{"gene":"L3MBTL3","run_date":"2026-06-10T02:59:49","timeline":{"discoveries":[{"year":2013,"finding":"L3MBTL3 binds mono- or dimethyllysine-containing peptides via its MBT domains (methyllysine reader function). X-ray crystallography revealed a unique 2:2 polyvalent mode of interaction between the chemical probe UNC1215 and L3MBTL3, with UNC1215 binding the Kme-binding pocket. Point mutants disrupting the Kme-binding function of L3MBTL3 phenocopy probe effects on cellular localization. The Kme-dependent interaction with BCLAF1 (implicated in DNA damage repair and apoptosis) was identified using this probe.","method":"X-ray crystallography, competitive displacement assay (Kd = 120 nM), point mutagenesis, cellular FRAP/GFP-mobility assay, chemical probe (UNC1215) pulldown of endogenous L3MBTL3","journal":"Nature chemical biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure, in vitro binding assay with mutagenesis, and cellular validation in a single rigorous study","pmids":["23292653"],"is_preprint":false},{"year":2013,"finding":"Structure-activity relationship studies established molecular requirements for potent L3MBTL3 binding by small-molecule Kme antagonists, confirming that UNC1215 and related compounds interact with endogenous L3MBTL3 in cells via the mono-/dimethyllysine reading function of the MBT domains.","method":"Medicinal chemistry SAR, in vitro binding assays, cellular target engagement assays","journal":"Journal of medicinal chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple in vitro and cellular assays in single lab, confirms binding mechanism","pmids":["24040942"],"is_preprint":false},{"year":2017,"finding":"L3MBTL3 acts as a transcriptional corepressor of Notch target genes by binding to the transcription factor RBPJ (CBF1/CSL) and competing with the Notch intracellular domain (NOTCH ICD) for RBPJ binding. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (LSD1) to enhancers of Notch target genes, leading to H3K4me2 demethylation and transcriptional repression. This functional link is evolutionarily conserved in Drosophila and C. elegans.","method":"Proteomic/co-immunoprecipitation identification of RBPJ-L3MBTL3 interaction, competitive binding assay (L3MBTL3 vs NOTCH ICD for RBPJ), chromatin immunoprecipitation (H3K4me2), in vivo genetic analyses in Drosophila and C. elegans","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP, competitive binding, and cross-species in vivo epistasis across multiple labs/organisms","pmids":["29030483"],"is_preprint":false},{"year":2018,"finding":"L3MBTL3 binds methylated lysine 142 of DNMT1 and recruits the CRL4DCAF5 ubiquitin E3 ligase complex to target DNMT1 for proteasomal degradation. LSD1 demethylates K142 to prevent this, while PHF20L1 also acts in S phase to protect DNMT1. Loss of L3MBTL3/MBT-1 in mice causes DNMT1 protein accumulation, increased genomic DNA methylation, and late embryonic lethality. L3MBTL3-CRL4DCAF5 similarly controls methylation-dependent degradation of E2F1.","method":"Co-immunoprecipitation, in vivo ubiquitination assay, mouse knockout (L3MBTL3/MBT-1 deletion), western blot for protein stability, genomic DNA methylation assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vivo ubiquitination, mouse KO with defined biochemical and developmental phenotype, multiple substrates tested","pmids":["29691401"],"is_preprint":false},{"year":2018,"finding":"L3MBTL3 preferentially binds methylated lysine 42 of SOX2 (with partial contribution from methylated K117) and recruits the CRL4DCAF5 ubiquitin E3 ligase to promote ubiquitin-dependent SOX2 proteolysis. Knockdown of L3MBTL3 or DCAF5 restores SOX2 protein levels and rescues self-renewal/pluripotency defects in mouse ES cells caused by LSD1 or PHF20L1 deficiency.","method":"Co-immunoprecipitation, site-directed mutagenesis (K42, K117), in vivo ubiquitination assay, siRNA knockdown, mouse ES cell self-renewal/pluripotency assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP with mutagenesis, in vivo ubiquitination, genetic rescue epistasis in ES cells, multiple orthogonal methods in single study","pmids":["30442713"],"is_preprint":false},{"year":2022,"finding":"The 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA ternary complex was determined. L3MBTL3 interacts with RBPJ via an unusual binding motif distinct from other RBPJ-binding partners. Structure-based mutations disrupting this interface impair RBPJ and L3MBTL3 function in cells, confirming the structural basis for L3MBTL3's role as a corepressor in Notch transcriptional regulation.","method":"X-ray crystallography (2.06 Å), structure-based mutagenesis, cellular functional assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure with comprehensive mutagenesis and cellular validation","pmids":["36477367"],"is_preprint":false},{"year":2023,"finding":"L3MBTL3 is transcriptionally induced by HIF-1α under hypoxia. In the nucleus, L3MBTL3 interacts with HIF-1α and promotes HIF-1α ubiquitination and degradation, forming a negative feedback loop to dampen hypoxic response.","method":"Co-immunoprecipitation (L3MBTL3-HIF-1α interaction), ubiquitination assay, knockdown/overexpression in hypoxia, reporter/western blot assays","journal":"Heliyon","confidence":"Medium","confidence_rationale":"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without in vitro reconstitution or mutagenesis; mechanistic claim supported by two methods but from one study","pmids":["36747531"],"is_preprint":false},{"year":2025,"finding":"L3MBTL3 interacts with STAT3 and recruits STAT3 to the SNAIL promoter to increase SNAIL transcription, promoting epithelial-mesenchymal transition and metastasis in breast cancer. The methylated lysine binding activity of L3MBTL3 is not required for this function; only the L3MBTL3-STAT3 interaction is required for SNAIL upregulation.","method":"Co-immunoprecipitation, chromatin immunoprecipitation (L3MBTL3 and STAT3 at SNAIL promoter), Kme-binding mutant analysis, knockdown/overexpression with metastasis assays","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ChIP, and Kme-binding mutant, single lab with multiple orthogonal methods","pmids":["39747894"],"is_preprint":false},{"year":2024,"finding":"L3MBTL3 was identified as a novel effector of DNA damage response. Using proximity ligation (TurboID tethered to γH2AX via MCPH1-BRCT probe), L3MBTL3 was found in the DNA damage-associated proteome across multiple genotoxic insults, and characterized as a 'methyl-binding and proteasome-recruiting protein' at DNA damage sites.","method":"Proximity ligation (TurboID-based proteomics at γH2AX-marked DNA damage sites), functional characterization of DNA damage response","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single proteomics screen with limited mechanistic follow-up on L3MBTL3 specifically","pmids":["bio_10.1101_2024.10.23.619792"],"is_preprint":true},{"year":2024,"finding":"A KRAB-L3MBTL3 fusion domain combination enhanced gene silencing up to 34-fold in dose-limited conditions compared to KRAB alone, demonstrating synergistic epigenetic repression activity when L3MBTL3 is fused to a KRAB domain in CRISPR-based epigenome editing.","method":"High-throughput combinatorial domain screening (COMBINE platform), endogenous gene transcription reporter assays","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, high-throughput functional screen but no mechanistic dissection of how L3MBTL3 contributes to the enhanced silencing","pmids":["bio_10.1101_2024.10.28.620683"],"is_preprint":true}],"current_model":"L3MBTL3 is a methyllysine reader protein (via its MBT domains) that functions as a transcriptional corepressor and proteolysis adapter: it binds mono-/dimethylated lysines on histones and non-histone proteins (DNMT1-K142me, SOX2-K42me, E2F1), recruiting the CRL4DCAF5 ubiquitin E3 ligase to degrade methylated substrates; it also binds RBPJ/CBF1 as a corepressor that recruits KDM1A/LSD1 to demethylate H3K4me2 at Notch target gene enhancers (structural basis defined by a 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex); additionally, it interacts with STAT3 to drive SNAIL transcription in a Kme-independent manner, and forms a negative feedback loop with HIF-1α by promoting its ubiquitination and degradation under hypoxia."},"narrative":{"mechanistic_narrative":"L3MBTL3 is a methyllysine-reader protein that couples recognition of mono-/dimethylated lysines to two distinct gene-regulatory outputs: targeted protein degradation and transcriptional corepression [PMID:23292653, PMID:29691401]. Through its MBT domains it engages mono- and dimethyllysine marks in a polyvalent binding mode, a function defined structurally and dissected with the chemical probe UNC1215 and Kme-binding point mutants [PMID:23292653]. As a proteolysis adapter, L3MBTL3 reads methylated lysines on non-histone substrates and recruits the CRL4DCAF5 ubiquitin E3 ligase to drive their proteasomal degradation: it binds DNMT1-K142me to control DNMT1 turnover, genomic DNA methylation, and embryonic viability, and similarly targets methylated SOX2 (K42) to regulate embryonic stem cell self-renewal and pluripotency [PMID:29691401, PMID:30442713]. Independently of its degradation role, L3MBTL3 is a transcriptional corepressor of Notch target genes, binding RBPJ/CBF1 via an unusual interface and competing with the Notch intracellular domain to instead recruit the histone demethylase KDM1A/LSD1 and demethylate H3K4me2 at target enhancers, with the structural basis defined by a 2.06 Å RBPJ-L3MBTL3-DNA crystal structure [PMID:29030483, PMID:36477367]. L3MBTL3 also supports transcriptional and degradative functions that do not require its methyllysine-reading activity, including a methylation-independent interaction with STAT3 that activates SNAIL transcription to promote epithelial-mesenchymal transition [PMID:39747894], and a hypoxia-induced negative feedback loop in which it promotes HIF-1α ubiquitination and degradation [PMID:36747531].","teleology":[{"year":2013,"claim":"Established that L3MBTL3 is a methyllysine reader, defining the biochemical activity underlying all its downstream roles and providing a chemical-genetic tool to probe it.","evidence":"X-ray crystallography of the L3MBTL3-UNC1215 complex, in vitro binding (Kd = 120 nM), point mutagenesis, and cellular probe pulldown of endogenous L3MBTL3","pmids":["23292653","24040942"],"confidence":"High","gaps":["Physiological methylated substrates were not yet identified at this stage","BCLAF1 interaction reported but not mechanistically pursued"]},{"year":2017,"claim":"Showed L3MBTL3 is a transcriptional corepressor that converts the reader activity into chromatin regulation, answering how RBPJ represses Notch targets in the absence of Notch signaling.","evidence":"Co-IP of RBPJ-L3MBTL3, competitive binding versus NOTCH ICD, H3K4me2 ChIP, and cross-species genetics in Drosophila and C. elegans","pmids":["29030483"],"confidence":"High","gaps":["Structural basis of the RBPJ interface not yet resolved","Whether MBT methyllysine reading is required for RBPJ corepression not delineated"]},{"year":2018,"claim":"Defined L3MBTL3 as a degradation adapter that reads methylated lysines on non-histone substrates and recruits CRL4DCAF5, linking lysine methylation to substrate proteostasis in development and pluripotency.","evidence":"Co-IP, site-directed mutagenesis of substrate lysines, in vivo ubiquitination, mouse knockout with DNA methylation/embryonic phenotype, and ES cell rescue assays for DNMT1, E2F1, and SOX2","pmids":["29691401","30442713"],"confidence":"High","gaps":["Full substrate repertoire beyond DNMT1, E2F1, and SOX2 not mapped","Determinants of substrate selectivity by L3MBTL3-CRL4DCAF5 not defined"]},{"year":2022,"claim":"Resolved the structural basis of corepression by showing L3MBTL3 binds RBPJ through an unusual motif distinct from other RBPJ partners.","evidence":"2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA ternary complex with structure-based mutagenesis and cellular validation","pmids":["36477367"],"confidence":"High","gaps":["How LSD1 recruitment is geometrically coordinated with the RBPJ interface not shown"]},{"year":2023,"claim":"Extended L3MBTL3's degradation role to hypoxia signaling, identifying a HIF-1α-induced negative feedback loop.","evidence":"Co-IP of L3MBTL3-HIF-1α, ubiquitination assay, and knockdown/overexpression under hypoxia","pmids":["36747531"],"confidence":"Medium","gaps":["No in vitro reconstitution or interface mutagenesis","Whether CRL4DCAF5 mediates HIF-1α degradation not established","Methyllysine dependence not tested"]},{"year":2025,"claim":"Demonstrated a methyllysine-independent function, showing L3MBTL3 can act through protein-protein interaction alone to drive oncogenic transcription.","evidence":"Co-IP and ChIP of L3MBTL3-STAT3 at the SNAIL promoter, Kme-binding mutant analysis, and metastasis assays in breast cancer","pmids":["39747894"],"confidence":"Medium","gaps":["Single-lab study","How L3MBTL3 promotes STAT3-driven transcription mechanistically is unclear"]},{"year":null,"claim":"It remains unknown what governs the choice between L3MBTL3's degradation-adapter, corepressor, and methyllysine-independent transcriptional outputs, and the breadth of its substrate and chromatin targets is incompletely mapped.","evidence":"","pmids":[],"confidence":"Low","gaps":["No unifying model for how reader activity is partitioned between proteolysis and corepression","Proposed role at DNA damage sites rests only on a preprint proximity-labeling screen","Engineered KRAB-L3MBTL3 silencing synergy lacks mechanistic dissection"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,2]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,7]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[3,4]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[3,4,6]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[6]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[2]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,7]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[3,4]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[2]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,6]}],"complexes":["CRL4DCAF5","RBPJ-L3MBTL3-DNA complex"],"partners":["RBPJ","DCAF5","DNMT1","SOX2","KDM1A","STAT3","HIF1A","E2F1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96JM7","full_name":"Lethal(3)malignant brain tumor-like protein 3","aliases":["MBT-1"],"length_aa":780,"mass_kda":88.3,"function":"Is a negative regulator of Notch target genes expression, required for RBPJ-mediated transcriptional repression (PubMed:29030483). It recruits KDM1A to Notch-responsive elements and promotes KDM1A-mediated H3K4me demethylation (PubMed:29030483). Involved in the regulation of ubiquitin-dependent degradation of a set of methylated non-histone proteins, including SOX2, DNMT1 and E2F1. It acts as an adapter recruiting the CRL4-DCAF5 E3 ubiquitin ligase complex to methylated target proteins (PubMed:29691401, PubMed:30442713). Required for normal maturation of myeloid progenitor cells (By similarity)","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/Q96JM7/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/L3MBTL3","classification":"Not Classified","n_dependent_lines":4,"n_total_lines":1208,"dependency_fraction":0.0033112582781456954},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CSNK2B","stoichiometry":0.2},{"gene":"HDAC1","stoichiometry":0.2},{"gene":"HDAC2","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/L3MBTL3","total_profiled":1310},"omim":[{"mim_id":"618844","title":"L3MBTL HISTONE METHYL-LYSINE-BINDING PROTEIN 3; L3MBTL3","url":"https://www.omim.org/entry/618844"},{"mim_id":"184429","title":"SRY-BOX 2; SOX2","url":"https://www.omim.org/entry/184429"},{"mim_id":"147183","title":"RECOMBINATION SIGNAL-BINDING PROTEIN FOR IMMUNOGLOBULIN KAPPA J REGION; RBPJ","url":"https://www.omim.org/entry/147183"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Nucleoli","reliability":"Supported"},{"location":"Vesicles","reliability":"Additional"},{"location":"Cytokinetic bridge","reliability":"Additional"},{"location":"Primary cilium","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/L3MBTL3"},"hgnc":{"alias_symbol":["KIAA1798"],"prev_symbol":[]},"alphafold":{"accession":"Q96JM7","domains":[{"cath_id":"-","chopping":"21-61","consensus_level":"high","plddt":81.2102,"start":21,"end":61},{"cath_id":"2.30.30.140","chopping":"261-363","consensus_level":"medium","plddt":96.0971,"start":261,"end":363},{"cath_id":"2.30.30.140","chopping":"380-425_427-462","consensus_level":"medium","plddt":95.0806,"start":380,"end":462},{"cath_id":"1.10.150.50","chopping":"687-778","consensus_level":"high","plddt":85.1634,"start":687,"end":778}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96JM7","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96JM7-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96JM7-F1-predicted_aligned_error_v6.png","plddt_mean":72.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=L3MBTL3","jax_strain_url":"https://www.jax.org/strain/search?query=L3MBTL3"},"sequence":{"accession":"Q96JM7","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96JM7.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96JM7/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96JM7"}},"corpus_meta":[{"pmid":"23292653","id":"PMC_23292653","title":"Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain.","date":"2013","source":"Nature chemical biology","url":"https://pubmed.ncbi.nlm.nih.gov/23292653","citation_count":129,"is_preprint":false},{"pmid":"29030483","id":"PMC_29030483","title":"RBPJ/CBF1 interacts with L3MBTL3/MBT1 to promote repression of Notch signaling via histone demethylase KDM1A/LSD1.","date":"2017","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/29030483","citation_count":60,"is_preprint":false},{"pmid":"29691401","id":"PMC_29691401","title":"Methylated DNMT1 and E2F1 are targeted for proteolysis by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.","date":"2018","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/29691401","citation_count":52,"is_preprint":false},{"pmid":"24040942","id":"PMC_24040942","title":"Small-molecule ligands of methyl-lysine binding proteins: optimization of selectivity for L3MBTL3.","date":"2013","source":"Journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24040942","citation_count":50,"is_preprint":false},{"pmid":"30442713","id":"PMC_30442713","title":"Proteolysis of methylated SOX2 protein is regulated by L3MBTL3 and CRL4DCAF5 ubiquitin ligase.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30442713","citation_count":39,"is_preprint":false},{"pmid":"39747894","id":"PMC_39747894","title":"L3MBTL3 and STAT3 collaboratively upregulate SNAIL expression to promote metastasis in female breast cancer.","date":"2025","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/39747894","citation_count":9,"is_preprint":false},{"pmid":"30456721","id":"PMC_30456721","title":"Association of SHMT1, MAZ, ERG, and L3MBTL3 Gene Polymorphisms with Susceptibility to Multiple Sclerosis.","date":"2018","source":"Biochemical genetics","url":"https://pubmed.ncbi.nlm.nih.gov/30456721","citation_count":9,"is_preprint":false},{"pmid":"36477367","id":"PMC_36477367","title":"The structure, binding and function of a Notch transcription complex involving RBPJ and the epigenetic reader protein L3MBTL3.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/36477367","citation_count":7,"is_preprint":false},{"pmid":"34373992","id":"PMC_34373992","title":"Effect of L3MBTL3/PTPN9 polymorphisms on risk to alcohol-induced ONFH in Chinese Han population.","date":"2021","source":"Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology","url":"https://pubmed.ncbi.nlm.nih.gov/34373992","citation_count":7,"is_preprint":false},{"pmid":"35088080","id":"PMC_35088080","title":"Identification of the genetic mechanism that associates L3MBTL3 to multiple sclerosis.","date":"2022","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35088080","citation_count":6,"is_preprint":false},{"pmid":"36747531","id":"PMC_36747531","title":"L3MBTL3 is induced by HIF-1α and fine tunes the HIF-1α degradation under hypoxia in vitro.","date":"2023","source":"Heliyon","url":"https://pubmed.ncbi.nlm.nih.gov/36747531","citation_count":5,"is_preprint":false},{"pmid":"37523690","id":"PMC_37523690","title":"Association of the L3MBTL3 rs1125970 and rs4897367 Gene Polymorphisms With Coronary Heart Disease Susceptibility in the Chinese Population: A Case-Control Study.","date":"2023","source":"Journal of cardiovascular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37523690","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.10.28.620683","title":"A combinatorial domain screening platform reveals epigenetic effector interactions for transcriptional perturbation","date":"2024-10-30","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.28.620683","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.10.23.619792","title":"Probing DNA damage sites reveals context-dependent and novel DNA damage response factors","date":"2024-10-25","source":"bioRxiv","url":"https://doi.org/10.1101/2024.10.23.619792","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.26.24310191","title":"Disentangling shared genetic etiologies for kidney function and cardiovascular diseases","date":"2024-07-27","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.26.24310191","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":9527,"output_tokens":3035,"usd":0.037053,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10298,"output_tokens":2841,"usd":0.061257,"stage2_stop_reason":"end_turn"},"total_usd":0.09831,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2013,\n      \"finding\": \"L3MBTL3 binds mono- or dimethyllysine-containing peptides via its MBT domains (methyllysine reader function). X-ray crystallography revealed a unique 2:2 polyvalent mode of interaction between the chemical probe UNC1215 and L3MBTL3, with UNC1215 binding the Kme-binding pocket. Point mutants disrupting the Kme-binding function of L3MBTL3 phenocopy probe effects on cellular localization. The Kme-dependent interaction with BCLAF1 (implicated in DNA damage repair and apoptosis) was identified using this probe.\",\n      \"method\": \"X-ray crystallography, competitive displacement assay (Kd = 120 nM), point mutagenesis, cellular FRAP/GFP-mobility assay, chemical probe (UNC1215) pulldown of endogenous L3MBTL3\",\n      \"journal\": \"Nature chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure, in vitro binding assay with mutagenesis, and cellular validation in a single rigorous study\",\n      \"pmids\": [\"23292653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Structure-activity relationship studies established molecular requirements for potent L3MBTL3 binding by small-molecule Kme antagonists, confirming that UNC1215 and related compounds interact with endogenous L3MBTL3 in cells via the mono-/dimethyllysine reading function of the MBT domains.\",\n      \"method\": \"Medicinal chemistry SAR, in vitro binding assays, cellular target engagement assays\",\n      \"journal\": \"Journal of medicinal chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple in vitro and cellular assays in single lab, confirms binding mechanism\",\n      \"pmids\": [\"24040942\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"L3MBTL3 acts as a transcriptional corepressor of Notch target genes by binding to the transcription factor RBPJ (CBF1/CSL) and competing with the Notch intracellular domain (NOTCH ICD) for RBPJ binding. In the absence of NOTCH ICD, RBPJ recruits L3MBTL3 and the histone demethylase KDM1A (LSD1) to enhancers of Notch target genes, leading to H3K4me2 demethylation and transcriptional repression. This functional link is evolutionarily conserved in Drosophila and C. elegans.\",\n      \"method\": \"Proteomic/co-immunoprecipitation identification of RBPJ-L3MBTL3 interaction, competitive binding assay (L3MBTL3 vs NOTCH ICD for RBPJ), chromatin immunoprecipitation (H3K4me2), in vivo genetic analyses in Drosophila and C. elegans\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP, competitive binding, and cross-species in vivo epistasis across multiple labs/organisms\",\n      \"pmids\": [\"29030483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"L3MBTL3 binds methylated lysine 142 of DNMT1 and recruits the CRL4DCAF5 ubiquitin E3 ligase complex to target DNMT1 for proteasomal degradation. LSD1 demethylates K142 to prevent this, while PHF20L1 also acts in S phase to protect DNMT1. Loss of L3MBTL3/MBT-1 in mice causes DNMT1 protein accumulation, increased genomic DNA methylation, and late embryonic lethality. L3MBTL3-CRL4DCAF5 similarly controls methylation-dependent degradation of E2F1.\",\n      \"method\": \"Co-immunoprecipitation, in vivo ubiquitination assay, mouse knockout (L3MBTL3/MBT-1 deletion), western blot for protein stability, genomic DNA methylation assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, in vivo ubiquitination, mouse KO with defined biochemical and developmental phenotype, multiple substrates tested\",\n      \"pmids\": [\"29691401\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"L3MBTL3 preferentially binds methylated lysine 42 of SOX2 (with partial contribution from methylated K117) and recruits the CRL4DCAF5 ubiquitin E3 ligase to promote ubiquitin-dependent SOX2 proteolysis. Knockdown of L3MBTL3 or DCAF5 restores SOX2 protein levels and rescues self-renewal/pluripotency defects in mouse ES cells caused by LSD1 or PHF20L1 deficiency.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (K42, K117), in vivo ubiquitination assay, siRNA knockdown, mouse ES cell self-renewal/pluripotency assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP with mutagenesis, in vivo ubiquitination, genetic rescue epistasis in ES cells, multiple orthogonal methods in single study\",\n      \"pmids\": [\"30442713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA ternary complex was determined. L3MBTL3 interacts with RBPJ via an unusual binding motif distinct from other RBPJ-binding partners. Structure-based mutations disrupting this interface impair RBPJ and L3MBTL3 function in cells, confirming the structural basis for L3MBTL3's role as a corepressor in Notch transcriptional regulation.\",\n      \"method\": \"X-ray crystallography (2.06 Å), structure-based mutagenesis, cellular functional assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure with comprehensive mutagenesis and cellular validation\",\n      \"pmids\": [\"36477367\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"L3MBTL3 is transcriptionally induced by HIF-1α under hypoxia. In the nucleus, L3MBTL3 interacts with HIF-1α and promotes HIF-1α ubiquitination and degradation, forming a negative feedback loop to dampen hypoxic response.\",\n      \"method\": \"Co-immunoprecipitation (L3MBTL3-HIF-1α interaction), ubiquitination assay, knockdown/overexpression in hypoxia, reporter/western blot assays\",\n      \"journal\": \"Heliyon\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Co-IP and ubiquitination assay without in vitro reconstitution or mutagenesis; mechanistic claim supported by two methods but from one study\",\n      \"pmids\": [\"36747531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"L3MBTL3 interacts with STAT3 and recruits STAT3 to the SNAIL promoter to increase SNAIL transcription, promoting epithelial-mesenchymal transition and metastasis in breast cancer. The methylated lysine binding activity of L3MBTL3 is not required for this function; only the L3MBTL3-STAT3 interaction is required for SNAIL upregulation.\",\n      \"method\": \"Co-immunoprecipitation, chromatin immunoprecipitation (L3MBTL3 and STAT3 at SNAIL promoter), Kme-binding mutant analysis, knockdown/overexpression with metastasis assays\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ChIP, and Kme-binding mutant, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"39747894\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"L3MBTL3 was identified as a novel effector of DNA damage response. Using proximity ligation (TurboID tethered to γH2AX via MCPH1-BRCT probe), L3MBTL3 was found in the DNA damage-associated proteome across multiple genotoxic insults, and characterized as a 'methyl-binding and proteasome-recruiting protein' at DNA damage sites.\",\n      \"method\": \"Proximity ligation (TurboID-based proteomics at γH2AX-marked DNA damage sites), functional characterization of DNA damage response\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single proteomics screen with limited mechanistic follow-up on L3MBTL3 specifically\",\n      \"pmids\": [\"bio_10.1101_2024.10.23.619792\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"A KRAB-L3MBTL3 fusion domain combination enhanced gene silencing up to 34-fold in dose-limited conditions compared to KRAB alone, demonstrating synergistic epigenetic repression activity when L3MBTL3 is fused to a KRAB domain in CRISPR-based epigenome editing.\",\n      \"method\": \"High-throughput combinatorial domain screening (COMBINE platform), endogenous gene transcription reporter assays\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, high-throughput functional screen but no mechanistic dissection of how L3MBTL3 contributes to the enhanced silencing\",\n      \"pmids\": [\"bio_10.1101_2024.10.28.620683\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"L3MBTL3 is a methyllysine reader protein (via its MBT domains) that functions as a transcriptional corepressor and proteolysis adapter: it binds mono-/dimethylated lysines on histones and non-histone proteins (DNMT1-K142me, SOX2-K42me, E2F1), recruiting the CRL4DCAF5 ubiquitin E3 ligase to degrade methylated substrates; it also binds RBPJ/CBF1 as a corepressor that recruits KDM1A/LSD1 to demethylate H3K4me2 at Notch target gene enhancers (structural basis defined by a 2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA complex); additionally, it interacts with STAT3 to drive SNAIL transcription in a Kme-independent manner, and forms a negative feedback loop with HIF-1α by promoting its ubiquitination and degradation under hypoxia.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"L3MBTL3 is a methyllysine-reader protein that couples recognition of mono-/dimethylated lysines to two distinct gene-regulatory outputs: targeted protein degradation and transcriptional corepression [#0, #3]. Through its MBT domains it engages mono- and dimethyllysine marks in a polyvalent binding mode, a function defined structurally and dissected with the chemical probe UNC1215 and Kme-binding point mutants [#0]. As a proteolysis adapter, L3MBTL3 reads methylated lysines on non-histone substrates and recruits the CRL4DCAF5 ubiquitin E3 ligase to drive their proteasomal degradation: it binds DNMT1-K142me to control DNMT1 turnover, genomic DNA methylation, and embryonic viability, and similarly targets methylated SOX2 (K42) to regulate embryonic stem cell self-renewal and pluripotency [#3, #4]. Independently of its degradation role, L3MBTL3 is a transcriptional corepressor of Notch target genes, binding RBPJ/CBF1 via an unusual interface and competing with the Notch intracellular domain to instead recruit the histone demethylase KDM1A/LSD1 and demethylate H3K4me2 at target enhancers, with the structural basis defined by a 2.06 Å RBPJ-L3MBTL3-DNA crystal structure [#2, #5]. L3MBTL3 also supports transcriptional and degradative functions that do not require its methyllysine-reading activity, including a methylation-independent interaction with STAT3 that activates SNAIL transcription to promote epithelial-mesenchymal transition [#7], and a hypoxia-induced negative feedback loop in which it promotes HIF-1α ubiquitination and degradation [#6].\",\n  \"teleology\": [\n    {\n      \"year\": 2013,\n      \"claim\": \"Established that L3MBTL3 is a methyllysine reader, defining the biochemical activity underlying all its downstream roles and providing a chemical-genetic tool to probe it.\",\n      \"evidence\": \"X-ray crystallography of the L3MBTL3-UNC1215 complex, in vitro binding (Kd = 120 nM), point mutagenesis, and cellular probe pulldown of endogenous L3MBTL3\",\n      \"pmids\": [\"23292653\", \"24040942\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological methylated substrates were not yet identified at this stage\", \"BCLAF1 interaction reported but not mechanistically pursued\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Showed L3MBTL3 is a transcriptional corepressor that converts the reader activity into chromatin regulation, answering how RBPJ represses Notch targets in the absence of Notch signaling.\",\n      \"evidence\": \"Co-IP of RBPJ-L3MBTL3, competitive binding versus NOTCH ICD, H3K4me2 ChIP, and cross-species genetics in Drosophila and C. elegans\",\n      \"pmids\": [\"29030483\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of the RBPJ interface not yet resolved\", \"Whether MBT methyllysine reading is required for RBPJ corepression not delineated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Defined L3MBTL3 as a degradation adapter that reads methylated lysines on non-histone substrates and recruits CRL4DCAF5, linking lysine methylation to substrate proteostasis in development and pluripotency.\",\n      \"evidence\": \"Co-IP, site-directed mutagenesis of substrate lysines, in vivo ubiquitination, mouse knockout with DNA methylation/embryonic phenotype, and ES cell rescue assays for DNMT1, E2F1, and SOX2\",\n      \"pmids\": [\"29691401\", \"30442713\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full substrate repertoire beyond DNMT1, E2F1, and SOX2 not mapped\", \"Determinants of substrate selectivity by L3MBTL3-CRL4DCAF5 not defined\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Resolved the structural basis of corepression by showing L3MBTL3 binds RBPJ through an unusual motif distinct from other RBPJ partners.\",\n      \"evidence\": \"2.06 Å crystal structure of the RBPJ-L3MBTL3-DNA ternary complex with structure-based mutagenesis and cellular validation\",\n      \"pmids\": [\"36477367\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How LSD1 recruitment is geometrically coordinated with the RBPJ interface not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Extended L3MBTL3's degradation role to hypoxia signaling, identifying a HIF-1α-induced negative feedback loop.\",\n      \"evidence\": \"Co-IP of L3MBTL3-HIF-1α, ubiquitination assay, and knockdown/overexpression under hypoxia\",\n      \"pmids\": [\"36747531\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No in vitro reconstitution or interface mutagenesis\", \"Whether CRL4DCAF5 mediates HIF-1α degradation not established\", \"Methyllysine dependence not tested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Demonstrated a methyllysine-independent function, showing L3MBTL3 can act through protein-protein interaction alone to drive oncogenic transcription.\",\n      \"evidence\": \"Co-IP and ChIP of L3MBTL3-STAT3 at the SNAIL promoter, Kme-binding mutant analysis, and metastasis assays in breast cancer\",\n      \"pmids\": [\"39747894\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"How L3MBTL3 promotes STAT3-driven transcription mechanistically is unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"It remains unknown what governs the choice between L3MBTL3's degradation-adapter, corepressor, and methyllysine-independent transcriptional outputs, and the breadth of its substrate and chromatin targets is incompletely mapped.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No unifying model for how reader activity is partitioned between proteolysis and corepression\", \"Proposed role at DNA damage sites rests only on a preprint proximity-labeling screen\", \"Engineered KRAB-L3MBTL3 silencing synergy lacks mechanistic dissection\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [3, 4, 6]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [6]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [2]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 7]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [3, 4]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [2]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 6]}\n    ],\n    \"complexes\": [\"CRL4DCAF5\", \"RBPJ-L3MBTL3-DNA complex\"],\n    \"partners\": [\"RBPJ\", \"DCAF5\", \"DNMT1\", \"SOX2\", \"KDM1A\", \"STAT3\", \"HIF1A\", \"E2F1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}