to the histone H3 K79 methyltransferase Dot1a, facilitates Dot1a nuclear export to reduce H3 K79 methylation and relieve Dot1a·AF9-mediated gene repression (notably of ENaC subunits in renal collecting duct cells), partners with ATF2 to establish active chromatin and drive DDIT3/4-dependent apoptosis, supports STAT1 signaling and IFN-γ-induced PD-L1 expression in cancer cells, and when fused to MLL via t(11;17) contributes to acute leukemia through its retained leucine-zipper dimerization domain.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MLLT6 (AF17) is a nuclear zinc finger/leucine-zipper protein that acts as a chromatin-associated transcriptional regulator [#0]. Its best-characterized mechanism is competition with AF9 for binding to the same domain of the H3 K79 methyltransferase Dot1a: MLLT6 redirects Dot1a from the nucleus to the cytoplasm in a leptomycin B-sensitive manner, lowers H3 K79 methylation, and relieves Dot1a·AF9-mediated gene repression [#4]. In renal collecting duct cells this axis derepresses the ENaC subunits (α, β, γ) and Sgk1 to enhance Na+ transport, and Af17-deficient mice display elevated H3 K79 dimethylation, reduced ENaC activity, increased sodium excretion, and lower blood pressure, with these effects compensated by high aldosterone [#5, #6]. MLLT6 also functions in transcriptional activation through chromatin remodeling: it complexes with ATF2 to establish active chromatin at the DDIT3/4 promoters and drive breast cancer cell apoptosis [#9], and it supports STAT1 signaling required for IFN-γ-induced PD-L1 expression and antigen-presentation gene induction, modulating CD8+ T cell-mediated cytolysis [#8]. The N-terminal cysteine-rich domain is retained alongside the leucine-zipper dimerization motif 3' of the MLL breakpoint in t(11;17) acute leukemia fusions, and the dimerization domain is implicated in leukemogenic activity of MLL-AF17 [#0, #2]. Beyond these contexts, MLLT6 is a β-catenin/TCF transcriptional target that promotes cell growth and G2-M progression [#3].\",\n  \"teleology\": [\n    {\n      \"year\": 1994,\n      \"claim\": \"Establishing the molecular identity of the t(11;17) MLL fusion partner defined MLLT6 as a leucine-zipper/zinc-finger protein and located the dimerization motif 3' of the breakpoint, framing a structural basis for its leukemogenic contribution.\",\n      \"evidence\": \"cDNA cloning and sequence/domain analysis of AF17 fused to MLL in t(11;17) AML\",\n      \"pmids\": [\"8058765\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional mutagenesis of the leucine zipper in this work\", \"DNA-binding or partner activity of the zinc-finger domain not tested\"]\n    },\n    {\n      \"year\": 1995,\n      \"claim\": \"Classifying the N-terminal cysteine-rich domain as a LAP zinc-finger motif shared with AF10 proposed a DNA-binding function for MLLT6.\",\n      \"evidence\": \"Sequence homology analysis of the LAP finger consensus across 25 proteins including MLLT6\",\n      \"pmids\": [\"7568208\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Computational prediction only — no biochemical validation of zinc or DNA binding for MLLT6\", \"No demonstration of a specific DNA target\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Placing MLLT6 downstream of Wnt signaling connected it to proliferative control, showing it is a β-catenin/TCF target that drives G2-M progression.\",\n      \"evidence\": \"cDNA microarray, β-catenin accumulation assays, overexpression and FACS cell-cycle analysis in NIH3T3 cells\",\n      \"pmids\": [\"11522623\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking MLLT6 to G2-M transition not defined\", \"No direct transcriptional targets identified in this context\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Ortholog genetics established an in vivo requirement for the AF17/AF10 family in maintaining cell-type-specific gene expression.\",\n      \"evidence\": \"P-element mutagenesis, immunostaining, and neuron-specific transgenic rescue of the Drosophila ortholog Dalf\",\n      \"pmids\": [\"11165485\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Molecular partners of Dalf not identified\", \"Relevance to mammalian MLLT6 mechanism inferred from orthology only\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Defining MLLT6 as a competitive Dot1a-binding partner of AF9 revealed its core mechanism: controlling Dot1a nucleocytoplasmic localization to set H3 K79 methylation and relieve gene repression.\",\n      \"evidence\": \"Competitive Co-IP/pulldown, luciferase reporters, RNAi, leptomycin B localization, and patch clamp in HEK 293T cells\",\n      \"pmids\": [\"19864429\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nuclear export signal/mechanism driving Dot1a relocalization not mapped\", \"Scope of repressed targets beyond α-ENaC not delineated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Tissue-level and in vivo work tied the MLLT6–Dot1a axis to ENaC-dependent sodium handling and blood pressure, with aldosterone acting as a compensating input.\",\n      \"evidence\": \"Reciprocal OE/knockdown with ChIP and electrophysiology in M-1 cells, plus Af17-knockout mice with blood pressure, sodium, and channel assays\",\n      \"pmids\": [\"22087315\", \"21546577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Crosstalk between MLLT6 and aldosterone signaling at the molecular level unresolved\", \"Whether the same axis operates in non-renal tissues not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"A CRISPR screen extended MLLT6 function into immune evasion, showing it supports STAT1 signaling and IFN-γ-induced PD-L1 and antigen-presentation gene expression in cancer cells.\",\n      \"evidence\": \"Genome-wide CRISPR screen, MLLT6 knockout, PD-L1 flow cytometry, STAT1 readouts, IFN-γ stimulation, and T cell cytolysis co-culture in human cancer cells\",\n      \"pmids\": [\"33063451\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular link between MLLT6 and STAT1 not defined\", \"Whether the Dot1a/H3 K79 mechanism underlies this immune phenotype unknown\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Identifying the MLLT6/ATF2 complex showed MLLT6 can act as a transcriptional activator establishing active chromatin to drive a pro-apoptotic DDIT3/4 program.\",\n      \"evidence\": \"Co-IP, chromatin assays at DDIT3/4 promoters, RNA profiling, stable knockdown with restoration, and in vivo colonization assays in breast cancer\",\n      \"pmids\": [\"38757913\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect nature of MLLT6 recruitment to DDIT3/4 promoters not resolved\", \"Reciprocal validation of the ATF2 interaction limited to single-lab Co-IP\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How MLLT6's distinct activities — Dot1a relocalization/repression relief, ATF2-driven activation, and STAT1 support — are integrated through its zinc-finger and leucine-zipper domains remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of MLLT6 domain function in any complex\", \"Direct DNA-binding activity of the LAP zinc finger never demonstrated\", \"Unifying mechanism across renal, oncogenic, and immune contexts unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [4, 5, 9]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 4, 7]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [4, 9]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [8]},\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [5, 6]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"DOT1L\", \"AF9\", \"ATF2\", \"STAT1\", \"KMT2A\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":6,"faith_total":6,"faith_pct":100.0}}