{"gene":"GRWD1","run_date":"2026-06-10T01:55:21","timeline":{"discoveries":[{"year":2015,"finding":"GRWD1 binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner; depletion of GRWD1 reduces MCM loading but not CDC6 or Cdt1 loading. Genome-wide ChIP-seq showed significant co-localization of GRWD1 with CDC6. GRWD1 possesses histone-binding activity and regulates chromatin openness at specific loci (by FAIRE-seq), facilitating MCM loading at replication origins.","method":"ChIP at replication origins, ChIP-seq, FAIRE-seq, FAIRE-qPCR, siRNA knockdown, histone-binding assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ChIP-seq, FAIRE-seq, histone binding, knockdown phenotype) in a single rigorous study establishing direct mechanism","pmids":["25990725"],"is_preprint":false},{"year":2016,"finding":"GRWD1 physically interacts with RPL11 (ribosomal protein L11). GRWD1 is localized to nucleoli and released into the nucleoplasm upon nucleolar stress. GRWD1 overexpression competitively inhibits the RPL11–MDM2 interaction and alleviates RPL11-mediated suppression of MDM2 ubiquitin ligase activity toward p53, thereby reducing p53 stability. The N-terminal acidic domain of GRWD1 mediates this interaction.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression, MDM2 ubiquitin ligase activity assay, immunofluorescence/subcellular fractionation","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, functional ubiquitin ligase assay, domain mapping, and rescue experiments in a single focused study","pmids":["27856536"],"is_preprint":false},{"year":2016,"finding":"GRWD1 promotes nucleosome disassembly in an ATP-independent manner, facilitating removal of H2A-H2B dimers to form hexasomes. The acidic domain of GRWD1 is required for efficient nucleosome disassembly (histone H2A-H2B eviction) but not for nucleosome assembly. In HeLa cells, the acidic domain is necessary for chromatin openness and efficient MCM loading at replication origins.","method":"In vitro reconstituted mononucleosome disassembly assay using recombinant histones, deletion mutagenesis, FAIRE-qPCR in HeLa cells","journal":"Biochimica et biophysica acta","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with deletion mutagenesis plus cellular FAIRE-qPCR validation in a single study","pmids":["27552915"],"is_preprint":false},{"year":2018,"finding":"GRWD1 interacts with RPL23 and with the E3 ubiquitin ligase EDD (UBR5). Co-expression of GRWD1 and EDD promotes RPL23 ubiquitylation and proteasomal degradation (rescued by MG132). GRWD1 knockdown upregulates RPL23. GRWD1-induced RPL23 proteolysis contributes to downregulation of p53 and promotes anchorage-independent growth.","method":"Proteomics/MS identification of interactors, Co-IP, ubiquitylation assay, proteasome inhibitor (MG132) rescue, siRNA knockdown, overexpression, colony formation assay","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 / Strong — MS interactome followed by reciprocal Co-IP, ubiquitylation assay, proteasome inhibitor rescue, and functional cellular readout in one study","pmids":["29991511"],"is_preprint":false},{"year":2020,"finding":"GRWD1 directly interacts with p53 via the p53 DNA-binding domain. Upon DNA damage, GRWD1 downregulation increases p21 expression. GRWD1 co-expression suppresses p53-regulated promoters (p21, MDM2) and these chromatin interactions require p53.","method":"Co-immunoprecipitation, ChIP at p21 and MDM2 promoters, siRNA knockdown, overexpression, reporter/promoter assays","journal":"Journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping and ChIP functional validation in a single lab study","pmids":["31545368"],"is_preprint":false},{"year":2004,"finding":"Yeast RRB1 (ortholog of human GRWD1) interacts with Yph1 (yeast pescadillo homologue), RPL3, ERB1, and ORC6, linking ribosome biogenesis to DNA replication. Inactivation of RRB1 in yeast alters chromosome segregation and blocks mitosis at the metaphase/anaphase transition. Transient depletion of the human homologue GRWD in human cells results in abnormal mitoses with binucleate/hyperploid cells, multipolar spindles, and aberrant metaphase plates.","method":"Yeast CIN indicator strain, two-hybrid/Co-IP interactions, siRNA knockdown in human cells, cell biology phenotyping (mitosis analysis)","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast genetics with direct interaction assays and human cell knockdown phenotype, two model systems, single lab","pmids":["15467761"],"is_preprint":false},{"year":2005,"finding":"GRWD1 co-sediments with preribosomal complexes and with Bop1 (a WD-repeat protein implicated in ribosome biogenesis) by nuclear fractionation. siRNA-mediated knockdown of GRWD1 decreases cellular proliferation and global protein synthesis (metabolic labeling).","method":"Nuclear fractionation/cosedimentation, siRNA knockdown, metabolic labeling of protein synthesis","journal":"Genomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct fractionation showing co-sedimentation with ribosomal complexes plus functional metabolic labeling readout, single lab","pmids":["15885502"],"is_preprint":false},{"year":2020,"finding":"The lncRNA PiHL promotes GRWD1 and RPL11 complex formation, which sequesters RPL11 from MDM2, thereby promoting p53 ubiquitination and reducing p53 stability in colorectal cancer cells.","method":"Co-IP (GRWD1–RPL11 complex), siRNA/overexpression, in vitro and in vivo tumor models","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — Co-IP demonstrating the GRWD1–RPL11 complex enhanced by PiHL, single lab","pmids":["31903119"],"is_preprint":false},{"year":2021,"finding":"GRWD1 interacts with WDR5 (core protein of H3K4 methyltransferase complex) and with MLL2 (H3K4me3 methyltransferase). GRWD1 knockdown causes global reduction of the H3K4me3 active histone mark in KSHV-transformed cells. ChIP-seq identified specific genomic loci where GRWD1, WDR5, and MLL2 co-regulate gene expression.","method":"CRISPR-Cas9 screen, Co-IP (GRWD1–WDR5–MLL2), ChIP-seq (H3K4me3), RNA-seq, siRNA knockdown, tumor formation assay","journal":"mBio","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP of the complex, genome-wide ChIP-seq, and functional tumor assay in a single study","pmids":["34933446"],"is_preprint":false},{"year":2026,"finding":"GRWD1 facilitates HSV-1 nuclear egress: GRWD1 knockdown traps nucleocapsids in the nucleus and suppresses viral replication. GRWD1 interacts with viral kinase US3 and partially colocalizes with UL34 (nuclear egress complex protein) at the nuclear membrane. GRWD1 promotes proteasomal degradation of Lamin A/C and directly binds Lamin A, suggesting it disrupts the nuclear lamina to facilitate capsid exit. GRWD1's pro-viral effect requires US3.","method":"siRNA knockdown, Co-IP (GRWD1–US3, GRWD1–Lamin A), immunofluorescence colocalization, western blot (Lamin A/C degradation), viral replication assay, US3-deficient infection","journal":"Microbiology spectrum","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, colocalization, knockdown, virus-deficient strain) in a single study","pmids":["41995456"],"is_preprint":false},{"year":2025,"finding":"Recurrent RPL3L variants associated with dilated cardiomyopathy exhibit increased affinity for GRWD1 (the RPL3/RPL3L chaperone), sequester 28S rRNA in the nucleus, disrupt ribosome biogenesis, and trigger cellular toxicity. This represents a gain-of-toxicity mechanism mediated by abnormal GRWD1–RPL3L interaction.","method":"Biochemical binding assay (increased affinity to GRWD1), nuclear rRNA localization, ribosome biogenesis assays, patient variant analysis","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, binding affinity assay without full mutagenesis or reconstitution validation","pmids":["bio_10.1101_2025.01.02.630345"],"is_preprint":true},{"year":2025,"finding":"GRWD1 is activated transcriptionally by the IL-6/STAT3 signaling pathway in colorectal cancer cells. GRWD1 then promotes degradation of p53 and upregulates GLUT1, facilitating aerobic glycolysis.","method":"IL-6 stimulation, STAT3 inhibition/activation, western blot, AOM/DSS mouse model, siRNA knockdown","journal":"Scientific reports","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, western blot-based pathway placement without direct binding or reconstitution assays","pmids":["41022893"],"is_preprint":false}],"current_model":"GRWD1 is a multifunctional WD40-repeat histone chaperone that (1) binds histones and disassembles nucleosomes (H2A-H2B eviction) at replication origins during G1 phase, thereby maintaining chromatin openness and facilitating MCM helicase loading in a Cdt1/CDC6-dependent manner; (2) negatively regulates the tumor suppressor p53 through at least three mechanisms — competing with MDM2 for RPL11 binding (preventing RPL11-mediated MDM2 inhibition), promoting EDD/UBR5-dependent ubiquitylation and proteasomal degradation of RPL23 (another MDM2 inhibitor), and directly binding the p53 DNA-binding domain to suppress its transcriptional activity; (3) resides in nucleoli and is released to the nucleoplasm under nucleolar stress; (4) forms a complex with WDR5 and MLL2 to regulate H3K4me3 methylation at specific genomic loci; and (5) facilitates HSV-1 nuclear egress by interacting with viral kinase US3, binding Lamin A, and promoting Lamin A/C proteasomal degradation to disrupt the nuclear lamina."},"narrative":{"mechanistic_narrative":"GRWD1 is a WD40-repeat protein that couples chromatin regulation, ribosome biogenesis, and p53 control to influence DNA replication licensing and cell proliferation [PMID:25990725, PMID:15885502]. At chromatin, GRWD1 binds histones and disassembles nucleosomes in an ATP-independent manner, evicting H2A-H2B dimers to form hexasomes through its N-terminal acidic domain; this activity maintains chromatin openness at replication origins and facilitates MCM helicase loading during G1 in a CDC6- and Cdt1-dependent manner [PMID:25990725, PMID:27552915]. GRWD1 localizes to nucleoli and is released to the nucleoplasm under nucleolar stress, where it negatively regulates the tumor suppressor p53 by multiple routes: it competes with MDM2 for binding to RPL11, relieving RPL11-mediated suppression of MDM2 ubiquitin ligase activity [PMID:27856536]; it cooperates with the E3 ligase EDD/UBR5 to drive ubiquitylation and proteasomal degradation of RPL23, removing another MDM2 inhibitor and promoting anchorage-independent growth [PMID:29991511]; and it directly binds the p53 DNA-binding domain to suppress transcription from p53 target promoters including p21 and MDM2 [PMID:31545368]. GRWD1 also associates with the H3K4 methyltransferase machinery via WDR5 and MLL2 to maintain H3K4me3 at specific loci [PMID:34933446], and it participates in HSV-1 nuclear egress through interaction with the viral kinase US3 and promotion of Lamin A/C degradation [PMID:41995456]. Its broader role in chromosome segregation and ribosome biogenesis is supported by genetic and biochemical evidence from the yeast ortholog RRB1 and human knockdown phenotypes [PMID:15467761, PMID:15885502].","teleology":[{"year":2004,"claim":"Established the first functional link between GRWD1 and the coordination of ribosome biogenesis with genome maintenance, situating the protein at the interface of these processes.","evidence":"Yeast RRB1 interaction assays (Yph1, RPL3, ERB1, ORC6) and human GRWD1 knockdown mitosis phenotyping","pmids":["15467761"],"confidence":"Medium","gaps":["Did not define the biochemical activity of GRWD1 on chromatin or replication origins","Mitotic defects not mechanistically connected to a specific molecular substrate"]},{"year":2005,"claim":"Showed GRWD1 physically co-sediments with preribosomal complexes and is required for proliferation and protein synthesis, supporting a role in ribosome biogenesis.","evidence":"Nuclear fractionation/cosedimentation with Bop1, siRNA knockdown, metabolic labeling of protein synthesis","pmids":["15885502"],"confidence":"Medium","gaps":["Co-sedimentation does not establish direct binding to ribosomal subunits","Did not separate ribosome biogenesis effects from replication or p53 roles"]},{"year":2015,"claim":"Defined GRWD1 as a replication-licensing factor that binds origins in G1 and promotes MCM loading by regulating chromatin openness, answering how it influences DNA replication.","evidence":"ChIP/ChIP-seq at origins, FAIRE-seq, histone-binding assay, siRNA knockdown in human cells","pmids":["25990725"],"confidence":"High","gaps":["Mechanism of how histone binding produces chromatin openness not yet resolved at this stage","Genome-wide origin specificity determinants unknown"]},{"year":2016,"claim":"Determined the biochemical basis of GRWD1's chromatin activity, showing it is an ATP-independent nucleosome disassembly factor whose acidic domain evicts H2A-H2B.","evidence":"In vitro reconstituted mononucleosome disassembly with recombinant histones, deletion mutagenesis, FAIRE-qPCR","pmids":["27552915"],"confidence":"High","gaps":["Does not explain how disassembly is targeted to specific origins in vivo","Fate of evicted H2A-H2B and reassembly partners undefined"]},{"year":2016,"claim":"Connected GRWD1 to p53 control via the ribosomal stress pathway, showing it competes with MDM2 for RPL11 to reduce p53 stability and responds to nucleolar stress.","evidence":"Reciprocal Co-IP, MDM2 ubiquitin ligase assay, domain mapping, subcellular fractionation/immunofluorescence","pmids":["27856536"],"confidence":"High","gaps":["Did not address whether GRWD1 acts on p53 through additional routes","In vivo tumor relevance not tested"]},{"year":2018,"claim":"Identified a second p53-suppressing mechanism in which GRWD1 recruits EDD/UBR5 to degrade RPL23, linking GRWD1 to oncogenic growth.","evidence":"MS interactome, reciprocal Co-IP, ubiquitylation assay, MG132 rescue, colony formation assay","pmids":["29991511"],"confidence":"High","gaps":["Relative contribution of RPL23 versus RPL11 routes to p53 control unquantified","Structural basis of GRWD1-EDD-RPL23 assembly unknown"]},{"year":2020,"claim":"Demonstrated a third, direct mode of p53 inhibition by GRWD1 binding the p53 DNA-binding domain and suppressing target promoters.","evidence":"Co-IP with domain mapping, ChIP at p21/MDM2 promoters, reporter assays, siRNA knockdown","pmids":["31545368"],"confidence":"Medium","gaps":["Single-lab study without reciprocal structural validation","Interplay between direct binding and ribosomal-protein-mediated routes not resolved"]},{"year":2020,"claim":"Placed GRWD1 downstream of a lncRNA regulator, showing PiHL enhances GRWD1-RPL11 complex formation to suppress p53 in colorectal cancer.","evidence":"Co-IP, siRNA/overexpression, in vitro and in vivo tumor models","pmids":["31903119"],"confidence":"Medium","gaps":["How PiHL physically promotes complex formation not mechanistically defined","Generality beyond colorectal cancer untested"]},{"year":2021,"claim":"Expanded GRWD1's chromatin role beyond replication, showing it associates with WDR5/MLL2 to maintain the H3K4me3 active mark at specific loci.","evidence":"CRISPR screen, Co-IP of GRWD1-WDR5-MLL2, H3K4me3 ChIP-seq, RNA-seq, tumor formation assay","pmids":["34933446"],"confidence":"Medium","gaps":["Stoichiometry and architecture of the GRWD1-WDR5-MLL2 complex unknown","Relationship between methyltransferase role and nucleosome disassembly activity unresolved"]},{"year":2026,"claim":"Revealed a host-pathogen role in which GRWD1 facilitates HSV-1 nuclear egress by degrading Lamin A/C in a US3-dependent manner.","evidence":"siRNA knockdown, Co-IP (US3, Lamin A), immunofluorescence colocalization with UL34, Lamin A/C degradation blots, US3-deficient infection, viral replication assay","pmids":["41995456"],"confidence":"Medium","gaps":["Mechanism by which GRWD1 promotes Lamin A/C proteasomal degradation undefined","Whether nuclear lamina disruption is direct or via recruited ligases unknown"]},{"year":2025,"claim":"Linked GRWD1 to disease through abnormal interaction with cardiomyopathy-associated RPL3L variants, proposing a gain-of-toxicity ribosome biogenesis defect.","evidence":"Binding affinity assay, nuclear rRNA localization, ribosome biogenesis assays, patient variant analysis (preprint)","pmids":["bio_10.1101_2025.01.02.630345"],"confidence":"Low","gaps":["Preprint with binding affinity data lacking mutagenesis or reconstitution validation","Causal role of GRWD1 in the disease phenotype not established"]},{"year":2025,"claim":"Positioned GRWD1 downstream of IL-6/STAT3 signaling driving p53 degradation and aerobic glycolysis in colorectal cancer.","evidence":"IL-6 stimulation, STAT3 modulation, western blot, AOM/DSS mouse model, siRNA knockdown","pmids":["41022893"],"confidence":"Low","gaps":["Pathway placement based on western blot without direct binding or reconstitution","Mechanism linking GRWD1 to GLUT1 upregulation undefined"]},{"year":null,"claim":"How GRWD1's distinct activities — origin licensing, nucleosome disassembly, p53 suppression, H3K4me3 maintenance, ribosome biogenesis — are coordinated and regulated within a single protein remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model integrating the WD40 repeats and N-terminal acidic domain functions","Determinants of nucleolar versus chromatin partitioning unknown","Whether the multiple p53-suppressing routes operate simultaneously or context-specifically is undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,2]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[3,9]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[1,4]}],"localization":[{"term_id":"GO:0005730","term_label":"nucleolus","supporting_discovery_ids":[1,6]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[1]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[0]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[5,6]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[2,8]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[1,3,4]}],"complexes":["GRWD1-WDR5-MLL2 H3K4 methyltransferase complex","preribosomal complex"],"partners":["RPL11","RPL23","UBR5","TP53","WDR5","MLL2","US3","LMNA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9BQ67","full_name":"Glutamate-rich WD repeat-containing protein 1","aliases":[],"length_aa":446,"mass_kda":49.4,"function":"Histone binding-protein that regulates chromatin dynamics and loading of minichromosome maintenance (MCM) complex at replication origins, possibly by promoting chromatin openness (PubMed:25990725). Plays a role in ribosomal biogenesis (PubMed:15885502)","subcellular_location":"Nucleus, nucleolus; Nucleus; Chromosome","url":"https://www.uniprot.org/uniprotkb/Q9BQ67/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/GRWD1","classification":"Common Essential","n_dependent_lines":1120,"n_total_lines":1208,"dependency_fraction":0.9271523178807947},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"ATL3","stoichiometry":0.2},{"gene":"IPO7","stoichiometry":0.2},{"gene":"NPM1","stoichiometry":0.2},{"gene":"SEC13","stoichiometry":0.2},{"gene":"SPTLC1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/GRWD1","total_profiled":1310},"omim":[{"mim_id":"621160","title":"DIARRHEA 14, CONGENITAL; DIAR14","url":"https://www.omim.org/entry/621160"},{"mim_id":"619494","title":"SMALL NUCLEOLAR RNA HOST GENE 11, NONCODING; SNHG11","url":"https://www.omim.org/entry/619494"},{"mim_id":"615255","title":"METHYLTRANSFERASE-LIKE 18; METTL18","url":"https://www.omim.org/entry/615255"},{"mim_id":"610597","title":"GLUTAMATE-RICH WD REPEAT-CONTAINING PROTEIN 1; GRWD1","url":"https://www.omim.org/entry/610597"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoli","reliability":"Enhanced"},{"location":"Nucleoli rim","reliability":"Enhanced"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/GRWD1"},"hgnc":{"alias_symbol":["WDR28","GRWD","RRB1"],"prev_symbol":[]},"alphafold":{"accession":"Q9BQ67","domains":[{"cath_id":"-","chopping":"56-116_137-173_175-211_423-446","consensus_level":"medium","plddt":96.5039,"start":56,"end":446}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BQ67","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BQ67-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BQ67-F1-predicted_aligned_error_v6.png","plddt_mean":88.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=GRWD1","jax_strain_url":"https://www.jax.org/strain/search?query=GRWD1"},"sequence":{"accession":"Q9BQ67","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BQ67.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BQ67/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BQ67"}},"corpus_meta":[{"pmid":"9271385","id":"PMC_9271385","title":"RRB1 and RRB2 encode maize retinoblastoma-related proteins that interact with a plant D-type cyclin and geminivirus replication protein.","date":"1997","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/9271385","citation_count":168,"is_preprint":false},{"pmid":"15467761","id":"PMC_15467761","title":"Inactivation of the RRB1-Pescadillo pathway involved in ribosome biogenesis induces chromosomal instability.","date":"2004","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/15467761","citation_count":58,"is_preprint":false},{"pmid":"31903119","id":"PMC_31903119","title":"Long noncoding RNA PiHL regulates p53 protein stability through GRWD1/RPL11/MDM2 axis in colorectal cancer.","date":"2020","source":"Theranostics","url":"https://pubmed.ncbi.nlm.nih.gov/31903119","citation_count":57,"is_preprint":false},{"pmid":"25990725","id":"PMC_25990725","title":"Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its 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Factors","date":"2025-09-12","source":"bioRxiv","url":"https://doi.org/10.1101/2025.09.07.674761","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":12768,"output_tokens":3361,"usd":0.04436,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10877,"output_tokens":4129,"usd":0.078805,"stage2_stop_reason":"end_turn"},"total_usd":0.123165,"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\": 2015,\n      \"finding\": \"GRWD1 binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner; depletion of GRWD1 reduces MCM loading but not CDC6 or Cdt1 loading. Genome-wide ChIP-seq showed significant co-localization of GRWD1 with CDC6. GRWD1 possesses histone-binding activity and regulates chromatin openness at specific loci (by FAIRE-seq), facilitating MCM loading at replication origins.\",\n      \"method\": \"ChIP at replication origins, ChIP-seq, FAIRE-seq, FAIRE-qPCR, siRNA knockdown, histone-binding assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ChIP-seq, FAIRE-seq, histone binding, knockdown phenotype) in a single rigorous study establishing direct mechanism\",\n      \"pmids\": [\"25990725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GRWD1 physically interacts with RPL11 (ribosomal protein L11). GRWD1 is localized to nucleoli and released into the nucleoplasm upon nucleolar stress. GRWD1 overexpression competitively inhibits the RPL11–MDM2 interaction and alleviates RPL11-mediated suppression of MDM2 ubiquitin ligase activity toward p53, thereby reducing p53 stability. The N-terminal acidic domain of GRWD1 mediates this interaction.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression, MDM2 ubiquitin ligase activity assay, immunofluorescence/subcellular fractionation\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, functional ubiquitin ligase assay, domain mapping, and rescue experiments in a single focused study\",\n      \"pmids\": [\"27856536\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"GRWD1 promotes nucleosome disassembly in an ATP-independent manner, facilitating removal of H2A-H2B dimers to form hexasomes. The acidic domain of GRWD1 is required for efficient nucleosome disassembly (histone H2A-H2B eviction) but not for nucleosome assembly. In HeLa cells, the acidic domain is necessary for chromatin openness and efficient MCM loading at replication origins.\",\n      \"method\": \"In vitro reconstituted mononucleosome disassembly assay using recombinant histones, deletion mutagenesis, FAIRE-qPCR in HeLa cells\",\n      \"journal\": \"Biochimica et biophysica acta\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with deletion mutagenesis plus cellular FAIRE-qPCR validation in a single study\",\n      \"pmids\": [\"27552915\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GRWD1 interacts with RPL23 and with the E3 ubiquitin ligase EDD (UBR5). Co-expression of GRWD1 and EDD promotes RPL23 ubiquitylation and proteasomal degradation (rescued by MG132). GRWD1 knockdown upregulates RPL23. GRWD1-induced RPL23 proteolysis contributes to downregulation of p53 and promotes anchorage-independent growth.\",\n      \"method\": \"Proteomics/MS identification of interactors, Co-IP, ubiquitylation assay, proteasome inhibitor (MG132) rescue, siRNA knockdown, overexpression, colony formation assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — MS interactome followed by reciprocal Co-IP, ubiquitylation assay, proteasome inhibitor rescue, and functional cellular readout in one study\",\n      \"pmids\": [\"29991511\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"GRWD1 directly interacts with p53 via the p53 DNA-binding domain. Upon DNA damage, GRWD1 downregulation increases p21 expression. GRWD1 co-expression suppresses p53-regulated promoters (p21, MDM2) and these chromatin interactions require p53.\",\n      \"method\": \"Co-immunoprecipitation, ChIP at p21 and MDM2 promoters, siRNA knockdown, overexpression, reporter/promoter assays\",\n      \"journal\": \"Journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping and ChIP functional validation in a single lab study\",\n      \"pmids\": [\"31545368\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Yeast RRB1 (ortholog of human GRWD1) interacts with Yph1 (yeast pescadillo homologue), RPL3, ERB1, and ORC6, linking ribosome biogenesis to DNA replication. Inactivation of RRB1 in yeast alters chromosome segregation and blocks mitosis at the metaphase/anaphase transition. Transient depletion of the human homologue GRWD in human cells results in abnormal mitoses with binucleate/hyperploid cells, multipolar spindles, and aberrant metaphase plates.\",\n      \"method\": \"Yeast CIN indicator strain, two-hybrid/Co-IP interactions, siRNA knockdown in human cells, cell biology phenotyping (mitosis analysis)\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast genetics with direct interaction assays and human cell knockdown phenotype, two model systems, single lab\",\n      \"pmids\": [\"15467761\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"GRWD1 co-sediments with preribosomal complexes and with Bop1 (a WD-repeat protein implicated in ribosome biogenesis) by nuclear fractionation. siRNA-mediated knockdown of GRWD1 decreases cellular proliferation and global protein synthesis (metabolic labeling).\",\n      \"method\": \"Nuclear fractionation/cosedimentation, siRNA knockdown, metabolic labeling of protein synthesis\",\n      \"journal\": \"Genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct fractionation showing co-sedimentation with ribosomal complexes plus functional metabolic labeling readout, single lab\",\n      \"pmids\": [\"15885502\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The lncRNA PiHL promotes GRWD1 and RPL11 complex formation, which sequesters RPL11 from MDM2, thereby promoting p53 ubiquitination and reducing p53 stability in colorectal cancer cells.\",\n      \"method\": \"Co-IP (GRWD1–RPL11 complex), siRNA/overexpression, in vitro and in vivo tumor models\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — Co-IP demonstrating the GRWD1–RPL11 complex enhanced by PiHL, single lab\",\n      \"pmids\": [\"31903119\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"GRWD1 interacts with WDR5 (core protein of H3K4 methyltransferase complex) and with MLL2 (H3K4me3 methyltransferase). GRWD1 knockdown causes global reduction of the H3K4me3 active histone mark in KSHV-transformed cells. ChIP-seq identified specific genomic loci where GRWD1, WDR5, and MLL2 co-regulate gene expression.\",\n      \"method\": \"CRISPR-Cas9 screen, Co-IP (GRWD1–WDR5–MLL2), ChIP-seq (H3K4me3), RNA-seq, siRNA knockdown, tumor formation assay\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP of the complex, genome-wide ChIP-seq, and functional tumor assay in a single study\",\n      \"pmids\": [\"34933446\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"GRWD1 facilitates HSV-1 nuclear egress: GRWD1 knockdown traps nucleocapsids in the nucleus and suppresses viral replication. GRWD1 interacts with viral kinase US3 and partially colocalizes with UL34 (nuclear egress complex protein) at the nuclear membrane. GRWD1 promotes proteasomal degradation of Lamin A/C and directly binds Lamin A, suggesting it disrupts the nuclear lamina to facilitate capsid exit. GRWD1's pro-viral effect requires US3.\",\n      \"method\": \"siRNA knockdown, Co-IP (GRWD1–US3, GRWD1–Lamin A), immunofluorescence colocalization, western blot (Lamin A/C degradation), viral replication assay, US3-deficient infection\",\n      \"journal\": \"Microbiology spectrum\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, colocalization, knockdown, virus-deficient strain) in a single study\",\n      \"pmids\": [\"41995456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Recurrent RPL3L variants associated with dilated cardiomyopathy exhibit increased affinity for GRWD1 (the RPL3/RPL3L chaperone), sequester 28S rRNA in the nucleus, disrupt ribosome biogenesis, and trigger cellular toxicity. This represents a gain-of-toxicity mechanism mediated by abnormal GRWD1–RPL3L interaction.\",\n      \"method\": \"Biochemical binding assay (increased affinity to GRWD1), nuclear rRNA localization, ribosome biogenesis assays, patient variant analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, binding affinity assay without full mutagenesis or reconstitution validation\",\n      \"pmids\": [\"bio_10.1101_2025.01.02.630345\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GRWD1 is activated transcriptionally by the IL-6/STAT3 signaling pathway in colorectal cancer cells. GRWD1 then promotes degradation of p53 and upregulates GLUT1, facilitating aerobic glycolysis.\",\n      \"method\": \"IL-6 stimulation, STAT3 inhibition/activation, western blot, AOM/DSS mouse model, siRNA knockdown\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, western blot-based pathway placement without direct binding or reconstitution assays\",\n      \"pmids\": [\"41022893\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"GRWD1 is a multifunctional WD40-repeat histone chaperone that (1) binds histones and disassembles nucleosomes (H2A-H2B eviction) at replication origins during G1 phase, thereby maintaining chromatin openness and facilitating MCM helicase loading in a Cdt1/CDC6-dependent manner; (2) negatively regulates the tumor suppressor p53 through at least three mechanisms — competing with MDM2 for RPL11 binding (preventing RPL11-mediated MDM2 inhibition), promoting EDD/UBR5-dependent ubiquitylation and proteasomal degradation of RPL23 (another MDM2 inhibitor), and directly binding the p53 DNA-binding domain to suppress its transcriptional activity; (3) resides in nucleoli and is released to the nucleoplasm under nucleolar stress; (4) forms a complex with WDR5 and MLL2 to regulate H3K4me3 methylation at specific genomic loci; and (5) facilitates HSV-1 nuclear egress by interacting with viral kinase US3, binding Lamin A, and promoting Lamin A/C proteasomal degradation to disrupt the nuclear lamina.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"GRWD1 is a WD40-repeat protein that couples chromatin regulation, ribosome biogenesis, and p53 control to influence DNA replication licensing and cell proliferation [#0, #6]. At chromatin, GRWD1 binds histones and disassembles nucleosomes in an ATP-independent manner, evicting H2A-H2B dimers to form hexasomes through its N-terminal acidic domain; this activity maintains chromatin openness at replication origins and facilitates MCM helicase loading during G1 in a CDC6- and Cdt1-dependent manner [#0, #2]. GRWD1 localizes to nucleoli and is released to the nucleoplasm under nucleolar stress, where it negatively regulates the tumor suppressor p53 by multiple routes: it competes with MDM2 for binding to RPL11, relieving RPL11-mediated suppression of MDM2 ubiquitin ligase activity [#1]; it cooperates with the E3 ligase EDD/UBR5 to drive ubiquitylation and proteasomal degradation of RPL23, removing another MDM2 inhibitor and promoting anchorage-independent growth [#3]; and it directly binds the p53 DNA-binding domain to suppress transcription from p53 target promoters including p21 and MDM2 [#4]. GRWD1 also associates with the H3K4 methyltransferase machinery via WDR5 and MLL2 to maintain H3K4me3 at specific loci [#8], and it participates in HSV-1 nuclear egress through interaction with the viral kinase US3 and promotion of Lamin A/C degradation [#9]. Its broader role in chromosome segregation and ribosome biogenesis is supported by genetic and biochemical evidence from the yeast ortholog RRB1 and human knockdown phenotypes [#5, #6].\",\n  \"teleology\": [\n    {\n      \"year\": 2004,\n      \"claim\": \"Established the first functional link between GRWD1 and the coordination of ribosome biogenesis with genome maintenance, situating the protein at the interface of these processes.\",\n      \"evidence\": \"Yeast RRB1 interaction assays (Yph1, RPL3, ERB1, ORC6) and human GRWD1 knockdown mitosis phenotyping\",\n      \"pmids\": [\"15467761\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not define the biochemical activity of GRWD1 on chromatin or replication origins\", \"Mitotic defects not mechanistically connected to a specific molecular substrate\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showed GRWD1 physically co-sediments with preribosomal complexes and is required for proliferation and protein synthesis, supporting a role in ribosome biogenesis.\",\n      \"evidence\": \"Nuclear fractionation/cosedimentation with Bop1, siRNA knockdown, metabolic labeling of protein synthesis\",\n      \"pmids\": [\"15885502\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Co-sedimentation does not establish direct binding to ribosomal subunits\", \"Did not separate ribosome biogenesis effects from replication or p53 roles\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined GRWD1 as a replication-licensing factor that binds origins in G1 and promotes MCM loading by regulating chromatin openness, answering how it influences DNA replication.\",\n      \"evidence\": \"ChIP/ChIP-seq at origins, FAIRE-seq, histone-binding assay, siRNA knockdown in human cells\",\n      \"pmids\": [\"25990725\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of how histone binding produces chromatin openness not yet resolved at this stage\", \"Genome-wide origin specificity determinants unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Determined the biochemical basis of GRWD1's chromatin activity, showing it is an ATP-independent nucleosome disassembly factor whose acidic domain evicts H2A-H2B.\",\n      \"evidence\": \"In vitro reconstituted mononucleosome disassembly with recombinant histones, deletion mutagenesis, FAIRE-qPCR\",\n      \"pmids\": [\"27552915\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not explain how disassembly is targeted to specific origins in vivo\", \"Fate of evicted H2A-H2B and reassembly partners undefined\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Connected GRWD1 to p53 control via the ribosomal stress pathway, showing it competes with MDM2 for RPL11 to reduce p53 stability and responds to nucleolar stress.\",\n      \"evidence\": \"Reciprocal Co-IP, MDM2 ubiquitin ligase assay, domain mapping, subcellular fractionation/immunofluorescence\",\n      \"pmids\": [\"27856536\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address whether GRWD1 acts on p53 through additional routes\", \"In vivo tumor relevance not tested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a second p53-suppressing mechanism in which GRWD1 recruits EDD/UBR5 to degrade RPL23, linking GRWD1 to oncogenic growth.\",\n      \"evidence\": \"MS interactome, reciprocal Co-IP, ubiquitylation assay, MG132 rescue, colony formation assay\",\n      \"pmids\": [\"29991511\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of RPL23 versus RPL11 routes to p53 control unquantified\", \"Structural basis of GRWD1-EDD-RPL23 assembly unknown\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Demonstrated a third, direct mode of p53 inhibition by GRWD1 binding the p53 DNA-binding domain and suppressing target promoters.\",\n      \"evidence\": \"Co-IP with domain mapping, ChIP at p21/MDM2 promoters, reporter assays, siRNA knockdown\",\n      \"pmids\": [\"31545368\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study without reciprocal structural validation\", \"Interplay between direct binding and ribosomal-protein-mediated routes not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Placed GRWD1 downstream of a lncRNA regulator, showing PiHL enhances GRWD1-RPL11 complex formation to suppress p53 in colorectal cancer.\",\n      \"evidence\": \"Co-IP, siRNA/overexpression, in vitro and in vivo tumor models\",\n      \"pmids\": [\"31903119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How PiHL physically promotes complex formation not mechanistically defined\", \"Generality beyond colorectal cancer untested\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Expanded GRWD1's chromatin role beyond replication, showing it associates with WDR5/MLL2 to maintain the H3K4me3 active mark at specific loci.\",\n      \"evidence\": \"CRISPR screen, Co-IP of GRWD1-WDR5-MLL2, H3K4me3 ChIP-seq, RNA-seq, tumor formation assay\",\n      \"pmids\": [\"34933446\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Stoichiometry and architecture of the GRWD1-WDR5-MLL2 complex unknown\", \"Relationship between methyltransferase role and nucleosome disassembly activity unresolved\"]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"Revealed a host-pathogen role in which GRWD1 facilitates HSV-1 nuclear egress by degrading Lamin A/C in a US3-dependent manner.\",\n      \"evidence\": \"siRNA knockdown, Co-IP (US3, Lamin A), immunofluorescence colocalization with UL34, Lamin A/C degradation blots, US3-deficient infection, viral replication assay\",\n      \"pmids\": [\"41995456\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which GRWD1 promotes Lamin A/C proteasomal degradation undefined\", \"Whether nuclear lamina disruption is direct or via recruited ligases unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Linked GRWD1 to disease through abnormal interaction with cardiomyopathy-associated RPL3L variants, proposing a gain-of-toxicity ribosome biogenesis defect.\",\n      \"evidence\": \"Binding affinity assay, nuclear rRNA localization, ribosome biogenesis assays, patient variant analysis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.01.02.630345\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint with binding affinity data lacking mutagenesis or reconstitution validation\", \"Causal role of GRWD1 in the disease phenotype not established\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Positioned GRWD1 downstream of IL-6/STAT3 signaling driving p53 degradation and aerobic glycolysis in colorectal cancer.\",\n      \"evidence\": \"IL-6 stimulation, STAT3 modulation, western blot, AOM/DSS mouse model, siRNA knockdown\",\n      \"pmids\": [\"41022893\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Pathway placement based on western blot without direct binding or reconstitution\", \"Mechanism linking GRWD1 to GLUT1 upregulation undefined\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How GRWD1's distinct activities — origin licensing, nucleosome disassembly, p53 suppression, H3K4me3 maintenance, ribosome biogenesis — are coordinated and regulated within a single protein remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model integrating the WD40 repeats and N-terminal acidic domain functions\", \"Determinants of nucleolar versus chromatin partitioning unknown\", \"Whether the multiple p53-suppressing routes operate simultaneously or context-specifically is undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 2]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [3, 9]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [1, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005730\", \"supporting_discovery_ids\": [1, 6]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [1]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [5, 6]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [2, 8]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [1, 3, 4]}\n    ],\n    \"complexes\": [\"GRWD1-WDR5-MLL2 H3K4 methyltransferase complex\", \"preribosomal complex\"],\n    \"partners\": [\"RPL11\", \"RPL23\", \"UBR5\", \"TP53\", \"WDR5\", \"MLL2\", \"US3\", \"LMNA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}