{"gene":"TG_261240","gene_symbol":"","run_date":"2026-09-11T11:01:00","timeline":{"discoveries":[{"year":2007,"finding":"Methylation and acetylation of histone H3 (and H4) mark active promoters in T. gondii tachyzoites, predicting promoter position and directionality with >95% accuracy; histone H3 arginine methylation distinguishes a subset of promoters, revealing complexity in the histone modification machinery.","method":"Custom oligonucleotide microarray ChIP (chromatin immunoprecipitation) across 1% of the T. gondii genome, integrated with gene expression and prediction data","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide ChIP with multiple histone marks, corroborated by expression data, multiple orthogonal datasets in one rigorous study","pmids":["17559302"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2003,"finding":"T. gondii encodes at least two histone H3 variants, H3 and H3.3, with novel and rare sequence features in their N-terminal domains compared to other eukaryotes; recombinant forms were expressed and purified as parasite-specific substrates for histone-modifying enzyme analysis.","method":"Gene cloning, sequence analysis, recombinant expression and purification","journal":"DNA sequence : the journal of DNA sequencing and mapping","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct cloning and recombinant protein production with sequence characterization, single lab","pmids":["14509838"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2008,"finding":"Coincident trimethylation of histone H3 lysine 4 (H3K4me3) and acetylation of histone H3 lysine 9 (H3K9ac) mark promoters of actively transcribed genes in T. gondii; their presence at some non-expressed genes implies additional layers of chromatin regulation.","method":"Genome-wide ChIP integrated with transcriptome expression data (epigenomics review synthesizing experimental datasets)","journal":"The Journal of eukaryotic microbiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — review synthesizing ChIP data from prior experimental work, single lab, consistent with primary data in PMID:17559302","pmids":["19120792"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"finding":"The lysine acetyltransferase GCN5b acetylates histone H3 at specific lysine residues in T. gondii; dominant-negative GCN5b (E703G point mutation abolishing enzymatic activity) reduces global H3 acetylation and arrests parasite replication.","method":"Inducible dominant-negative (destabilization domain-fused) GCN5b expression, ChIP-chip, proteomics (Co-IP/MS), active-site point mutagenesis (E703G)","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — active-site mutagenesis combined with ChIP, proteomics, and cellular phenotype in one rigorous study","pmids":["24391497"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"finding":"Garcinol inhibits TgGCN5b lysine acetyltransferase activity, leading to reduced global lysine acetylation particularly on histone H3 in T. gondii tachyzoites and arresting replication.","method":"Drug treatment with garcinol, immunoblot for histone H3 acetylation, RNA-seq transcriptome analysis, ChIP-chip cross-reference","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — pharmacological inhibition with biochemical readout (histone H3 acetylation) and transcriptome, single lab","pmids":["26810649"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"T. gondii infection of macrophages blocks LPS-induced Ser10 phosphorylation and Lys9/Lys14 acetylation of histone H3 at the host TNF-alpha promoter, preventing chromatin remodeling required for TNF-alpha transcription.","method":"Chromatin immunoprecipitation (ChIP) at the host TNF-alpha promoter in infected macrophages; LPS stimulation assay","journal":"Journal of immunology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP with specific histone H3 modification antibodies at a defined promoter, single lab","pmids":["19109180"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"finding":"T. gondii infection inhibits histone H3 Ser10 phosphorylation and Lys9/14 acetylation at the host IL-10 promoter when macrophages are stimulated through combined TLR4 and FcγR signaling, suppressing high-level IL-10 production.","method":"ChIP at the host IL-10 promoter in infected macrophages; combined TLR4/FcγR stimulation assay; cytokine measurement","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP with histone H3 modification antibodies at a defined promoter locus, single lab","pmids":["19859561"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"finding":"Resveratrol treatment of intracellular T. gondii tachyzoites induces deacetylation of histone H3 (and H4K16) and increases phosphorylation of histone H2A.X at the SQE motif (γH2A.X), indicating induction of DNA damage responses alongside histone modification changes.","method":"Immunoblot and immunofluorescence for H3 acetylation, H4K16ac, and γH2A.X in drug-treated intracellular tachyzoites","journal":"BMC research notes","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single immunoblot/immunofluorescence readout, single lab, no mechanistic follow-up on H3 modification pathway","pmids":["33413578"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"finding":"The parasite-specific bromodomain protein TgBDP4 binds acetylated histone H3 (reader interaction); conditional knockdown of TgBDP4 is lethal to T. gondii in culture and in a mouse model, establishing its essential role in parasite survival.","method":"Protein-peptide interaction assay, pull-down with acetylated histone H3 peptides, conditional knockdown (TetO/Tet-regulatable system), parasite growth assay in culture and mouse infection model","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding assay (pull-down with acetylated H3) combined with conditional KD and in vivo phenotype, single lab","pmids":["42140435"],"is_preprint":false,"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"current_model":"In Toxoplasma gondii, histone H3 exists as two variants (H3 and H3.3) with apicomplexan-specific sequence features; its N-terminal tail undergoes combinatorial covalent modifications (particularly H3K4me3, H3K9ac, and arginine methylation) that demarcate active promoters, while the acetyltransferase GCN5b is the principal writer of H3 acetylation required for tachyzoite gene expression and replication, and bromodomain protein TgBDP4 acts as an essential reader of H3 acetylation; the parasite also exploits H3 modification pathways in host macrophages—blocking LPS/TLR-induced H3 Ser10 phosphorylation and Lys9/14 acetylation at cytokine promoters, and deploying ROP16 to drive repressive H3 deacetylation/methylation at the host cyclin B1 promoter to arrest the host cell cycle."},"narrative":{"mechanistic_narrative":"In Toxoplasma gondii, histone H3 is the substrate for a combinatorial code of N-terminal tail modifications that governs transcriptional activity in tachyzoites [PMID:17559302]. The parasite encodes two H3 variants, H3 and H3.3, whose N-terminal domains carry apicomplexan-specific sequence features [PMID:14509838]. Coincident H3K4 trimethylation and H3K9 acetylation, along with arginine methylation of a distinct promoter subset, demarcate active and directional promoters across the genome [PMID:17559302, PMID:19120792]. The lysine acetyltransferase GCN5b is the principal writer of H3 acetylation: catalytically dead GCN5b (E703G) reduces global H3 acetylation and arrests parasite replication, and pharmacological GCN5b inhibition by garcinol reproduces this loss of H3 acetylation and replication block [PMID:24391497, PMID:26810649]. Acetylated H3 is decoded by the parasite-specific bromodomain reader TgBDP4, which binds acetyl-H3 peptides and is essential for parasite survival in culture and in mice [PMID:42140435]. Beyond writing and reading its own chromatin, T. gondii manipulates host H3 modifications: infection blocks LPS/TLR-driven H3 Ser10 phosphorylation and Lys9/14 acetylation at the host TNF-alpha and IL-10 promoters to suppress cytokine transcription [PMID:19109180, PMID:19859561].","teleology":[{"year":2003,"claim":"Before enzymatic studies were possible, it was unknown whether T. gondii H3 differed from canonical eukaryotic H3; cloning revealed two variants with parasite-specific tail features, providing authentic substrates for downstream modification analysis.","evidence":"Gene cloning, sequence analysis, and recombinant expression/purification of H3 and H3.3","pmids":["14509838"],"confidence":"Medium","gaps":["Functional distinction between H3 and H3.3 not established","Modification sites on the parasite-specific tail not mapped in this study"],"attribution":[{"species":"Toxoplasma gondii","strain":""},{"species":"Plasmodium falciparum","strain":""}]},{"year":2007,"claim":"It was unclear whether histone modifications organize the T. gondii genome as in other eukaryotes; genome-wide ChIP showed H3/H4 methylation and acetylation mark active promoters with high accuracy and that H3 arginine methylation defines a promoter subset, establishing a functional histone code in the parasite.","evidence":"Custom oligonucleotide ChIP-chip across 1% of the genome integrated with expression and prediction data","pmids":["17559302"],"confidence":"High","gaps":["Enzymes writing these marks not identified here","Only 1% of genome surveyed"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2008,"claim":"Refining which specific marks define activity, coincident H3K4me3 and H3K9ac were shown to mark actively transcribed promoters, while their presence at some silent genes implied additional regulatory layers.","evidence":"Genome-wide ChIP integrated with transcriptome data (review synthesizing experimental datasets)","pmids":["19120792"],"confidence":"Medium","gaps":["Basis for marks at non-expressed genes unresolved","Synthesizes prior ChIP rather than new primary data"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2014,"claim":"The writer responsible for H3 acetylation and its functional requirement were unknown; active-site mutagenesis of GCN5b (E703G) showed it acetylates H3 and that its activity is required for parasite replication.","evidence":"Inducible dominant-negative GCN5b, ChIP-chip, Co-IP/MS, and E703G active-site point mutagenesis with replication readout","pmids":["24391497"],"confidence":"High","gaps":["Precise H3 lysine specificity of GCN5b not fully resolved","Direct versus indirect contribution of other HATs not delineated"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2016,"claim":"Extending the genetic finding pharmacologically, garcinol was shown to inhibit TgGCN5b, reduce global H3 acetylation, and arrest replication, validating H3 acetylation as a druggable dependency.","evidence":"Garcinol treatment with immunoblot for H3 acetylation, RNA-seq, and ChIP-chip cross-reference","pmids":["26810649"],"confidence":"Medium","gaps":["Off-target effects of garcinol not excluded","Selectivity for GCN5b over other acetyltransferases not quantified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2009,"claim":"Whether T. gondii manipulates host H3 to evade immunity was untested; infection was shown to block LPS-induced H3 Ser10 phosphorylation and Lys9/14 acetylation at the TNF-alpha promoter and at the IL-10 promoter under combined TLR4/FcgammaR signaling, linking host H3 modification to cytokine suppression.","evidence":"ChIP with H3 modification antibodies at host TNF-alpha and IL-10 promoters in stimulated infected macrophages, with cytokine measurement","pmids":["19109180","19859561"],"confidence":"Medium","gaps":["Parasite effector driving the block not identified in these studies","Mechanism connecting infection to loss of the host modifying enzymes unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2021,"claim":"Whether stress agents perturb parasite H3 was addressed by showing resveratrol induces H3 (and H4K16) deacetylation alongside gammaH2A.X, coupling histone modification change to a DNA damage response.","evidence":"Immunoblot and immunofluorescence for H3ac, H4K16ac, and gammaH2A.X in drug-treated intracellular tachyzoites","pmids":["33413578"],"confidence":"Low","gaps":["Single immunoblot/IF readout without mechanistic follow-up on the H3 pathway","Whether H3 deacetylation is cause or consequence of DNA damage unresolved"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":2026,"claim":"The reader that decodes acetylated H3 and its essentiality were unknown; the parasite-specific bromodomain protein TgBDP4 was shown to bind acetyl-H3 and to be essential in culture and in mice, closing the write-read loop for H3 acetylation.","evidence":"Pull-down with acetylated H3 peptides, conditional TetO knockdown, and growth assays in culture and mouse infection","pmids":["42140435"],"confidence":"Medium","gaps":["Specific acetyl-lysine residues bound by TgBDP4 not delineated","Downstream transcriptional targets of TgBDP4 not mapped"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]},{"year":null,"claim":"How the distinct functions of H3 versus H3.3 variants, and the full set of enzymes writing/erasing the combinatorial marks in T. gondii, are coordinated remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["Functional division of labor between H3 and H3.3 not established","Complete writer/eraser/reader inventory incomplete","Arginine methyltransferase responsible for promoter subset marking not identified"],"attribution":[{"species":"Toxoplasma gondii","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":[],"partners":["GCN5B (TGGCN5B) — LYSINE ACETYLTRANSFERASE WRITER OF H3 ACETYLATION","TGBDP4 — BROMODOMAIN READER BINDING ACETYLATED H3"],"other_free_text":["H3 exists as two variants, H3 and H3.3, with apicomplexan-specific N-terminal tail features [#1].","Active promoters are marked by coincident H3K4me3 and H3K9ac; a promoter subset is distinguished by H3 arginine methylation [#0, #2].","GCN5b-dependent H3 acetylation is required for tachyzoite replication and is pharmacologically inhibitable by garcinol [#3, #4].","T. gondii manipulates host H3 modifications at TNF-alpha, IL-10, and cyclin B1 promoters to suppress cytokines and arrest the host cell cycle [#5, #6, #7]."]},"strain_conflicts":[]},"prefetch_data":{},"corpus_meta":[{"pmid":"17559302","id":"PMC_17559302","title":"Epigenomic modifications predict active promoters and gene structure in Toxoplasma gondii.","date":"2007","source":"PLoS pathogens","url":"https://pubmed.ncbi.nlm.nih.gov/17559302","citation_count":97,"is_preprint":false},{"pmid":"19501055","id":"PMC_19501055","title":"Drug discovery targeting epigenetic codes: the great potential of UHRF1, which links DNA methylation and histone modifications, as a drug target in cancers and toxoplasmosis.","date":"2009","source":"Biochemical pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/19501055","citation_count":92,"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":"19109180","id":"PMC_19109180","title":"Toxoplasma gondii prevents chromatin remodeling initiated by TLR-triggered macrophage activation.","date":"2009","source":"Journal of immunology (Baltimore, Md. : 1950)","url":"https://pubmed.ncbi.nlm.nih.gov/19109180","citation_count":52,"is_preprint":false},{"pmid":"28669381","id":"PMC_28669381","title":"Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle.","date":"2017","source":"Veterinary immunology and immunopathology","url":"https://pubmed.ncbi.nlm.nih.gov/28669381","citation_count":39,"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":"19859561","id":"PMC_19859561","title":"Toxoplasma gondii inhibits covalent modification of histone H3 at the IL-10 promoter in infected macrophages.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19859561","citation_count":30,"is_preprint":false},{"pmid":"14509838","id":"PMC_14509838","title":"Histone H3 and H3.3 variants in the protozoan pathogens Plasmodium falciparum and Toxoplasma gondii.","date":"2003","source":"DNA sequence : the journal of DNA sequencing and mapping","url":"https://pubmed.ncbi.nlm.nih.gov/14509838","citation_count":19,"is_preprint":false},{"pmid":"31492965","id":"PMC_31492965","title":"Toxoplasma gondii ROP16 kinase silences the cyclin B1 gene promoter by hijacking host cell UHRF1-dependent epigenetic pathways.","date":"2019","source":"Cellular and molecular life sciences : CMLS","url":"https://pubmed.ncbi.nlm.nih.gov/31492965","citation_count":17,"is_preprint":false},{"pmid":"33413578","id":"PMC_33413578","title":"Resveratrol induces H3 and H4K16 deacetylation and H2A.X phosphorylation in Toxoplasma gondii.","date":"2021","source":"BMC research notes","url":"https://pubmed.ncbi.nlm.nih.gov/33413578","citation_count":11,"is_preprint":false},{"pmid":"19120792","id":"PMC_19120792","title":"How epigenomics contributes to the understanding of gene regulation in Toxoplasma gondii.","date":"2008","source":"The Journal of eukaryotic microbiology","url":"https://pubmed.ncbi.nlm.nih.gov/19120792","citation_count":9,"is_preprint":false},{"pmid":"42140435","id":"PMC_42140435","title":"Parasite-specific essential bromodomain protein TgBDP4 is a key epigenetic reader and a potential drug target for the parasite Toxoplasma gondii.","date":"2026","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/42140435","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8163,"output_tokens":2757,"usd":0.032922,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8395,"output_tokens":4195,"usd":0.073425,"stage2_stop_reason":"end_turn"},"total_usd":0.106347,"stage1_batch_id":"msgbatch_01TEPNZ4MCzJ7xtsNwosiDRk","stage2_batch_id":"msgbatch_01FQShM7n1sfCr4pWTDShmki","note":"batch pricing = 50% of standard","hand_review_patch":"2026-09-11 AK hand-review verdicts (handoff/tools_2026-09-11/patch)","hand_review_verdict":"edit"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2007,\n      \"finding\": \"Methylation and acetylation of histone H3 (and H4) mark active promoters in T. gondii tachyzoites, predicting promoter position and directionality with >95% accuracy; histone H3 arginine methylation distinguishes a subset of promoters, revealing complexity in the histone modification machinery.\",\n      \"method\": \"Custom oligonucleotide microarray ChIP (chromatin immunoprecipitation) across 1% of the T. gondii genome, integrated with gene expression and prediction data\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide ChIP with multiple histone marks, corroborated by expression data, multiple orthogonal datasets in one rigorous study\",\n      \"pmids\": [\"17559302\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"T. gondii encodes at least two histone H3 variants, H3 and H3.3, with novel and rare sequence features in their N-terminal domains compared to other eukaryotes; recombinant forms were expressed and purified as parasite-specific substrates for histone-modifying enzyme analysis.\",\n      \"method\": \"Gene cloning, sequence analysis, recombinant expression and purification\",\n      \"journal\": \"DNA sequence : the journal of DNA sequencing and mapping\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct cloning and recombinant protein production with sequence characterization, single lab\",\n      \"pmids\": [\"14509838\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Coincident trimethylation of histone H3 lysine 4 (H3K4me3) and acetylation of histone H3 lysine 9 (H3K9ac) mark promoters of actively transcribed genes in T. gondii; their presence at some non-expressed genes implies additional layers of chromatin regulation.\",\n      \"method\": \"Genome-wide ChIP integrated with transcriptome expression data (epigenomics review synthesizing experimental datasets)\",\n      \"journal\": \"The Journal of eukaryotic microbiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — review synthesizing ChIP data from prior experimental work, single lab, consistent with primary data in PMID:17559302\",\n      \"pmids\": [\"19120792\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"The lysine acetyltransferase GCN5b acetylates histone H3 at specific lysine residues in T. gondii; dominant-negative GCN5b (E703G point mutation abolishing enzymatic activity) reduces global H3 acetylation and arrests parasite replication.\",\n      \"method\": \"Inducible dominant-negative (destabilization domain-fused) GCN5b expression, ChIP-chip, proteomics (Co-IP/MS), active-site point mutagenesis (E703G)\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — active-site mutagenesis combined with ChIP, proteomics, and cellular phenotype in one rigorous study\",\n      \"pmids\": [\"24391497\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Garcinol inhibits TgGCN5b lysine acetyltransferase activity, leading to reduced global lysine acetylation particularly on histone H3 in T. gondii tachyzoites and arresting replication.\",\n      \"method\": \"Drug treatment with garcinol, immunoblot for histone H3 acetylation, RNA-seq transcriptome analysis, ChIP-chip cross-reference\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — pharmacological inhibition with biochemical readout (histone H3 acetylation) and transcriptome, single lab\",\n      \"pmids\": [\"26810649\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"T. gondii infection of macrophages blocks LPS-induced Ser10 phosphorylation and Lys9/Lys14 acetylation of histone H3 at the host TNF-alpha promoter, preventing chromatin remodeling required for TNF-alpha transcription.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP) at the host TNF-alpha promoter in infected macrophages; LPS stimulation assay\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with specific histone H3 modification antibodies at a defined promoter, single lab\",\n      \"pmids\": [\"19109180\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"T. gondii infection inhibits histone H3 Ser10 phosphorylation and Lys9/14 acetylation at the host IL-10 promoter when macrophages are stimulated through combined TLR4 and FcγR signaling, suppressing high-level IL-10 production.\",\n      \"method\": \"ChIP at the host IL-10 promoter in infected macrophages; combined TLR4/FcγR stimulation assay; cytokine measurement\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP with histone H3 modification antibodies at a defined promoter locus, single lab\",\n      \"pmids\": [\"19859561\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"T. gondii ROP16 kinase phosphorylates host UHRF1, which then recruits a repressive multienzymatic complex to the cyclin B1 promoter, leading to deacetylation and methylation of histone H3 surrounding that promoter and silencing cyclin B1 transcription to arrest the host cell cycle.\",\n      \"method\": \"Chromatin remodeling assay at cyclin B1 promoter (ChIP), ROP16 strain-dependent analysis, UHRF1 knockdown, DNMT activity measurement, protein complex characterization\",\n      \"journal\": \"Cellular and molecular life sciences : CMLS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP plus biochemical/genetic epistasis (ROP16→UHRF1→H3 modification), single lab, multiple complementary methods\",\n      \"pmids\": [\"31492955\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Resveratrol treatment of intracellular T. gondii tachyzoites induces deacetylation of histone H3 (and H4K16) and increases phosphorylation of histone H2A.X at the SQE motif (γH2A.X), indicating induction of DNA damage responses alongside histone modification changes.\",\n      \"method\": \"Immunoblot and immunofluorescence for H3 acetylation, H4K16ac, and γH2A.X in drug-treated intracellular tachyzoites\",\n      \"journal\": \"BMC research notes\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single immunoblot/immunofluorescence readout, single lab, no mechanistic follow-up on H3 modification pathway\",\n      \"pmids\": [\"33413578\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"The parasite-specific bromodomain protein TgBDP4 binds acetylated histone H3 (reader interaction); conditional knockdown of TgBDP4 is lethal to T. gondii in culture and in a mouse model, establishing its essential role in parasite survival.\",\n      \"method\": \"Protein-peptide interaction assay, pull-down with acetylated histone H3 peptides, conditional knockdown (TetO/Tet-regulatable system), parasite growth assay in culture and mouse infection model\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding assay (pull-down with acetylated H3) combined with conditional KD and in vivo phenotype, single lab\",\n      \"pmids\": [\"42140435\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"In Toxoplasma gondii, histone H3 exists as two variants (H3 and H3.3) with apicomplexan-specific sequence features; its N-terminal tail undergoes combinatorial covalent modifications (particularly H3K4me3, H3K9ac, and arginine methylation) that demarcate active promoters, while the acetyltransferase GCN5b is the principal writer of H3 acetylation required for tachyzoite gene expression and replication, and bromodomain protein TgBDP4 acts as an essential reader of H3 acetylation; the parasite also exploits H3 modification pathways in host macrophages—blocking LPS/TLR-induced H3 Ser10 phosphorylation and Lys9/14 acetylation at cytokine promoters, and deploying ROP16 to drive repressive H3 deacetylation/methylation at the host cyclin B1 promoter to arrest the host cell cycle.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"In Toxoplasma gondii, histone H3 is the substrate for a combinatorial code of N-terminal tail modifications that governs transcriptional activity in tachyzoites [#0]. The parasite encodes two H3 variants, H3 and H3.3, whose N-terminal domains carry apicomplexan-specific sequence features [#1]. Coincident H3K4 trimethylation and H3K9 acetylation, along with arginine methylation of a distinct promoter subset, demarcate active and directional promoters across the genome [#0, #2]. The lysine acetyltransferase GCN5b is the principal writer of H3 acetylation: catalytically dead GCN5b (E703G) reduces global H3 acetylation and arrests parasite replication, and pharmacological GCN5b inhibition by garcinol reproduces this loss of H3 acetylation and replication block [#3, #4]. Acetylated H3 is decoded by the parasite-specific bromodomain reader TgBDP4, which binds acetyl-H3 peptides and is essential for parasite survival in culture and in mice [#9]. Beyond writing and reading its own chromatin, T. gondii manipulates host H3 modifications: infection blocks LPS/TLR-driven H3 Ser10 phosphorylation and Lys9/14 acetylation at the host TNF-alpha and IL-10 promoters to suppress cytokine transcription [#5, #6], and the secreted kinase ROP16 phosphorylates host UHRF1 to recruit a repressive complex that deacetylates and methylates H3 at the cyclin B1 promoter, arresting the host cell cycle [#7].\",\n  \"teleology\": [\n    {\n      \"year\": 2003,\n      \"claim\": \"Before enzymatic studies were possible, it was unknown whether T. gondii H3 differed from canonical eukaryotic H3; cloning revealed two variants with parasite-specific tail features, providing authentic substrates for downstream modification analysis.\",\n      \"evidence\": \"Gene cloning, sequence analysis, and recombinant expression/purification of H3 and H3.3\",\n      \"pmids\": [\"14509838\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional distinction between H3 and H3.3 not established\", \"Modification sites on the parasite-specific tail not mapped in this study\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}, {\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"It was unclear whether histone modifications organize the T. gondii genome as in other eukaryotes; genome-wide ChIP showed H3/H4 methylation and acetylation mark active promoters with high accuracy and that H3 arginine methylation defines a promoter subset, establishing a functional histone code in the parasite.\",\n      \"evidence\": \"Custom oligonucleotide ChIP-chip across 1% of the genome integrated with expression and prediction data\",\n      \"pmids\": [\"17559302\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Enzymes writing these marks not identified here\", \"Only 1% of genome surveyed\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Refining which specific marks define activity, coincident H3K4me3 and H3K9ac were shown to mark actively transcribed promoters, while their presence at some silent genes implied additional regulatory layers.\",\n      \"evidence\": \"Genome-wide ChIP integrated with transcriptome data (review synthesizing experimental datasets)\",\n      \"pmids\": [\"19120792\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Basis for marks at non-expressed genes unresolved\", \"Synthesizes prior ChIP rather than new primary data\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"The writer responsible for H3 acetylation and its functional requirement were unknown; active-site mutagenesis of GCN5b (E703G) showed it acetylates H3 and that its activity is required for parasite replication.\",\n      \"evidence\": \"Inducible dominant-negative GCN5b, ChIP-chip, Co-IP/MS, and E703G active-site point mutagenesis with replication readout\",\n      \"pmids\": [\"24391497\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Precise H3 lysine specificity of GCN5b not fully resolved\", \"Direct versus indirect contribution of other HATs not delineated\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Extending the genetic finding pharmacologically, garcinol was shown to inhibit TgGCN5b, reduce global H3 acetylation, and arrest replication, validating H3 acetylation as a druggable dependency.\",\n      \"evidence\": \"Garcinol treatment with immunoblot for H3 acetylation, RNA-seq, and ChIP-chip cross-reference\",\n      \"pmids\": [\"26810649\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Off-target effects of garcinol not excluded\", \"Selectivity for GCN5b over other acetyltransferases not quantified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Whether T. gondii manipulates host H3 to evade immunity was untested; infection was shown to block LPS-induced H3 Ser10 phosphorylation and Lys9/14 acetylation at the TNF-alpha promoter and at the IL-10 promoter under combined TLR4/FcgammaR signaling, linking host H3 modification to cytokine suppression.\",\n      \"evidence\": \"ChIP with H3 modification antibodies at host TNF-alpha and IL-10 promoters in stimulated infected macrophages, with cytokine measurement\",\n      \"pmids\": [\"19109180\", \"19859561\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Parasite effector driving the block not identified in these studies\", \"Mechanism connecting infection to loss of the host modifying enzymes unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"The effector and pathway for host cell-cycle arrest via H3 were unknown; ROP16 was shown to phosphorylate host UHRF1, recruiting a repressive complex that deacetylates and methylates H3 at the cyclin B1 promoter to silence transcription and arrest the host cell cycle.\",\n      \"evidence\": \"ChIP at cyclin B1 promoter, ROP16 strain-dependent analysis, UHRF1 knockdown, DNMT activity assay, and complex characterization\",\n      \"pmids\": [\"31492955\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of all enzymes in the recruited repressive complex not fully defined\", \"Strain-dependence of ROP16 effect not detailed here\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Whether stress agents perturb parasite H3 was addressed by showing resveratrol induces H3 (and H4K16) deacetylation alongside gammaH2A.X, coupling histone modification change to a DNA damage response.\",\n      \"evidence\": \"Immunoblot and immunofluorescence for H3ac, H4K16ac, and gammaH2A.X in drug-treated intracellular tachyzoites\",\n      \"pmids\": [\"33413578\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single immunoblot/IF readout without mechanistic follow-up on the H3 pathway\", \"Whether H3 deacetylation is cause or consequence of DNA damage unresolved\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"claim\": \"The reader that decodes acetylated H3 and its essentiality were unknown; the parasite-specific bromodomain protein TgBDP4 was shown to bind acetyl-H3 and to be essential in culture and in mice, closing the write-read loop for H3 acetylation.\",\n      \"evidence\": \"Pull-down with acetylated H3 peptides, conditional TetO knockdown, and growth assays in culture and mouse infection\",\n      \"pmids\": [\"42140435\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific acetyl-lysine residues bound by TgBDP4 not delineated\", \"Downstream transcriptional targets of TgBDP4 not mapped\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How the distinct functions of H3 versus H3.3 variants, and the full set of enzymes writing/erasing the combinatorial marks in T. gondii, are coordinated remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Functional division of labor between H3 and H3.3 not established\", \"Complete writer/eraser/reader inventory incomplete\", \"Arginine methyltransferase responsible for promoter subset marking not identified\"],\n      \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0031492\", \"supporting_discovery_ids\": [1], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0140566\", \"supporting_discovery_ids\": [0, 2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0000786\", \"supporting_discovery_ids\": [0, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006325\", \"supporting_discovery_ids\": [0, 2, 3], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0045893\", \"supporting_discovery_ids\": [0, 2], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]},\n      {\"term_id\": \"GO:0000122\", \"supporting_discovery_ids\": [5, 6, 7], \"attribution\": [{\"species\": \"Toxoplasma gondii\", \"strain\": \"\"}]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"GCN5b (TgGCN5b) — lysine acetyltransferase writer of H3 acetylation\",\n      \"TgBDP4 — bromodomain reader binding acetylated H3\",\n      \"ROP16 — parasite kinase driving repressive H3 modification at host cyclin B1 via UHRF1\",\n      \"host UHRF1 — phosphorylated by ROP16 to recruit repressive complex\"\n    ],\n    \"other_free_text\": [\n      \"H3 exists as two variants, H3 and H3.3, with apicomplexan-specific N-terminal tail features [#1].\",\n      \"Active promoters are marked by coincident H3K4me3 and H3K9ac; a promoter subset is distinguished by H3 arginine methylation [#0, #2].\",\n      \"GCN5b-dependent H3 acetylation is required for tachyzoite replication and is pharmacologically inhibitable by garcinol [#3, #4].\",\n      \"T. gondii manipulates host H3 modifications at TNF-alpha, IL-10, and cyclin B1 promoters to suppress cytokines and arrest the host cell cycle [#5, #6, #7].\"\n    ]\n  },\n  \"strain_conflicts\": []\n}","audit_flag":null,"strains":[{"strain":"ME49","gene_id":"TGME49_261240","name":null,"product":"histone H3","uniprot_acc":"A0A125YSM1","chromosome":"TGME49_chrVIIb","start_pos":1807866},{"strain":"GT1","gene_id":"TGGT1_261240","name":null,"product":"histone H3","uniprot_acc":null,"chromosome":"TGGT1_chrVIIb","start_pos":1780702},{"strain":"RH88","gene_id":"TGRH88_069360","name":null,"product":"histone H3","uniprot_acc":null,"chromosome":"CM023089","start_pos":1826311},{"strain":"VEG","gene_id":"TGVEG_261240","name":null,"product":"histone H3","uniprot_acc":"A0A0F7UZ25","chromosome":"TGVEG_chrVIIb","start_pos":1822729}]}