{"gene":"ANKRA2","run_date":"2026-06-09T22:02:43","timeline":{"discoveries":[{"year":2005,"finding":"Class IIa HDACs (HDAC4 and HDAC5) physically associate with the ankyrin repeat domain of ANKRA2, and through association with the paralog RFXANK, repress MHC II promoter activation and endogenous HLA-DRA gene expression induced by CIITA. Phosphorylation of class II HDACs by CaMK results in CRM1-dependent nuclear export of HDAC/RFXANK complexes.","method":"Co-immunoprecipitation, reporter gene assays, RNAi knockdown, nuclear export assays","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP with functional reporter assays and CaMK/export experiments, single lab with multiple orthogonal methods","pmids":["16236793"],"is_preprint":false},{"year":2012,"finding":"The ankyrin repeat domain of ANKRA2 recognizes a PxLPxI/L motif found in HDAC4, HDAC5, HDAC9, megalin, and RFX5, using a tumbler-lock binding mode where each of the middle three ankyrin repeats contacts one residue of the motif. Crystal structures of ANKRA2 ankyrin repeats in complex with binding peptides defined this recognition mechanism. Phosphorylation of Ser350 within the PxLPxI/L motif of HDAC4 impairs ANKRA2 binding while generating a 14-3-3 docking site.","method":"X-ray crystallography, isothermal titration calorimetry, mutagenesis, phosphopeptide binding assays","journal":"Science signaling","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structures with bound peptides, mutagenesis of binding interface, multiple orthogonal binding assays in a single rigorous study","pmids":["22649097"],"is_preprint":false},{"year":2007,"finding":"ANKRA2 was identified as a binding partner of the AhR repressor (AhRR) C-terminal repression domain via yeast two-hybrid screening. ANKRA2 recruits HDAC4 and HDAC5 as corepressors for AhRR-mediated transcriptional repression of CYP1A1; siRNA knockdown of ANKRA2 reduces AhRR repression activity.","method":"Yeast two-hybrid, RNAi knockdown, reporter gene assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid interaction confirmed with siRNA functional assay, single lab with two orthogonal methods","pmids":["17949687"],"is_preprint":false},{"year":2009,"finding":"SUMOylation of AhRR at Lys-542, Lys-583, and Lys-660 is required for the interaction between AhRR and ANKRA2 (as well as HDAC4 and HDAC5); arginine mutation of these residues reduces both SUMOylation and the AhRR–ANKRA2 interaction, impairing transcriptional repression.","method":"In vivo SUMOylation assays, site-directed mutagenesis, co-immunoprecipitation, reporter gene assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with Co-IP and functional reporter assays, single lab","pmids":["19251700"],"is_preprint":false},{"year":2005,"finding":"The ankyrin repeat domain (ARD) of ANKRA2 can substitute for RFXANK in activating MHC II gene expression, as demonstrated by complementation of a bare lymphocyte syndrome cell line deficient in RFX-B (RFXANK). Mouse and Xenopus RFXANK orthologues complement this deficiency but ANKRA2 does so only through its ARD.","method":"Complementation assay in BLS patient-derived cell line, domain-swap experiments","journal":"Immunogenetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional complementation with domain mapping, single lab","pmids":["15655668"],"is_preprint":false},{"year":2005,"finding":"ANKRA2 ankyrin repeat domain mediates interaction with RFX5, and high-resolution mutagenesis of the closely related RFXANK ARD mapped the RFX5 interaction surface; ANKRA2 can substitute for RFXANK in MHC-II enhanceosome assembly through its ARD.","method":"Mutagenesis, complementation assay in BLS cell line, in vivo chromatin occupancy assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis combined with functional complementation and chromatin assays, single lab with multiple methods","pmids":["16166641"],"is_preprint":false},{"year":2015,"finding":"The ankyrin repeats of ANKRA2 recognize a PxLPxL motif at the C-terminal region of CCDC8 (a 3M syndrome protein), establishing CCDC8 as a major cellular partner of ANKRA2 but not RFXANK. The N-terminal part of CCDC8 interacts with OBSL1 to form a CUL7 ligase complex, linking ANKRA2 to the 3M syndrome complex.","method":"Co-immunoprecipitation (cellular), X-ray crystallography (structural analysis of ANKRA2 ARD–CCDC8 peptide complex), binding assays","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure of complex combined with cellular Co-IP and binding assays; novel partner defined with structural and biochemical orthogonal methods","pmids":["25752541"],"is_preprint":false},{"year":2019,"finding":"Crystal structures of ANKRA2 ankyrin domain bound to an RFX7 fragment revealed that ANKRA2 recognizes the PxLPxL motif of RFX7 and flanking sequences via extensive hydrophobic interactions, with higher binding affinity than RFXANK for RFX7.","method":"X-ray crystallography, binding affinity measurements","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with quantitative binding comparison; single lab but structural evidence is direct","pmids":["31864703"],"is_preprint":false},{"year":2024,"finding":"ANKRA2 is a direct transcriptional target of p53, and functions as a critical cofactor of the tumor suppressor transcription factor RFX7. Mass spectrometry identified ANKRA2 binding to the X-box motif of the PDCD4 promoter together with RFX5, RFXAP, RFXANK, and RFX7. Transcriptome analyses showed ANKRA2 regulates a gene set overlapping with RFX7 targets, distinct from RFXANK-regulated genes.","method":"Reporter gene assay, mass spectrometry of promoter-bound proteins, siRNA knockdown with transcriptome analysis, p53 target gene validation","journal":"Cell death discovery","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry of DNA-bound complex combined with transcriptome knockdown analysis and reporter assays; single lab with multiple orthogonal methods","pmids":["39181888"],"is_preprint":false}],"current_model":"ANKRA2 is an ankyrin repeat scaffold protein whose ARD recognizes PxLPxI/L linear motifs (via a tumbler-lock mechanism defined by crystal structures) in diverse partners including HDAC4, HDAC5, HDAC9, megalin, RFX5, RFX7, and CCDC8; it functions as a signal-responsive transcriptional corepressor that recruits class IIa HDACs to repress MHC II, AhRR-target, and RFX7-target genes, with complex formation regulated by CaMK-driven phosphorylation of HDAC4-Ser350 (which disrupts ANKRA2 binding and promotes 14-3-3 sequestration) and by SUMOylation of binding partners; ANKRA2 is itself a direct p53 target gene and a cofactor of the tumor suppressor RFX7."},"narrative":{"mechanistic_narrative":"ANKRA2 is an ankyrin-repeat scaffold protein that functions as a signal-responsive transcriptional corepressor by reading short linear PxLPxI/L motifs in diverse partner proteins [PMID:22649097]. Crystal structures of its ankyrin repeat domain bound to target peptides defined a tumbler-lock recognition mode in which each of the middle three repeats engages one motif residue, and showed that this interface accommodates motifs from HDAC4, HDAC5, HDAC9, megalin, and RFX5 [PMID:22649097]. Through this domain ANKRA2 binds class IIa HDACs (HDAC4, HDAC5) and recruits them as corepressors: it represses CIITA-induced MHC II and HLA-DRA expression in conjunction with the paralog RFXANK [PMID:16236793], and is recruited to the AhR repressor C-terminal domain to repress CYP1A1, an interaction that depends on SUMOylation of AhRR [PMID:17949687, PMID:19251700]. The same ankyrin domain can substitute for RFXANK in MHC II enhanceosome assembly by contacting RFX5, complementing RFXANK-deficient bare lymphocyte syndrome cells [PMID:15655668, PMID:16166641]. Beyond immune gene regulation, ANKRA2 binds the PxLPxL motif of the 3M-syndrome protein CCDC8, linking it to the OBSL1/CUL7 ligase complex [PMID:25752541], and serves as a direct p53 target gene and a high-affinity cofactor of the tumor suppressor transcription factor RFX7, regulating RFX7-overlapping targets such as PDCD4 [PMID:31864703, PMID:39181888]. HDAC4 recruitment is switched off by CaMK-driven phosphorylation of Ser350 within its PxLPxI/L motif, which disrupts ANKRA2 binding and creates a 14-3-3 docking site, coupling complex assembly to calcium signaling [PMID:16236793, PMID:22649097].","teleology":[{"year":2005,"claim":"Established that ANKRA2's ankyrin repeat domain physically recruits class IIa HDACs to repress MHC II transcription and that this is reversed by calcium signaling, defining ANKRA2 as a signal-responsive corepressor scaffold.","evidence":"Co-IP, reporter assays, RNAi, and nuclear export assays in cells","pmids":["16236793"],"confidence":"Medium","gaps":["Did not define the structural basis of HDAC recognition","Endogenous physiological context of CaMK-driven export not fully mapped"]},{"year":2005,"claim":"Showed the ankyrin repeat domain of ANKRA2 can functionally substitute for RFXANK in MHC II enhanceosome assembly and RFX5 binding, establishing ANKRA2 as a RFXANK paralog with shared recognition surface.","evidence":"Complementation in RFXANK-deficient BLS cells, domain-swap and mutagenesis, chromatin occupancy","pmids":["15655668","16166641"],"confidence":"Medium","gaps":["Did not establish whether ANKRA2 substitutes for RFXANK under endogenous conditions","Distinct vs overlapping target genes of the two paralogs unresolved"]},{"year":2007,"claim":"Extended ANKRA2's corepressor role beyond MHC II by identifying it as a recruiter of HDAC4/HDAC5 for AhRR-mediated repression of CYP1A1.","evidence":"Yeast two-hybrid, siRNA knockdown, reporter gene assay","pmids":["17949687"],"confidence":"Medium","gaps":["Direct binding interface with AhRR not structurally defined","In vivo relevance to xenobiotic response not tested"]},{"year":2009,"claim":"Revealed that AhRR SUMOylation is a prerequisite for assembling the AhRR–ANKRA2–HDAC repressor complex, adding a post-translational control layer to ANKRA2 recruitment.","evidence":"In vivo SUMOylation assays, mutagenesis, Co-IP, reporter assay","pmids":["19251700"],"confidence":"Medium","gaps":["Whether SUMO acts directly on the interaction interface or indirectly is unclear","SUMO regulation of other ANKRA2 partners not addressed"]},{"year":2012,"claim":"Defined the molecular logic of ANKRA2 partner recognition: a tumbler-lock readout of the PxLPxI/L motif by the central ankyrin repeats, and explained signal regulation by showing Ser350 phosphorylation disrupts binding while creating a 14-3-3 site.","evidence":"X-ray crystallography of peptide complexes, ITC, mutagenesis, phosphopeptide binding","pmids":["22649097"],"confidence":"High","gaps":["Structures are of peptides, not full-length partners","Does not establish which motif-bearing partners predominate in a given cell type"]},{"year":2015,"claim":"Identified CCDC8 as a major ANKRA2-specific (non-RFXANK) cellular partner via its PxLPxL motif, linking ANKRA2 to the OBSL1/CUL7 3M-syndrome ligase complex.","evidence":"Cellular Co-IP, X-ray crystallography of ARD–CCDC8 peptide complex, binding assays","pmids":["25752541"],"confidence":"High","gaps":["Functional consequence of ANKRA2 in CUL7 ligase activity not established","No phenotypic link to 3M syndrome demonstrated for ANKRA2"]},{"year":2019,"claim":"Structurally established ANKRA2 as a higher-affinity reader of the RFX7 PxLPxL motif than RFXANK, distinguishing ANKRA2's partner preference at the structural level.","evidence":"X-ray crystallography of ARD–RFX7 fragment, binding affinity measurements","pmids":["31864703"],"confidence":"High","gaps":["Cellular consequence of preferential RFX7 binding not tested in this study","Whether RFX7 engagement is corepressive or coactivating left open"]},{"year":2024,"claim":"Placed ANKRA2 in a tumor-suppressor circuit by showing it is a direct p53 target and an RFX7 cofactor that occupies the PDCD4 X-box and regulates an RFX7-overlapping, RFXANK-distinct gene set.","evidence":"Mass spectrometry of promoter-bound complex, siRNA knockdown with transcriptome analysis, reporter and p53 target validation","pmids":["39181888"],"confidence":"Medium","gaps":["Whether ANKRA2 activates or represses RFX7 targets mechanistically not fully resolved","Tumor-suppressive phenotype in vivo not demonstrated"]},{"year":null,"claim":"How ANKRA2 partner selection is governed across cell types and how its corepressor versus RFX7-cofactor roles are integrated remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structure of ANKRA2 with full-length partners or within an intact DNA-bound enhanceosome","Determinants choosing among HDAC4/5/9, RFX5, RFX7, CCDC8 partners in a given context unknown","Physiological loss-of-function phenotype of ANKRA2 uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,8]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,1]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,5,8]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,8]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[0,4,5]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,2]}],"complexes":["MHC II enhanceosome (RFX complex)"],"partners":["HDAC4","HDAC5","RFX5","RFX7","CCDC8","AHRR","RFXANK"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9H9E1","full_name":"Ankyrin repeat family A protein 2","aliases":["RFXANK-like protein 2"],"length_aa":313,"mass_kda":34.3,"function":"May regulate the interaction between the 3M complex and the histone deacetylases HDAC4 and HDAC5 (PubMed:25752541). May also regulate LRP2/megalin (By similarity)","subcellular_location":"Cytoplasm, cytoskeleton; Membrane","url":"https://www.uniprot.org/uniprotkb/Q9H9E1/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/ANKRA2","classification":"Not Classified","n_dependent_lines":96,"n_total_lines":1208,"dependency_fraction":0.07947019867549669},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/ANKRA2","total_profiled":1310},"omim":[{"mim_id":"605787","title":"ANKYRIN REPEAT-CONTAINING PROTEIN, FAMILY A, MEMBER 2; ANKRA2","url":"https://www.omim.org/entry/605787"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Plasma membrane","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"},{"location":"Cell Junctions","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/ANKRA2"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"Q9H9E1","domains":[],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H9E1","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H9E1-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9H9E1-F1-predicted_aligned_error_v6.png","plddt_mean":67.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=ANKRA2","jax_strain_url":"https://www.jax.org/strain/search?query=ANKRA2"},"sequence":{"accession":"Q9H9E1","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9H9E1.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9H9E1/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9H9E1"}},"corpus_meta":[{"pmid":"16236793","id":"PMC_16236793","title":"Class II histone deacetylases confer signal responsiveness to the ankyrin-repeat proteins ANKRA2 and RFXANK.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16236793","citation_count":48,"is_preprint":false},{"pmid":"22649097","id":"PMC_22649097","title":"Sequence-specific recognition of a PxLPxI/L motif by an ankyrin repeat tumbler lock.","date":"2012","source":"Science signaling","url":"https://pubmed.ncbi.nlm.nih.gov/22649097","citation_count":44,"is_preprint":false},{"pmid":"17949687","id":"PMC_17949687","title":"Molecular mechanism of transcriptional repression of AhR repressor involving ANKRA2, HDAC4, and HDAC5.","date":"2007","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/17949687","citation_count":32,"is_preprint":false},{"pmid":"24007313","id":"PMC_24007313","title":"Whole transcriptome sequencing identifies tumor-specific mutations in human oral squamous cell carcinoma.","date":"2013","source":"BMC medical genomics","url":"https://pubmed.ncbi.nlm.nih.gov/24007313","citation_count":29,"is_preprint":false},{"pmid":"19251700","id":"PMC_19251700","title":"SUMO modification regulates the transcriptional repressor function of aryl hydrocarbon receptor repressor.","date":"2009","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/19251700","citation_count":26,"is_preprint":false},{"pmid":"16166641","id":"PMC_16166641","title":"New functions of the major histocompatibility complex class II-specific transcription factor RFXANK revealed by a high-resolution mutagenesis study.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16166641","citation_count":17,"is_preprint":false},{"pmid":"25752541","id":"PMC_25752541","title":"Ankyrin repeats of ANKRA2 recognize a PxLPxL motif on the 3M syndrome protein CCDC8.","date":"2015","source":"Structure (London, England : 1993)","url":"https://pubmed.ncbi.nlm.nih.gov/25752541","citation_count":16,"is_preprint":false},{"pmid":"22034997","id":"PMC_22034997","title":"Association of single nucleotide polymorphisms in the ANKRA2 and CD180 genes with bovine respiratory disease and presence of Mycobacterium avium subsp. paratuberculosis(1).","date":"2011","source":"Animal genetics","url":"https://pubmed.ncbi.nlm.nih.gov/22034997","citation_count":14,"is_preprint":false},{"pmid":"36848325","id":"PMC_36848325","title":"gBLUP-GWAS identifies candidate genes, signaling pathways, and putative functional polymorphisms for age at puberty in gilts.","date":"2023","source":"Journal of animal science","url":"https://pubmed.ncbi.nlm.nih.gov/36848325","citation_count":11,"is_preprint":false},{"pmid":"15655668","id":"PMC_15655668","title":"Evolutionary conservation and characterization of the bare lymphocyte syndrome transcription factor RFX-B and its paralogue ANKRA2.","date":"2005","source":"Immunogenetics","url":"https://pubmed.ncbi.nlm.nih.gov/15655668","citation_count":10,"is_preprint":false},{"pmid":"39181888","id":"PMC_39181888","title":"p53 target ANKRA2 cooperates with RFX7 to regulate tumor suppressor genes.","date":"2024","source":"Cell death discovery","url":"https://pubmed.ncbi.nlm.nih.gov/39181888","citation_count":7,"is_preprint":false},{"pmid":"22140378","id":"PMC_22140378","title":"FOXD1 Duplication Causes Branchial Defects and Interacts with the TFAP2A Gene Implicated in the Branchio-Oculo-Facial Syndrome in Causing Eye Effects in Zebrafish.","date":"2011","source":"Molecular syndromology","url":"https://pubmed.ncbi.nlm.nih.gov/22140378","citation_count":6,"is_preprint":false},{"pmid":"31864703","id":"PMC_31864703","title":"Structural basis for the recognition of RFX7 by ANKRA2 and RFXANK.","date":"2019","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/31864703","citation_count":5,"is_preprint":false},{"pmid":"41959735","id":"PMC_41959735","title":"Receptor-mediated endocytosis by Megalin: Exploring its role in ligand interaction and disease mechanisms.","date":"2025","source":"Genes & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/41959735","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8320,"output_tokens":2374,"usd":0.030285,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9453,"output_tokens":3173,"usd":0.063295,"stage2_stop_reason":"end_turn"},"total_usd":0.09358,"stage1_batch_id":"msgbatch_0179Z2pxRYBsXi2xADneafRw","stage2_batch_id":"msgbatch_01LaY9ZaVdgjWArcZo9eqQE5","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"Class IIa HDACs (HDAC4 and HDAC5) physically associate with the ankyrin repeat domain of ANKRA2, and through association with the paralog RFXANK, repress MHC II promoter activation and endogenous HLA-DRA gene expression induced by CIITA. Phosphorylation of class II HDACs by CaMK results in CRM1-dependent nuclear export of HDAC/RFXANK complexes.\",\n      \"method\": \"Co-immunoprecipitation, reporter gene assays, RNAi knockdown, nuclear export assays\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP with functional reporter assays and CaMK/export experiments, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"16236793\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The ankyrin repeat domain of ANKRA2 recognizes a PxLPxI/L motif found in HDAC4, HDAC5, HDAC9, megalin, and RFX5, using a tumbler-lock binding mode where each of the middle three ankyrin repeats contacts one residue of the motif. Crystal structures of ANKRA2 ankyrin repeats in complex with binding peptides defined this recognition mechanism. Phosphorylation of Ser350 within the PxLPxI/L motif of HDAC4 impairs ANKRA2 binding while generating a 14-3-3 docking site.\",\n      \"method\": \"X-ray crystallography, isothermal titration calorimetry, mutagenesis, phosphopeptide binding assays\",\n      \"journal\": \"Science signaling\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structures with bound peptides, mutagenesis of binding interface, multiple orthogonal binding assays in a single rigorous study\",\n      \"pmids\": [\"22649097\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"ANKRA2 was identified as a binding partner of the AhR repressor (AhRR) C-terminal repression domain via yeast two-hybrid screening. ANKRA2 recruits HDAC4 and HDAC5 as corepressors for AhRR-mediated transcriptional repression of CYP1A1; siRNA knockdown of ANKRA2 reduces AhRR repression activity.\",\n      \"method\": \"Yeast two-hybrid, RNAi knockdown, reporter gene assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid interaction confirmed with siRNA functional assay, single lab with two orthogonal methods\",\n      \"pmids\": [\"17949687\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"SUMOylation of AhRR at Lys-542, Lys-583, and Lys-660 is required for the interaction between AhRR and ANKRA2 (as well as HDAC4 and HDAC5); arginine mutation of these residues reduces both SUMOylation and the AhRR–ANKRA2 interaction, impairing transcriptional repression.\",\n      \"method\": \"In vivo SUMOylation assays, site-directed mutagenesis, co-immunoprecipitation, reporter gene assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with Co-IP and functional reporter assays, single lab\",\n      \"pmids\": [\"19251700\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The ankyrin repeat domain (ARD) of ANKRA2 can substitute for RFXANK in activating MHC II gene expression, as demonstrated by complementation of a bare lymphocyte syndrome cell line deficient in RFX-B (RFXANK). Mouse and Xenopus RFXANK orthologues complement this deficiency but ANKRA2 does so only through its ARD.\",\n      \"method\": \"Complementation assay in BLS patient-derived cell line, domain-swap experiments\",\n      \"journal\": \"Immunogenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional complementation with domain mapping, single lab\",\n      \"pmids\": [\"15655668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"ANKRA2 ankyrin repeat domain mediates interaction with RFX5, and high-resolution mutagenesis of the closely related RFXANK ARD mapped the RFX5 interaction surface; ANKRA2 can substitute for RFXANK in MHC-II enhanceosome assembly through its ARD.\",\n      \"method\": \"Mutagenesis, complementation assay in BLS cell line, in vivo chromatin occupancy assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis combined with functional complementation and chromatin assays, single lab with multiple methods\",\n      \"pmids\": [\"16166641\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The ankyrin repeats of ANKRA2 recognize a PxLPxL motif at the C-terminal region of CCDC8 (a 3M syndrome protein), establishing CCDC8 as a major cellular partner of ANKRA2 but not RFXANK. The N-terminal part of CCDC8 interacts with OBSL1 to form a CUL7 ligase complex, linking ANKRA2 to the 3M syndrome complex.\",\n      \"method\": \"Co-immunoprecipitation (cellular), X-ray crystallography (structural analysis of ANKRA2 ARD–CCDC8 peptide complex), binding assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure of complex combined with cellular Co-IP and binding assays; novel partner defined with structural and biochemical orthogonal methods\",\n      \"pmids\": [\"25752541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Crystal structures of ANKRA2 ankyrin domain bound to an RFX7 fragment revealed that ANKRA2 recognizes the PxLPxL motif of RFX7 and flanking sequences via extensive hydrophobic interactions, with higher binding affinity than RFXANK for RFX7.\",\n      \"method\": \"X-ray crystallography, binding affinity measurements\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with quantitative binding comparison; single lab but structural evidence is direct\",\n      \"pmids\": [\"31864703\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"ANKRA2 is a direct transcriptional target of p53, and functions as a critical cofactor of the tumor suppressor transcription factor RFX7. Mass spectrometry identified ANKRA2 binding to the X-box motif of the PDCD4 promoter together with RFX5, RFXAP, RFXANK, and RFX7. Transcriptome analyses showed ANKRA2 regulates a gene set overlapping with RFX7 targets, distinct from RFXANK-regulated genes.\",\n      \"method\": \"Reporter gene assay, mass spectrometry of promoter-bound proteins, siRNA knockdown with transcriptome analysis, p53 target gene validation\",\n      \"journal\": \"Cell death discovery\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry of DNA-bound complex combined with transcriptome knockdown analysis and reporter assays; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39181888\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"ANKRA2 is an ankyrin repeat scaffold protein whose ARD recognizes PxLPxI/L linear motifs (via a tumbler-lock mechanism defined by crystal structures) in diverse partners including HDAC4, HDAC5, HDAC9, megalin, RFX5, RFX7, and CCDC8; it functions as a signal-responsive transcriptional corepressor that recruits class IIa HDACs to repress MHC II, AhRR-target, and RFX7-target genes, with complex formation regulated by CaMK-driven phosphorylation of HDAC4-Ser350 (which disrupts ANKRA2 binding and promotes 14-3-3 sequestration) and by SUMOylation of binding partners; ANKRA2 is itself a direct p53 target gene and a cofactor of the tumor suppressor RFX7.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"ANKRA2 is an ankyrin-repeat scaffold protein that functions as a signal-responsive transcriptional corepressor by reading short linear PxLPxI/L motifs in diverse partner proteins [#1]. Crystal structures of its ankyrin repeat domain bound to target peptides defined a tumbler-lock recognition mode in which each of the middle three repeats engages one motif residue, and showed that this interface accommodates motifs from HDAC4, HDAC5, HDAC9, megalin, and RFX5 [#1]. Through this domain ANKRA2 binds class IIa HDACs (HDAC4, HDAC5) and recruits them as corepressors: it represses CIITA-induced MHC II and HLA-DRA expression in conjunction with the paralog RFXANK [#0], and is recruited to the AhR repressor C-terminal domain to repress CYP1A1, an interaction that depends on SUMOylation of AhRR [#2, #3]. The same ankyrin domain can substitute for RFXANK in MHC II enhanceosome assembly by contacting RFX5, complementing RFXANK-deficient bare lymphocyte syndrome cells [#4, #5]. Beyond immune gene regulation, ANKRA2 binds the PxLPxL motif of the 3M-syndrome protein CCDC8, linking it to the OBSL1/CUL7 ligase complex [#6], and serves as a direct p53 target gene and a high-affinity cofactor of the tumor suppressor transcription factor RFX7, regulating RFX7-overlapping targets such as PDCD4 [#7, #8]. HDAC4 recruitment is switched off by CaMK-driven phosphorylation of Ser350 within its PxLPxI/L motif, which disrupts ANKRA2 binding and creates a 14-3-3 docking site, coupling complex assembly to calcium signaling [#0, #1].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that ANKRA2's ankyrin repeat domain physically recruits class IIa HDACs to repress MHC II transcription and that this is reversed by calcium signaling, defining ANKRA2 as a signal-responsive corepressor scaffold.\",\n      \"evidence\": \"Co-IP, reporter assays, RNAi, and nuclear export assays in cells\",\n      \"pmids\": [\"16236793\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the structural basis of HDAC recognition\", \"Endogenous physiological context of CaMK-driven export not fully mapped\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed the ankyrin repeat domain of ANKRA2 can functionally substitute for RFXANK in MHC II enhanceosome assembly and RFX5 binding, establishing ANKRA2 as a RFXANK paralog with shared recognition surface.\",\n      \"evidence\": \"Complementation in RFXANK-deficient BLS cells, domain-swap and mutagenesis, chromatin occupancy\",\n      \"pmids\": [\"15655668\", \"16166641\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not establish whether ANKRA2 substitutes for RFXANK under endogenous conditions\", \"Distinct vs overlapping target genes of the two paralogs unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extended ANKRA2's corepressor role beyond MHC II by identifying it as a recruiter of HDAC4/HDAC5 for AhRR-mediated repression of CYP1A1.\",\n      \"evidence\": \"Yeast two-hybrid, siRNA knockdown, reporter gene assay\",\n      \"pmids\": [\"17949687\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct binding interface with AhRR not structurally defined\", \"In vivo relevance to xenobiotic response not tested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Revealed that AhRR SUMOylation is a prerequisite for assembling the AhRR–ANKRA2–HDAC repressor complex, adding a post-translational control layer to ANKRA2 recruitment.\",\n      \"evidence\": \"In vivo SUMOylation assays, mutagenesis, Co-IP, reporter assay\",\n      \"pmids\": [\"19251700\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SUMO acts directly on the interaction interface or indirectly is unclear\", \"SUMO regulation of other ANKRA2 partners not addressed\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Defined the molecular logic of ANKRA2 partner recognition: a tumbler-lock readout of the PxLPxI/L motif by the central ankyrin repeats, and explained signal regulation by showing Ser350 phosphorylation disrupts binding while creating a 14-3-3 site.\",\n      \"evidence\": \"X-ray crystallography of peptide complexes, ITC, mutagenesis, phosphopeptide binding\",\n      \"pmids\": [\"22649097\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structures are of peptides, not full-length partners\", \"Does not establish which motif-bearing partners predominate in a given cell type\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified CCDC8 as a major ANKRA2-specific (non-RFXANK) cellular partner via its PxLPxL motif, linking ANKRA2 to the OBSL1/CUL7 3M-syndrome ligase complex.\",\n      \"evidence\": \"Cellular Co-IP, X-ray crystallography of ARD–CCDC8 peptide complex, binding assays\",\n      \"pmids\": [\"25752541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of ANKRA2 in CUL7 ligase activity not established\", \"No phenotypic link to 3M syndrome demonstrated for ANKRA2\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Structurally established ANKRA2 as a higher-affinity reader of the RFX7 PxLPxL motif than RFXANK, distinguishing ANKRA2's partner preference at the structural level.\",\n      \"evidence\": \"X-ray crystallography of ARD–RFX7 fragment, binding affinity measurements\",\n      \"pmids\": [\"31864703\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular consequence of preferential RFX7 binding not tested in this study\", \"Whether RFX7 engagement is corepressive or coactivating left open\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Placed ANKRA2 in a tumor-suppressor circuit by showing it is a direct p53 target and an RFX7 cofactor that occupies the PDCD4 X-box and regulates an RFX7-overlapping, RFXANK-distinct gene set.\",\n      \"evidence\": \"Mass spectrometry of promoter-bound complex, siRNA knockdown with transcriptome analysis, reporter and p53 target validation\",\n      \"pmids\": [\"39181888\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether ANKRA2 activates or represses RFX7 targets mechanistically not fully resolved\", \"Tumor-suppressive phenotype in vivo not demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How ANKRA2 partner selection is governed across cell types and how its corepressor versus RFX7-cofactor roles are integrated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structure of ANKRA2 with full-length partners or within an intact DNA-bound enhanceosome\", \"Determinants choosing among HDAC4/5/9, RFX5, RFX7, CCDC8 partners in a given context unknown\", \"Physiological loss-of-function phenotype of ANKRA2 uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 8]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 5, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [0, 4, 5]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 2]}\n    ],\n    \"complexes\": [\"MHC II enhanceosome (RFX complex)\"],\n    \"partners\": [\"HDAC4\", \"HDAC5\", \"RFX5\", \"RFX7\", \"CCDC8\", \"AHRR\", \"RFXANK\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}