{"gene":"CC2D1B","run_date":"2026-06-09T22:57:17","timeline":{"discoveries":[{"year":2018,"finding":"CC2D1B coordinates ESCRT-III activity during mitotic nuclear envelope (NE) reformation by regulating the spatiotemporal distribution of the CHMP7-ESCRT-III axis. CC2D1B depletion uncouples ESCRT activity from Spastin-mediated spindle microtubule severing, resulting in persisting microtubules that compromise nuclear morphology. CC2D1B also coordinates ER membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE.","method":"siRNA depletion in human cells with live imaging, immunofluorescence, and functional readouts of nuclear compartmentalization and microtubule dynamics","journal":"Developmental Cell","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean loss-of-function (depletion) with multiple defined cellular phenotypes and pathway placement within ESCRT-III/NE reformation, single lab but multiple orthogonal readouts","pmids":["30513301"],"is_preprint":false},{"year":2009,"finding":"Human Freud-2/CC2D1B binds specifically to the dual repressor element (DRE) of the 5-HT1A receptor gene promoter (at a site adjacent to the Freud-1/CC2D1A binding site) and represses 5-HT1A receptor transcription in non-serotonergic cells and neurons. siRNA knockdown of CC2D1B de-represses 5-HT1A promoter activity and increases 5-HT1A receptor protein levels in SK-N-SH cells.","method":"EMSA, reporter assay, siRNA knockdown with Western blot and promoter activity readout, immunohistochemistry, subcellular fractionation (nuclear enrichment)","journal":"Biological Psychiatry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (EMSA, reporter assay, siRNA KD with protein-level readout) replicated across two independent papers (PMID 19423080 and 21155902)","pmids":["19423080"],"is_preprint":false},{"year":2010,"finding":"Mouse Freud-2/CC2D1B binds specifically to the rat 5-HT1A dual repressor element (DRE) at a site adjacent to, and partially overlapping, the Freud-1/CC2D1A binding site. Supershift assay distinguished Freud-2-DRE complexes from Freud-1-DRE complexes. Freud-2 represses 5-HT1A promoter-reporter constructs in a DRE-dependent manner; siRNA knockdown reduces Freud-2 binding to the DRE (by ChIP) and increases 5-HT1A promoter activity and protein levels in neuronal cells.","method":"EMSA, supershift assay, reporter assay, ChIP, siRNA knockdown with Western blot","journal":"European Journal of Neuroscience","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (EMSA/supershift, reporter, ChIP, KD) in two independent studies confirming DRE binding and repressor function","pmids":["21155902"],"is_preprint":false},{"year":2016,"finding":"CC2D1B (together with CC2D1A) prevents premature sorting of EGFR and TLR4 to intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Depletion of CC2D1A and CC2D1B accelerates lysosomal degradation of EGFR and TLR4 and causes rapid termination of downstream ERK1/2 signaling. CC2D1A binds to CHMP4B polymers formed on endosomes to regulate this endosomal sorting pathway.","method":"siRNA knockdown of CC2D1A/B with EGFR/TLR4 degradation assays, ERK1/2 signaling assays, and pulldown/binding assay for CC2D1A–CHMP4B interaction","journal":"Biochemical and Biophysical Research Communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean loss-of-function with defined molecular phenotypes (receptor degradation, signaling), but single lab and CC2D1B effects reported jointly with CC2D1A without full separation","pmids":["27769858"],"is_preprint":false},{"year":2015,"finding":"CC2D1B localizes on endosomes (together with CHMP4B) in MEF cells when VPS4 activity is reduced, indicating cycling between cytosol and endosomal membrane. Cc2d1b knockout mice show no increase in endosome size (unlike Cc2d1a KO), indicating CC2D1B is not individually required for endosomal morphogenesis. CC2D1B can functionally replace the Drosophila ortholog Lgd in rescue experiments, supporting conservation of ESCRT-III-related function.","method":"Conditional knockout mouse generation, electron microscopy of endosomes, immunofluorescence localization with VPS4 dominant-negative, Drosophila rescue assay","journal":"PLoS Genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (KO mouse, EM, localization, cross-species rescue) but CC2D1B-specific phenotype is negative for endosome size; cross-species rescue provides positive functional evidence","pmids":["26720614"],"is_preprint":false},{"year":2019,"finding":"CC2D1B expression in Schwann cells is regulated downstream of YAP/TAZ. Silencing of Cc2d1b limits the formation of myelin segments in Schwann cells in vitro, establishing a role for CC2D1B in peripheral nervous system myelination.","method":"YAP/TAZ ablation (expression analysis), siRNA silencing of Cc2d1b with quantification of myelin segment formation","journal":"Frontiers in Molecular Neuroscience","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — loss-of-function with defined cellular phenotype (myelination), but single lab, in vitro, and limited mechanistic depth","pmids":["31379499"],"is_preprint":false},{"year":2022,"finding":"In Cc2d1b constitutive knockout mice, large-diameter myelinated fibers in the optic nerve show hypomyelination (g-ratio 0.844 vs. 0.832 in controls), while peripheral nervous system myelination and CNS oligodendrocyte numbers/myelin protein levels in cortex, corpus callosum, and striatum are unaffected, establishing a selective role for CC2D1B in CNS developmental myelination.","method":"Constitutive Cc2d1b knockout mouse, morphological analysis of semithin sections and electron micrographs, immunohistology","journal":"Frontiers in Molecular Neuroscience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean in vivo KO with defined morphological phenotype in optic nerve, single lab but robust quantitative EM analysis","pmids":["35592111"],"is_preprint":false},{"year":2025,"finding":"CC2D1A and CC2D1B (its only paralog) physically interact in mouse brain (hippocampus), confirmed by co-immunoprecipitation. The two paralogs localize to different synaptic compartments: CC2D1A is uniquely enriched in the post-synapse, while CC2D1B distribution differs, suggesting distinct synaptic roles despite their capacity to interact.","method":"Co-immunoprecipitation in mouse hippocampus (with Cc2d1a hypomorph controls), synaptic fractionation","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, Co-IP and fractionation; CC2D1B-specific mechanistic detail is secondary to the CC2D1A-focused study","pmids":["bio_10.1101_2025.06.26.661826"],"is_preprint":true}],"current_model":"CC2D1B (Freud-2/Lgd1) is a multifunctional scaffold protein that (1) acts as a transcriptional repressor of the serotonin-1A receptor gene by binding a dual repressor element (DRE) in the promoter, (2) coordinates ESCRT-III activity during mitotic nuclear envelope reformation by regulating the CHMP7-ESCRT-III axis and coupling it to Spastin-mediated microtubule severing, (3) restrains premature endosomal sorting of transmembrane receptors (EGFR, TLR4) to intraluminal vesicles via interaction with CHMP4B polymers, and (4) is required for normal CNS developmental myelination in the optic nerve; it physically interacts with its paralog CC2D1A in the brain but occupies distinct synaptic compartments."},"narrative":{"mechanistic_narrative":"CC2D1B (Freud-2/Lgd1) is a multifunctional scaffold protein that operates in both nuclear transcriptional repression and ESCRT-III-dependent membrane remodeling [PMID:19423080, PMID:30513301]. In the nucleus it binds the dual repressor element (DRE) of the serotonin-1A (5-HT1A) receptor gene promoter, at a site adjacent to that bound by its paralog CC2D1A/Freud-1, and represses 5-HT1A receptor transcription; depletion de-represses promoter activity and raises receptor protein levels in neuronal cells [PMID:19423080, PMID:21155902]. At cellular membranes CC2D1B coordinates ESCRT-III activity: during mitotic nuclear envelope reformation it regulates the spatiotemporal distribution of the CHMP7-ESCRT-III axis and couples ESCRT activity to Spastin-mediated spindle microtubule severing, and on endosomes it restrains premature sorting of transmembrane receptors EGFR and TLR4 into intraluminal vesicles, thereby sustaining downstream ERK1/2 signaling, acting alongside CC2D1A which binds CHMP4B polymers [PMID:30513301, PMID:27769858]. CC2D1B cycles between cytosol and endosomal membranes in a VPS4-sensitive manner and can functionally substitute for the Drosophila ortholog Lgd, indicating conserved ESCRT-III-related function [PMID:26720614]. In the nervous system CC2D1B is required for normal CNS developmental myelination, with constitutive knockout mice showing selective hypomyelination of large-diameter optic nerve fibers [PMID:35592111]. It physically interacts with CC2D1A in the brain while occupying distinct synaptic compartments [PMID:bio_10.1101_2025.06.26.661826].","teleology":[{"year":2009,"claim":"Established CC2D1B as a sequence-specific transcriptional repressor, answering whether the Freud-1 paralog also controls 5-HT1A receptor gene expression.","evidence":"EMSA, reporter assays, and siRNA knockdown with Western blot in human SK-N-SH cells","pmids":["19423080"],"confidence":"High","gaps":["Did not define co-repressor partners or chromatin-modifying machinery recruited to the DRE","Did not resolve how nuclear versus cytoplasmic functions are partitioned"]},{"year":2010,"claim":"Confirmed conserved, DRE-dependent repression across species and distinguished CC2D1B-DRE complexes from CC2D1A-DRE complexes, clarifying that the two paralogs bind adjacent overlapping sites.","evidence":"EMSA/supershift, reporter assay, ChIP, and siRNA knockdown in neuronal cells (mouse/rat sequences)","pmids":["21155902"],"confidence":"High","gaps":["Functional consequence of adjacent CC2D1A/CC2D1B binding (cooperation vs redundancy) not resolved","In vivo transcriptional role in serotonergic circuits not tested"]},{"year":2015,"claim":"Placed CC2D1B in the ESCRT-III pathway and tested its individual requirement for endosomal morphogenesis, showing it cycles to endosomes but is dispensable for endosome size unlike CC2D1A.","evidence":"Conditional knockout mice, EM of endosomes, VPS4 dominant-negative localization, and Drosophila Lgd rescue assay","pmids":["26720614"],"confidence":"Medium","gaps":["CC2D1B-specific endosomal phenotype was negative, leaving its non-redundant membrane role undefined","Mechanism of cytosol-to-membrane cycling not characterized"]},{"year":2016,"claim":"Defined a functional output for CC2D1A/B at endosomes — restraining premature receptor sorting to ILVs — linking the scaffold to receptor signaling duration.","evidence":"siRNA knockdown with EGFR/TLR4 degradation and ERK1/2 signaling assays plus CC2D1A-CHMP4B pulldown","pmids":["27769858"],"confidence":"Medium","gaps":["CC2D1B contribution reported jointly with CC2D1A without full separation","Direct CC2D1B-CHMP4B binding not demonstrated independently of CC2D1A"]},{"year":2018,"claim":"Identified a distinct membrane-remodeling role in mitosis, showing CC2D1B coordinates the CHMP7-ESCRT-III axis with Spastin-mediated microtubule severing during nuclear envelope reformation.","evidence":"siRNA depletion with live imaging, immunofluorescence, and readouts of nuclear compartmentalization and microtubule dynamics in human cells","pmids":["30513301"],"confidence":"High","gaps":["Direct biochemical interactions of CC2D1B with CHMP7 or Spastin not mapped","How CC2D1B times ESCRT recruitment relative to ER deposition not mechanistically resolved"]},{"year":2019,"claim":"Connected CC2D1B to glial myelination, placing it downstream of YAP/TAZ in Schwann cells.","evidence":"YAP/TAZ ablation expression analysis and siRNA silencing with myelin segment quantification in vitro","pmids":["31379499"],"confidence":"Medium","gaps":["In vitro only; in vivo PNS requirement not established here","Molecular link between CC2D1B and myelin gene programs unknown"]},{"year":2022,"claim":"Resolved the in vivo myelination role, showing a selective CNS requirement (optic nerve large-fiber hypomyelination) rather than a general PNS or oligodendrocyte defect.","evidence":"Constitutive Cc2d1b knockout mouse with quantitative EM g-ratio analysis and immunohistology","pmids":["35592111"],"confidence":"Medium","gaps":["Cell-autonomous mechanism in oligodendrocytes vs axons not determined","Relationship between myelination role and ESCRT/transcriptional functions unknown"]},{"year":2025,"claim":"Tested the paralog relationship in brain, showing CC2D1A and CC2D1B physically interact yet occupy distinct synaptic compartments.","evidence":"Co-immunoprecipitation and synaptic fractionation in mouse hippocampus (preprint)","pmids":["bio_10.1101_2025.06.26.661826"],"confidence":"Low","gaps":["Preprint, single lab; reciprocal validation and CC2D1B-specific synaptic function not established","Functional consequence of heterotypic CC2D1A/B interaction unknown"]},{"year":null,"claim":"It remains unknown how CC2D1B's distinct activities — DRE-bound transcriptional repression, ESCRT-III coordination at the nuclear envelope and endosomes, and CNS myelination — are integrated within a single protein and partitioned across subcellular compartments.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural basis for substrate/partner selection across functions","No direct CC2D1B-CHMP4B/CHMP7 binding map separate from CC2D1A","No defined disease-causing CC2D1B mutation in the corpus"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,2]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,2]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,3]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[1,2]},{"term_id":"GO:0005768","term_label":"endosome","supporting_discovery_ids":[3,4]},{"term_id":"GO:0005635","term_label":"nuclear envelope","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[1,2]},{"term_id":"R-HSA-5653656","term_label":"Vesicle-mediated transport","supporting_discovery_ids":[3]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[6]}],"complexes":["ESCRT-III"],"partners":["CC2D1A","CHMP4B","CHMP7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5T0F9","full_name":"Coiled-coil and C2 domain-containing protein 1B","aliases":["Five prime repressor element under dual repression-binding protein 2","FRE under dual repression-binding protein 2","Freud-2"],"length_aa":858,"mass_kda":94.2,"function":"Transcription factor that binds specifically to the DRE (dual repressor element) and represses HTR1A gene transcription in neuronal cells","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q5T0F9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CC2D1B","classification":"Not Classified","n_dependent_lines":26,"n_total_lines":1208,"dependency_fraction":0.02152317880794702},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CHMP4B","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CC2D1B","total_profiled":1310},"omim":[{"mim_id":"610055","title":"COILED-COIL AND C2 DOMAINS-CONTAINING PROTEIN 1A; CC2D1A","url":"https://www.omim.org/entry/610055"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CC2D1B"},"hgnc":{"alias_symbol":["KIAA1836","Freud-2","Lgd1"],"prev_symbol":[]},"alphafold":{"accession":"Q5T0F9","domains":[{"cath_id":"-","chopping":"164-221","consensus_level":"high","plddt":88.2124,"start":164,"end":221},{"cath_id":"-","chopping":"282-322","consensus_level":"medium","plddt":89.4029,"start":282,"end":322},{"cath_id":"2.60.40.150","chopping":"701-842","consensus_level":"high","plddt":92.1677,"start":701,"end":842},{"cath_id":"1.10.287","chopping":"385-439","consensus_level":"medium","plddt":88.9407,"start":385,"end":439},{"cath_id":"1.10.287","chopping":"532-591","consensus_level":"high","plddt":86.8537,"start":532,"end":591},{"cath_id":"1.10.287","chopping":"614-679","consensus_level":"high","plddt":90.1861,"start":614,"end":679}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T0F9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T0F9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T0F9-F1-predicted_aligned_error_v6.png","plddt_mean":70.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CC2D1B","jax_strain_url":"https://www.jax.org/strain/search?query=CC2D1B"},"sequence":{"accession":"Q5T0F9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5T0F9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5T0F9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T0F9"}},"corpus_meta":[{"pmid":"30513301","id":"PMC_30513301","title":"CC2D1B Coordinates ESCRT-III Activity during the Mitotic Reformation of the Nuclear Envelope.","date":"2018","source":"Developmental cell","url":"https://pubmed.ncbi.nlm.nih.gov/30513301","citation_count":64,"is_preprint":false},{"pmid":"26720614","id":"PMC_26720614","title":"The Mammalian Orthologs of Drosophila Lgd, CC2D1A and CC2D1B, Function in the Endocytic Pathway, but Their Individual Loss of Function Does Not Affect Notch Signalling.","date":"2015","source":"PLoS genetics","url":"https://pubmed.ncbi.nlm.nih.gov/26720614","citation_count":29,"is_preprint":false},{"pmid":"19423080","id":"PMC_19423080","title":"Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.","date":"2009","source":"Biological psychiatry","url":"https://pubmed.ncbi.nlm.nih.gov/19423080","citation_count":26,"is_preprint":false},{"pmid":"29552027","id":"PMC_29552027","title":"Loss of the Intellectual Disability and Autism Gene Cc2d1a and Its Homolog Cc2d1b Differentially Affect Spatial Memory, Anxiety, and Hyperactivity.","date":"2018","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/29552027","citation_count":19,"is_preprint":false},{"pmid":"22409537","id":"PMC_22409537","title":"Rice LGD1 containing RNA binding activity affects growth and development through alternative promoters.","date":"2012","source":"The Plant journal : for cell and molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/22409537","citation_count":17,"is_preprint":false},{"pmid":"27769858","id":"PMC_27769858","title":"CC2D1A and CC2D1B regulate degradation and signaling of EGFR and TLR4.","date":"2016","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/27769858","citation_count":13,"is_preprint":false},{"pmid":"21155902","id":"PMC_21155902","title":"Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.","date":"2010","source":"The European journal of neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/21155902","citation_count":12,"is_preprint":false},{"pmid":"31379499","id":"PMC_31379499","title":"YAP and TAZ Regulate Cc2d1b and Purβ in Schwann Cells.","date":"2019","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/31379499","citation_count":11,"is_preprint":false},{"pmid":"31680860","id":"PMC_31680860","title":"Corrigendum: YAP and TAZ Regulate Cc2d1b and Purβ in Schwann Cells.","date":"2019","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/31680860","citation_count":11,"is_preprint":false},{"pmid":"35592111","id":"PMC_35592111","title":"Cc2d1b Contributes to the Regulation of Developmental Myelination in the Central Nervous System.","date":"2022","source":"Frontiers in molecular neuroscience","url":"https://pubmed.ncbi.nlm.nih.gov/35592111","citation_count":8,"is_preprint":false},{"pmid":"27150226","id":"PMC_27150226","title":"5-HT1A receptor gene silencers Freud-1 and Freud-2 are differently expressed in the brain of rats with genetically determined high level of fear-induced aggression or its absence.","date":"2016","source":"Behavioural brain research","url":"https://pubmed.ncbi.nlm.nih.gov/27150226","citation_count":6,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.06.26.661826","title":"Interactome Analysis of the CC2D1A Scaffold Reveals Novel Neuronal Interactions and a Postsynaptic Role","date":"2025-06-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.06.26.661826","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8384,"output_tokens":2477,"usd":0.031154,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9542,"output_tokens":3214,"usd":0.06403,"stage2_stop_reason":"end_turn"},"total_usd":0.095184,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2018,\n      \"finding\": \"CC2D1B coordinates ESCRT-III activity during mitotic nuclear envelope (NE) reformation by regulating the spatiotemporal distribution of the CHMP7-ESCRT-III axis. CC2D1B depletion uncouples ESCRT activity from Spastin-mediated spindle microtubule severing, resulting in persisting microtubules that compromise nuclear morphology. CC2D1B also coordinates ER membrane deposition around chromatin disks with ESCRT-III recruitment to the reforming NE.\",\n      \"method\": \"siRNA depletion in human cells with live imaging, immunofluorescence, and functional readouts of nuclear compartmentalization and microtubule dynamics\",\n      \"journal\": \"Developmental Cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean loss-of-function (depletion) with multiple defined cellular phenotypes and pathway placement within ESCRT-III/NE reformation, single lab but multiple orthogonal readouts\",\n      \"pmids\": [\"30513301\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Human Freud-2/CC2D1B binds specifically to the dual repressor element (DRE) of the 5-HT1A receptor gene promoter (at a site adjacent to the Freud-1/CC2D1A binding site) and represses 5-HT1A receptor transcription in non-serotonergic cells and neurons. siRNA knockdown of CC2D1B de-represses 5-HT1A promoter activity and increases 5-HT1A receptor protein levels in SK-N-SH cells.\",\n      \"method\": \"EMSA, reporter assay, siRNA knockdown with Western blot and promoter activity readout, immunohistochemistry, subcellular fractionation (nuclear enrichment)\",\n      \"journal\": \"Biological Psychiatry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (EMSA, reporter assay, siRNA KD with protein-level readout) replicated across two independent papers (PMID 19423080 and 21155902)\",\n      \"pmids\": [\"19423080\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Mouse Freud-2/CC2D1B binds specifically to the rat 5-HT1A dual repressor element (DRE) at a site adjacent to, and partially overlapping, the Freud-1/CC2D1A binding site. Supershift assay distinguished Freud-2-DRE complexes from Freud-1-DRE complexes. Freud-2 represses 5-HT1A promoter-reporter constructs in a DRE-dependent manner; siRNA knockdown reduces Freud-2 binding to the DRE (by ChIP) and increases 5-HT1A promoter activity and protein levels in neuronal cells.\",\n      \"method\": \"EMSA, supershift assay, reporter assay, ChIP, siRNA knockdown with Western blot\",\n      \"journal\": \"European Journal of Neuroscience\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (EMSA/supershift, reporter, ChIP, KD) in two independent studies confirming DRE binding and repressor function\",\n      \"pmids\": [\"21155902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"CC2D1B (together with CC2D1A) prevents premature sorting of EGFR and TLR4 to intraluminal vesicles (ILVs) of multivesicular bodies (MVBs). Depletion of CC2D1A and CC2D1B accelerates lysosomal degradation of EGFR and TLR4 and causes rapid termination of downstream ERK1/2 signaling. CC2D1A binds to CHMP4B polymers formed on endosomes to regulate this endosomal sorting pathway.\",\n      \"method\": \"siRNA knockdown of CC2D1A/B with EGFR/TLR4 degradation assays, ERK1/2 signaling assays, and pulldown/binding assay for CC2D1A–CHMP4B interaction\",\n      \"journal\": \"Biochemical and Biophysical Research Communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean loss-of-function with defined molecular phenotypes (receptor degradation, signaling), but single lab and CC2D1B effects reported jointly with CC2D1A without full separation\",\n      \"pmids\": [\"27769858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"CC2D1B localizes on endosomes (together with CHMP4B) in MEF cells when VPS4 activity is reduced, indicating cycling between cytosol and endosomal membrane. Cc2d1b knockout mice show no increase in endosome size (unlike Cc2d1a KO), indicating CC2D1B is not individually required for endosomal morphogenesis. CC2D1B can functionally replace the Drosophila ortholog Lgd in rescue experiments, supporting conservation of ESCRT-III-related function.\",\n      \"method\": \"Conditional knockout mouse generation, electron microscopy of endosomes, immunofluorescence localization with VPS4 dominant-negative, Drosophila rescue assay\",\n      \"journal\": \"PLoS Genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (KO mouse, EM, localization, cross-species rescue) but CC2D1B-specific phenotype is negative for endosome size; cross-species rescue provides positive functional evidence\",\n      \"pmids\": [\"26720614\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"CC2D1B expression in Schwann cells is regulated downstream of YAP/TAZ. Silencing of Cc2d1b limits the formation of myelin segments in Schwann cells in vitro, establishing a role for CC2D1B in peripheral nervous system myelination.\",\n      \"method\": \"YAP/TAZ ablation (expression analysis), siRNA silencing of Cc2d1b with quantification of myelin segment formation\",\n      \"journal\": \"Frontiers in Molecular Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — loss-of-function with defined cellular phenotype (myelination), but single lab, in vitro, and limited mechanistic depth\",\n      \"pmids\": [\"31379499\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In Cc2d1b constitutive knockout mice, large-diameter myelinated fibers in the optic nerve show hypomyelination (g-ratio 0.844 vs. 0.832 in controls), while peripheral nervous system myelination and CNS oligodendrocyte numbers/myelin protein levels in cortex, corpus callosum, and striatum are unaffected, establishing a selective role for CC2D1B in CNS developmental myelination.\",\n      \"method\": \"Constitutive Cc2d1b knockout mouse, morphological analysis of semithin sections and electron micrographs, immunohistology\",\n      \"journal\": \"Frontiers in Molecular Neuroscience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean in vivo KO with defined morphological phenotype in optic nerve, single lab but robust quantitative EM analysis\",\n      \"pmids\": [\"35592111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"CC2D1A and CC2D1B (its only paralog) physically interact in mouse brain (hippocampus), confirmed by co-immunoprecipitation. The two paralogs localize to different synaptic compartments: CC2D1A is uniquely enriched in the post-synapse, while CC2D1B distribution differs, suggesting distinct synaptic roles despite their capacity to interact.\",\n      \"method\": \"Co-immunoprecipitation in mouse hippocampus (with Cc2d1a hypomorph controls), synaptic fractionation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, Co-IP and fractionation; CC2D1B-specific mechanistic detail is secondary to the CC2D1A-focused study\",\n      \"pmids\": [\"bio_10.1101_2025.06.26.661826\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"CC2D1B (Freud-2/Lgd1) is a multifunctional scaffold protein that (1) acts as a transcriptional repressor of the serotonin-1A receptor gene by binding a dual repressor element (DRE) in the promoter, (2) coordinates ESCRT-III activity during mitotic nuclear envelope reformation by regulating the CHMP7-ESCRT-III axis and coupling it to Spastin-mediated microtubule severing, (3) restrains premature endosomal sorting of transmembrane receptors (EGFR, TLR4) to intraluminal vesicles via interaction with CHMP4B polymers, and (4) is required for normal CNS developmental myelination in the optic nerve; it physically interacts with its paralog CC2D1A in the brain but occupies distinct synaptic compartments.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"CC2D1B (Freud-2/Lgd1) is a multifunctional scaffold protein that operates in both nuclear transcriptional repression and ESCRT-III-dependent membrane remodeling [#1, #0]. In the nucleus it binds the dual repressor element (DRE) of the serotonin-1A (5-HT1A) receptor gene promoter, at a site adjacent to that bound by its paralog CC2D1A/Freud-1, and represses 5-HT1A receptor transcription; depletion de-represses promoter activity and raises receptor protein levels in neuronal cells [#1, #2]. At cellular membranes CC2D1B coordinates ESCRT-III activity: during mitotic nuclear envelope reformation it regulates the spatiotemporal distribution of the CHMP7-ESCRT-III axis and couples ESCRT activity to Spastin-mediated spindle microtubule severing, and on endosomes it restrains premature sorting of transmembrane receptors EGFR and TLR4 into intraluminal vesicles, thereby sustaining downstream ERK1/2 signaling, acting alongside CC2D1A which binds CHMP4B polymers [#0, #3]. CC2D1B cycles between cytosol and endosomal membranes in a VPS4-sensitive manner and can functionally substitute for the Drosophila ortholog Lgd, indicating conserved ESCRT-III-related function [#4]. In the nervous system CC2D1B is required for normal CNS developmental myelination, with constitutive knockout mice showing selective hypomyelination of large-diameter optic nerve fibers [#6]. It physically interacts with CC2D1A in the brain while occupying distinct synaptic compartments [#7].\"\n,\n  \"teleology\": [\n    {\n      \"year\": 2009,\n      \"claim\": \"Established CC2D1B as a sequence-specific transcriptional repressor, answering whether the Freud-1 paralog also controls 5-HT1A receptor gene expression.\",\n      \"evidence\": \"EMSA, reporter assays, and siRNA knockdown with Western blot in human SK-N-SH cells\",\n      \"pmids\": [\"19423080\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define co-repressor partners or chromatin-modifying machinery recruited to the DRE\", \"Did not resolve how nuclear versus cytoplasmic functions are partitioned\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Confirmed conserved, DRE-dependent repression across species and distinguished CC2D1B-DRE complexes from CC2D1A-DRE complexes, clarifying that the two paralogs bind adjacent overlapping sites.\",\n      \"evidence\": \"EMSA/supershift, reporter assay, ChIP, and siRNA knockdown in neuronal cells (mouse/rat sequences)\",\n      \"pmids\": [\"21155902\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of adjacent CC2D1A/CC2D1B binding (cooperation vs redundancy) not resolved\", \"In vivo transcriptional role in serotonergic circuits not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Placed CC2D1B in the ESCRT-III pathway and tested its individual requirement for endosomal morphogenesis, showing it cycles to endosomes but is dispensable for endosome size unlike CC2D1A.\",\n      \"evidence\": \"Conditional knockout mice, EM of endosomes, VPS4 dominant-negative localization, and Drosophila Lgd rescue assay\",\n      \"pmids\": [\"26720614\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CC2D1B-specific endosomal phenotype was negative, leaving its non-redundant membrane role undefined\", \"Mechanism of cytosol-to-membrane cycling not characterized\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined a functional output for CC2D1A/B at endosomes — restraining premature receptor sorting to ILVs — linking the scaffold to receptor signaling duration.\",\n      \"evidence\": \"siRNA knockdown with EGFR/TLR4 degradation and ERK1/2 signaling assays plus CC2D1A-CHMP4B pulldown\",\n      \"pmids\": [\"27769858\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"CC2D1B contribution reported jointly with CC2D1A without full separation\", \"Direct CC2D1B-CHMP4B binding not demonstrated independently of CC2D1A\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a distinct membrane-remodeling role in mitosis, showing CC2D1B coordinates the CHMP7-ESCRT-III axis with Spastin-mediated microtubule severing during nuclear envelope reformation.\",\n      \"evidence\": \"siRNA depletion with live imaging, immunofluorescence, and readouts of nuclear compartmentalization and microtubule dynamics in human cells\",\n      \"pmids\": [\"30513301\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct biochemical interactions of CC2D1B with CHMP7 or Spastin not mapped\", \"How CC2D1B times ESCRT recruitment relative to ER deposition not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Connected CC2D1B to glial myelination, placing it downstream of YAP/TAZ in Schwann cells.\",\n      \"evidence\": \"YAP/TAZ ablation expression analysis and siRNA silencing with myelin segment quantification in vitro\",\n      \"pmids\": [\"31379499\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro only; in vivo PNS requirement not established here\", \"Molecular link between CC2D1B and myelin gene programs unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the in vivo myelination role, showing a selective CNS requirement (optic nerve large-fiber hypomyelination) rather than a general PNS or oligodendrocyte defect.\",\n      \"evidence\": \"Constitutive Cc2d1b knockout mouse with quantitative EM g-ratio analysis and immunohistology\",\n      \"pmids\": [\"35592111\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-autonomous mechanism in oligodendrocytes vs axons not determined\", \"Relationship between myelination role and ESCRT/transcriptional functions unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Tested the paralog relationship in brain, showing CC2D1A and CC2D1B physically interact yet occupy distinct synaptic compartments.\",\n      \"evidence\": \"Co-immunoprecipitation and synaptic fractionation in mouse hippocampus (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.06.26.661826\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, single lab; reciprocal validation and CC2D1B-specific synaptic function not established\", \"Functional consequence of heterotypic CC2D1A/B interaction unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown how CC2D1B's distinct activities — DRE-bound transcriptional repression, ESCRT-III coordination at the nuclear envelope and endosomes, and CNS myelination — are integrated within a single protein and partitioned across subcellular compartments.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural basis for substrate/partner selection across functions\", \"No direct CC2D1B-CHMP4B/CHMP7 binding map separate from CC2D1A\", \"No defined disease-causing CC2D1B mutation in the corpus\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"GO:0005768\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0005635\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [1, 2]},\n      {\"term_id\": \"R-HSA-5653656\", \"supporting_discovery_ids\": [3]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [6]}\n    ],\n    \"complexes\": [\"ESCRT-III\"],\n    \"partners\": [\"CC2D1A\", \"CHMP4B\", \"CHMP7\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}