{"gene":"HCFC2","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":1999,"finding":"HCF-2 was identified as a second human HCF-like protein sharing three regions of strong amino acid sequence homology with HCF-1, including the beta-propeller (kelch repeat) domain required for VP16 association. HCF-2 is expressed in many tissues (especially testis), can associate with VP16 and support complex assembly with Oct-1 and DNA, but is significantly less efficient than HCF-1 at doing so. Analysis of chimeric proteins showed that differences between the fifth and sixth kelch repeats of the beta-propeller domains of HCF-1 and HCF-2 dictate this selectivity. HCF-2 also shows a more dynamic pattern of subcellular localization than HCF-1.","method":"Sequence homology cloning, co-immunoprecipitation, gel-shift/complex assembly assay, chimeric protein mutagenesis, subcellular localization imaging","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1-2 — original identification with chimeric mutagenesis pinpointing the structural determinant of selectivity, plus multiple orthogonal methods in one study","pmids":["10196288"],"is_preprint":false},{"year":2000,"finding":"HCF-2, like HCF-1, contains a functional SAS1 (self-association sequence 1) element despite not being proteolytically processed, indicating that this association element does not function solely to maintain HCF-1 N- and C-terminal subunit association. The SAS1 module in HCF-1 consists of a short 43-amino-acid region on the N-terminal subunit that associates with a C-terminal region composed of tandem fibronectin type 3 (FnIII) repeats.","method":"Deletion/mutagenesis analysis, co-immunoprecipitation, domain mapping","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal domain mapping with mutagenesis in single study","pmids":["10958670"],"is_preprint":false},{"year":2001,"finding":"HCF-2 can promote VP16-induced complex formation (stabilizing the VP16-Oct-1-DNA complex), indicating that VP16 targets a conserved function shared by all HCF family members. However, unlike HCF-1, HCF-2 fails to support VP16 transcriptional activation effectively, demonstrating that stabilization of the VP16-induced complex and its transcriptional activity are separable functions within the HCF protein family.","method":"Co-immunoprecipitation, electrophoretic mobility shift assay (EMSA), transcriptional reporter assay","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal functional assays separating two distinct activities of HCF-2","pmids":["11711630"],"is_preprint":false},{"year":2002,"finding":"The highly conserved C-terminal WYF domain of HCF-1 interacts with the MYND domain of PDCD2, and this interaction is conserved between human HCF-1 and HCF-2 (as well as C. elegans HCF). PDCD2, which associates with the N-CoR/mSin3A corepressor complexes, acts as a negative regulator of HCF-1 complementation activity; overexpression of interfering domains of either PDCD2 or HCF-1 enhances HCF-1 function.","method":"Co-immunoprecipitation, complementation assay (tsBN67 temperature-sensitive cell line), overexpression/dominant-negative analysis","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2-3 — interaction and functional complementation assay; HCF-2 conservation shown by co-IP but detailed functional consequences for HCF-2 itself are inferred","pmids":["12149646"],"is_preprint":false},{"year":2004,"finding":"HCF-2 was identified as a component of the ~1-MDa MLL histone methyltransferase complex, alongside HCF-1. Both HCF-1 and HCF-2 specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL, suggesting a potential mechanism for regulating MLL's transcriptional properties.","method":"Biochemical purification (affinity chromatography/co-purification of endogenous complex), mass spectrometry identification, co-immunoprecipitation","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1-2 — biochemical purification of endogenous complex plus co-IP confirmation; foundational study with >500 citations","pmids":["15199122"],"is_preprint":false},{"year":2013,"finding":"Quantitative mass spectrometry of human SET1/MLL complexes confirmed HCF-2 as a component of these histone H3K4 methyltransferase complexes, with determined stoichiometry.","method":"Label-free quantitative mass spectrometry after affinity purification of tagged complex subunits","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 — quantitative proteomics with stoichiometry determination, single study","pmids":["23508102"],"is_preprint":false},{"year":2017,"finding":"HCFC2 is required for IRF1- and IRF2-dependent transcription of Tlr3 in mouse macrophages. Three independent ENU-induced mutations in Hcfc2 each abrogated macrophage responses to poly(I:C). HCFC2 promotes binding of IRF1 and IRF2 to the Tlr3 promoter; without HCFC2, inflammatory cytokine and type I IFN responses to dsRNA analogue are reduced. HCFC2 is also necessary for transcription of a large subset of other IRF2-dependent interferon-regulated genes. Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections in mice.","method":"ENU mutagenesis screen, chromatin immunoprecipitation (ChIP) for IRF1/IRF2 at Tlr3 promoter, cytokine/IFN response assays in macrophages, in vivo infection survival assays, genetic epistasis","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1-2 — three independent alleles, ChIP demonstrating direct promoter occupancy dependence, in vivo phenotype; strong mechanistic evidence from multiple orthogonal methods","pmids":["28970238"],"is_preprint":false},{"year":2015,"finding":"High-throughput AP-MS (BioPlex) identified interaction partners of HCFC2 in HEK293T cells, placing it in protein-interaction communities consistent with transcriptional regulation.","method":"Affinity purification mass spectrometry (AP-MS)","journal":"Cell","confidence":"Low","confidence_rationale":"Tier 3 — single AP-MS dataset, no functional validation specific to HCFC2","pmids":["26186194"],"is_preprint":false},{"year":2019,"finding":"A mass-spectrometry-based interactome survey of interferon-stimulated genes (ISGs) identified HCFC2 among the ISGs with characterized interaction partners, integrating it into the innate immune protein-interaction network.","method":"Affinity purification mass spectrometry of ISG-interaction network","journal":"Nature immunology","confidence":"Low","confidence_rationale":"Tier 3 — large-scale AP-MS survey; no HCFC2-specific functional follow-up reported","pmids":["30833792"],"is_preprint":false}],"current_model":"HCFC2 is a nuclear transcriptional coregulator that promotes IRF1/IRF2 binding to innate immune gene promoters (including Tlr3) to drive type I interferon and inflammatory cytokine responses to viral dsRNA, participates as a subunit of the MLL histone H3K4 methyltransferase complex via a conserved binding motif in MLL(N), and can support VP16-induced complex stabilization (but not transcriptional activation) through its conserved kelch/beta-propeller domain, with selectivity relative to HCF-1 determined by differences in the fifth and sixth kelch repeats."},"narrative":{"teleology":[{"year":1999,"claim":"Identification of HCFC2 as a second HCF family member established that the kelch-repeat beta-propeller domain is a conserved VP16-interaction module, and chimeric analysis pinpointed the fifth and sixth repeats as the determinant of reduced VP16-association efficiency relative to HCF-1.","evidence":"Sequence homology cloning, co-IP, gel-shift, chimeric mutagenesis, subcellular localization imaging in human cells","pmids":["10196288"],"confidence":"High","gaps":["Endogenous transcriptional targets of HCFC2 were unknown","Whether HCFC2 participates in chromatin-modifying complexes was untested"]},{"year":2000,"claim":"Demonstration that HCFC2 contains a functional SAS1 self-association element, despite not undergoing proteolytic processing, showed that this module serves a broader association function beyond simply linking HCF-1 subunits.","evidence":"Deletion/mutagenesis analysis and co-IP domain mapping","pmids":["10958670"],"confidence":"Medium","gaps":["The physiological binding partners recruited through the HCFC2 SAS1 element remain uncharacterized","No in vivo functional assay for HCFC2 SAS1 activity"]},{"year":2001,"claim":"Separation of VP16-induced complex stabilization from transcriptional activation revealed that HCFC2 supports complex assembly but lacks the ability to drive transcription, demonstrating that these are genetically separable HCF functions.","evidence":"EMSA, co-IP, and transcriptional reporter assays","pmids":["11711630"],"confidence":"High","gaps":["The structural basis for why HCFC2 fails to activate VP16-dependent transcription was not determined","Whether HCFC2 has independent transcriptional activation roles outside the VP16 context was unknown"]},{"year":2002,"claim":"Conservation of the WYF–PDCD2 interaction between HCF-1 and HCFC2 linked HCFC2 to corepressor complex regulation, suggesting a shared mechanism through which PDCD2 modulates HCF family activity.","evidence":"Co-IP and complementation assay in tsBN67 cells with dominant-negative constructs","pmids":["12149646"],"confidence":"Medium","gaps":["Functional consequences of PDCD2 interaction for HCFC2-specific targets were inferred from HCF-1 data rather than directly shown","No endogenous HCFC2-PDCD2 complex was isolated"]},{"year":2004,"claim":"Biochemical purification of the MLL histone methyltransferase complex identified HCFC2 as a bona fide subunit, establishing its role in H3K4 methylation-associated transcriptional regulation.","evidence":"Endogenous complex purification by affinity chromatography, mass spectrometry, and co-IP from human cells","pmids":["15199122"],"confidence":"High","gaps":["Whether HCFC2 is required for MLL complex enzymatic activity or target-gene selectivity was not tested","Relative contributions of HCFC2 versus HCF-1 within the complex were unclear"]},{"year":2013,"claim":"Quantitative stoichiometry determination of human SET1/MLL complexes confirmed HCFC2 as a substoichiometric component, refining the model of complex composition.","evidence":"Label-free quantitative mass spectrometry of affinity-purified tagged complexes","pmids":["23508102"],"confidence":"Medium","gaps":["Functional impact of HCFC2 stoichiometry on complex activity was not addressed","Whether HCFC2 and HCF-1 occupy the same or distinct MLL complexes was unresolved"]},{"year":2017,"claim":"Three independent ENU alleles demonstrated that HCFC2 is essential for IRF1/IRF2-dependent transcription of Tlr3 and a broad set of interferon-regulated genes, directly linking it to innate antiviral immunity and in vivo host defense.","evidence":"Forward genetic ENU screen in mice, ChIP for IRF1/IRF2 at Tlr3 promoter, cytokine/IFN assays in macrophages, survival studies during influenza and HSV-1 infection","pmids":["28970238"],"confidence":"High","gaps":["Whether HCFC2 recruits IRF1/IRF2 directly or via chromatin remodeling intermediates is unknown","The relationship between HCFC2's MLL complex membership and its IRF-dependent promoter function has not been tested","Human genetic validation of HCFC2 deficiency in immunodeficiency is lacking"]},{"year":null,"claim":"The mechanism by which HCFC2 facilitates IRF1/IRF2 binding — whether through direct protein–protein interaction, chromatin modification via MLL-mediated H3K4 methylation, or another route — remains unresolved, as does the structural basis for functional divergence from HCF-1.","evidence":"","pmids":[],"confidence":"High","gaps":["No crystal or cryo-EM structure of HCFC2 or its complexes","No separation-of-function alleles distinguishing MLL complex roles from IRF coregulatory roles","No human disease mutations reported"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,6]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[6,8]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[4,6]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[4,5]}],"complexes":["MLL/SET1 histone H3K4 methyltransferase complex"],"partners":["MLL","HCF1","PDCD2","IRF1","IRF2"],"other_free_text":[]},"mechanistic_narrative":"HCFC2 is a nuclear transcriptional coregulator that functions in both chromatin modification and innate immune gene regulation. It is a subunit of the MLL/SET1 histone H3K4 methyltransferase complex, where it interacts with a conserved binding motif in the MLL(N) subunit [PMID:15199122, PMID:23508102]. HCFC2 promotes IRF1 and IRF2 binding to innate immune gene promoters, including Tlr3, and is required for type I interferon and inflammatory cytokine responses to viral dsRNA; loss-of-function mutations in Hcfc2 compromise antiviral defense in vivo [PMID:28970238]. Its kelch/beta-propeller domain supports VP16-induced complex assembly but, unlike HCF-1, does not efficiently drive VP16-dependent transcriptional activation, with selectivity determined by differences in the fifth and sixth kelch repeats [PMID:10196288, PMID:11711630]."},"prefetch_data":{"uniprot":{"accession":"Q9Y5Z7","full_name":"Host cell factor 2","aliases":["C2 factor"],"length_aa":792,"mass_kda":86.8,"function":"","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q9Y5Z7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HCFC2","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HIST2H2BE","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HCFC2","total_profiled":1310},"omim":[{"mim_id":"607926","title":"HOST CELL FACTOR C2; HCFC2","url":"https://www.omim.org/entry/607926"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HCFC2"},"hgnc":{"alias_symbol":["HCF-2"],"prev_symbol":[]},"alphafold":{"accession":"Q9Y5Z7","domains":[{"cath_id":"2.120.10.80","chopping":"16-181","consensus_level":"medium","plddt":94.6505,"start":16,"end":181},{"cath_id":"2.120.10.80","chopping":"200-208_236-268_280-356","consensus_level":"medium","plddt":88.3434,"start":200,"end":356},{"cath_id":"2.60.40.10","chopping":"357-395_598-620_637-675","consensus_level":"medium","plddt":90.3171,"start":357,"end":675},{"cath_id":"2.60.40.10","chopping":"686-783","consensus_level":"high","plddt":84.6582,"start":686,"end":783}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y5Z7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y5Z7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9Y5Z7-F1-predicted_aligned_error_v6.png","plddt_mean":72.88},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HCFC2","jax_strain_url":"https://www.jax.org/strain/search?query=HCFC2"},"sequence":{"accession":"Q9Y5Z7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9Y5Z7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9Y5Z7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9Y5Z7"}},"corpus_meta":[{"pmid":"21392734","id":"PMC_21392734","title":"Genetic 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HCF-2 associates with VP16 and can support complex assembly with Oct-1 and DNA, but is significantly less efficient than HCF-1. Differences between the fifth and sixth kelch repeats of the beta-propeller domains from HCF-1 and HCF-2 dictate this selectivity. HCF-2 shows a more dynamic pattern of subcellular localization than HCF-1 and is expressed in many tissues, especially the testis.\",\n      \"method\": \"Sequence homology analysis, co-immunoprecipitation, gel shift/EMSA, chimeric protein analysis, subcellular localization by immunofluorescence\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple orthogonal methods (binding assays, chimeric mutants, localization) in the founding characterization paper\",\n      \"pmids\": [\"10196288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HCF-2 contains a functional SAS1 self-association element (a short 43-amino-acid region) analogous to that in HCF-1, which mediates association between N- and C-terminal subunits. Unlike HCF-1, HCF-2 is not proteolyzed, yet it retains this self-association motif, suggesting SAS1 does not function solely to maintain subunit association after cleavage.\",\n      \"method\": \"Deletion/chimeric protein analysis, co-immunoprecipitation, domain mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — domain mutagenesis and reciprocal interaction assays in a rigorous mechanistic study\",\n      \"pmids\": [\"10958670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HCF-2, like HCF-1, interacts with the MYND domain of PDCD2 through the conserved C-terminal WYF domain. Overexpression of PDCD2 suppresses HCF-1 complementation of a temperature-sensitive cell proliferation lesion, and overexpression of domains that interfere with this interaction enhances complementation, indicating PDCD2 is a negative regulator of HCF-1 (and by extension HCF-2 via the same conserved interaction).\",\n      \"method\": \"Co-immunoprecipitation, domain interaction mapping, functional complementation assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — co-IP plus functional complementation, but HCF-2-specific functional consequence not directly tested\",\n      \"pmids\": [\"12149646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HCF-2 can promote VP16-induced complex formation (i.e., stabilize the VP16/Oct-1/DNA complex), but unlike HCF-1 and CeHCF, HCF-2 fails to support VP16 transcriptional activation effectively, indicating that complex stabilization and transcriptional co-activation are separable functions within the HCF family.\",\n      \"method\": \"EMSA, transcriptional reporter assays, comparison of HCF family members\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — functional dissection using multiple orthogonal assays distinguishing stabilization from transcriptional activation\",\n      \"pmids\": [\"11711630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HCFC2 is required for IRF1- and IRF2-dependent transcription of Tlr3 in macrophages. Three independent ENU-induced loss-of-function mutations in Hcfc2 abrogated macrophage responses to poly(I:C). HCFC2 promotes the binding of IRF1 and IRF2 to the Tlr3 promoter; without HCFC2, inflammatory cytokine and type I IFN responses to double-stranded RNA are reduced. HCFC2 is also necessary for transcription of a large subset of other IRF2-dependent interferon-regulated genes. Loss of Hcfc2 compromised survival during influenza virus and herpes simplex virus 1 infections in mice.\",\n      \"method\": \"ENU mutagenesis screen, chromatin immunoprecipitation (ChIP), cytokine/IFN response assays, in vivo infection survival studies, genetic epistasis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — three independent alleles, ChIP demonstrating promoter binding, and in vivo functional consequence; strong evidence from multiple orthogonal approaches\",\n      \"pmids\": [\"28970238\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HCFC2 is a nuclear co-factor that, through its conserved kelch/beta-propeller domain, associates with VP16 to stabilize transcriptional complexes (but does not support VP16-driven transcriptional activation), and in innate immunity it acts as an essential cofactor enabling IRF1 and IRF2 to bind the Tlr3 promoter and drive expression of a broad subset of interferon-regulated genes, such that its loss abrogates macrophage poly(I:C) responses and compromises survival during viral infections.\"\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\nI'll classify each paper first, then extract discoveries only from KEPT papers.\n\n**Classification:**\n\nPapers about canonical HCFC2 protein:\n- PMID:28970238 (Sun et al., 2017) — KEEP: directly about mouse Hcfc2/HCFC2 protein function in innate immunity\n- PMID:10196288 (Johnson et al., 1999) — KEEP: describes identification of HCF-2 protein, its interaction with VP16, tissue expression, subcellular localization, and domain function\n- PMID:10958670 (Wilson et al., 2000) — KEEP: describes HCF-2 SAS1 association element and structural features\n- PMID:12149646 (Scarr & Sharp, 2002) — KEEP: describes PDCD2 interaction with HCF-1 C-terminal WYF domain, interaction conserved with HCF-2\n- PMID:11341844 (Wysocka et al., 2001) — KEEP: discusses HCF-1/HCF-2 family phosphorylation and VP16 association\n- PMID:11711630 (Lee & Herr, 2001) — KEEP: directly characterizes HCF-2 in VP16 complex formation and transcriptional activity\n- PMID:15199122 (Yokoyama et al., 2004) — KEEP: identifies HCF-2 as component of MLL complex\n- PMID:12494450 (Mahajan et al., 2003) — KEEP: discusses HCF-2 SAS1 domains in context of HCF family\n- PMID:14629117 (Izeta et al., 2003) — KEEP: describes CeHCF (closely related to HCF-2) subcellular localization; CeHCF is ortholog\n- PMID:26186194 (BioPlex, 2015) — KEEP: identifies HCFC2 protein interactions in human cells\n- PMID:28514442 (BioPlex 2.0, 2017) — KEEP: HCFC2 interactions\n- PMID:33961781 (BioPlex 3.0, 2021) — KEEP: HCFC2 interactions\n- PMID:21903422 (Li et al., 2011) — KEEP: innate immunity interactome including HCFC2\n- PMID:30833792 (Hubel et al., 2019) — KEEP: ISG interaction network, HCFC2 is ISG\n- PMID:35271311 (OpenCell, 2022) — KEEP: localization data for human proteins including HCFC2\n\nPapers about HCf-2 (Cladosporium fulvum hydrophobin) — SYMBOL COLLISION (fungal protein):\n- PMID:10394901, PMID:11343402, PMID:18958901 — EXCLUDE\n\nPapers about hCF-2 (human chromosome fragment) — SYMBOL COLLISION:\n- PMID:11196134 — EXCLUDE\n\nPapers about hcf-2 (maize nuclear mutant) — SYMBOL COLLISION (plant):\n- PMID:16663238 — EXCLUDE\n\nPapers about HCF2 chemistry (difluoromethyl radical/reagent) — SYMBOL COLLISION (chemical):\n- PMID:11456769, PMID:31184157, PMID:28981298, PMID:33532577, PMID:36867562, PMID:35166558, PMID:38115769, PMID:37183760, PMID:34618445, PMID:29870013 — EXCLUDE\n\nPapers about hCFC2/hCR-1 (Cripto-1, EGF-CFC protein) — ALIAS COLLISION (different gene, CFC2):\n- PMID:18930707 — EXCLUDE\n\nPapers with only expression/survival correlation (no mechanistic content about HCFC2):\n- PMID:21392734, PMID:36038535, PMID:35045690, PMID:33221751, PMID:35284334 — EXCLUDE (expression/prognostic only)\n\nGeneral interactome/cDNA papers (HCFC2 mentioned but no mechanistic details extracted beyond interaction lists captured in BioPlex entries):\n- PMID:12477932, PMID:15489334, PMID:16344560, PMID:8125298 — EXCLUDE (no specific HCFC2 mechanism)\n- PMID:21873635 — EXCLUDE\n- PMID:26496610 — KEEP for interaction data\n- PMID:29395067 — check... mentions mRNA biology proteins; HCFC2 not specifically highlighted — EXCLUDE\n- PMID:34079125 — KEEP for localization\n- PMID:25036637 — EXCLUDE (chaperone network, HCFC2 not specifically featured)\n- PMID:27705803 — EXCLUDE (PcG complexome)\n- PMID:23508102 — KEEP: MLL/SET1 complexes, HCF-2 identified as component\n- PMID:26886794 — EXCLUDE (MLL structure, HCF-2 not specifically featured)\n- PMID:30804502 — EXCLUDE (BRCA1-BARD1)\n- PMID:24981860 — EXCLUDE (demethylase complex)\n- PMID:20305087 — KEEP: MLL complex including HCF-2\n- PMID:22379092 — EXCLUDE (LANA interactors)\n- PMID:31804488 — EXCLUDE (MLL1 cryo-EM, HCF-2 not featured)\n- PMID:26841866 — EXCLUDE (EMSY complex)\n- PMID:34857952 — EXCLUDE (DUSP4/6)\n- PMID:32694731 — EXCLUDE (fatty acid synthesis)\n- PMID:35140242 — KEEP: TF interaction network\n- PMID:18029348 — KEEP: subcellular localization atlas\n- PMID:21491926 — EXCLUDE (solar cell chemistry)\n\n```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1999,\n      \"finding\": \"HCF-2 was identified as a second human HCF-like protein sharing three regions of strong amino acid sequence homology with HCF-1, including the beta-propeller (kelch repeat) domain required for VP16 association. HCF-2 is expressed in many tissues (especially testis), can associate with VP16 and support complex assembly with Oct-1 and DNA, but is significantly less efficient than HCF-1 at doing so. Analysis of chimeric proteins showed that differences between the fifth and sixth kelch repeats of the beta-propeller domains of HCF-1 and HCF-2 dictate this selectivity. HCF-2 also shows a more dynamic pattern of subcellular localization than HCF-1.\",\n      \"method\": \"Sequence homology cloning, co-immunoprecipitation, gel-shift/complex assembly assay, chimeric protein mutagenesis, subcellular localization imaging\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — original identification with chimeric mutagenesis pinpointing the structural determinant of selectivity, plus multiple orthogonal methods in one study\",\n      \"pmids\": [\"10196288\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"HCF-2, like HCF-1, contains a functional SAS1 (self-association sequence 1) element despite not being proteolytically processed, indicating that this association element does not function solely to maintain HCF-1 N- and C-terminal subunit association. The SAS1 module in HCF-1 consists of a short 43-amino-acid region on the N-terminal subunit that associates with a C-terminal region composed of tandem fibronectin type 3 (FnIII) repeats.\",\n      \"method\": \"Deletion/mutagenesis analysis, co-immunoprecipitation, domain mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal domain mapping with mutagenesis in single study\",\n      \"pmids\": [\"10958670\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"HCF-2 can promote VP16-induced complex formation (stabilizing the VP16-Oct-1-DNA complex), indicating that VP16 targets a conserved function shared by all HCF family members. However, unlike HCF-1, HCF-2 fails to support VP16 transcriptional activation effectively, demonstrating that stabilization of the VP16-induced complex and its transcriptional activity are separable functions within the HCF protein family.\",\n      \"method\": \"Co-immunoprecipitation, electrophoretic mobility shift assay (EMSA), transcriptional reporter assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal functional assays separating two distinct activities of HCF-2\",\n      \"pmids\": [\"11711630\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"The highly conserved C-terminal WYF domain of HCF-1 interacts with the MYND domain of PDCD2, and this interaction is conserved between human HCF-1 and HCF-2 (as well as C. elegans HCF). PDCD2, which associates with the N-CoR/mSin3A corepressor complexes, acts as a negative regulator of HCF-1 complementation activity; overexpression of interfering domains of either PDCD2 or HCF-1 enhances HCF-1 function.\",\n      \"method\": \"Co-immunoprecipitation, complementation assay (tsBN67 temperature-sensitive cell line), overexpression/dominant-negative analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — interaction and functional complementation assay; HCF-2 conservation shown by co-IP but detailed functional consequences for HCF-2 itself are inferred\",\n      \"pmids\": [\"12149646\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"HCF-2 was identified as a component of the ~1-MDa MLL histone methyltransferase complex, alongside HCF-1. Both HCF-1 and HCF-2 specifically interact with a conserved binding motif in the MLL(N) (p300) subunit of MLL, suggesting a potential mechanism for regulating MLL's transcriptional properties.\",\n      \"method\": \"Biochemical purification (affinity chromatography/co-purification of endogenous complex), mass spectrometry identification, co-immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — biochemical purification of endogenous complex plus co-IP confirmation; foundational study with >500 citations\",\n      \"pmids\": [\"15199122\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Quantitative mass spectrometry of human SET1/MLL complexes confirmed HCF-2 as a component of these histone H3K4 methyltransferase complexes, with determined stoichiometry.\",\n      \"method\": \"Label-free quantitative mass spectrometry after affinity purification of tagged complex subunits\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — quantitative proteomics with stoichiometry determination, single study\",\n      \"pmids\": [\"23508102\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HCFC2 is required for IRF1- and IRF2-dependent transcription of Tlr3 in mouse macrophages. Three independent ENU-induced mutations in Hcfc2 each abrogated macrophage responses to poly(I:C). HCFC2 promotes binding of IRF1 and IRF2 to the Tlr3 promoter; without HCFC2, inflammatory cytokine and type I IFN responses to dsRNA analogue are reduced. HCFC2 is also necessary for transcription of a large subset of other IRF2-dependent interferon-regulated genes. Hcfc2 mutations compromised survival during influenza virus and herpes simplex virus 1 infections in mice.\",\n      \"method\": \"ENU mutagenesis screen, chromatin immunoprecipitation (ChIP) for IRF1/IRF2 at Tlr3 promoter, cytokine/IFN response assays in macrophages, in vivo infection survival assays, genetic epistasis\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — three independent alleles, ChIP demonstrating direct promoter occupancy dependence, in vivo phenotype; strong mechanistic evidence from multiple orthogonal methods\",\n      \"pmids\": [\"28970238\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"High-throughput AP-MS (BioPlex) identified interaction partners of HCFC2 in HEK293T cells, placing it in protein-interaction communities consistent with transcriptional regulation.\",\n      \"method\": \"Affinity purification mass spectrometry (AP-MS)\",\n      \"journal\": \"Cell\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single AP-MS dataset, no functional validation specific to HCFC2\",\n      \"pmids\": [\"26186194\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"A mass-spectrometry-based interactome survey of interferon-stimulated genes (ISGs) identified HCFC2 among the ISGs with characterized interaction partners, integrating it into the innate immune protein-interaction network.\",\n      \"method\": \"Affinity purification mass spectrometry of ISG-interaction network\",\n      \"journal\": \"Nature immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — large-scale AP-MS survey; no HCFC2-specific functional follow-up reported\",\n      \"pmids\": [\"30833792\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HCFC2 is a nuclear transcriptional coregulator that promotes IRF1/IRF2 binding to innate immune gene promoters (including Tlr3) to drive type I interferon and inflammatory cytokine responses to viral dsRNA, participates as a subunit of the MLL histone H3K4 methyltransferase complex via a conserved binding motif in MLL(N), and can support VP16-induced complex stabilization (but not transcriptional activation) through its conserved kelch/beta-propeller domain, with selectivity relative to HCF-1 determined by differences in the fifth and sixth kelch repeats.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HCFC2 is a nuclear transcriptional cofactor of the host cell factor family that functions both in herpesvirus-related transcriptional complex assembly and in innate immune gene regulation. HCFC2 contains a conserved kelch-repeat (beta-propeller) domain that mediates association with VP16 and supports VP16/Oct-1/DNA complex formation, but unlike HCF-1, HCFC2 fails to support VP16-driven transcriptional activation, demonstrating that complex stabilization and co-activation are separable functions [PMID:10196288, PMID:11711630]. HCFC2 also contains a functional SAS1 self-association element and a C-terminal WYF domain that interacts with the MYND domain of PDCD2, a negative regulator of HCF family function [PMID:10958670, PMID:12149646]. In macrophages, HCFC2 is essential for IRF1- and IRF2-dependent transcription of Tlr3 and a broad subset of interferon-regulated genes; three independent loss-of-function alleles abolish poly(I:C)-induced cytokine and type I interferon responses and compromise survival during influenza and herpes simplex virus infections in mice [PMID:28970238].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of HCFC2 as a second HCF family member established that the kelch-repeat beta-propeller domain mediates VP16 association and complex assembly with Oct-1, but with reduced efficiency compared to HCF-1, attributable to divergence in kelch repeats 5–6.\",\n      \"evidence\": \"Sequence homology, co-immunoprecipitation, EMSA, chimeric protein analysis, and immunofluorescence in mammalian cells\",\n      \"pmids\": [\"10196288\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Structural basis for the reduced efficiency of HCFC2 in VP16 complex assembly not resolved at atomic level\",\n        \"Endogenous physiological role of HCFC2 beyond VP16-related complex formation unknown\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that HCFC2 retains a functional SAS1 self-association element despite not undergoing proteolytic cleavage revealed that this motif has an HCF-family-wide function beyond post-cleavage subunit maintenance.\",\n      \"evidence\": \"Deletion and chimeric protein analysis with co-immunoprecipitation\",\n      \"pmids\": [\"10958670\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Functional role of SAS1-mediated self-association in intact HCFC2 not determined\",\n        \"No structural model of HCFC2 SAS1 element\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Separating complex stabilization from transcriptional activation showed that HCFC2 can promote VP16-induced complex formation but cannot drive VP16-dependent transcription, defining a functional split within the HCF family and raising questions about what additional domains or cofactors enable co-activation.\",\n      \"evidence\": \"EMSA and transcriptional reporter assays comparing HCF-1, HCF-2, and CeHCF\",\n      \"pmids\": [\"11711630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Domains or cofactors responsible for HCF-1's transcriptional activation that HCFC2 lacks remain unidentified\",\n        \"Whether HCFC2 co-activates any endogenous mammalian promoter was unknown\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Identification of the PDCD2 MYND domain as a direct interactor of the HCFC2 WYF domain suggested a conserved negative regulatory axis, though the functional consequence was demonstrated only for HCF-1.\",\n      \"evidence\": \"Co-immunoprecipitation, domain interaction mapping, and HCF-1 complementation assay\",\n      \"pmids\": [\"12149646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"HCFC2-specific functional impact of PDCD2 interaction not directly tested\",\n        \"In vivo relevance of PDCD2–HCFC2 interaction uncharacterized\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Three independent ENU alleles established HCFC2 as an essential cofactor for IRF1/IRF2-dependent transcription of Tlr3 and a broad set of interferon-regulated genes, linking HCFC2 to innate antiviral immunity and in vivo host defense.\",\n      \"evidence\": \"Forward genetic ENU screen, ChIP showing HCFC2-dependent IRF1/IRF2 binding to the Tlr3 promoter, cytokine assays, and survival studies during influenza and HSV-1 infection in mice\",\n      \"pmids\": [\"28970238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Mechanism by which HCFC2 promotes IRF1/IRF2 binding to DNA is unknown (direct bridging vs. chromatin remodeling)\",\n        \"Whether HCFC2 functions similarly in human macrophages has not been demonstrated\",\n        \"Relationship between HCFC2's VP16-complex role and its innate-immune role unclear\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The molecular mechanism by which HCFC2 enables IRF1/IRF2 promoter occupancy — whether through direct protein–protein contact, chromatin accessibility changes, or recruitment of additional cofactors — remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No structural model of HCFC2 in any complex\",\n        \"No genome-wide binding profile (ChIP-seq) for HCFC2\",\n        \"Whether HCFC2 has transcription-factor-independent chromatin functions is untested\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [4]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [3, 4]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"VP16\",\n      \"IRF1\",\n      \"IRF2\",\n      \"OCT1\",\n      \"PDCD2\",\n      \"HCFC1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\n--- ROUND 2 (R2/R3, augmented corpus) ---\n\n```json\n{\n  \"mechanistic_narrative\": \"HCFC2 is a nuclear transcriptional coregulator that functions in both chromatin modification and innate immune gene regulation. It is a subunit of the MLL/SET1 histone H3K4 methyltransferase complex, where it interacts with a conserved binding motif in the MLL(N) subunit [PMID:15199122, PMID:23508102]. HCFC2 promotes IRF1 and IRF2 binding to innate immune gene promoters, including Tlr3, and is required for type I interferon and inflammatory cytokine responses to viral dsRNA; loss-of-function mutations in Hcfc2 compromise antiviral defense in vivo [PMID:28970238]. Its kelch/beta-propeller domain supports VP16-induced complex assembly but, unlike HCF-1, does not efficiently drive VP16-dependent transcriptional activation, with selectivity determined by differences in the fifth and sixth kelch repeats [PMID:10196288, PMID:11711630].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Identification of HCFC2 as a second HCF family member established that the kelch-repeat beta-propeller domain is a conserved VP16-interaction module, and chimeric analysis pinpointed the fifth and sixth repeats as the determinant of reduced VP16-association efficiency relative to HCF-1.\",\n      \"evidence\": \"Sequence homology cloning, co-IP, gel-shift, chimeric mutagenesis, subcellular localization imaging in human cells\",\n      \"pmids\": [\"10196288\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Endogenous transcriptional targets of HCFC2 were unknown\",\n        \"Whether HCFC2 participates in chromatin-modifying complexes was untested\"\n      ]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstration that HCFC2 contains a functional SAS1 self-association element, despite not undergoing proteolytic processing, showed that this module serves a broader association function beyond simply linking HCF-1 subunits.\",\n      \"evidence\": \"Deletion/mutagenesis analysis and co-IP domain mapping\",\n      \"pmids\": [\"10958670\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"The physiological binding partners recruited through the HCFC2 SAS1 element remain uncharacterized\",\n        \"No in vivo functional assay for HCFC2 SAS1 activity\"\n      ]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Separation of VP16-induced complex stabilization from transcriptional activation revealed that HCFC2 supports complex assembly but lacks the ability to drive transcription, demonstrating that these are genetically separable HCF functions.\",\n      \"evidence\": \"EMSA, co-IP, and transcriptional reporter assays\",\n      \"pmids\": [\"11711630\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"The structural basis for why HCFC2 fails to activate VP16-dependent transcription was not determined\",\n        \"Whether HCFC2 has independent transcriptional activation roles outside the VP16 context was unknown\"\n      ]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Conservation of the WYF–PDCD2 interaction between HCF-1 and HCFC2 linked HCFC2 to corepressor complex regulation, suggesting a shared mechanism through which PDCD2 modulates HCF family activity.\",\n      \"evidence\": \"Co-IP and complementation assay in tsBN67 cells with dominant-negative constructs\",\n      \"pmids\": [\"12149646\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional consequences of PDCD2 interaction for HCFC2-specific targets were inferred from HCF-1 data rather than directly shown\",\n        \"No endogenous HCFC2-PDCD2 complex was isolated\"\n      ]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Biochemical purification of the MLL histone methyltransferase complex identified HCFC2 as a bona fide subunit, establishing its role in H3K4 methylation-associated transcriptional regulation.\",\n      \"evidence\": \"Endogenous complex purification by affinity chromatography, mass spectrometry, and co-IP from human cells\",\n      \"pmids\": [\"15199122\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether HCFC2 is required for MLL complex enzymatic activity or target-gene selectivity was not tested\",\n        \"Relative contributions of HCFC2 versus HCF-1 within the complex were unclear\"\n      ]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Quantitative stoichiometry determination of human SET1/MLL complexes confirmed HCFC2 as a substoichiometric component, refining the model of complex composition.\",\n      \"evidence\": \"Label-free quantitative mass spectrometry of affinity-purified tagged complexes\",\n      \"pmids\": [\"23508102\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\n        \"Functional impact of HCFC2 stoichiometry on complex activity was not addressed\",\n        \"Whether HCFC2 and HCF-1 occupy the same or distinct MLL complexes was unresolved\"\n      ]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Three independent ENU alleles demonstrated that HCFC2 is essential for IRF1/IRF2-dependent transcription of Tlr3 and a broad set of interferon-regulated genes, directly linking it to innate antiviral immunity and in vivo host defense.\",\n      \"evidence\": \"Forward genetic ENU screen in mice, ChIP for IRF1/IRF2 at Tlr3 promoter, cytokine/IFN assays in macrophages, survival studies during influenza and HSV-1 infection\",\n      \"pmids\": [\"28970238\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"Whether HCFC2 recruits IRF1/IRF2 directly or via chromatin remodeling intermediates is unknown\",\n        \"The relationship between HCFC2's MLL complex membership and its IRF-dependent promoter function has not been tested\",\n        \"Human genetic validation of HCFC2 deficiency in immunodeficiency is lacking\"\n      ]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The mechanism by which HCFC2 facilitates IRF1/IRF2 binding — whether through direct protein–protein interaction, chromatin modification via MLL-mediated H3K4 methylation, or another route — remains unresolved, as does the structural basis for functional divergence from HCF-1.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\n        \"No crystal or cryo-EM structure of HCFC2 or its complexes\",\n        \"No separation-of-function alleles distinguishing MLL complex roles from IRF coregulatory roles\",\n        \"No human disease mutations reported\"\n      ]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 6]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [6, 8]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [4, 6]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [4, 5]}\n    ],\n    \"complexes\": [\n      \"MLL/SET1 histone H3K4 methyltransferase complex\"\n    ],\n    \"partners\": [\n      \"MLL\",\n      \"HCF1\",\n      \"PDCD2\",\n      \"IRF1\",\n      \"IRF2\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}