{"gene":"POLR2M","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2012,"finding":"Gdown1 specifically blocks elongation stimulation by TFIIF in vitro, inhibits the termination activity of TTF2, and stabilizes promoter-proximally paused Pol II in the presence of nuclear extract. P-TEFb can overcome the Gdown1-mediated block, and ChIP-Seq confirms Gdown1 co-localizes with essentially all poised Pol II across the human genome.","method":"In vitro transcription assays, ChIP-Seq","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal in vitro assays combined with genome-wide ChIP-Seq; replicated in a companion paper from an independent lab (PMID:22244332)","pmids":["22244331"],"is_preprint":false},{"year":2012,"finding":"Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, inhibiting TFIIF function in preinitiation complex assembly. Mediator can facilitate Pol II(G) binding to promoters and overcome the Gdown1-mediated block, establishing Pol II(G) as a Mediator-dependent form of Pol II.","method":"In vitro transcription with purified factors, ChIP, RNAi","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 1-2 / Strong — multiple orthogonal methods (biochemical competition, ChIP, RNAi), replicated independently by PMID:22244331","pmids":["22244332"],"is_preprint":false},{"year":2012,"finding":"Cryo-EM maps of RNAPII(G) at ~19 Å localized Gdown1 primarily to the RPB5 shelf–RPB1 jaw region of Pol II, with Gdown1 binding sites overlapping extensively with TFIIF binding sites. Competition assays by size exclusion chromatography confirmed that Gdown1 sterically excludes TFIIF from RNAPII.","method":"Single-particle cryo-EM, antibody labeling, size exclusion chromatography competition assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structural mapping combined with orthogonal biochemical competition assays in one study","pmids":["22850672"],"is_preprint":false},{"year":2014,"finding":"Two functional domains of Gdown1 were identified: the N-terminal domain is responsible for blocking TTF2 (via a conserved LPDKG motif) and the C-terminal domain blocks TFIIF. Both domains are required for tight Pol II association. Gdown1 is phosphorylated at Ser-270 during mitosis, which reduces its affinity for Pol II and consequently reduces its ability to block both TTF2 and TFIIF. An S270A mutation abolishes this phosphorylation and S270E partially mimics phospho-Gdown1.","method":"In vitro transcription assays, deletion mutagenesis, partial purification of a mitotic kinase from HeLa nuclear extract, mass spectrometry, site-directed mutagenesis (S270A, S270E)","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — reconstitution-based in vitro assays, domain dissection, site-directed mutagenesis, and MS in a single rigorous study","pmids":["24634214"],"is_preprint":false},{"year":2014,"finding":"Gdown1 does not functionally associate with Pol II preinitiation complexes (PICs) that contain TFIIF, despite completely displacing TFIIF from free Pol II and elongating Pol II. Gdown1 can associate with Pol II during early transcript elongation in competition with TFIIF; phosphorylation of TFIIF provides a pathway for efficient Gdown1 loading early in elongation.","method":"In vitro transcription with purified factors, phosphorylation of TFIIF, functional association assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified factors, multiple mechanistic conditions tested in one study","pmids":["24596085"],"is_preprint":false},{"year":2016,"finding":"Gdown1 cannot functionally associate with Pol II in open complexes or during abortive synthesis of very short RNAs. Efficient Gdown1 association occurs only after the upstream portion of the transcription bubble reanneals (early elongation complexes with 5–9 nt RNAs), and Gdown1 displaces TFIIF slowly (~5 min), suggesting Gdown1 locks in the paused state after pausing has already been initiated by other factors (DSIF, NELF).","method":"In vitro transcription assays with promoter variants, timed competition assays with purified Gdown1 and TFIIF","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous in vitro mechanistic dissection but single lab, single study","pmids":["27716820"],"is_preprint":false},{"year":2018,"finding":"Although Gdown1 is intrinsically disordered, its Pol II-interacting domains were mapped and shown to occlude both TFIIF and TFIIB binding sites on Pol II. Robust Pol II binding requires cooperative interactions of a strong binding region and two weaker modulatory regions. In Drosophila embryos, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei but re-localizes to the cytoplasm upon zygotic genome activation, demonstrating a physiological role in transcription repression.","method":"Integrative structural modeling (cross-linking/MS, SAXS, negative-stain EM), domain mutagenesis, immunofluorescence in Drosophila embryos","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — multiple orthogonal structural and biochemical methods plus in vivo functional validation across species in one rigorous study","pmids":["30190596"],"is_preprint":false},{"year":2020,"finding":"Hepatocyte-specific ablation of Gdown1 in mice leads to down-regulation of highly expressed metabolic genes (plasma protein synthesis), reduced Pol II recruitment to these genes, and concomitant cell cycle re-entry with induction of cyclin D1 and p21 via p53 signaling. In the absence of p53, Gdown1-deficient hepatocytes display dysregulated mitosis and premalignant transformation. Gdown1 is associated with elongating (not initiating) Pol II on these highly expressed genes in vivo.","method":"Hepatocyte-specific conditional knockout mouse, ChIP, gene expression profiling, p53 pathway analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Moderate — clean conditional KO in defined cell type with ChIP-based pathway placement and genetic epistasis via p53 deletion","pmids":["32381628"],"is_preprint":false},{"year":2022,"finding":"Gdown1 localizes predominantly in the cytoplasm of interphase somatic cells, regulated by CRM1-dependent nuclear export signals (NES) and a novel cytoplasmic anchoring signal (CAS) that retains it outside the nuclear pore complex. Mutagenesis of CAS increases nuclear Gdown1 accumulation, causing a drastic reduction in Pol II levels and global transcription. Gdown1 translocates to the nucleus in response to oxidative stress, and Gdown1 ablation weakens cellular stress tolerance.","method":"Mutagenesis of NES and CAS, live-cell microscopy/immunofluorescence, CRM1 inhibition, global transcription assays, oxidative stress experiments in human cell lines","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Moderate — genetic (mutagenesis) and pharmacological (CRM1 inhibition) dissection of localization signals combined with functional transcription readout and stress biology, single lab but multiple orthogonal approaches","pmids":["36476745"],"is_preprint":false},{"year":2022,"finding":"Gdown1 resides predominantly in the cytoplasm of interphase cells and enters the nucleus at mitotic onset. Acute depletion of Gdown1 in human cell lines has minimal direct effects on interphase transcription but is associated with partial de-repression of mitotic transcription and aberrant mitoses coupled to p53 pathway activation. In vitro, Gdown1 modulates the combined functions of elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb.","method":"Genetic depletion (acute and KO), microscopy, multi-omics (ChIP-seq, RNA-seq), in vitro reconstitution with purified elongation factors","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal approaches (genetic depletion, imaging, multi-omics, in vitro reconstitution) in three human cell lines, single lab","pmids":["35048979"],"is_preprint":false},{"year":2021,"finding":"A genome-wide CRISPR-Cas9 knockout screen identified POLR2M as a regulatory factor for MIR139 silencing in MLL-AF9 AML, operating downstream of MLL-AF9 and PRC2. POLR2M knockout de-repressed MIR139 expression, placing POLR2M in the epigenetic silencing pathway for this tumor suppressor locus.","method":"Genome-wide CRISPR-Cas9 knockout screen, genetic validation in human AML cell lines","journal":"Leukemia","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — CRISPR screen with genetic validation, but mechanistic details of how POLR2M mediates MIR139 silencing are not resolved in the abstract","pmids":["34741119"],"is_preprint":false}],"current_model":"POLR2M/Gdown1 is a substoichiometric, intrinsically disordered subunit that tightly binds Pol II via cooperative interactions across RPB1 and RPB5, sterically occluding both TFIIF and TFIIB to block transcription initiation and early elongation; it loads onto Pol II after promoter clearance and bubble collapse to stabilize promoter-proximal pausing while maintaining responsiveness to P-TEFb, is itself inactivated by mitotic phosphorylation at Ser-270, and is sequestered in the cytoplasm of interphase cells via CRM1-dependent nuclear export and a cytoplasmic anchoring signal (CAS), entering the nucleus at mitosis onset and in response to oxidative stress to repress global transcription and safeguard genome integrity."},"narrative":{"mechanistic_narrative":"POLR2M (Gdown1) is a substoichiometric, intrinsically disordered subunit of RNA polymerase II that represses transcription by sterically gating the polymerase's interactions with general factors [PMID:22244331, PMID:30190596]. It binds Pol II through cooperative contacts that occlude the binding sites of both TFIIF and TFIIB, with structural mapping placing it on the RPB5 shelf–RPB1 jaw region directly overlapping the TFIIF footprint [PMID:22850672, PMID:30190596]. Functionally, Gdown1 blocks TFIIF-stimulated elongation and inhibits TTF2-mediated termination, and it stabilizes promoter-proximally paused Pol II while remaining responsive to P-TEFb, which overcomes the block; genome-wide it co-localizes with essentially all poised Pol II [PMID:22244331]. These two repressive activities map to separable domains—an N-terminal domain bearing a conserved LPDKG motif that blocks TTF2 and a C-terminal domain that blocks TFIIF—both required for tight Pol II association [PMID:24634214]. Gdown1 cannot load onto preinitiation complexes or open complexes; efficient association occurs only after promoter clearance and reannealing of the upstream transcription bubble in early elongation complexes, where it slowly displaces TFIIF to lock in the paused state downstream of pause initiation by DSIF and NELF [PMID:24596085, PMID:27716820]. In vivo, Gdown1 associates with elongating Pol II on highly expressed genes, and its hepatocyte-specific loss reduces Pol II recruitment to metabolic genes and triggers cell-cycle re-entry and, absent p53, premalignant transformation [PMID:32381628]. Its activity is spatially and temporally controlled: Ser-270 phosphorylation during mitosis lowers Pol II affinity and relieves repression [PMID:24634214], while in interphase Gdown1 is held in the cytoplasm by CRM1-dependent nuclear export and a cytoplasmic anchoring signal, entering the nucleus at mitotic onset and upon oxidative stress to repress global transcription and support stress tolerance [PMID:36476745, PMID:35048979]. POLR2M was also identified as an epigenetic silencing factor for the MIR139 tumor-suppressor locus in MLL-AF9 AML [PMID:34741119].","teleology":[{"year":2012,"claim":"Established that Gdown1 is a transcriptional repressor that stabilizes promoter-proximal pausing by antagonizing TFIIF-stimulated elongation while remaining a target for P-TEFb-mediated release.","evidence":"In vitro transcription assays, ChIP-Seq, and biochemical competition with purified factors in human systems","pmids":["22244331","22244332"],"confidence":"High","gaps":["Did not resolve the structural basis of TFIIF competition","Did not establish how Gdown1 is loaded onto Pol II in vivo"]},{"year":2012,"claim":"Resolved where Gdown1 sits on Pol II, mapping it to the RPB5 shelf–RPB1 jaw region overlapping the TFIIF footprint and explaining the steric exclusion mechanism.","evidence":"Single-particle cryo-EM with antibody labeling and size-exclusion competition assays","pmids":["22850672"],"confidence":"High","gaps":["Low resolution (~19 Å) limited atomic interpretation","Did not address TFIIB occlusion"]},{"year":2014,"claim":"Dissected Gdown1 into separable TTF2-blocking (N-terminal LPDKG) and TFIIF-blocking (C-terminal) domains and identified mitotic Ser-270 phosphorylation as a switch that releases repression.","evidence":"In vitro transcription, deletion and site-directed mutagenesis, partial kinase purification, and mass spectrometry","pmids":["24634214"],"confidence":"High","gaps":["Did not identify the physiological mitotic kinase definitively","In vivo consequences of S270 phosphorylation not tested"]},{"year":2014,"claim":"Showed Gdown1 is excluded from TFIIF-containing PICs but can engage Pol II during early elongation, defining a TFIIF-phosphorylation-gated loading pathway.","evidence":"In vitro reconstitution with purified factors and TFIIF phosphorylation","pmids":["24596085"],"confidence":"High","gaps":["Did not identify the kinase phosphorylating TFIIF in vivo","Timing relative to other elongation factors unresolved"]},{"year":2016,"claim":"Defined the precise transcription stage at which Gdown1 loads, showing engagement requires bubble reannealing in early elongation complexes and that it locks in a pause initiated by other factors.","evidence":"In vitro transcription with promoter variants and timed competition assays","pmids":["27716820"],"confidence":"Medium","gaps":["Single lab, single study","Did not directly test DSIF/NELF dependence in the same assay"]},{"year":2018,"claim":"Provided an integrative structural model showing Gdown1 occludes both TFIIF and TFIIB sites via cooperative binding, and demonstrated a physiological repressive role through Pol II co-localization in transcriptionally silent Drosophila nuclei.","evidence":"Cross-linking/MS, SAXS, negative-stain EM, mutagenesis, and immunofluorescence in Drosophila embryos","pmids":["30190596"],"confidence":"High","gaps":["Disordered regions limit high-resolution structure","Mechanism linking nuclear localization to genome activation not resolved"]},{"year":2020,"claim":"Established an in vivo physiological function, showing Gdown1 supports expression of and associates with elongating Pol II on highly expressed metabolic genes and restrains cell-cycle re-entry via p53.","evidence":"Hepatocyte-specific conditional knockout mouse with ChIP, expression profiling, and p53 epistasis","pmids":["32381628"],"confidence":"High","gaps":["Mechanism by which a repressor supports high gene expression unclear","Tissue specificity of these effects not generalized"]},{"year":2021,"claim":"Implicated POLR2M in an epigenetic silencing pathway, identifying it as a regulator of MIR139 silencing downstream of MLL-AF9 and PRC2 in AML.","evidence":"Genome-wide CRISPR-Cas9 knockout screen with genetic validation in human AML cell lines","pmids":["34741119"],"confidence":"Medium","gaps":["Mechanism of how POLR2M mediates MIR139 silencing not resolved","Connection to its Pol II pausing role not established"]},{"year":2022,"claim":"Defined the spatial control of Gdown1, showing CRM1-dependent export and a cytoplasmic anchoring signal sequester it in interphase, with nuclear entry upon oxidative stress driving global transcriptional repression and stress tolerance.","evidence":"NES/CAS mutagenesis, CRM1 inhibition, live-cell imaging, and oxidative stress assays in human cells","pmids":["36476745"],"confidence":"High","gaps":["Molecular nature of the CAS anchor unknown","Signal coupling stress to nuclear import unresolved"]},{"year":2022,"claim":"Showed Gdown1 enters the nucleus at mitotic onset to repress mitotic transcription and modulates a panel of elongation factors, linking its repression to mitotic fidelity and p53.","evidence":"Acute and KO depletion, imaging, multi-omics, and in vitro reconstitution with PAF1C, RTF1, SPT6, DSIF, and P-TEFb","pmids":["35048979"],"confidence":"High","gaps":["Minimal interphase transcription effect leaves its main interphase role unclear","How it integrates multiple elongation factors mechanistically not resolved"]},{"year":null,"claim":"How Gdown1's biochemical pausing/repression activity is reconciled with its in vivo requirement for high-level gene expression and its role in epigenetic locus silencing remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking pausing, mitotic repression, and supporting high gene expression","Mechanism of MIR139 silencing undefined","Identity of physiological mitotic and TFIIF kinases unconfirmed"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,6,7]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,3,4]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[6,8,9]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[8,9]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,7]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[8]}],"complexes":["RNA polymerase II"],"partners":["POLR2A","POLR2E","TFIIF","TFIIB"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P0CAP2","full_name":"DNA-directed RNA polymerase II subunit GRINL1A","aliases":["DNA-directed RNA polymerase II subunit M","Glutamate receptor-like protein 1A"],"length_aa":368,"mass_kda":41.7,"function":"Appears to be a stable component of the Pol II(G) complex form of RNA polymerase II (Pol II). Pol II synthesizes mRNA precursors and many functional non-coding RNAs and is the central component of the basal RNA polymerase II transcription machinery. May play a role in the Mediator complex-dependent regulation of transcription activation. Acts as a negative regulator of transcriptional activation; this repression is relieved by the Mediator complex, which restores Pol II(G) activator-dependent transcription to a level equivalent to that of Pol II","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P0CAP2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/POLR2M","classification":"Not Classified","n_dependent_lines":254,"n_total_lines":1208,"dependency_fraction":0.21026490066225165},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/POLR2M","total_profiled":1310},"omim":[{"mim_id":"620894","title":"CARDIOMYOPATHY, DILATED, 2K; CMD2K","url":"https://www.omim.org/entry/620894"},{"mim_id":"614071","title":"MYOCARDIAL ZONULA ADHERENS PROTEIN; MYZAP","url":"https://www.omim.org/entry/614071"},{"mim_id":"606485","title":"POLYMERASE II, RNA, SUBUNIT M; POLR2M","url":"https://www.omim.org/entry/606485"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/POLR2M"},"hgnc":{"alias_symbol":["Gdown","Gdown1"],"prev_symbol":["GRINL1A"]},"alphafold":{"accession":"P0CAP2","domains":[{"cath_id":"1.10.287","chopping":"20-76","consensus_level":"high","plddt":88.4507,"start":20,"end":76}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P0CAP2","model_url":"https://alphafold.ebi.ac.uk/files/AF-P0CAP2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P0CAP2-F1-predicted_aligned_error_v6.png","plddt_mean":66.19},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=POLR2M","jax_strain_url":"https://www.jax.org/strain/search?query=POLR2M"},"sequence":{"accession":"P0CAP2","fasta_url":"https://rest.uniprot.org/uniprotkb/P0CAP2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P0CAP2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P0CAP2"}},"corpus_meta":[{"pmid":"22244331","id":"PMC_22244331","title":"Functional association of Gdown1 with RNA polymerase II poised on human genes.","date":"2012","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/22244331","citation_count":109,"is_preprint":false},{"pmid":"22244332","id":"PMC_22244332","title":"Transcriptional regulation by Pol II(G) involving mediator and competitive interactions of Gdown1 and TFIIF with Pol II.","date":"2012","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/22244332","citation_count":60,"is_preprint":false},{"pmid":"30190596","id":"PMC_30190596","title":"Architecture of Pol II(G) and molecular mechanism of transcription regulation by Gdown1.","date":"2018","source":"Nature structural & molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/30190596","citation_count":27,"is_preprint":false},{"pmid":"22850672","id":"PMC_22850672","title":"Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM.","date":"2012","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/22850672","citation_count":24,"is_preprint":false},{"pmid":"24634214","id":"PMC_24634214","title":"Regulation of RNA polymerase II termination by phosphorylation of Gdown1.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24634214","citation_count":22,"is_preprint":false},{"pmid":"34741119","id":"PMC_34741119","title":"The tumor suppressor MIR139 is silenced by POLR2M to promote AML oncogenesis.","date":"2021","source":"Leukemia","url":"https://pubmed.ncbi.nlm.nih.gov/34741119","citation_count":13,"is_preprint":false},{"pmid":"27716820","id":"PMC_27716820","title":"Gdown1 Associates Efficiently with RNA Polymerase II after Promoter Clearance and Displaces TFIIF during Transcript Elongation.","date":"2016","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/27716820","citation_count":13,"is_preprint":false},{"pmid":"24596085","id":"PMC_24596085","title":"Functional interactions of the RNA polymerase II-interacting proteins Gdown1 and TFIIF.","date":"2014","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/24596085","citation_count":11,"is_preprint":false},{"pmid":"34899865","id":"PMC_34899865","title":"GRINL1A Complex Transcription Unit Containing GCOM1, MYZAP, and POLR2M Genes Associates with Fully Penetrant Recessive Dilated Cardiomyopathy.","date":"2021","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/34899865","citation_count":11,"is_preprint":false},{"pmid":"22244325","id":"PMC_22244325","title":"Get back TFIIF, don't let me Gdown1.","date":"2012","source":"Molecular cell","url":"https://pubmed.ncbi.nlm.nih.gov/22244325","citation_count":11,"is_preprint":false},{"pmid":"35048979","id":"PMC_35048979","title":"Nuclear export restricts Gdown1 to a mitotic function.","date":"2022","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/35048979","citation_count":6,"is_preprint":false},{"pmid":"36476745","id":"PMC_36476745","title":"Overcoming the cytoplasmic retention of GDOWN1 modulates global transcription and facilitates stress adaptation.","date":"2022","source":"eLife","url":"https://pubmed.ncbi.nlm.nih.gov/36476745","citation_count":6,"is_preprint":false},{"pmid":"22771989","id":"PMC_22771989","title":"Gdown1: making a link between mediator and RNA polymerase II elongation control.","date":"2012","source":"Transcription","url":"https://pubmed.ncbi.nlm.nih.gov/22771989","citation_count":6,"is_preprint":false},{"pmid":"32381628","id":"PMC_32381628","title":"Transcriptional down-regulation of metabolic genes by Gdown1 ablation induces quiescent cell re-entry into the cell cycle.","date":"2020","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/32381628","citation_count":4,"is_preprint":false},{"pmid":"33238793","id":"PMC_33238793","title":"Regulation of hepatocyte cell cycle re-entry by RNA polymerase II-associated Gdown1.","date":"2020","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/33238793","citation_count":3,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8169,"output_tokens":3047,"usd":0.035106,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10417,"output_tokens":3520,"usd":0.070042,"stage2_stop_reason":"end_turn"},"total_usd":0.105148,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2012,\n      \"finding\": \"Gdown1 specifically blocks elongation stimulation by TFIIF in vitro, inhibits the termination activity of TTF2, and stabilizes promoter-proximally paused Pol II in the presence of nuclear extract. P-TEFb can overcome the Gdown1-mediated block, and ChIP-Seq confirms Gdown1 co-localizes with essentially all poised Pol II across the human genome.\",\n      \"method\": \"In vitro transcription assays, ChIP-Seq\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal in vitro assays combined with genome-wide ChIP-Seq; replicated in a companion paper from an independent lab (PMID:22244332)\",\n      \"pmids\": [\"22244331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, inhibiting TFIIF function in preinitiation complex assembly. Mediator can facilitate Pol II(G) binding to promoters and overcome the Gdown1-mediated block, establishing Pol II(G) as a Mediator-dependent form of Pol II.\",\n      \"method\": \"In vitro transcription with purified factors, ChIP, RNAi\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Strong — multiple orthogonal methods (biochemical competition, ChIP, RNAi), replicated independently by PMID:22244331\",\n      \"pmids\": [\"22244332\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Cryo-EM maps of RNAPII(G) at ~19 Å localized Gdown1 primarily to the RPB5 shelf–RPB1 jaw region of Pol II, with Gdown1 binding sites overlapping extensively with TFIIF binding sites. Competition assays by size exclusion chromatography confirmed that Gdown1 sterically excludes TFIIF from RNAPII.\",\n      \"method\": \"Single-particle cryo-EM, antibody labeling, size exclusion chromatography competition assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structural mapping combined with orthogonal biochemical competition assays in one study\",\n      \"pmids\": [\"22850672\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Two functional domains of Gdown1 were identified: the N-terminal domain is responsible for blocking TTF2 (via a conserved LPDKG motif) and the C-terminal domain blocks TFIIF. Both domains are required for tight Pol II association. Gdown1 is phosphorylated at Ser-270 during mitosis, which reduces its affinity for Pol II and consequently reduces its ability to block both TTF2 and TFIIF. An S270A mutation abolishes this phosphorylation and S270E partially mimics phospho-Gdown1.\",\n      \"method\": \"In vitro transcription assays, deletion mutagenesis, partial purification of a mitotic kinase from HeLa nuclear extract, mass spectrometry, site-directed mutagenesis (S270A, S270E)\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstitution-based in vitro assays, domain dissection, site-directed mutagenesis, and MS in a single rigorous study\",\n      \"pmids\": [\"24634214\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Gdown1 does not functionally associate with Pol II preinitiation complexes (PICs) that contain TFIIF, despite completely displacing TFIIF from free Pol II and elongating Pol II. Gdown1 can associate with Pol II during early transcript elongation in competition with TFIIF; phosphorylation of TFIIF provides a pathway for efficient Gdown1 loading early in elongation.\",\n      \"method\": \"In vitro transcription with purified factors, phosphorylation of TFIIF, functional association assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified factors, multiple mechanistic conditions tested in one study\",\n      \"pmids\": [\"24596085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Gdown1 cannot functionally associate with Pol II in open complexes or during abortive synthesis of very short RNAs. Efficient Gdown1 association occurs only after the upstream portion of the transcription bubble reanneals (early elongation complexes with 5–9 nt RNAs), and Gdown1 displaces TFIIF slowly (~5 min), suggesting Gdown1 locks in the paused state after pausing has already been initiated by other factors (DSIF, NELF).\",\n      \"method\": \"In vitro transcription assays with promoter variants, timed competition assays with purified Gdown1 and TFIIF\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous in vitro mechanistic dissection but single lab, single study\",\n      \"pmids\": [\"27716820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Although Gdown1 is intrinsically disordered, its Pol II-interacting domains were mapped and shown to occlude both TFIIF and TFIIB binding sites on Pol II. Robust Pol II binding requires cooperative interactions of a strong binding region and two weaker modulatory regions. In Drosophila embryos, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei but re-localizes to the cytoplasm upon zygotic genome activation, demonstrating a physiological role in transcription repression.\",\n      \"method\": \"Integrative structural modeling (cross-linking/MS, SAXS, negative-stain EM), domain mutagenesis, immunofluorescence in Drosophila embryos\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — multiple orthogonal structural and biochemical methods plus in vivo functional validation across species in one rigorous study\",\n      \"pmids\": [\"30190596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Hepatocyte-specific ablation of Gdown1 in mice leads to down-regulation of highly expressed metabolic genes (plasma protein synthesis), reduced Pol II recruitment to these genes, and concomitant cell cycle re-entry with induction of cyclin D1 and p21 via p53 signaling. In the absence of p53, Gdown1-deficient hepatocytes display dysregulated mitosis and premalignant transformation. Gdown1 is associated with elongating (not initiating) Pol II on these highly expressed genes in vivo.\",\n      \"method\": \"Hepatocyte-specific conditional knockout mouse, ChIP, gene expression profiling, p53 pathway analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean conditional KO in defined cell type with ChIP-based pathway placement and genetic epistasis via p53 deletion\",\n      \"pmids\": [\"32381628\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Gdown1 localizes predominantly in the cytoplasm of interphase somatic cells, regulated by CRM1-dependent nuclear export signals (NES) and a novel cytoplasmic anchoring signal (CAS) that retains it outside the nuclear pore complex. Mutagenesis of CAS increases nuclear Gdown1 accumulation, causing a drastic reduction in Pol II levels and global transcription. Gdown1 translocates to the nucleus in response to oxidative stress, and Gdown1 ablation weakens cellular stress tolerance.\",\n      \"method\": \"Mutagenesis of NES and CAS, live-cell microscopy/immunofluorescence, CRM1 inhibition, global transcription assays, oxidative stress experiments in human cell lines\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic (mutagenesis) and pharmacological (CRM1 inhibition) dissection of localization signals combined with functional transcription readout and stress biology, single lab but multiple orthogonal approaches\",\n      \"pmids\": [\"36476745\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Gdown1 resides predominantly in the cytoplasm of interphase cells and enters the nucleus at mitotic onset. Acute depletion of Gdown1 in human cell lines has minimal direct effects on interphase transcription but is associated with partial de-repression of mitotic transcription and aberrant mitoses coupled to p53 pathway activation. In vitro, Gdown1 modulates the combined functions of elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb.\",\n      \"method\": \"Genetic depletion (acute and KO), microscopy, multi-omics (ChIP-seq, RNA-seq), in vitro reconstitution with purified elongation factors\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal approaches (genetic depletion, imaging, multi-omics, in vitro reconstitution) in three human cell lines, single lab\",\n      \"pmids\": [\"35048979\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"A genome-wide CRISPR-Cas9 knockout screen identified POLR2M as a regulatory factor for MIR139 silencing in MLL-AF9 AML, operating downstream of MLL-AF9 and PRC2. POLR2M knockout de-repressed MIR139 expression, placing POLR2M in the epigenetic silencing pathway for this tumor suppressor locus.\",\n      \"method\": \"Genome-wide CRISPR-Cas9 knockout screen, genetic validation in human AML cell lines\",\n      \"journal\": \"Leukemia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — CRISPR screen with genetic validation, but mechanistic details of how POLR2M mediates MIR139 silencing are not resolved in the abstract\",\n      \"pmids\": [\"34741119\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"POLR2M/Gdown1 is a substoichiometric, intrinsically disordered subunit that tightly binds Pol II via cooperative interactions across RPB1 and RPB5, sterically occluding both TFIIF and TFIIB to block transcription initiation and early elongation; it loads onto Pol II after promoter clearance and bubble collapse to stabilize promoter-proximal pausing while maintaining responsiveness to P-TEFb, is itself inactivated by mitotic phosphorylation at Ser-270, and is sequestered in the cytoplasm of interphase cells via CRM1-dependent nuclear export and a cytoplasmic anchoring signal (CAS), entering the nucleus at mitosis onset and in response to oxidative stress to repress global transcription and safeguard genome integrity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"POLR2M (Gdown1) is a substoichiometric, intrinsically disordered subunit of RNA polymerase II that represses transcription by sterically gating the polymerase's interactions with general factors [#0, #6]. It binds Pol II through cooperative contacts that occlude the binding sites of both TFIIF and TFIIB, with structural mapping placing it on the RPB5 shelf\\u2013RPB1 jaw region directly overlapping the TFIIF footprint [#2, #6]. Functionally, Gdown1 blocks TFIIF-stimulated elongation and inhibits TTF2-mediated termination, and it stabilizes promoter-proximally paused Pol II while remaining responsive to P-TEFb, which overcomes the block; genome-wide it co-localizes with essentially all poised Pol II [#0]. These two repressive activities map to separable domains\\u2014an N-terminal domain bearing a conserved LPDKG motif that blocks TTF2 and a C-terminal domain that blocks TFIIF\\u2014both required for tight Pol II association [#3]. Gdown1 cannot load onto preinitiation complexes or open complexes; efficient association occurs only after promoter clearance and reannealing of the upstream transcription bubble in early elongation complexes, where it slowly displaces TFIIF to lock in the paused state downstream of pause initiation by DSIF and NELF [#4, #5]. In vivo, Gdown1 associates with elongating Pol II on highly expressed genes, and its hepatocyte-specific loss reduces Pol II recruitment to metabolic genes and triggers cell-cycle re-entry and, absent p53, premalignant transformation [#7]. Its activity is spatially and temporally controlled: Ser-270 phosphorylation during mitosis lowers Pol II affinity and relieves repression [#3], while in interphase Gdown1 is held in the cytoplasm by CRM1-dependent nuclear export and a cytoplasmic anchoring signal, entering the nucleus at mitotic onset and upon oxidative stress to repress global transcription and support stress tolerance [#8, #9]. POLR2M was also identified as an epigenetic silencing factor for the MIR139 tumor-suppressor locus in MLL-AF9 AML [#10].\",\n  \"teleology\": [\n    {\n      \"year\": 2012,\n      \"claim\": \"Established that Gdown1 is a transcriptional repressor that stabilizes promoter-proximal pausing by antagonizing TFIIF-stimulated elongation while remaining a target for P-TEFb-mediated release.\",\n      \"evidence\": \"In vitro transcription assays, ChIP-Seq, and biochemical competition with purified factors in human systems\",\n      \"pmids\": [\"22244331\", \"22244332\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of TFIIF competition\", \"Did not establish how Gdown1 is loaded onto Pol II in vivo\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Resolved where Gdown1 sits on Pol II, mapping it to the RPB5 shelf\\u2013RPB1 jaw region overlapping the TFIIF footprint and explaining the steric exclusion mechanism.\",\n      \"evidence\": \"Single-particle cryo-EM with antibody labeling and size-exclusion competition assays\",\n      \"pmids\": [\"22850672\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Low resolution (~19 \\u00c5) limited atomic interpretation\", \"Did not address TFIIB occlusion\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Dissected Gdown1 into separable TTF2-blocking (N-terminal LPDKG) and TFIIF-blocking (C-terminal) domains and identified mitotic Ser-270 phosphorylation as a switch that releases repression.\",\n      \"evidence\": \"In vitro transcription, deletion and site-directed mutagenesis, partial kinase purification, and mass spectrometry\",\n      \"pmids\": [\"24634214\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the physiological mitotic kinase definitively\", \"In vivo consequences of S270 phosphorylation not tested\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showed Gdown1 is excluded from TFIIF-containing PICs but can engage Pol II during early elongation, defining a TFIIF-phosphorylation-gated loading pathway.\",\n      \"evidence\": \"In vitro reconstitution with purified factors and TFIIF phosphorylation\",\n      \"pmids\": [\"24596085\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the kinase phosphorylating TFIIF in vivo\", \"Timing relative to other elongation factors unresolved\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Defined the precise transcription stage at which Gdown1 loads, showing engagement requires bubble reannealing in early elongation complexes and that it locks in a pause initiated by other factors.\",\n      \"evidence\": \"In vitro transcription with promoter variants and timed competition assays\",\n      \"pmids\": [\"27716820\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab, single study\", \"Did not directly test DSIF/NELF dependence in the same assay\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Provided an integrative structural model showing Gdown1 occludes both TFIIF and TFIIB sites via cooperative binding, and demonstrated a physiological repressive role through Pol II co-localization in transcriptionally silent Drosophila nuclei.\",\n      \"evidence\": \"Cross-linking/MS, SAXS, negative-stain EM, mutagenesis, and immunofluorescence in Drosophila embryos\",\n      \"pmids\": [\"30190596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Disordered regions limit high-resolution structure\", \"Mechanism linking nuclear localization to genome activation not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Established an in vivo physiological function, showing Gdown1 supports expression of and associates with elongating Pol II on highly expressed metabolic genes and restrains cell-cycle re-entry via p53.\",\n      \"evidence\": \"Hepatocyte-specific conditional knockout mouse with ChIP, expression profiling, and p53 epistasis\",\n      \"pmids\": [\"32381628\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which a repressor supports high gene expression unclear\", \"Tissue specificity of these effects not generalized\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Implicated POLR2M in an epigenetic silencing pathway, identifying it as a regulator of MIR139 silencing downstream of MLL-AF9 and PRC2 in AML.\",\n      \"evidence\": \"Genome-wide CRISPR-Cas9 knockout screen with genetic validation in human AML cell lines\",\n      \"pmids\": [\"34741119\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of how POLR2M mediates MIR139 silencing not resolved\", \"Connection to its Pol II pausing role not established\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Defined the spatial control of Gdown1, showing CRM1-dependent export and a cytoplasmic anchoring signal sequester it in interphase, with nuclear entry upon oxidative stress driving global transcriptional repression and stress tolerance.\",\n      \"evidence\": \"NES/CAS mutagenesis, CRM1 inhibition, live-cell imaging, and oxidative stress assays in human cells\",\n      \"pmids\": [\"36476745\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular nature of the CAS anchor unknown\", \"Signal coupling stress to nuclear import unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Showed Gdown1 enters the nucleus at mitotic onset to repress mitotic transcription and modulates a panel of elongation factors, linking its repression to mitotic fidelity and p53.\",\n      \"evidence\": \"Acute and KO depletion, imaging, multi-omics, and in vitro reconstitution with PAF1C, RTF1, SPT6, DSIF, and P-TEFb\",\n      \"pmids\": [\"35048979\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Minimal interphase transcription effect leaves its main interphase role unclear\", \"How it integrates multiple elongation factors mechanistically not resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How Gdown1's biochemical pausing/repression activity is reconciled with its in vivo requirement for high-level gene expression and its role in epigenetic locus silencing remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking pausing, mitotic repression, and supporting high gene expression\", \"Mechanism of MIR139 silencing undefined\", \"Identity of physiological mitotic and TFIIF kinases unconfirmed\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 6, 7]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 3, 4]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [6, 8, 9]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [8, 9]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 7]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [8]}\n    ],\n    \"complexes\": [\"RNA polymerase II\"],\n    \"partners\": [\"POLR2A\", \"POLR2E\", \"TFIIF\", \"TFIIB\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}