{"gene":"BEND3","run_date":"2026-06-09T22:02:44","timeline":{"discoveries":[{"year":2014,"finding":"BEND3 is enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, and facilitates Polycomb recruitment and H3K27Me3 deposition at pericentromeric regions, providing a redundant pathway to generate repressive chromatin and mediating a switch from constitutive to facultative heterochromatin.","method":"Quantitative locus purification of pericentromeric chromatin-associated proteins in DNA methyltransferase-deficient and H3K9Me3 methyltransferase-deficient mouse embryonic stem cells","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct chromatin purification from genetic loss-of-function ESCs, replicated in multiple methylation-deficient contexts, mechanistic pathway placement established","pmids":["25457167"],"is_preprint":false},{"year":2022,"finding":"BEND3 is a CpG island (CGI)-binding protein that binds regulatory elements in a DNA methylation-sensitive manner; it is required for stable association of PRC2 at bivalent genes to maintain H3K27me3 and prevent premature activation of these genes during differentiation. Bend3 null mouse embryos die at pregastrulation, and null ESCs show premature activation of hundreds of CGI-containing bivalent genes. The co-crystal structure of BEND3 with its target DNA reveals the structural basis for methylation-sensitive binding.","method":"Co-crystal structure of BEND3-DNA complex; Bend3 knockout mouse embryos and ESCs; ChIP-seq for H3K27me3 and PRC2; RNA-seq in null ESCs during differentiation","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure plus genetic KO with defined molecular and developmental phenotypes, multiple orthogonal methods in a single rigorous study","pmids":["35143257"],"is_preprint":false},{"year":2015,"finding":"BEND3 localizes to nucleoli and binds ribosomal DNA (rDNA) promoters to repress rRNA gene transcription. Loss of BEND3 increases H3K4me3 and decreases rDNA promoter DNA methylation. BEND3 associates with the nucleolar remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TIP5 (TTF-1-interacting protein 5) via interaction with the deubiquitinase USP21.","method":"ChIP at rDNA promoters; co-immunoprecipitation of BEND3 with NoRC and USP21; BEND3 knockdown with measurement of H3K4me3 and DNA methylation at rDNA; immunofluorescence localization to nucleoli","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP for complex identification, ChIP with epigenetic readouts, subcellular localization with functional consequence, multiple orthogonal methods in single study","pmids":["26100909"],"is_preprint":false},{"year":2022,"finding":"BEND3 binds promoters of differentiation-associated genes (including CDKN1A/p21) and represses them by enhancing H3K27me3 decoration at these promoters in pluripotent cells. Removal of BEND3 from pluripotent cells causes upregulation of differentiation-inducing gene expression.","method":"ChIP-seq for BEND3 and H3K27me3; RNA-seq in BEND3-depleted pluripotent cells; reporter and knockdown assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Moderate — direct ChIP-seq evidence of promoter binding, H3K27me3 changes upon KD, RNA-seq, multiple orthogonal methods","pmids":["35217604"],"is_preprint":false},{"year":2017,"finding":"BEND3 interacts with the DNA translocase PICH in mitosis. The interaction occurs via a novel TPR domain in PICH and the first BEN domain (BD1) of BEND3. The crystal structure of the NTPR-BD1 complex was solved at 2.2 Å resolution, and structure-guided mutagenesis identified residues critical for the interaction and for the functional interaction of full-length proteins.","method":"Co-immunoprecipitation in human cells; in vitro biochemical interaction with purified full-length proteins; crystal structure of TPR-BEN domain complex at 2.2 Å; site-directed mutagenesis of interface residues","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with in vitro reconstitution of interaction, mutagenesis validation, and cellular Co-IP; multiple orthogonal methods","pmids":["28977671"],"is_preprint":false},{"year":2016,"finding":"The NTPR domain of PICH and BD1 domain of BEND3 form a heterodimeric complex that crystallizes in space group P6122 with unit-cell parameters a=b=47.28, c=431.58 Å, providing atomic-level structural data for the PICH-BEND3 interaction interface.","method":"Protein crystallography (MAD at 2.2 Å resolution) of purified recombinant NTPR and BD1 domain complex","journal":"Acta crystallographica. Section F, Structural biology communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination, corroborates PMID 28977671 findings with structural data","pmids":["27487930"],"is_preprint":false},{"year":2015,"finding":"Three basic amino acid residues in the N-terminal region of BEND3 (Lys56-Arg57-Lys58, KRK) are essential for its nuclear localization, functioning as a nuclear localization signal (NLS).","method":"Immunofluorescence assay of deletion and site-directed mutants of human BEND3 expressed in cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — direct localization experiment with mutagenesis in a single study, but single lab with single method","pmids":["25600804"],"is_preprint":false},{"year":2015,"finding":"BEND3 binds to the human-specific C-allele (rs138452745) at position -220 of the CALR gene promoter as shown by DNA pulldown assay, whereas NFI family members (NF1A, B, C, X) bind the ancestral A-allele, consistent with BEND3 acting as a transcriptional repressor at this locus.","method":"DNA pulldown assay from SK-N-BE and HeLa cell nuclear extracts using biotinylated C- and A-allele oligonucleotides, followed by protein identification","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single pulldown method, two cell lines, no functional reporter in same paper; single lab","pmids":["26481236"],"is_preprint":false},{"year":2025,"finding":"Preprint data indicate that PRC1 and PRC2 recruitment to hypomethylated pericentromeric heterochromatin in mESCs does not depend on BEND3 (nor on SCML2, KDM2b, or TET enzymes), suggesting that the permissive chromatin environment from DNA hypomethylation is the principal facilitator of Polycomb spreading independently of BEND3.","method":"Doxycycline-inducible Dnmt1 modulation in mESCs; ChIP-seq for H2AK119ub1 and H3K27me3 at PCH in BEND3-depleted cells","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint, single lab, negative result (BEND3 not required) contradicting prior models; requires peer review validation","pmids":["bio_10.1101_2025.11.14.688451"],"is_preprint":true}],"current_model":"BEND3 is a quadruple BEN domain-containing nuclear protein that functions as a transcriptional repressor by binding CpG island-containing regulatory elements in a DNA methylation-sensitive manner; it stabilizes PRC2 association at bivalent genes to maintain H3K27me3 and prevent premature gene activation, recruits Polycomb to pericentromeric chromatin in the absence of DNA methylation or H3K9me3, represses rDNA transcription by associating with the NoRC complex (stabilizing TIP5 via the deubiquitinase USP21 through a SUMOylation-dependent mechanism), and interacts with the mitotic DNA translocase PICH via a structurally defined novel TPR–BEN domain interface, while its nuclear localization is mediated by an N-terminal KRK motif."},"narrative":{"mechanistic_narrative":"BEND3 is a methylation-sensitive, DNA-binding nuclear protein that links chromatin recognition to Polycomb-mediated transcriptional repression across multiple genomic compartments [PMID:35143257, PMID:35217604]. It binds CpG island regulatory elements in a DNA methylation-sensitive manner—a specificity defined at atomic resolution by a co-crystal structure with its target DNA—and is required for stable PRC2 association at bivalent genes, maintaining H3K27me3 and preventing premature activation of developmental genes; Bend3 loss causes pregastrulation lethality in mouse embryos and derepression of hundreds of bivalent CGI genes [PMID:35143257]. In pluripotent cells, BEND3 occupies and represses differentiation-associated promoters including CDKN1A/p21 by enhancing H3K27me3 deposition [PMID:35217604], and at pericentromeric chromatin lacking DNA methylation or H3K9me3 it provides a redundant route to repressive chromatin by facilitating Polycomb recruitment and H3K27me3, driving a switch from constitutive to facultative heterochromatin [PMID:25457167]. BEND3 also localizes to nucleoli and represses rDNA transcription by binding rDNA promoters and associating with the NoRC complex, where SUMOylated BEND3 stabilizes the NoRC subunit TIP5 via the deubiquitinase USP21 [PMID:26100909]. Independently of its repressive roles, BEND3 engages the mitotic DNA translocase PICH through a structurally defined interface between PICH's TPR domain and the first BEN domain of BEND3 [PMID:28977671, PMID:27487930]. Nuclear localization is mediated by an N-terminal KRK motif [PMID:25600804].","teleology":[{"year":2014,"claim":"Established that BEND3 provides a redundant, DNA-methylation-independent pathway to generate repressive chromatin, answering how heterochromatin is maintained when canonical methylation marks are absent.","evidence":"Quantitative locus purification of pericentromeric chromatin from Dnmt- and Suv39-deficient mouse ESCs","pmids":["25457167"],"confidence":"High","gaps":["Mechanism by which BEND3 recruits Polycomb at pericentromeres not resolved at the molecular level","Direct versus indirect chromatin recruitment not distinguished"]},{"year":2015,"claim":"Defined BEND3 as a nucleolar repressor of rRNA genes and connected it to NoRC, showing how it stabilizes the complex through a deubiquitinase- and SUMO-dependent route.","evidence":"ChIP at rDNA promoters, reciprocal Co-IP of BEND3 with NoRC and USP21, knockdown with epigenetic readouts, immunofluorescence","pmids":["26100909"],"confidence":"High","gaps":["Direct enzymatic role of BEND3 in SUMOylation/deubiquitination not established","Whether rDNA binding is direct sequence recognition unclear"]},{"year":2015,"claim":"Mapped the determinant of BEND3 nuclear import, identifying a KRK basic motif as the NLS.","evidence":"Immunofluorescence of deletion and point mutants of human BEND3","pmids":["25600804"],"confidence":"Medium","gaps":["Single lab, single method","Import receptor mediating recognition not identified"]},{"year":2015,"claim":"Provided an allele-specific binding example consistent with BEND3 acting as a repressor at a regulatory polymorphism.","evidence":"DNA pulldown of BEND3 onto the CALR promoter C-allele from cell nuclear extracts","pmids":["26481236"],"confidence":"Medium","gaps":["No functional reporter in same study","Single pulldown method, no in vivo validation"]},{"year":2016,"claim":"Delivered atomic-level structural data for the PICH NTPR–BEND3 BD1 interface, establishing the physical basis of a mitotic interaction.","evidence":"MAD crystallography of purified recombinant NTPR–BD1 complex at 2.2 Å","pmids":["27487930"],"confidence":"High","gaps":["Structure alone does not define the cellular function of the interaction"]},{"year":2017,"claim":"Validated the BEND3–PICH interaction functionally, mapping it to a novel TPR–BEN interface and confirming interface residues by mutagenesis.","evidence":"Cellular Co-IP, in vitro reconstitution with full-length proteins, crystal structure, structure-guided mutagenesis","pmids":["28977671"],"confidence":"High","gaps":["Downstream mitotic consequence of the BEND3–PICH interaction not fully defined","Whether this role is separable from BEND3's repressive functions unclear"]},{"year":2022,"claim":"Unified BEND3's repressive role at the genome scale by showing it is a methylation-sensitive CGI binder required for PRC2 stability at bivalent genes, with structural and developmental consequences defined.","evidence":"Co-crystal of BEND3-DNA, Bend3 KO embryos and ESCs, ChIP-seq for PRC2/H3K27me3, RNA-seq during differentiation","pmids":["35143257"],"confidence":"High","gaps":["How BEND3 mechanistically stabilizes PRC2 at chromatin not detailed","Relationship between CGI binding and pericentromeric recruitment not integrated"]},{"year":2022,"claim":"Showed BEND3 restrains differentiation in pluripotent cells by repressing differentiation-inducing genes including CDKN1A via H3K27me3.","evidence":"ChIP-seq for BEND3 and H3K27me3, RNA-seq in BEND3-depleted pluripotent cells, reporter/knockdown assays","pmids":["35217604"],"confidence":"High","gaps":["Direct versus indirect repression of individual targets not fully resolved"]},{"year":2025,"claim":"Challenged the requirement of BEND3 for Polycomb spreading at hypomethylated pericentromeres, attributing recruitment instead to the permissive chromatin environment.","evidence":"Inducible Dnmt1 modulation and ChIP-seq for H2AK119ub1/H3K27me3 in BEND3-depleted mESCs (preprint)","pmids":["bio_10.1101_2025.11.14.688451"],"confidence":"Low","gaps":["Preprint, not peer-reviewed; negative result contradicting prior model","Single lab","Does not address BEND3's CGI/bivalent gene roles"]},{"year":null,"claim":"How BEND3 physically bridges DNA recognition to PRC2 stabilization, and whether its pericentromeric, CGI, nucleolar, and mitotic functions share a common molecular mechanism, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No defined molecular mechanism for PRC2 recruitment/stabilization by BEND3","Conflicting evidence on pericentromeric requirement","Integration of distinct genomic roles into one model lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[1,2,3,7]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[1,2,3]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[2]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,1,3]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,3]}],"complexes":["NoRC"],"partners":["TIP5","USP21","PICH","PRC2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5T5X7","full_name":"BEN domain-containing protein 3","aliases":[],"length_aa":828,"mass_kda":94.5,"function":"Transcriptional repressor which associates with the NoRC (nucleolar remodeling complex) complex and plays a key role in repressing rDNA transcription. The sumoylated form modulates the stability of the NoRC complex component BAZ2A/TIP5 by controlling its USP21-mediated deubiquitination (PubMed:21914818, PubMed:26100909). Binds to unmethylated major satellite DNA and is involved in the recruitment of the Polycomb repressive complex 2 (PRC2) to major satellites (By similarity). Stimulates the ERCC6L translocase and ATPase activities (PubMed:28977671)","subcellular_location":"Nucleus; Nucleus, nucleolus","url":"https://www.uniprot.org/uniprotkb/Q5T5X7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/BEND3","classification":"Not Classified","n_dependent_lines":57,"n_total_lines":1208,"dependency_fraction":0.04718543046357616},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"HDAC1","stoichiometry":0.2},{"gene":"HDAC2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/BEND3","total_profiled":1310},"omim":[{"mim_id":"621197","title":"BEN DOMAIN-CONTAINING PROTEIN 4; BEND4","url":"https://www.omim.org/entry/621197"},{"mim_id":"616374","title":"BEN DOMAIN-CONTAINING PROTEIN 3; BEND3","url":"https://www.omim.org/entry/616374"},{"mim_id":"300687","title":"ERCC EXCISION REPAIR 6-LIKE, SPINDLE ASSEMBLY CHECKPOINT HELICASE; ERCC6L","url":"https://www.omim.org/entry/300687"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in some","driving_tissues":[],"url":"https://www.proteinatlas.org/search/BEND3"},"hgnc":{"alias_symbol":[],"prev_symbol":["KIAA1553"]},"alphafold":{"accession":"Q5T5X7","domains":[{"cath_id":"-","chopping":"251-281_292-345","consensus_level":"high","plddt":88.0896,"start":251,"end":345},{"cath_id":"-","chopping":"395-482","consensus_level":"high","plddt":86.7764,"start":395,"end":482},{"cath_id":"-","chopping":"537-646","consensus_level":"high","plddt":89.2062,"start":537,"end":646},{"cath_id":"-","chopping":"704-828","consensus_level":"high","plddt":85.5907,"start":704,"end":828}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T5X7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T5X7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5T5X7-F1-predicted_aligned_error_v6.png","plddt_mean":64.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=BEND3","jax_strain_url":"https://www.jax.org/strain/search?query=BEND3"},"sequence":{"accession":"Q5T5X7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5T5X7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5T5X7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5T5X7"}},"corpus_meta":[{"pmid":"25457167","id":"PMC_25457167","title":"Redundant mechanisms to form silent chromatin at pericentromeric regions rely on BEND3 and DNA methylation.","date":"2014","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/25457167","citation_count":179,"is_preprint":false},{"pmid":"25056869","id":"PMC_25056869","title":"Hydrogen Sulfide Epigenetically Attenuates Homocysteine-Induced Mitochondrial Toxicity Mediated Through NMDA Receptor in Mouse Brain Endothelial (bEnd3) Cells.","date":"2015","source":"Journal of cellular physiology","url":"https://pubmed.ncbi.nlm.nih.gov/25056869","citation_count":78,"is_preprint":false},{"pmid":"35143257","id":"PMC_35143257","title":"Highly enriched BEND3 prevents the premature activation of bivalent genes during differentiation.","date":"2022","source":"Science (New York, N.Y.)","url":"https://pubmed.ncbi.nlm.nih.gov/35143257","citation_count":65,"is_preprint":false},{"pmid":"29059256","id":"PMC_29059256","title":"Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier.","date":"2017","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29059256","citation_count":61,"is_preprint":false},{"pmid":"26100909","id":"PMC_26100909","title":"BEND3 represses rDNA transcription by stabilizing a NoRC component via USP21 deubiquitinase.","date":"2015","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/26100909","citation_count":37,"is_preprint":false},{"pmid":"35358795","id":"PMC_35358795","title":"Treponema pallidum Tp0751 alters the expression of tight junction proteins by promoting bEnd3 cell apoptosis and IL-6 secretion.","date":"2022","source":"International journal of medical microbiology : IJMM","url":"https://pubmed.ncbi.nlm.nih.gov/35358795","citation_count":22,"is_preprint":false},{"pmid":"35217604","id":"PMC_35217604","title":"BEND3 safeguards pluripotency by repressing differentiation-associated genes.","date":"2022","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/35217604","citation_count":20,"is_preprint":false},{"pmid":"29683403","id":"PMC_29683403","title":"Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells.","date":"2018","source":"Neurological research","url":"https://pubmed.ncbi.nlm.nih.gov/29683403","citation_count":19,"is_preprint":false},{"pmid":"26507581","id":"PMC_26507581","title":"BEND3 mediates transcriptional repression and heterochromatin organization.","date":"2015","source":"Transcription","url":"https://pubmed.ncbi.nlm.nih.gov/26507581","citation_count":17,"is_preprint":false},{"pmid":"30867725","id":"PMC_30867725","title":"Epidermal growth factor treatment has protective effects on the integrity of the blood-brain barrier against cerebral ischemia injury in bEnd3 cells.","date":"2019","source":"Experimental and therapeutic medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30867725","citation_count":15,"is_preprint":false},{"pmid":"28977671","id":"PMC_28977671","title":"A novel TPR-BEN domain interaction mediates PICH-BEND3 association.","date":"2017","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/28977671","citation_count":14,"is_preprint":false},{"pmid":"26481236","id":"PMC_26481236","title":"BEND3 is involved in the human-specific repression of calreticulin: Implication for the evolution of higher brain functions in human.","date":"2015","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/26481236","citation_count":8,"is_preprint":false},{"pmid":"27487930","id":"PMC_27487930","title":"Characterization of the NTPR and BD1 interacting domains of the human PICH-BEND3 complex.","date":"2016","source":"Acta crystallographica. Section F, Structural biology communications","url":"https://pubmed.ncbi.nlm.nih.gov/27487930","citation_count":5,"is_preprint":false},{"pmid":"40158797","id":"PMC_40158797","title":"BEND3 promotes hepatocellular carcinoma progression and metastasis by activating the PI3K/AKT/mTOR pathway and inducing epithelial-mesenchymal transition.","date":"2025","source":"Clinics and research in hepatology and gastroenterology","url":"https://pubmed.ncbi.nlm.nih.gov/40158797","citation_count":4,"is_preprint":false},{"pmid":"25600804","id":"PMC_25600804","title":"Basic amino acid residues located in the N-terminal region of BEND3 are essential for its nuclear localization.","date":"2015","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/25600804","citation_count":4,"is_preprint":false},{"pmid":"16247462","id":"PMC_16247462","title":"Adenoviral modification of mouse brain derived endothelial cells, bEnd3, to induce apoptosis by vascular endothelial growth factor.","date":"2006","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/16247462","citation_count":3,"is_preprint":false},{"pmid":"39717669","id":"PMC_39717669","title":"Exosomes from IH- Induced bEnd3 Cells Promote OSA Cognitive Impairment via miR-20a-5p/MFN2 Mediated Pyroptosis of HT22 Cells.","date":"2024","source":"Nature and science of sleep","url":"https://pubmed.ncbi.nlm.nih.gov/39717669","citation_count":3,"is_preprint":false},{"pmid":"25400923","id":"PMC_25400923","title":"Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.","date":"2014","source":"Immunity, inflammation and disease","url":"https://pubmed.ncbi.nlm.nih.gov/25400923","citation_count":3,"is_preprint":false},{"pmid":"37509346","id":"PMC_37509346","title":"Role of Transcription Factor BEND3 and Its Potential Effect on Cancer Progression.","date":"2023","source":"Cancers","url":"https://pubmed.ncbi.nlm.nih.gov/37509346","citation_count":2,"is_preprint":false},{"pmid":"29360423","id":"PMC_29360423","title":"Efficacy for lung metastasis induced by the allogeneic bEnd3 vaccine in mice.","date":"2018","source":"Human vaccines & immunotherapeutics","url":"https://pubmed.ncbi.nlm.nih.gov/29360423","citation_count":2,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.11.14.688451","title":"Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells","date":"2025-11-14","source":"bioRxiv","url":"https://doi.org/10.1101/2025.11.14.688451","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":11874,"output_tokens":2514,"usd":0.036666,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":9638,"output_tokens":3061,"usd":0.062358,"stage2_stop_reason":"end_turn"},"total_usd":0.099024,"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\": 2014,\n      \"finding\": \"BEND3 is enriched on pericentromeric chromatin in the absence of DNA methylation or H3K9Me3, and facilitates Polycomb recruitment and H3K27Me3 deposition at pericentromeric regions, providing a redundant pathway to generate repressive chromatin and mediating a switch from constitutive to facultative heterochromatin.\",\n      \"method\": \"Quantitative locus purification of pericentromeric chromatin-associated proteins in DNA methyltransferase-deficient and H3K9Me3 methyltransferase-deficient mouse embryonic stem cells\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct chromatin purification from genetic loss-of-function ESCs, replicated in multiple methylation-deficient contexts, mechanistic pathway placement established\",\n      \"pmids\": [\"25457167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BEND3 is a CpG island (CGI)-binding protein that binds regulatory elements in a DNA methylation-sensitive manner; it is required for stable association of PRC2 at bivalent genes to maintain H3K27me3 and prevent premature activation of these genes during differentiation. Bend3 null mouse embryos die at pregastrulation, and null ESCs show premature activation of hundreds of CGI-containing bivalent genes. The co-crystal structure of BEND3 with its target DNA reveals the structural basis for methylation-sensitive binding.\",\n      \"method\": \"Co-crystal structure of BEND3-DNA complex; Bend3 knockout mouse embryos and ESCs; ChIP-seq for H3K27me3 and PRC2; RNA-seq in null ESCs during differentiation\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure plus genetic KO with defined molecular and developmental phenotypes, multiple orthogonal methods in a single rigorous study\",\n      \"pmids\": [\"35143257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"BEND3 localizes to nucleoli and binds ribosomal DNA (rDNA) promoters to repress rRNA gene transcription. Loss of BEND3 increases H3K4me3 and decreases rDNA promoter DNA methylation. BEND3 associates with the nucleolar remodeling complex (NoRC), and SUMOylated BEND3 stabilizes NoRC component TIP5 (TTF-1-interacting protein 5) via interaction with the deubiquitinase USP21.\",\n      \"method\": \"ChIP at rDNA promoters; co-immunoprecipitation of BEND3 with NoRC and USP21; BEND3 knockdown with measurement of H3K4me3 and DNA methylation at rDNA; immunofluorescence localization to nucleoli\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP for complex identification, ChIP with epigenetic readouts, subcellular localization with functional consequence, multiple orthogonal methods in single study\",\n      \"pmids\": [\"26100909\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"BEND3 binds promoters of differentiation-associated genes (including CDKN1A/p21) and represses them by enhancing H3K27me3 decoration at these promoters in pluripotent cells. Removal of BEND3 from pluripotent cells causes upregulation of differentiation-inducing gene expression.\",\n      \"method\": \"ChIP-seq for BEND3 and H3K27me3; RNA-seq in BEND3-depleted pluripotent cells; reporter and knockdown assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct ChIP-seq evidence of promoter binding, H3K27me3 changes upon KD, RNA-seq, multiple orthogonal methods\",\n      \"pmids\": [\"35217604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"BEND3 interacts with the DNA translocase PICH in mitosis. The interaction occurs via a novel TPR domain in PICH and the first BEN domain (BD1) of BEND3. The crystal structure of the NTPR-BD1 complex was solved at 2.2 Å resolution, and structure-guided mutagenesis identified residues critical for the interaction and for the functional interaction of full-length proteins.\",\n      \"method\": \"Co-immunoprecipitation in human cells; in vitro biochemical interaction with purified full-length proteins; crystal structure of TPR-BEN domain complex at 2.2 Å; site-directed mutagenesis of interface residues\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with in vitro reconstitution of interaction, mutagenesis validation, and cellular Co-IP; multiple orthogonal methods\",\n      \"pmids\": [\"28977671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The NTPR domain of PICH and BD1 domain of BEND3 form a heterodimeric complex that crystallizes in space group P6122 with unit-cell parameters a=b=47.28, c=431.58 Å, providing atomic-level structural data for the PICH-BEND3 interaction interface.\",\n      \"method\": \"Protein crystallography (MAD at 2.2 Å resolution) of purified recombinant NTPR and BD1 domain complex\",\n      \"journal\": \"Acta crystallographica. Section F, Structural biology communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination, corroborates PMID 28977671 findings with structural data\",\n      \"pmids\": [\"27487930\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Three basic amino acid residues in the N-terminal region of BEND3 (Lys56-Arg57-Lys58, KRK) are essential for its nuclear localization, functioning as a nuclear localization signal (NLS).\",\n      \"method\": \"Immunofluorescence assay of deletion and site-directed mutants of human BEND3 expressed in cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — direct localization experiment with mutagenesis in a single study, but single lab with single method\",\n      \"pmids\": [\"25600804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"BEND3 binds to the human-specific C-allele (rs138452745) at position -220 of the CALR gene promoter as shown by DNA pulldown assay, whereas NFI family members (NF1A, B, C, X) bind the ancestral A-allele, consistent with BEND3 acting as a transcriptional repressor at this locus.\",\n      \"method\": \"DNA pulldown assay from SK-N-BE and HeLa cell nuclear extracts using biotinylated C- and A-allele oligonucleotides, followed by protein identification\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single pulldown method, two cell lines, no functional reporter in same paper; single lab\",\n      \"pmids\": [\"26481236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Preprint data indicate that PRC1 and PRC2 recruitment to hypomethylated pericentromeric heterochromatin in mESCs does not depend on BEND3 (nor on SCML2, KDM2b, or TET enzymes), suggesting that the permissive chromatin environment from DNA hypomethylation is the principal facilitator of Polycomb spreading independently of BEND3.\",\n      \"method\": \"Doxycycline-inducible Dnmt1 modulation in mESCs; ChIP-seq for H2AK119ub1 and H3K27me3 at PCH in BEND3-depleted cells\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint, single lab, negative result (BEND3 not required) contradicting prior models; requires peer review validation\",\n      \"pmids\": [\"bio_10.1101_2025.11.14.688451\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"BEND3 is a quadruple BEN domain-containing nuclear protein that functions as a transcriptional repressor by binding CpG island-containing regulatory elements in a DNA methylation-sensitive manner; it stabilizes PRC2 association at bivalent genes to maintain H3K27me3 and prevent premature gene activation, recruits Polycomb to pericentromeric chromatin in the absence of DNA methylation or H3K9me3, represses rDNA transcription by associating with the NoRC complex (stabilizing TIP5 via the deubiquitinase USP21 through a SUMOylation-dependent mechanism), and interacts with the mitotic DNA translocase PICH via a structurally defined novel TPR–BEN domain interface, while its nuclear localization is mediated by an N-terminal KRK motif.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"BEND3 is a methylation-sensitive, DNA-binding nuclear protein that links chromatin recognition to Polycomb-mediated transcriptional repression across multiple genomic compartments [#1, #3]. It binds CpG island regulatory elements in a DNA methylation-sensitive manner—a specificity defined at atomic resolution by a co-crystal structure with its target DNA—and is required for stable PRC2 association at bivalent genes, maintaining H3K27me3 and preventing premature activation of developmental genes; Bend3 loss causes pregastrulation lethality in mouse embryos and derepression of hundreds of bivalent CGI genes [#1]. In pluripotent cells, BEND3 occupies and represses differentiation-associated promoters including CDKN1A/p21 by enhancing H3K27me3 deposition [#3], and at pericentromeric chromatin lacking DNA methylation or H3K9me3 it provides a redundant route to repressive chromatin by facilitating Polycomb recruitment and H3K27me3, driving a switch from constitutive to facultative heterochromatin [#0]. BEND3 also localizes to nucleoli and represses rDNA transcription by binding rDNA promoters and associating with the NoRC complex, where SUMOylated BEND3 stabilizes the NoRC subunit TIP5 via the deubiquitinase USP21 [#2]. Independently of its repressive roles, BEND3 engages the mitotic DNA translocase PICH through a structurally defined interface between PICH's TPR domain and the first BEN domain of BEND3 [#4, #5]. Nuclear localization is mediated by an N-terminal KRK motif [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2014,\n      \"claim\": \"Established that BEND3 provides a redundant, DNA-methylation-independent pathway to generate repressive chromatin, answering how heterochromatin is maintained when canonical methylation marks are absent.\",\n      \"evidence\": \"Quantitative locus purification of pericentromeric chromatin from Dnmt- and Suv39-deficient mouse ESCs\",\n      \"pmids\": [\"25457167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which BEND3 recruits Polycomb at pericentromeres not resolved at the molecular level\", \"Direct versus indirect chromatin recruitment not distinguished\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined BEND3 as a nucleolar repressor of rRNA genes and connected it to NoRC, showing how it stabilizes the complex through a deubiquitinase- and SUMO-dependent route.\",\n      \"evidence\": \"ChIP at rDNA promoters, reciprocal Co-IP of BEND3 with NoRC and USP21, knockdown with epigenetic readouts, immunofluorescence\",\n      \"pmids\": [\"26100909\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct enzymatic role of BEND3 in SUMOylation/deubiquitination not established\", \"Whether rDNA binding is direct sequence recognition unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Mapped the determinant of BEND3 nuclear import, identifying a KRK basic motif as the NLS.\",\n      \"evidence\": \"Immunofluorescence of deletion and point mutants of human BEND3\",\n      \"pmids\": [\"25600804\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single method\", \"Import receptor mediating recognition not identified\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Provided an allele-specific binding example consistent with BEND3 acting as a repressor at a regulatory polymorphism.\",\n      \"evidence\": \"DNA pulldown of BEND3 onto the CALR promoter C-allele from cell nuclear extracts\",\n      \"pmids\": [\"26481236\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No functional reporter in same study\", \"Single pulldown method, no in vivo validation\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Delivered atomic-level structural data for the PICH NTPR–BEND3 BD1 interface, establishing the physical basis of a mitotic interaction.\",\n      \"evidence\": \"MAD crystallography of purified recombinant NTPR–BD1 complex at 2.2 Å\",\n      \"pmids\": [\"27487930\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure alone does not define the cellular function of the interaction\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Validated the BEND3–PICH interaction functionally, mapping it to a novel TPR–BEN interface and confirming interface residues by mutagenesis.\",\n      \"evidence\": \"Cellular Co-IP, in vitro reconstitution with full-length proteins, crystal structure, structure-guided mutagenesis\",\n      \"pmids\": [\"28977671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream mitotic consequence of the BEND3–PICH interaction not fully defined\", \"Whether this role is separable from BEND3's repressive functions unclear\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Unified BEND3's repressive role at the genome scale by showing it is a methylation-sensitive CGI binder required for PRC2 stability at bivalent genes, with structural and developmental consequences defined.\",\n      \"evidence\": \"Co-crystal of BEND3-DNA, Bend3 KO embryos and ESCs, ChIP-seq for PRC2/H3K27me3, RNA-seq during differentiation\",\n      \"pmids\": [\"35143257\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How BEND3 mechanistically stabilizes PRC2 at chromatin not detailed\", \"Relationship between CGI binding and pericentromeric recruitment not integrated\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed BEND3 restrains differentiation in pluripotent cells by repressing differentiation-inducing genes including CDKN1A via H3K27me3.\",\n      \"evidence\": \"ChIP-seq for BEND3 and H3K27me3, RNA-seq in BEND3-depleted pluripotent cells, reporter/knockdown assays\",\n      \"pmids\": [\"35217604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect repression of individual targets not fully resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Challenged the requirement of BEND3 for Polycomb spreading at hypomethylated pericentromeres, attributing recruitment instead to the permissive chromatin environment.\",\n      \"evidence\": \"Inducible Dnmt1 modulation and ChIP-seq for H2AK119ub1/H3K27me3 in BEND3-depleted mESCs (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.11.14.688451\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, not peer-reviewed; negative result contradicting prior model\", \"Single lab\", \"Does not address BEND3's CGI/bivalent gene roles\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How BEND3 physically bridges DNA recognition to PRC2 stabilization, and whether its pericentromeric, CGI, nucleolar, and mitotic functions share a common molecular mechanism, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No defined molecular mechanism for PRC2 recruitment/stabilization by BEND3\", \"Conflicting evidence on pericentromeric requirement\", \"Integration of distinct genomic roles into one model lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [1, 2, 3, 7]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [1, 2, 3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 1, 3]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 3]}\n    ],\n    \"complexes\": [\"NoRC\"],\n    \"partners\": [\"TIP5\", \"USP21\", \"PICH\", \"PRC2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}