{"gene":"HMG20B","run_date":"2026-06-10T01:55:22","timeline":{"discoveries":[{"year":2002,"finding":"BRAF35 (HMG20B) is a subunit of a six-subunit BHC (BRAF-histone deacetylase) complex that contains HDAC1/2, CoREST, BHC80, a PHD zinc-finger subunit, and an MTA-like subunit, and this complex deacetylates histones and mediates repression of neuron-specific genes via the RE1/NRS cis-regulatory element; a single point mutation in the HMG domain of BRAF35 abrogated REST-mediated transcriptional repression.","method":"Biochemical isolation of complex from human cells, histone deacetylase assay, chromatin immunoprecipitation, point mutation analysis of HMG domain","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — complex reconstitution, enzymatic assay, ChIP, and mutagenesis in one study; foundational paper replicated by subsequent work","pmids":["12032298"],"is_preprint":false},{"year":2012,"finding":"Sumoylation of BRAF35 (HMG20B) is required for full repression of neuron-specific genes, for occupancy of the LSD1-CoREST complex at target genes, and for its antineurogenic activity; its paralogue iBraf forms heterodimers with Braf35, impairing Braf35 interaction with the LSD1-CoREST complex and inhibiting Braf35 sumoylation.","method":"Sumoylation assays, co-immunoprecipitation of Braf35/iBraf heterodimers, chromatin immunoprecipitation, knockdown/overexpression in P19 cells and chicken embryo neural tube","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ChIP, in vivo neuronal differentiation assays, sumoylation assays) in one study; replicated mechanistic context from prior BHC work","pmids":["22570500"],"is_preprint":false},{"year":2011,"finding":"HMG20B depletion by RNAi disturbs completion of cell division (cytokinesis); in vitro, HMG20B binds directly to the BRC repeats of BRCA2, with highest affinity for BRC5; in vivo BRC5 overexpression inhibits the BRCA2-HMG20B interaction and recapitulates cytokinesis defects, separating this function from the BRC4-RAD51-DNA recombination pathway.","method":"RNAi depletion, in vitro direct binding assay (BRC repeats vs HMG20B), overexpression of BRC peptides in vivo, cell division completion assays","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vitro binding assay combined with RNAi phenotype and BRC5 competition experiment providing mechanistic pathway placement; replicated by follow-up study (PMID 25486196)","pmids":["21399666"],"is_preprint":false},{"year":2014,"finding":"A C-terminal region of HMG20b (residues 173–317) is necessary and sufficient for localization to cytokinetic structures and for interaction with BRCA2; a cancer-associated non-conservative substitution Ala247Pro in this region disrupts both activities and impairs cytokinesis in a trans-dominant manner.","method":"Domain truncation and expression, cytokinesis rescue assays in HMG20b-depleted cells, co-immunoprecipitation, localization imaging, cancer mutation analysis","journal":"Cell cycle (Georgetown, Tex.)","confidence":"High","confidence_rationale":"Tier 2 / Strong — structure-function dissection with mutagenesis, rescue experiments, and mechanistic epistasis; builds on replicated BRCA2-HMG20B interaction","pmids":["25486196"],"is_preprint":false},{"year":2011,"finding":"Hmg20b depletion in erythroblasts induces spontaneous erythroid differentiation and predominantly up-regulates target genes (85% of deregulated genes), including embryonic β-like globins and Hrasls3; Gfi1b (a known repressor of erythroid differentiation) is down-regulated, placing Hmg20b as a repressor of erythroid differentiation acting through the CoREST complex.","method":"shRNA knockdown in mouse fetal liver cell line and primary fetal liver cells, microarray gene expression profiling, globin gene expression assays, functional analysis of target gene Hrasls3","journal":"Haematologica","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KD with defined differentiation phenotype and microarray target identification in two cell systems; single lab, no reconstitution","pmids":["21606163"],"is_preprint":false},{"year":2017,"finding":"The E3 ubiquitin ligase MID1/TRIM18 ubiquitinates BRAF35 (HMG20B) via atypical K6-, K27-, and K29-linked poly-ubiquitin chains; MID1 depletion alters BRAF35 localization in cytoplasmic bodies and increases BRAF35 stability, affecting its cytoplasmic abundance.","method":"Co-immunoprecipitation, ubiquitination assays with linkage-specific analysis, MID1 depletion, subcellular fractionation and co-localization imaging","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and localization change upon depletion; single lab with two orthogonal methods","pmids":["28760657"],"is_preprint":false},{"year":2022,"finding":"HMG20B co-localizes genome-wide on chromatin with GFI1 and LSD1; HMG20B depletion induces AML leukemia cell differentiation; HMG20B is required for GFI1 transcription repressor activity by stabilizing LSD1 on chromatin at GFI1-binding sites; HMG20B interacts with LSD1 through its coiled-coil domain; LSD1 inhibitor treatment disrupts the HMG20B–LSD1 interaction.","method":"Mass spectrometry of LSD1 complex after LSD1 inhibitor treatment, ChIP-seq for HMG20B/GFI1/LSD1, HMG20B depletion with differentiation readout, co-immunoprecipitation domain mapping","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — mass spectrometry, genome-wide ChIP-seq, functional KD with differentiation readout, and domain-level Co-IP in one study; multiple orthogonal methods","pmids":["36171271"],"is_preprint":false},{"year":2002,"finding":"Human SMARCE1r/HMG20B protein is associated with the nuclear matrix and possesses DNA-binding activity demonstrated by DNA-affinity chromatography and EMSA; it contains a nuclear localization signal, an HMG domain, and a coiled-coil domain.","method":"Subcellular fractionation, DNA-affinity column chromatography, electrophoretic mobility shift assay (EMSA), Western blot","journal":"Biochimica et biophysica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct biochemical assays (EMSA and affinity chromatography) for DNA binding and fractionation for nuclear matrix association; single lab","pmids":["11997092"],"is_preprint":false},{"year":2022,"finding":"Dp71 point mutants (C272Y, E299del) acquire dominant-negative function that impairs the nuclear distribution of BRAF35 (HMG20B) and iBRAF in neuronal cells, linking Dp71 to the regulation of BRAF35/iBRAF localization and downstream heterochromatin marker H3K9me2 organization.","method":"Expression of GFP-tagged Dp71 point mutants in neuronal cell lines (N1E-115, SH-SY5Y), immunofluorescence localization, H3K9me2 immunostaining","journal":"International journal of molecular sciences","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, localization imaging with dominant-negative overexpression; no direct biochemical interaction assay with HMG20B reported","pmids":["36233175"],"is_preprint":false},{"year":2024,"finding":"CPSF4 inhibits an alternative 3′ splice site event of HMG20B in triple-negative breast cancer cells, and HMG20B siRNA knockdown reduces cell proliferation, migration, and invasion, indicating that CPSF4-regulated splicing of HMG20B contributes to TNBC cell growth.","method":"RIP-seq and RNA-seq for CPSF4-regulated splicing events, qRT-PCR validation of alternative splicing, HMG20B siRNA with CCK-8 and Transwell assays","journal":"Journal of translational medicine","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, functional knockdown with proliferation/migration readout but limited molecular mechanism; splicing event identified by sequencing without structural/biochemical mechanistic detail","pmids":["39731153"],"is_preprint":false}],"current_model":"HMG20B (BRAF35) is a nuclear HMG-box/coiled-coil protein that functions as a core subunit of the LSD1-CoREST repressor complex, where it stabilizes LSD1 on chromatin at GFI1 and REST binding sites to silence neuronal and differentiation genes; it is post-translationally regulated by sumoylation (required for full repressive activity and antineurogenic function), MID1-mediated atypical K6/K27/K29-linked polyubiquitination (controlling its cytoplasmic stability and localization), and direct interaction with BRCA2 BRC5 repeats through its C-terminal coiled-coil domain (residues 173–317), which localizes HMG20B to cytokinetic structures and is required for the completion of cytokinesis."},"narrative":{"mechanistic_narrative":"HMG20B (BRAF35/SMARCE1r) is a nuclear HMG-box and coiled-coil protein that functions as a core subunit of histone deacetylase/LSD1-CoREST repressor complexes to silence neuronal and differentiation gene programs [PMID:12032298, PMID:36171271]. It was first isolated as a subunit of the six-protein BHC complex containing HDAC1/2, CoREST, BHC80, a PHD zinc-finger subunit and an MTA-like subunit, where it deacetylates histones and mediates REST-dependent repression of neuron-specific genes via the RE1/NRS element; a single point mutation in its HMG domain abrogates this repression [PMID:12032298]. HMG20B associates with the nuclear matrix and binds DNA directly through its HMG domain [PMID:11997092]. Genome-wide, it co-localizes with GFI1 and LSD1, and contacts LSD1 through its coiled-coil domain to stabilize LSD1 on chromatin at GFI1-binding sites, thereby enabling GFI1 repressor activity; depletion drives differentiation of AML and erythroid cells, identifying HMG20B as a repressor of hematopoietic differentiation acting through CoREST and downregulating Gfi1b [PMID:36171271, PMID:21606163]. Its repressive function is regulated post-translationally: sumoylation is required for full target-gene repression, complex occupancy and antineurogenic activity, and is antagonized by heterodimerization with its paralogue iBraf, which displaces HMG20B from the LSD1-CoREST complex [PMID:22570500]. Independently of its chromatin role, a C-terminal coiled-coil region (residues 173–317) binds the BRC5 repeat of BRCA2 and localizes HMG20B to cytokinetic structures, an activity required for completion of cytokinesis and separable from the BRC4-RAD51 recombination pathway; a cancer-associated Ala247Pro substitution disrupts both BRCA2 binding and cytokinesis in a trans-dominant manner [PMID:21399666, PMID:25486196]. The E3 ligase MID1/TRIM18 modifies HMG20B with atypical K6/K27/K29-linked polyubiquitin chains that control its cytoplasmic stability and localization [PMID:28760657].","teleology":[{"year":2002,"claim":"Established HMG20B as a functional chromatin-repressor subunit rather than an isolated HMG protein, placing it within an HDAC-containing complex that silences neuronal genes.","evidence":"Biochemical isolation of the BHC complex, histone deacetylase assay, ChIP, and HMG-domain point mutation in human cells","pmids":["12032298"],"confidence":"High","gaps":["Direct contribution of the HMG domain to chromatin engagement not structurally resolved","Whether HMG20B is obligate or accessory to deacetylase activity unclear"]},{"year":2002,"claim":"Defined the biochemical activities of the protein — nuclear matrix association and intrinsic DNA binding — and its domain architecture (NLS, HMG box, coiled-coil).","evidence":"Subcellular fractionation, DNA-affinity chromatography, and EMSA on the human protein","pmids":["11997092"],"confidence":"Medium","gaps":["No sequence-specific DNA target identified","Functional consequence of nuclear matrix association not tested"]},{"year":2011,"claim":"Revealed an unexpected non-chromatin function: HMG20B is required for cytokinesis through a direct interaction with BRCA2 BRC repeats, distinct from BRCA2's recombination role.","evidence":"RNAi depletion, in vitro BRC-repeat binding assays, and BRC5 competition in cells","pmids":["21399666"],"confidence":"High","gaps":["Molecular events downstream of HMG20B at the cleavage furrow unknown","How a nuclear repressor reaches cytokinetic structures unresolved"]},{"year":2011,"claim":"Extended HMG20B's repressor role to hematopoiesis, showing it restrains erythroid differentiation predominantly by repressing target genes via CoREST.","evidence":"shRNA knockdown in mouse fetal liver cells and primary cells with microarray profiling and globin/Gfi1b readouts","pmids":["21606163"],"confidence":"Medium","gaps":["Direct vs indirect regulation of Gfi1b not distinguished","Single lab, no complex reconstitution"]},{"year":2012,"claim":"Identified sumoylation as a switch controlling HMG20B repressive output and complex occupancy, and defined iBraf as a heterodimeric antagonist.","evidence":"Sumoylation assays, Braf35/iBraf Co-IP, ChIP, and neuronal differentiation assays in P19 cells and chick neural tube","pmids":["22570500"],"confidence":"High","gaps":["SUMO ligase/site stoichiometry not fully mapped","How sumoylation mechanistically enhances chromatin occupancy unclear"]},{"year":2014,"claim":"Mapped the C-terminal coiled-coil region (173–317) as necessary and sufficient for BRCA2 binding and cytokinetic localization, and linked a cancer mutation to loss of this function.","evidence":"Domain truncation, cytokinesis rescue, Co-IP, localization imaging, and Ala247Pro mutation analysis","pmids":["25486196"],"confidence":"High","gaps":["Structural basis of coiled-coil/BRC5 recognition not solved","Trans-dominant mechanism of the mutant not detailed"]},{"year":2017,"claim":"Showed HMG20B is targeted by MID1/TRIM18 with atypical polyubiquitin chains that govern its cytoplasmic stability and localization, adding a second post-translational control layer.","evidence":"Co-IP, linkage-specific ubiquitination assays, MID1 depletion, fractionation and co-localization imaging","pmids":["28760657"],"confidence":"Medium","gaps":["Functional consequence of ubiquitination for repressor or cytokinesis roles untested","Single lab, ubiquitination site not mapped"]},{"year":2022,"claim":"Provided genome-wide mechanistic placement: HMG20B stabilizes LSD1 on chromatin at GFI1 sites via its coiled-coil domain to sustain GFI1 repression, and its loss drives leukemic differentiation.","evidence":"LSD1-complex mass spectrometry, HMG20B/GFI1/LSD1 ChIP-seq, depletion with differentiation readout, and domain-mapping Co-IP in AML cells","pmids":["36171271"],"confidence":"High","gaps":["Whether HMG20B directly recruits or only stabilizes LSD1 unresolved","Generality across other CoREST target genes not established"]},{"year":2022,"claim":"Linked Dp71 to regulation of HMG20B nuclear distribution and downstream heterochromatin marks in neuronal cells.","evidence":"Overexpression of dominant-negative GFP-Dp71 mutants in neuronal cell lines with immunofluorescence and H3K9me2 staining","pmids":["36233175"],"confidence":"Low","gaps":["No direct biochemical interaction between Dp71 and HMG20B demonstrated","Localization-only readout in overexpression context","Mechanism connecting Dp71 to HMG20B unclear"]},{"year":2024,"claim":"Connected CPSF4-regulated alternative splicing of HMG20B to triple-negative breast cancer cell growth.","evidence":"RIP-seq/RNA-seq for splicing events, qRT-PCR validation, and HMG20B siRNA with proliferation/migration/invasion assays","pmids":["39731153"],"confidence":"Low","gaps":["Functional difference between the spliced isoforms not characterized","Single lab, limited molecular mechanism","Whether the chromatin or cytokinesis function drives the phenotype unknown"]},{"year":null,"claim":"How HMG20B's two functional arms — chromatin repression via LSD1-CoREST and BRCA2-dependent cytokinesis — are coordinated, and how its post-translational modifications partition it between these roles, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of the HMG20B coiled-coil bound to either LSD1 or BRCA2","Interplay between sumoylation and MID1 ubiquitination not tested","Whether the same molecules switch between nuclear and cytokinetic pools unknown"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[7]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[6,2]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,7]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[5]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[6,0]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,6]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,6]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[2,3]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,1]}],"complexes":["BHC / LSD1-CoREST complex"],"partners":["LSD1","COREST","BRCA2","GFI1","IBRAF","MID1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9P0W2","full_name":"SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1-related","aliases":["BRCA2-associated factor 35","HMG box-containing protein 20B","HMG domain-containing protein 2","HMG domain-containing protein HMGX2","Sox-like transcriptional factor","Structural DNA-binding protein BRAF35"],"length_aa":317,"mass_kda":35.8,"function":"Required for correct progression through G2 phase of the cell cycle and entry into mitosis. Required for RCOR1/CoREST mediated repression of neuronal specific gene promoters","subcellular_location":"Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q9P0W2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HMG20B","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":"HDAC1","stoichiometry":0.2},{"gene":"HDAC2","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/HMG20B","total_profiled":1310},"omim":[{"mim_id":"609132","title":"LYSINE DEMETHYLASE 1A; KDM1A","url":"https://www.omim.org/entry/609132"},{"mim_id":"605535","title":"HIGH MOBILITY GROUP PROTEIN 20B; HMG20B","url":"https://www.omim.org/entry/605535"},{"mim_id":"605534","title":"HIGH MOBILITY GROUP PROTEIN 20A; HMG20A","url":"https://www.omim.org/entry/605534"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HMG20B"},"hgnc":{"alias_symbol":["SOXL","HMGX2","BRAF35","SMARCE1r","BRAF25","HMGXB2"],"prev_symbol":[]},"alphafold":{"accession":"Q9P0W2","domains":[{"cath_id":"1.10.30.10","chopping":"69-139","consensus_level":"medium","plddt":91.3854,"start":69,"end":139},{"cath_id":"-","chopping":"249-315","consensus_level":"medium","plddt":92.0475,"start":249,"end":315}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P0W2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P0W2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9P0W2-F1-predicted_aligned_error_v6.png","plddt_mean":77.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HMG20B","jax_strain_url":"https://www.jax.org/strain/search?query=HMG20B"},"sequence":{"accession":"Q9P0W2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9P0W2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9P0W2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9P0W2"}},"corpus_meta":[{"pmid":"12032298","id":"PMC_12032298","title":"A core-BRAF35 complex containing histone deacetylase mediates repression of neuronal-specific genes.","date":"2002","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/12032298","citation_count":257,"is_preprint":false},{"pmid":"22570500","id":"PMC_22570500","title":"Control of neuronal differentiation by sumoylation of BRAF35, a subunit of the LSD1-CoREST histone demethylase complex.","date":"2012","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/22570500","citation_count":61,"is_preprint":false},{"pmid":"10773667","id":"PMC_10773667","title":"HMG20A and HMG20B map to human chromosomes 15q24 and 19p13.3 and constitute a distinct class of HMG-box genes with ubiquitous expression.","date":"2000","source":"Cytogenetics and cell genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10773667","citation_count":32,"is_preprint":false},{"pmid":"21399666","id":"PMC_21399666","title":"A mitotic function for the high-mobility group protein HMG20b regulated by its interaction with the BRC repeats of the BRCA2 tumor suppressor.","date":"2011","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/21399666","citation_count":26,"is_preprint":false},{"pmid":"21606163","id":"PMC_21606163","title":"The DNA binding factor Hmg20b is a repressor of erythroid differentiation.","date":"2011","source":"Haematologica","url":"https://pubmed.ncbi.nlm.nih.gov/21606163","citation_count":15,"is_preprint":false},{"pmid":"28760657","id":"PMC_28760657","title":"The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2-associated factor 35, BRAF35.","date":"2017","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/28760657","citation_count":11,"is_preprint":false},{"pmid":"36171271","id":"PMC_36171271","title":"HMG20B stabilizes association of LSD1 with GFI1 on chromatin to confer transcription repression and leukemia cell differentiation block.","date":"2022","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/36171271","citation_count":10,"is_preprint":false},{"pmid":"25486196","id":"PMC_25486196","title":"A cancer-associated mutation inactivates a region of the high-mobility group protein HMG20b essential for cytokinesis.","date":"2014","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/25486196","citation_count":7,"is_preprint":false},{"pmid":"11997092","id":"PMC_11997092","title":"Characterization of human SMARCE1r high-mobility-group protein.","date":"2002","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11997092","citation_count":5,"is_preprint":false},{"pmid":"12083780","id":"PMC_12083780","title":"Immunohistological detection of BRAF25 in human prostate tumor and cancer specimens.","date":"2002","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/12083780","citation_count":4,"is_preprint":false},{"pmid":"23053932","id":"PMC_23053932","title":"Structural insight into the mode of interactions of SoxL from Allochromatium vinosum in the global sulfur oxidation cycle.","date":"2012","source":"Molecular biology reports","url":"https://pubmed.ncbi.nlm.nih.gov/23053932","citation_count":4,"is_preprint":false},{"pmid":"36233175","id":"PMC_36233175","title":"Dp71 Point Mutations Induce Protein Aggregation, Loss of Nuclear Lamina Integrity and Impaired Braf35 and Ibraf Function in Neuronal Cells.","date":"2022","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/36233175","citation_count":3,"is_preprint":false},{"pmid":"39731153","id":"PMC_39731153","title":"CPSF4-mediated regulation of alternative splicing of HMG20B facilitates the progression of triple-negative breast cancer.","date":"2024","source":"Journal of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39731153","citation_count":2,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8259,"output_tokens":2906,"usd":0.034183,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10114,"output_tokens":3746,"usd":0.07211,"stage2_stop_reason":"end_turn"},"total_usd":0.106293,"stage1_batch_id":"msgbatch_01DUPACZi7AAWwuZiUjseQF4","stage2_batch_id":"msgbatch_018yt7chE3f48PEFtDwtj6AE","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"BRAF35 (HMG20B) is a subunit of a six-subunit BHC (BRAF-histone deacetylase) complex that contains HDAC1/2, CoREST, BHC80, a PHD zinc-finger subunit, and an MTA-like subunit, and this complex deacetylates histones and mediates repression of neuron-specific genes via the RE1/NRS cis-regulatory element; a single point mutation in the HMG domain of BRAF35 abrogated REST-mediated transcriptional repression.\",\n      \"method\": \"Biochemical isolation of complex from human cells, histone deacetylase assay, chromatin immunoprecipitation, point mutation analysis of HMG domain\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — complex reconstitution, enzymatic assay, ChIP, and mutagenesis in one study; foundational paper replicated by subsequent work\",\n      \"pmids\": [\"12032298\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Sumoylation of BRAF35 (HMG20B) is required for full repression of neuron-specific genes, for occupancy of the LSD1-CoREST complex at target genes, and for its antineurogenic activity; its paralogue iBraf forms heterodimers with Braf35, impairing Braf35 interaction with the LSD1-CoREST complex and inhibiting Braf35 sumoylation.\",\n      \"method\": \"Sumoylation assays, co-immunoprecipitation of Braf35/iBraf heterodimers, chromatin immunoprecipitation, knockdown/overexpression in P19 cells and chicken embryo neural tube\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ChIP, in vivo neuronal differentiation assays, sumoylation assays) in one study; replicated mechanistic context from prior BHC work\",\n      \"pmids\": [\"22570500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"HMG20B depletion by RNAi disturbs completion of cell division (cytokinesis); in vitro, HMG20B binds directly to the BRC repeats of BRCA2, with highest affinity for BRC5; in vivo BRC5 overexpression inhibits the BRCA2-HMG20B interaction and recapitulates cytokinesis defects, separating this function from the BRC4-RAD51-DNA recombination pathway.\",\n      \"method\": \"RNAi depletion, in vitro direct binding assay (BRC repeats vs HMG20B), overexpression of BRC peptides in vivo, cell division completion assays\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vitro binding assay combined with RNAi phenotype and BRC5 competition experiment providing mechanistic pathway placement; replicated by follow-up study (PMID 25486196)\",\n      \"pmids\": [\"21399666\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"A C-terminal region of HMG20b (residues 173–317) is necessary and sufficient for localization to cytokinetic structures and for interaction with BRCA2; a cancer-associated non-conservative substitution Ala247Pro in this region disrupts both activities and impairs cytokinesis in a trans-dominant manner.\",\n      \"method\": \"Domain truncation and expression, cytokinesis rescue assays in HMG20b-depleted cells, co-immunoprecipitation, localization imaging, cancer mutation analysis\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — structure-function dissection with mutagenesis, rescue experiments, and mechanistic epistasis; builds on replicated BRCA2-HMG20B interaction\",\n      \"pmids\": [\"25486196\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Hmg20b depletion in erythroblasts induces spontaneous erythroid differentiation and predominantly up-regulates target genes (85% of deregulated genes), including embryonic β-like globins and Hrasls3; Gfi1b (a known repressor of erythroid differentiation) is down-regulated, placing Hmg20b as a repressor of erythroid differentiation acting through the CoREST complex.\",\n      \"method\": \"shRNA knockdown in mouse fetal liver cell line and primary fetal liver cells, microarray gene expression profiling, globin gene expression assays, functional analysis of target gene Hrasls3\",\n      \"journal\": \"Haematologica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KD with defined differentiation phenotype and microarray target identification in two cell systems; single lab, no reconstitution\",\n      \"pmids\": [\"21606163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The E3 ubiquitin ligase MID1/TRIM18 ubiquitinates BRAF35 (HMG20B) via atypical K6-, K27-, and K29-linked poly-ubiquitin chains; MID1 depletion alters BRAF35 localization in cytoplasmic bodies and increases BRAF35 stability, affecting its cytoplasmic abundance.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assays with linkage-specific analysis, MID1 depletion, subcellular fractionation and co-localization imaging\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and localization change upon depletion; single lab with two orthogonal methods\",\n      \"pmids\": [\"28760657\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"HMG20B co-localizes genome-wide on chromatin with GFI1 and LSD1; HMG20B depletion induces AML leukemia cell differentiation; HMG20B is required for GFI1 transcription repressor activity by stabilizing LSD1 on chromatin at GFI1-binding sites; HMG20B interacts with LSD1 through its coiled-coil domain; LSD1 inhibitor treatment disrupts the HMG20B–LSD1 interaction.\",\n      \"method\": \"Mass spectrometry of LSD1 complex after LSD1 inhibitor treatment, ChIP-seq for HMG20B/GFI1/LSD1, HMG20B depletion with differentiation readout, co-immunoprecipitation domain mapping\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mass spectrometry, genome-wide ChIP-seq, functional KD with differentiation readout, and domain-level Co-IP in one study; multiple orthogonal methods\",\n      \"pmids\": [\"36171271\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Human SMARCE1r/HMG20B protein is associated with the nuclear matrix and possesses DNA-binding activity demonstrated by DNA-affinity chromatography and EMSA; it contains a nuclear localization signal, an HMG domain, and a coiled-coil domain.\",\n      \"method\": \"Subcellular fractionation, DNA-affinity column chromatography, electrophoretic mobility shift assay (EMSA), Western blot\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct biochemical assays (EMSA and affinity chromatography) for DNA binding and fractionation for nuclear matrix association; single lab\",\n      \"pmids\": [\"11997092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Dp71 point mutants (C272Y, E299del) acquire dominant-negative function that impairs the nuclear distribution of BRAF35 (HMG20B) and iBRAF in neuronal cells, linking Dp71 to the regulation of BRAF35/iBRAF localization and downstream heterochromatin marker H3K9me2 organization.\",\n      \"method\": \"Expression of GFP-tagged Dp71 point mutants in neuronal cell lines (N1E-115, SH-SY5Y), immunofluorescence localization, H3K9me2 immunostaining\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, localization imaging with dominant-negative overexpression; no direct biochemical interaction assay with HMG20B reported\",\n      \"pmids\": [\"36233175\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"CPSF4 inhibits an alternative 3′ splice site event of HMG20B in triple-negative breast cancer cells, and HMG20B siRNA knockdown reduces cell proliferation, migration, and invasion, indicating that CPSF4-regulated splicing of HMG20B contributes to TNBC cell growth.\",\n      \"method\": \"RIP-seq and RNA-seq for CPSF4-regulated splicing events, qRT-PCR validation of alternative splicing, HMG20B siRNA with CCK-8 and Transwell assays\",\n      \"journal\": \"Journal of translational medicine\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, functional knockdown with proliferation/migration readout but limited molecular mechanism; splicing event identified by sequencing without structural/biochemical mechanistic detail\",\n      \"pmids\": [\"39731153\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HMG20B (BRAF35) is a nuclear HMG-box/coiled-coil protein that functions as a core subunit of the LSD1-CoREST repressor complex, where it stabilizes LSD1 on chromatin at GFI1 and REST binding sites to silence neuronal and differentiation genes; it is post-translationally regulated by sumoylation (required for full repressive activity and antineurogenic function), MID1-mediated atypical K6/K27/K29-linked polyubiquitination (controlling its cytoplasmic stability and localization), and direct interaction with BRCA2 BRC5 repeats through its C-terminal coiled-coil domain (residues 173–317), which localizes HMG20B to cytokinetic structures and is required for the completion of cytokinesis.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"HMG20B (BRAF35/SMARCE1r) is a nuclear HMG-box and coiled-coil protein that functions as a core subunit of histone deacetylase/LSD1-CoREST repressor complexes to silence neuronal and differentiation gene programs [#0, #6]. It was first isolated as a subunit of the six-protein BHC complex containing HDAC1/2, CoREST, BHC80, a PHD zinc-finger subunit and an MTA-like subunit, where it deacetylates histones and mediates REST-dependent repression of neuron-specific genes via the RE1/NRS element; a single point mutation in its HMG domain abrogates this repression [#0]. HMG20B associates with the nuclear matrix and binds DNA directly through its HMG domain [#7]. Genome-wide, it co-localizes with GFI1 and LSD1, and contacts LSD1 through its coiled-coil domain to stabilize LSD1 on chromatin at GFI1-binding sites, thereby enabling GFI1 repressor activity; depletion drives differentiation of AML and erythroid cells, identifying HMG20B as a repressor of hematopoietic differentiation acting through CoREST and downregulating Gfi1b [#6, #4]. Its repressive function is regulated post-translationally: sumoylation is required for full target-gene repression, complex occupancy and antineurogenic activity, and is antagonized by heterodimerization with its paralogue iBraf, which displaces HMG20B from the LSD1-CoREST complex [#1]. Independently of its chromatin role, a C-terminal coiled-coil region (residues 173\\u2013317) binds the BRC5 repeat of BRCA2 and localizes HMG20B to cytokinetic structures, an activity required for completion of cytokinesis and separable from the BRC4-RAD51 recombination pathway; a cancer-associated Ala247Pro substitution disrupts both BRCA2 binding and cytokinesis in a trans-dominant manner [#2, #3]. The E3 ligase MID1/TRIM18 modifies HMG20B with atypical K6/K27/K29-linked polyubiquitin chains that control its cytoplasmic stability and localization [#5].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established HMG20B as a functional chromatin-repressor subunit rather than an isolated HMG protein, placing it within an HDAC-containing complex that silences neuronal genes.\",\n      \"evidence\": \"Biochemical isolation of the BHC complex, histone deacetylase assay, ChIP, and HMG-domain point mutation in human cells\",\n      \"pmids\": [\"12032298\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct contribution of the HMG domain to chromatin engagement not structurally resolved\", \"Whether HMG20B is obligate or accessory to deacetylase activity unclear\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined the biochemical activities of the protein \\u2014 nuclear matrix association and intrinsic DNA binding \\u2014 and its domain architecture (NLS, HMG box, coiled-coil).\",\n      \"evidence\": \"Subcellular fractionation, DNA-affinity chromatography, and EMSA on the human protein\",\n      \"pmids\": [\"11997092\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No sequence-specific DNA target identified\", \"Functional consequence of nuclear matrix association not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed an unexpected non-chromatin function: HMG20B is required for cytokinesis through a direct interaction with BRCA2 BRC repeats, distinct from BRCA2's recombination role.\",\n      \"evidence\": \"RNAi depletion, in vitro BRC-repeat binding assays, and BRC5 competition in cells\",\n      \"pmids\": [\"21399666\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular events downstream of HMG20B at the cleavage furrow unknown\", \"How a nuclear repressor reaches cytokinetic structures unresolved\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extended HMG20B's repressor role to hematopoiesis, showing it restrains erythroid differentiation predominantly by repressing target genes via CoREST.\",\n      \"evidence\": \"shRNA knockdown in mouse fetal liver cells and primary cells with microarray profiling and globin/Gfi1b readouts\",\n      \"pmids\": [\"21606163\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect regulation of Gfi1b not distinguished\", \"Single lab, no complex reconstitution\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identified sumoylation as a switch controlling HMG20B repressive output and complex occupancy, and defined iBraf as a heterodimeric antagonist.\",\n      \"evidence\": \"Sumoylation assays, Braf35/iBraf Co-IP, ChIP, and neuronal differentiation assays in P19 cells and chick neural tube\",\n      \"pmids\": [\"22570500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO ligase/site stoichiometry not fully mapped\", \"How sumoylation mechanistically enhances chromatin occupancy unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapped the C-terminal coiled-coil region (173\\u2013317) as necessary and sufficient for BRCA2 binding and cytokinetic localization, and linked a cancer mutation to loss of this function.\",\n      \"evidence\": \"Domain truncation, cytokinesis rescue, Co-IP, localization imaging, and Ala247Pro mutation analysis\",\n      \"pmids\": [\"25486196\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of coiled-coil/BRC5 recognition not solved\", \"Trans-dominant mechanism of the mutant not detailed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed HMG20B is targeted by MID1/TRIM18 with atypical polyubiquitin chains that govern its cytoplasmic stability and localization, adding a second post-translational control layer.\",\n      \"evidence\": \"Co-IP, linkage-specific ubiquitination assays, MID1 depletion, fractionation and co-localization imaging\",\n      \"pmids\": [\"28760657\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional consequence of ubiquitination for repressor or cytokinesis roles untested\", \"Single lab, ubiquitination site not mapped\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Provided genome-wide mechanistic placement: HMG20B stabilizes LSD1 on chromatin at GFI1 sites via its coiled-coil domain to sustain GFI1 repression, and its loss drives leukemic differentiation.\",\n      \"evidence\": \"LSD1-complex mass spectrometry, HMG20B/GFI1/LSD1 ChIP-seq, depletion with differentiation readout, and domain-mapping Co-IP in AML cells\",\n      \"pmids\": [\"36171271\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HMG20B directly recruits or only stabilizes LSD1 unresolved\", \"Generality across other CoREST target genes not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Linked Dp71 to regulation of HMG20B nuclear distribution and downstream heterochromatin marks in neuronal cells.\",\n      \"evidence\": \"Overexpression of dominant-negative GFP-Dp71 mutants in neuronal cell lines with immunofluorescence and H3K9me2 staining\",\n      \"pmids\": [\"36233175\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No direct biochemical interaction between Dp71 and HMG20B demonstrated\", \"Localization-only readout in overexpression context\", \"Mechanism connecting Dp71 to HMG20B unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Connected CPSF4-regulated alternative splicing of HMG20B to triple-negative breast cancer cell growth.\",\n      \"evidence\": \"RIP-seq/RNA-seq for splicing events, qRT-PCR validation, and HMG20B siRNA with proliferation/migration/invasion assays\",\n      \"pmids\": [\"39731153\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Functional difference between the spliced isoforms not characterized\", \"Single lab, limited molecular mechanism\", \"Whether the chromatin or cytokinesis function drives the phenotype unknown\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How HMG20B's two functional arms \\u2014 chromatin repression via LSD1-CoREST and BRCA2-dependent cytokinesis \\u2014 are coordinated, and how its post-translational modifications partition it between these roles, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of the HMG20B coiled-coil bound to either LSD1 or BRCA2\", \"Interplay between sumoylation and MID1 ubiquitination not tested\", \"Whether the same molecules switch between nuclear and cytokinetic pools unknown\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [7]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [6, 2]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 7]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [5]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [6, 0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 6]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 6]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [2, 3]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 1]}\n    ],\n    \"complexes\": [\"BHC / LSD1-CoREST complex\"],\n    \"partners\": [\"LSD1\", \"CoREST\", \"BRCA2\", \"GFI1\", \"iBraf\", \"MID1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}