{"gene":"TG_243440","gene_symbol":"GCN5B","run_date":"2026-09-10T21:09:19","timeline":{"discoveries":[{"year":2006,"finding":"TgGCN5-B acetylates multiple lysine residues in the H3 tail (broader substrate specificity than TgGCN5-A, which exclusively targets H3K18), and harbors a divergent N-terminal domain required for nuclear localization. TgGCN5-B also interacts with a distinct ADA2 homologue compared to TgGCN5-A.","method":"In vitro histone acetyltransferase assays with histone substrates; co-immunoprecipitation of ADA2 homologues; domain deletion/nuclear localization experiments","journal":"Eukaryotic cell","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic assays with mutagenesis-level substrate specificity mapping, reciprocal co-IP for ADA2 interactions, localization experiments; single lab but multiple orthogonal methods","pmids":["16400169"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2014,"finding":"GCN5b is essential for Toxoplasma tachyzoite viability; expression of a dominant-negative catalytically dead point mutant (E703G) causes rapid arrest in parasite replication, decreased H3 acetylation at specific lysine residues, and reduced expression of GCN5b target genes. GCN5b forms a complex with AP2-domain transcription factors, the co-activator ADA2-A, TFIID subunits, LEO1 (Paf1 subunit), and RRM proteins.","method":"Dominant-negative inducible expression (destabilization domain strategy); ChIP-chip to identify target genes; mass spectrometry proteomics (Co-IP) to identify complex members; global histone acetylation assessment","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — dominant-negative with defined catalytic mutation, ChIP-chip for target genes, MS-based interactome, multiple orthogonal methods in single study; replicated by subsequent work","pmids":["24391497"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2010,"finding":"TgGCN5-B contains a novel nuclear localization signal (RPAENKKRGR) located within an intrinsically disordered region of its N-terminus, distinct from the NLS of TgGCN5-A; functionality of this NLS was experimentally validated.","method":"Computational prediction of intrinsically disordered regions combined with experimental validation of NLS activity (GFP-fusion localization assays)","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2-3 / Moderate — localization assay validated NLS function; single lab, but computational prediction confirmed by direct experiment","pmids":["21055425"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Garcinol inhibits TgGCN5b lysine acetyltransferase activity in vitro and in vivo, reducing global H3 acetylation and acetylation of GCN5b itself, and dysregulating GCN5b target genes to block tachyzoite replication. Genes most affected by garcinol treatment overlap with GCN5b ChIP-chip targets.","method":"In vitro KAT inhibition assay; global histone acetylation western blot; RNA-seq transcriptome analysis; comparison with prior ChIP-chip data","journal":"Antimicrobial agents and chemotherapy","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — in vitro enzymatic assay for inhibition, in vivo histone acetylation measurement, transcriptome analysis; multiple orthogonal methods in one study","pmids":["26810649"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"finding":"GCN5b associates with at least two distinct complexes characterized by different pairs of AP2 transcription factors (AP2IX-7 and AP2XII-4) under normal and bradyzoite-induction conditions. The composition of the GCN5b complex shifts during bradyzoite induction, and reciprocal co-IPs with AP2IX-7 and AP2XII-4 delineate their respective interactomes.","method":"Co-immunoprecipitation under tachyzoite and bradyzoite-inducing conditions; mass spectrometry; reciprocal co-IP with AP2IX-7 and AP2XII-4","journal":"Molecular and biochemical parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal co-IP with MS identification; single lab, two orthogonal methods","pmids":["31381949"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"finding":"The bromodomain of GCN5b is required for Toxoplasma viability; dominant-negative bromodomain disruption arrests parasite growth. The GCN5-family bromodomain inhibitor L-Moses blocks GCN5b bromodomain binding to acetylated histone residues in vitro, and kills tachyzoites in a GCN5b-dependent manner (over-expression of GCN5b overcomes L-Moses toxicity).","method":"Inducible dominant-negative bromodomain strategy; in vitro binding assay of bromodomain to acetylated histone peptides; parasite growth assay with GCN5b over-expression rescue","journal":"Experimental parasitology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — dominant-negative with defined domain, in vitro binding assay, over-expression rescue; single lab, multiple orthogonal methods","pmids":["32119930"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"finding":"GCN5b localizes to coding regions of the genome (not telomeres), as determined by CUT&Tag chromatin occupancy mapping, distinguishing its genomic targets from those of GCN5a (which localizes exclusively to telomeres). GCN5b interacts with the essential kinase TgGSK, and chemical inhibition of GCN5b leads to specific degradation of TgGSK protein.","method":"CUT&Tag chromatin occupancy mapping; co-immunoprecipitation (GCN5b–TgGSK interaction); chemical inhibition of GCN5b with monitoring of TgGSK protein levels","journal":"mSphere","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — CUT&Tag for genomic localization; co-IP for TgGSK interaction; chemical perturbation with protein level readout; single lab, multiple orthogonal methods","pmids":["40879678","40152591"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":"Pru"}]}],"current_model":"TgGCN5b is an essential nuclear lysine acetyltransferase (KAT) in Toxoplasma gondii that acetylates multiple H3 tail lysines, occupies coding-region promoters genome-wide, and assembles into at least two distinct multi-protein complexes with different pairs of AP2-domain transcription factors, the co-activator ADA2-A, TFIID subunits, LEO1, and RRM proteins; its catalytic KAT domain and bromodomain are both required for parasite viability, its nuclear entry is mediated by an intrinsically disordered NLS, and it interacts with the essential kinase TgGSK whose stability depends on GCN5b acetyltransferase activity."},"narrative":{"mechanistic_narrative":"TgGCN5b is an essential nuclear lysine acetyltransferase (KAT) of Toxoplasma gondii that drives transcriptional gene regulation by acetylating histone H3 tail lysines and occupying protein-coding gene loci genome-wide [PMID:16400169, PMID:24391497, PMID:40879678, PMID:40152591]. Unlike its paralogue GCN5-A, which shows a narrow bias for H3K18 and localizes to telomeres, GCN5b acetylates multiple H3 tail lysines and its chromatin occupancy maps to coding regions of the genome [PMID:16400169, PMID:40879678, PMID:40152591]. Its catalytic activity is required for tachyzoite viability: a catalytically dead point mutant (E703G) arrests replication, lowers H3 acetylation, and downregulates GCN5b target genes, and small-molecule inhibition of its KAT activity by garcinol reproduces this transcriptional dysregulation and growth block [PMID:24391497, PMID:26810649]. GCN5b assembles into at least two distinct multi-protein complexes distinguished by different pairs of AP2-domain transcription factors (AP2IX-7 and AP2XII-4) together with the co-activator ADA2-A, TFIID subunits, LEO1/Paf1, and RRM proteins, and this composition shifts during bradyzoite induction [PMID:24391497, PMID:31381949]. Beyond its catalytic KAT domain, its bromodomain—which reads acetylated histone residues—is independently required for viability [PMID:32119930]. Nuclear entry is mediated by a novel NLS (RPAENKKRGR) embedded in an intrinsically disordered N-terminal region [PMID:21055425]. GCN5b also interacts with the essential kinase TgGSK, whose protein stability depends on GCN5b acetyltransferase activity [PMID:40879678, PMID:40152591].","teleology":[{"year":2006,"claim":"Distinguishing GCN5b from GCN5-A—broader H3 tail acetylation, a divergent nuclear-targeting N-terminus, and a distinct ADA2 partner—defined GCN5b as a functionally separate enzyme rather than a redundant paralogue.","evidence":"In vitro HAT assays, co-IP of ADA2 homologues, domain-deletion localization experiments","pmids":["16400169"],"confidence":"High","gaps":["Genome-wide targets not yet mapped","Essentiality not tested","Full complex composition unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2010,"claim":"Localizing GCN5b's NLS to an intrinsically disordered N-terminal region, distinct from the GCN5-A basic NLS, explained how the two paralogues use different import determinants.","evidence":"Computational disorder prediction with GFP-fusion localization validation","pmids":["21055425"],"confidence":"Medium","gaps":["Import receptor for this NLS not identified","Functional consequence of disorder region beyond import unknown"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"Demonstrating GCN5b essentiality and mapping its interactome and target genes converted it from a biochemical curiosity into a validated regulator: its catalysis is required for viability, target-gene expression, and H3 acetylation, and it works within AP2/ADA2-A/TFIID/LEO1/RRM complexes.","evidence":"Inducible dominant-negative catalytic mutant (E703G), ChIP-chip target mapping, MS interactome, global acetylation assessment","pmids":["24391497"],"confidence":"High","gaps":["Which acetylation events are direct vs indirect not fully resolved","Roles of individual complex members unclear"],"attribution":[{"species":"Toxoplasma gondii","strain":"RH"}]},{"year":2016,"claim":"Chemical inhibition by garcinol confirmed that GCN5b KAT activity is pharmacologically druggable and that blocking it phenocopies genetic disruption, reinforcing GCN5b as an antiparasitic target.","evidence":"In vitro KAT inhibition assay, global H3 acetylation western blot, RNA-seq, overlap with prior ChIP-chip targets","pmids":["26810649"],"confidence":"High","gaps":["Garcinol specificity for GCN5b vs other KATs not fully delineated","Self-acetylation function not defined"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2019,"claim":"Resolving that GCN5b partitions into at least two complexes defined by distinct AP2 factor pairs, with composition shifting during bradyzoite induction, established a mechanism for stage-specific transcriptional control.","evidence":"Co-IP under tachyzoite and bradyzoite-inducing conditions, MS, reciprocal co-IP with AP2IX-7 and AP2XII-4","pmids":["31381949"],"confidence":"Medium","gaps":["How AP2 factors direct GCN5b to specific loci not shown","Functional consequence of the composition shift on differentiation not established"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2020,"claim":"Showing the bromodomain is independently essential and druggable established that GCN5b requires both writing (KAT) and reading (bromodomain) of acetyl marks for function.","evidence":"Inducible dominant-negative bromodomain disruption, in vitro acetyl-histone peptide binding assay, GCN5b over-expression rescue of L-Moses toxicity","pmids":["32119930"],"confidence":"Medium","gaps":["In vivo acetyl-lysine targets read by the bromodomain not mapped","Whether reading and writing are coupled at chromatin unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2025,"claim":"Genome-wide CUT&Tag mapping placed GCN5b at protein-coding loci (versus GCN5a at telomeres) and linked its catalytic activity to stabilization of the essential kinase TgGSK, extending GCN5b's role from histone acetylation to control of a signaling kinase.","evidence":"CUT&Tag occupancy mapping, co-IP of GCN5b-TgGSK, chemical inhibition with TgGSK protein-level readout","pmids":["40879678","40152591"],"confidence":"Medium","gaps":["Whether TgGSK is a direct acetylation substrate of GCN5b not demonstrated","Mechanism coupling GCN5b activity to TgGSK stability unresolved","Single strain (Pru)"],"attribution":[{"species":"Toxoplasma gondii","strain":"Pru"}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["GCN5b–AP2IX-7 complex (with ADA2-A, TFIID subunits, LEO1/Paf1, RRM proteins)","GCN5b–AP2XII-4 complex (bradyzoite-associated composition)"],"partners":["ADA2-A (CO-ACTIVATOR)","AP2IX-7 (AP2-DOMAIN TRANSCRIPTION FACTOR)","AP2XII-4 (AP2-DOMAIN TRANSCRIPTION FACTOR)","TFIID SUBUNITS","LEO1 / PAF1 SUBUNIT","RRM PROTEINS","TGGSK (ESSENTIAL KINASE)"],"other_free_text":["Catalytically dead point mutant E703G acts as dominant-negative and arrests tachyzoite replication","KAT domain and bromodomain are each independently required for viability","Inhibited by garcinol (KAT activity) and L-Moses (bromodomain)","Nuclear import mediated by a novel NLS (RPAENKKRGR) within an intrinsically disordered N-terminal region, distinct from GCN5-A","GCN5b KAT activity is required for stability of the TgGSK protein"]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"16287846","id":"PMC_16287846","title":"Histone-modifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16287846","citation_count":160,"is_preprint":false},{"pmid":"24391497","id":"PMC_24391497","title":"Lysine acetyltransferase GCN5b interacts with AP2 factors and is required for Toxoplasma gondii proliferation.","date":"2014","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/24391497","citation_count":63,"is_preprint":false},{"pmid":"16400169","id":"PMC_16400169","title":"Pair of unusual GCN5 histone acetyltransferases and ADA2 homologues in the protozoan parasite Toxoplasma gondii.","date":"2006","source":"Eukaryotic cell","url":"https://pubmed.ncbi.nlm.nih.gov/16400169","citation_count":44,"is_preprint":false},{"pmid":"15591057","id":"PMC_15591057","title":"Histone acetylase GCN5 enters the nucleus via importin-alpha in protozoan parasite Toxoplasma gondii.","date":"2004","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15591057","citation_count":37,"is_preprint":false},{"pmid":"26810649","id":"PMC_26810649","title":"Garcinol Inhibits GCN5-Mediated Lysine Acetyltransferase Activity and Prevents Replication of the Parasite Toxoplasma gondii.","date":"2016","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/26810649","citation_count":36,"is_preprint":false},{"pmid":"10536141","id":"PMC_10536141","title":"Cloning and analysis of a Toxoplasma gondii histone acetyltransferase: a novel chromatin remodelling factor in Apicomplexan parasites.","date":"1999","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/10536141","citation_count":25,"is_preprint":false},{"pmid":"17178801","id":"PMC_17178801","title":"Quinoline derivative MC1626, a putative GCN5 histone acetyltransferase (HAT) inhibitor, exhibits HAT-independent activity against Toxoplasma gondii.","date":"2006","source":"Antimicrobial agents and chemotherapy","url":"https://pubmed.ncbi.nlm.nih.gov/17178801","citation_count":24,"is_preprint":false},{"pmid":"10721712","id":"PMC_10721712","title":"Cloning and characterization of a novel histone acetyltransferase homologue from the protozoan parasite Toxoplasma gondii reveals a distinct GCN5 family member.","date":"2000","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/10721712","citation_count":23,"is_preprint":false},{"pmid":"31381949","id":"PMC_31381949","title":"A novel GCN5b lysine acetyltransferase complex associates with distinct transcription factors in the protozoan parasite Toxoplasma gondii.","date":"2019","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/31381949","citation_count":20,"is_preprint":false},{"pmid":"21055425","id":"PMC_21055425","title":"Regions of intrinsic disorder help identify a novel nuclear localization signal in Toxoplasma gondii histone acetyltransferase TgGCN5-B.","date":"2010","source":"Molecular and biochemical parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/21055425","citation_count":15,"is_preprint":false},{"pmid":"32119930","id":"PMC_32119930","title":"Evaluating the GCN5b bromodomain as a novel therapeutic target against the parasite Toxoplasma gondii.","date":"2020","source":"Experimental parasitology","url":"https://pubmed.ncbi.nlm.nih.gov/32119930","citation_count":13,"is_preprint":false},{"pmid":"40152591","id":"PMC_40152591","title":"The essential kinase TgGSK regulates centrosome segregation and endodyogeny in Toxoplasma gondii.","date":"2025","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/40152591","citation_count":4,"is_preprint":false},{"pmid":"39386585","id":"PMC_39386585","title":"The essential kinase TgGSK regulates centrosome division and endodyogeny in Toxoplasma gondii.","date":"2024","source":"bioRxiv : the preprint server for biology","url":"https://pubmed.ncbi.nlm.nih.gov/39386585","citation_count":2,"is_preprint":false},{"pmid":"40879678","id":"PMC_40879678","title":"GCN5a is a telomeric lysine acetyltransferase whose loss primes Toxoplasma gondii for latency.","date":"2025","source":"mSphere","url":"https://pubmed.ncbi.nlm.nih.gov/40879678","citation_count":1,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":10084,"output_tokens":2974,"usd":0.037431,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8698,"output_tokens":4597,"usd":0.079208,"stage2_stop_reason":"end_turn"},"total_usd":0.123224,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard","corrective_rerun":"2026-09-10 wrong-gene rerun (round-4 rules)","corrective_reason":"summary amended"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2006,\n      \"finding\": \"TgGCN5-B acetylates multiple lysine residues in the H3 tail (broader substrate specificity than TgGCN5-A, which exclusively targets H3K18), and harbors a divergent N-terminal domain required for nuclear localization. TgGCN5-B also interacts with a distinct ADA2 homologue compared to TgGCN5-A.\",\n      \"method\": \"In vitro histone acetyltransferase assays with histone substrates; co-immunoprecipitation of ADA2 homologues; domain deletion/nuclear localization experiments\",\n      \"journal\": \"Eukaryotic cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic assays with mutagenesis-level substrate specificity mapping, reciprocal co-IP for ADA2 interactions, localization experiments; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"16400169\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"TgGCN5 (the founding GCN5 in Toxoplasma, later designated GCN5-A) exhibits an unusual bias for H3K18 acetylation in vitro, and its chromatin occupancy at target promoters is stage-specific.\",\n      \"method\": \"In vitro histone acetyltransferase assay; chromatin immunoprecipitation (ChIP) at stage-specific promoters\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic assay for substrate specificity; ChIP for promoter occupancy; single lab, two orthogonal methods\",\n      \"pmids\": [\"16287846\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"GCN5b is essential for Toxoplasma tachyzoite viability; expression of a dominant-negative catalytically dead point mutant (E703G) causes rapid arrest in parasite replication, decreased H3 acetylation at specific lysine residues, and reduced expression of GCN5b target genes. GCN5b forms a complex with AP2-domain transcription factors, the co-activator ADA2-A, TFIID subunits, LEO1 (Paf1 subunit), and RRM proteins.\",\n      \"method\": \"Dominant-negative inducible expression (destabilization domain strategy); ChIP-chip to identify target genes; mass spectrometry proteomics (Co-IP) to identify complex members; global histone acetylation assessment\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — dominant-negative with defined catalytic mutation, ChIP-chip for target genes, MS-based interactome, multiple orthogonal methods in single study; replicated by subsequent work\",\n      \"pmids\": [\"24391497\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"TgGCN5 (GCN5-A) enters the nucleus via a six-amino acid basic NLS (RKRVKR) in its N-terminus that directly interacts with TgIMPalpha (importin-alpha); deletion of the NLS abolishes the interaction and nuclear import.\",\n      \"method\": \"GFP-fusion nuclear localization experiments; in vitro co-immunoprecipitation of TgGCN5 with TgIMPalpha; NLS deletion mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro co-IP with deletion mutagenesis and localization confirmation; single lab, two orthogonal methods\",\n      \"pmids\": [\"15591057\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"TgGCN5-B contains a novel nuclear localization signal (RPAENKKRGR) located within an intrinsically disordered region of its N-terminus, distinct from the NLS of TgGCN5-A; functionality of this NLS was experimentally validated.\",\n      \"method\": \"Computational prediction of intrinsically disordered regions combined with experimental validation of NLS activity (GFP-fusion localization assays)\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 / Moderate — localization assay validated NLS function; single lab, but computational prediction confirmed by direct experiment\",\n      \"pmids\": [\"21055425\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Garcinol inhibits TgGCN5b lysine acetyltransferase activity in vitro and in vivo, reducing global H3 acetylation and acetylation of GCN5b itself, and dysregulating GCN5b target genes to block tachyzoite replication. Genes most affected by garcinol treatment overlap with GCN5b ChIP-chip targets.\",\n      \"method\": \"In vitro KAT inhibition assay; global histone acetylation western blot; RNA-seq transcriptome analysis; comparison with prior ChIP-chip data\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — in vitro enzymatic assay for inhibition, in vivo histone acetylation measurement, transcriptome analysis; multiple orthogonal methods in one study\",\n      \"pmids\": [\"26810649\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"GCN5b associates with at least two distinct complexes characterized by different pairs of AP2 transcription factors (AP2IX-7 and AP2XII-4) under normal and bradyzoite-induction conditions. The composition of the GCN5b complex shifts during bradyzoite induction, and reciprocal co-IPs with AP2IX-7 and AP2XII-4 delineate their respective interactomes.\",\n      \"method\": \"Co-immunoprecipitation under tachyzoite and bradyzoite-inducing conditions; mass spectrometry; reciprocal co-IP with AP2IX-7 and AP2XII-4\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal co-IP with MS identification; single lab, two orthogonal methods\",\n      \"pmids\": [\"31381949\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The bromodomain of GCN5b is required for Toxoplasma viability; dominant-negative bromodomain disruption arrests parasite growth. The GCN5-family bromodomain inhibitor L-Moses blocks GCN5b bromodomain binding to acetylated histone residues in vitro, and kills tachyzoites in a GCN5b-dependent manner (over-expression of GCN5b overcomes L-Moses toxicity).\",\n      \"method\": \"Inducible dominant-negative bromodomain strategy; in vitro binding assay of bromodomain to acetylated histone peptides; parasite growth assay with GCN5b over-expression rescue\",\n      \"journal\": \"Experimental parasitology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — dominant-negative with defined domain, in vitro binding assay, over-expression rescue; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"32119930\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"GCN5b localizes to coding regions of the genome (not telomeres), as determined by CUT&Tag chromatin occupancy mapping, distinguishing its genomic targets from those of GCN5a (which localizes exclusively to telomeres). GCN5b interacts with the essential kinase TgGSK, and chemical inhibition of GCN5b leads to specific degradation of TgGSK protein.\",\n      \"method\": \"CUT&Tag chromatin occupancy mapping; co-immunoprecipitation (GCN5b–TgGSK interaction); chemical inhibition of GCN5b with monitoring of TgGSK protein levels\",\n      \"journal\": \"mSphere\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — CUT&Tag for genomic localization; co-IP for TgGSK interaction; chemical perturbation with protein level readout; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"40879678\", \"40152591\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"TgGCN5 (original designation, subsequently GCN5-A) is a functional histone acetyltransferase; overexpression of the full-length protein is toxic unless the HAT activity is disrupted by a single point mutation. The HAT domain can functionally complement yeast gcn5− when fused to the remainder of yeast GCN5, but the full Toxoplasma protein cannot.\",\n      \"method\": \"Yeast complementation assay; point mutagenesis of HAT catalytic residue; overexpression toxicity assay in Toxoplasma tachyzoites\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — enzymatic activity established by mutagenesis and heterologous complementation; single lab, two orthogonal methods\",\n      \"pmids\": [\"10536141\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"TgGCN5b is an essential nuclear lysine acetyltransferase (KAT) in Toxoplasma gondii that acetylates multiple H3 tail lysines, occupies coding-region promoters genome-wide, and assembles into at least two distinct multi-protein complexes with different pairs of AP2-domain transcription factors, the co-activator ADA2-A, TFIID subunits, LEO1, and RRM proteins; its catalytic KAT domain and bromodomain are both required for parasite viability, its nuclear entry is mediated by an intrinsically disordered NLS, and it interacts with the essential kinase TgGSK whose stability depends on GCN5b acetyltransferase activity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"TgGCN5b is an essential nuclear lysine acetyltransferase (KAT) of Toxoplasma gondii that drives transcriptional gene regulation by acetylating histone H3 tail lysines and occupying protein-coding gene loci genome-wide [#0, #2, #8]. Unlike its paralogue GCN5-A, which shows a narrow bias for H3K18 and localizes to telomeres, GCN5b acetylates multiple H3 tail lysines and its chromatin occupancy maps to coding regions of the genome [#0, #8]. Its catalytic activity is required for tachyzoite viability: a catalytically dead point mutant (E703G) arrests replication, lowers H3 acetylation, and downregulates GCN5b target genes, and small-molecule inhibition of its KAT activity by garcinol reproduces this transcriptional dysregulation and growth block [#2, #5]. GCN5b assembles into at least two distinct multi-protein complexes distinguished by different pairs of AP2-domain transcription factors (AP2IX-7 and AP2XII-4) together with the co-activator ADA2-A, TFIID subunits, LEO1/Paf1, and RRM proteins, and this composition shifts during bradyzoite induction [#0, #2, #6]. Beyond its catalytic KAT domain, its bromodomain—which reads acetylated histone residues—is independently required for viability [#7]. Nuclear entry is mediated by a novel NLS embedded in an intrinsically disordered N-terminal region, distinct from the importin-alpha–dependent basic NLS of GCN5-A [#4, #3]. GCN5b also interacts with the essential kinase TgGSK, whose protein stability depends on GCN5b acetyltransferase activity [#8].\",\n  \"teleology\": [\n    {\n      \"year\": 1999,\n      \"claim\": \"Establishing that the founding Toxoplasma GCN5 was a bona fide histone acetyltransferase set the biochemical foundation for a parasite HAT system, showing catalytic activity was toxic when unregulated and could complement yeast gcn5-.\",\n      \"evidence\": \"Yeast complementation, catalytic point mutagenesis, and overexpression toxicity assay in tachyzoites\",\n      \"pmids\": [\"10536141\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Concerns the GCN5-A paralogue, not GCN5b specifically\", \"Does not define histone substrate specificity in the parasite\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defining how GCN5-A reaches the nucleus—via a six-residue basic NLS binding importin-alpha—provided the framework against which GCN5b's distinct import mechanism would later be contrasted.\",\n      \"evidence\": \"GFP-fusion localization, in vitro co-IP with TgIMPalpha, NLS deletion mutagenesis\",\n      \"pmids\": [\"15591057\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Applies to GCN5-A, not GCN5b\", \"Import receptor for GCN5b not addressed here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showing GCN5-A has an unusual H3K18 bias and stage-specific promoter occupancy established that Toxoplasma GCN5 enzymes have divergent, specialized substrate and targeting behavior.\",\n      \"evidence\": \"In vitro HAT assay and ChIP at stage-specific promoters\",\n      \"pmids\": [\"16287846\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Characterizes GCN5-A, leaving GCN5b substrate range undefined\", \"No genome-wide occupancy map\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Distinguishing GCN5b from GCN5-A—broader H3 tail acetylation, a divergent nuclear-targeting N-terminus, and a distinct ADA2 partner—defined GCN5b as a functionally separate enzyme rather than a redundant paralogue.\",\n      \"evidence\": \"In vitro HAT assays, co-IP of ADA2 homologues, domain-deletion localization experiments\",\n      \"pmids\": [\"16400169\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Genome-wide targets not yet mapped\", \"Essentiality not tested\", \"Full complex composition unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Localizing GCN5b's NLS to an intrinsically disordered N-terminal region, distinct from the GCN5-A basic NLS, explained how the two paralogues use different import determinants.\",\n      \"evidence\": \"Computational disorder prediction with GFP-fusion localization validation\",\n      \"pmids\": [\"21055425\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Import receptor for this NLS not identified\", \"Functional consequence of disorder region beyond import unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Demonstrating GCN5b essentiality and mapping its interactome and target genes converted it from a biochemical curiosity into a validated regulator: its catalysis is required for viability, target-gene expression, and H3 acetylation, and it works within AP2/ADA2-A/TFIID/LEO1/RRM complexes.\",\n      \"evidence\": \"Inducible dominant-negative catalytic mutant (E703G), ChIP-chip target mapping, MS interactome, global acetylation assessment\",\n      \"pmids\": [\"24391497\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Which acetylation events are direct vs indirect not fully resolved\", \"Roles of individual complex members unclear\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Chemical inhibition by garcinol confirmed that GCN5b KAT activity is pharmacologically druggable and that blocking it phenocopies genetic disruption, reinforcing GCN5b as an antiparasitic target.\",\n      \"evidence\": \"In vitro KAT inhibition assay, global H3 acetylation western blot, RNA-seq, overlap with prior ChIP-chip targets\",\n      \"pmids\": [\"26810649\"],\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Garcinol specificity for GCN5b vs other KATs not fully delineated\", \"Self-acetylation function not defined\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Resolving that GCN5b partitions into at least two complexes defined by distinct AP2 factor pairs, with composition shifting during bradyzoite induction, established a mechanism for stage-specific transcriptional control.\",\n      \"evidence\": \"Co-IP under tachyzoite and bradyzoite-inducing conditions, MS, reciprocal co-IP with AP2IX-7 and AP2XII-4\",\n      \"pmids\": [\"31381949\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"How AP2 factors direct GCN5b to specific loci not shown\", \"Functional consequence of the composition shift on differentiation not established\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Showing the bromodomain is independently essential and druggable established that GCN5b requires both writing (KAT) and reading (bromodomain) of acetyl marks for function.\",\n      \"evidence\": \"Inducible dominant-negative bromodomain disruption, in vitro acetyl-histone peptide binding assay, GCN5b over-expression rescue of L-Moses toxicity\",\n      \"pmids\": [\"32119930\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"In vivo acetyl-lysine targets read by the bromodomain not mapped\", \"Whether reading and writing are coupled at chromatin unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Genome-wide CUT&Tag mapping placed GCN5b at protein-coding loci (versus GCN5a at telomeres) and linked its catalytic activity to stabilization of the essential kinase TgGSK, extending GCN5b's role from histone acetylation to control of a signaling kinase.\",\n      \"evidence\": \"CUT&Tag occupancy mapping, co-IP of GCN5b-TgGSK, chemical inhibition with TgGSK protein-level readout\",\n      \"pmids\": [\"40879678\", \"40152591\"],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"Whether TgGSK is a direct acetylation substrate of GCN5b not demonstrated\", \"Mechanism coupling GCN5b activity to TgGSK stability unresolved\", \"Single strain (Pru)\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How AP2 transcription factors direct GCN5b complexes to specific coding loci, and whether TgGSK and other non-histone proteins are direct acetylation substrates, remain open.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"\",\n      \"gaps\": [\"No direct non-histone substrate confirmed\", \"Locus-selection mechanism by AP2 partners unresolved\", \"Structural basis of complex assembly unknown\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004402\", \"supporting_discovery_ids\": [0, 2, 5, 9], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0043998\", \"supporting_discovery_ids\": [0], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0070577\", \"supporting_discovery_ids\": [7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 3, 4], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000785\", \"supporting_discovery_ids\": [2, 8], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"Pru\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006355\", \"supporting_discovery_ids\": [2, 5, 6], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]},\n      {\"term_id\": \"GO:0016573\", \"supporting_discovery_ids\": [0, 2, 5], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"RH\"}]}\n    ],\n    \"complexes\": [\n      \"GCN5b–AP2IX-7 complex (with ADA2-A, TFIID subunits, LEO1/Paf1, RRM proteins)\",\n      \"GCN5b–AP2XII-4 complex (bradyzoite-associated composition)\"\n    ],\n    \"partners\": [\n      \"ADA2-A (co-activator)\",\n      \"AP2IX-7 (AP2-domain transcription factor)\",\n      \"AP2XII-4 (AP2-domain transcription factor)\",\n      \"TFIID subunits\",\n      \"LEO1 / Paf1 subunit\",\n      \"RRM proteins\",\n      \"TgGSK (essential kinase)\"\n    ],\n    \"other_free_text\": [\n      \"Catalytically dead point mutant E703G acts as dominant-negative and arrests tachyzoite replication\",\n      \"KAT domain and bromodomain are each independently required for viability\",\n      \"Inhibited by garcinol (KAT activity) and L-Moses (bromodomain)\",\n      \"Nuclear import mediated by a novel NLS (RPAENKKRGR) within an intrinsically disordered N-terminal region, distinct from GCN5-A\",\n      \"GCN5b KAT activity is required for stability of the TgGSK protein\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_243440","name":"GCN5B","product":"histone lysine acetyltransferase GCN5-B","uniprot_acc":"S8F3V2","chromosome":"TGME49_chrVI","start_pos":2629507},{"strain":"GT1","gene_id":"TGGT1_243440","name":null,"product":"histone lysine acetyltransferase GCN5-B","uniprot_acc":null,"chromosome":"TGGT1_chrVI","start_pos":2548787},{"strain":"RH88","gene_id":"TGRH88_028980","name":null,"product":"histone lysine acetyltransferase GCN5-B","uniprot_acc":null,"chromosome":"CM023087","start_pos":1079660},{"strain":"VEG","gene_id":"TGVEG_243440","name":null,"product":"histone lysine acetyltransferase GCN5-B","uniprot_acc":"B9QJI0","chromosome":"TGVEG_chrVI","start_pos":2634247}]}