{"gene":"HDAC8","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":2000,"finding":"HDAC8 was cloned and characterized as a new class I HDAC with trichostatin A- and sodium butyrate-inhibitable deacetylase activity on histone H4 peptide substrates and core histones; mutation of two adjacent histidine residues in the predicted active site severely decreased activity, confirming these residues as essential for catalysis.","method":"Expression in cell systems, immunopurification, in vitro HDAC activity assay, active-site histidine mutagenesis","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro enzymatic assay plus mutagenesis in the original characterization paper","pmids":["10926844"],"is_preprint":false},{"year":2014,"finding":"HDAC8 is the Zn2+-dependent SMC3 (cohesin subunit) lysine deacetylase required for cohesin recycling during the cell cycle; HDAC8 inhibition leads to accumulation of acetylated SMC3, delays cell cycle progression, and suppresses proliferation without affecting cohesin-dependent transcription of estrogen-responsive genes.","method":"HDAC8-specific inhibitor (PCI-34051), Western blot for ac-SMC3, ChIP, siRNA knockdown, cell cycle analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (inhibitor, siRNA, ChIP) in a single rigorous study, consistent with structural studies on CdLS mutations","pmids":["27072133"],"is_preprint":false},{"year":2014,"finding":"HDAC8 physically interacts with EZH2 to concordantly repress Wnt antagonists via histone H4 deacetylation and H3K27 trimethylation in NAFLD-associated hepatocellular carcinoma cells.","method":"Co-immunoprecipitation, ChIP, lentiviral knockdown in vivo, Western blot","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2/3 — reciprocal co-IP plus in vivo knockdown, single lab","pmids":["26383163"],"is_preprint":false},{"year":2014,"finding":"HDAC8 and STAT3 directly repress the BMF gene promoter; HDAC8 occupancy at the BMF promoter was identified as a direct transcriptional target, and overexpression of HDAC8 interfered with BMF induction whereas HDAC8 RNAi activated it.","method":"ChIP, RNAi knockdown, transient overexpression, p300 inhibitor treatment, reporter assay","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2/3 — ChIP plus functional RNAi/overexpression, single lab","pmids":["25321483"],"is_preprint":false},{"year":2014,"finding":"X-ray crystal structures of five CdLS-associated HDAC8 missense mutants (C153F, A188T, I243N, T311M, H334R) revealed local structural changes that compromise catalysis and/or thermostability; the catalytic activity of these mutants could be partially or fully rescued in vitro by the HDAC8 activator N-(phenylcarbamothioyl)benzamide.","method":"X-ray crystallography, enzymatic activity assays, thermostability assays, activator rescue experiments","journal":"ACS chemical biology","confidence":"High","confidence_rationale":"Tier 1 — crystal structures plus in vitro enzymatic characterization with mutagenesis and activator rescue","pmids":["25075551"],"is_preprint":false},{"year":2015,"finding":"X-ray crystal structures of additional CdLS HDAC8 mutants (G117E, P91L-Y306F, D233G-Y306F) showed that CdLS mutations disrupt key hydrogen bond networks and active-site residue positioning, compromising catalysis and thermostability; molecular dynamics simulations of H180R and G304R showed bulky arginine side chains protruding into the substrate binding site.","method":"X-ray crystallography, molecular dynamics simulations, enzymatic activity assays, thermostability assays","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple crystal structures plus MD simulations and enzymatic assays","pmids":["26463496"],"is_preprint":false},{"year":2016,"finding":"Structure-based identification of novel HDAC8 non-histone substrates using Rosetta FlexPepBind revealed that SMC3, ERRα, and ARID1A are among known non-histone HDAC8 substrates, and the in silico approach identified many additional acetyl-lysine-containing peptides as HDAC8 substrates in vitro.","method":"Structure-based computational docking (Rosetta FlexPepBind), in vitro peptide deacetylation assays","journal":"Structure","confidence":"Medium","confidence_rationale":"Tier 1/3 — in vitro substrate assays validated computationally, single lab","pmids":["26933971"],"is_preprint":false},{"year":2017,"finding":"HDAC8 protein interacts with p53 and modulates p53 activity via deacetylation; HDAC8-deficient LT-HSCs displayed hyperactivation of p53, increased apoptosis under stress, and compromised long-term repopulating activity, all of which were rescued by genetic inactivation of p53.","method":"Co-immunoprecipitation, Hdac8 conditional knockout, serial repopulation assays, genetic epistasis (p53 double KO), Western blot","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — reciprocal co-IP, clean conditional KO with defined phenotype, and genetic epistasis rescue","pmids":["29084772"],"is_preprint":false},{"year":2019,"finding":"HDAC8 deacetylates c-Jun at lysine 273, decreasing c-Jun acetylation and increasing its transcriptional activity; HDAC8-mediated BRAF inhibitor resistance in melanoma is mediated through receptor tyrosine kinase activation and MAPK/AP-1 signaling.","method":"Mass spectrometry-based phosphoproteomics, HDAC8 introduction into drug-naïve cells, in vitro and in vivo xenograft studies, c-Jun K273 acetylation site mutagenesis","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1/2 — MS-based substrate identification, site mutagenesis, in vivo validation","pmids":["30987999"],"is_preprint":false},{"year":2019,"finding":"HDAC8 interacts with the proteasome receptor ADRM1 to regulate MGMT protein levels independent of MGMT promoter methylation in glioblastoma cells; HDAC8 inhibition or shRNA decreased MGMT levels and increased DNA damage and cell cycle arrest.","method":"Co-immunoprecipitation, HDAC8-specific inhibitor (PCI34051), shRNA knockdown, Western blot","journal":"Genes & cancer","confidence":"Medium","confidence_rationale":"Tier 3 — single co-IP and pharmacological inhibition, single lab","pmids":["31798765"],"is_preprint":false},{"year":2019,"finding":"HDAC8 binds the ID2 enhancer and represses ID2 transcription; lncRNA ID2-AS1 suppresses HCC metastasis by blocking HDAC8 binding to the ID2 enhancer, thereby increasing H3K27 acetylation and ID2 expression.","method":"ChIP, RNAi knockdown, overexpression, migration/invasion assays in vitro and in vivo","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2/3 — ChIP demonstrating HDAC8 occupancy and functional KD, single lab","pmids":["31730902"],"is_preprint":false},{"year":2020,"finding":"FLT3 inhibition induces HDAC8 upregulation through FOXO1- and FOXO3-mediated transactivation; upregulated HDAC8 deacetylates and inactivates p53, promoting FLT3-ITD+ AML maintenance and TKI resistance.","method":"Genetic/pharmacological HDAC8 inhibition, FOXO1/3 ChIP, p53 acetylation Western blot, patient-derived xenograft models","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 — ChIP for transcriptional regulation, p53 deacetylation shown, in vivo PDX validation","pmids":["32315388"],"is_preprint":false},{"year":2020,"finding":"AMPK phosphorylates HDAC8 in response to glucose deprivation, stimulating HDAC8 translocation from the nucleus to the cytoplasm and disrupting HDAC8 binding to histone H3, thereby derepressing PGM1 expression in lung cancer cells.","method":"Co-immunoprecipitation, nuclear/cytoplasmic fractionation, Western blot, AMPK inhibition/activation","journal":"Cancer letters","confidence":"Medium","confidence_rationale":"Tier 2/3 — fractionation with functional consequence, single lab","pmids":["32171858"],"is_preprint":false},{"year":2020,"finding":"HDAC8 binds and deacetylates PKM2 at the K62 residue; K62 deacetylation facilitates PKM2 nuclear translocation where it binds β-catenin to promote CCND1 transcription and cell cycle progression in hepatocellular carcinoma.","method":"Co-immunoprecipitation, site-directed mutagenesis (K62), subcellular fractionation, ChIP, enzymatic activity assays","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1/2 — in vitro deacetylation assay, mutagenesis, ChIP, and nuclear localization with functional readout","pmids":["33279948"],"is_preprint":false},{"year":2021,"finding":"HDAC8 is an H3K27-specific deacetylase in HCC; pharmacological inhibition of HDAC8 increases global and enhancer H3K27 acetylation, reactivating T cell-trafficking chemokines and relieving T cell exclusion.","method":"Chromatin profiling (ChIP-Seq), selective HDAC8 inhibitor treatment, humanized and immunodeficient mouse models","journal":"Science translational medicine","confidence":"High","confidence_rationale":"Tier 1/2 — genome-wide chromatin profiling plus multiple in vivo models demonstrating H3K27 substrate specificity","pmids":["33827976"],"is_preprint":false},{"year":2021,"finding":"HDAC8 interacts with HDAC9 and the FACT complex (SSRP1 and SUPT16H) to regulate H3K9 and H3K27 acetylation at the MAP2K3 promoter, thereby suppressing MAP2K3 expression and maintaining cutaneous innate immune tolerance in keratinocytes.","method":"Proteomic analysis (HDAC8/9 interactors), ChIP for histone marks, HDAC8/9 siRNA, keratinocyte-specific KO mice, cytokine assays","journal":"Science immunology","confidence":"High","confidence_rationale":"Tier 2 — proteomics identified complex, ChIP confirmed histone marks, KO mouse with in vivo phenotype","pmids":["34021025"],"is_preprint":false},{"year":2018,"finding":"HDAC8 associates with α-smooth muscle actin (α-SMA) in TGFβ1-treated lung fibroblasts as shown by co-immunoprecipitation; HDAC8 inhibition represses TGFβ1-induced fibroblast contraction, α-SMA expression, and fibrotic gene expression, and increases H3K27ac at the PPARγ gene enhancer.","method":"Co-immunoprecipitation, HDAC8-selective inhibitor (NCC170), siRNA knockdown, ChIP-qPCR (H3K27ac), collagen gel contraction assay, bleomycin mouse model","journal":"American journal of physiology. Lung cellular and molecular physiology","confidence":"High","confidence_rationale":"Tier 2 — co-IP, ChIP, selective inhibitor, siRNA, and in vivo mouse model with multiple orthogonal methods","pmids":["30358439"],"is_preprint":false},{"year":2014,"finding":"DEC1 interacts with HDAC8 and recruits HDAC8 to the TAp73 promoter to enhance TAp73 expression; HDAC8 is required for DEC1-mediated transcriptional activation of TAp73 but not ΔNp73.","method":"Co-immunoprecipitation, ChIP, RNAi knockdown, reporter assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 3 — co-IP and ChIP demonstrating recruitment, single lab","pmids":["24404147"],"is_preprint":false},{"year":2008,"finding":"HDAC8 localizes in the nucleus of HEK293 cells and binds both CREB and PP1; expression of HDAC8 decreases CREB phosphorylation at S133 and CREB-mediated gene transcription in response to forskolin.","method":"Co-immunoprecipitation, subcellular localization, overexpression, luciferase reporter assay","journal":"Biochemical and biophysical research communications","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP/overexpression, single lab, single method","pmids":["19070599"],"is_preprint":false},{"year":2016,"finding":"HDAC8 maintains Notch1 protein stability in breast cancer cells through a non-epigenetic mechanism; HDAC8 inhibition promotes Notch1 proteasomal degradation via Fbxw7, although HDAC8 does not form a complex with Notch1 and inhibition does not affect Notch1 acetylation.","method":"Co-immunoprecipitation, siRNA/shRNA knockdown, proteasome inhibitor (MG132), Fbxw7 siRNA, xenograft model","journal":"Oncotarget","confidence":"Medium","confidence_rationale":"Tier 2/3 — multiple genetic approaches and proteasome rescue, but mechanism linking HDAC8 to Notch1 stability remains indirect","pmids":["26625202"],"is_preprint":false},{"year":2023,"finding":"HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation; increased HDAC8 activity leads to EP300 binding at c-Jun transcriptional sites (increasing chromatin accessibility) and away from MITF sites, driving a neural crest-stem cell state that promotes melanoma brain metastasis.","method":"ATAC-Seq, ChIP-Seq (H3K27ac), mass spectrometry, HDAC8 inhibition, in vivo brain metastasis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1/2 — MS-based substrate identification (EP300 deacetylation), ATAC-Seq, ChIP-Seq, in vivo model with multiple orthogonal methods","pmids":["38030596"],"is_preprint":false},{"year":2023,"finding":"Simultaneous PROTAC-mediated degradation of HDAC3 and HDAC8 (YX968) does not induce global histone hyperacetylation or broad transcriptomic perturbation, suggesting that histone hyperacetylation is a major factor driving transcriptional changes induced by HDAC inhibitors rather than loss of HDAC3/8 catalytic activity per se.","method":"PROTAC-induced protein degradation, quantitative proteomics, histone acetylation Western blot, RNA-Seq, cell viability assays","journal":"Cell chemical biology","confidence":"Medium","confidence_rationale":"Tier 1/2 — selective protein degradation with unbiased quantitative proteomics confirming selectivity, single lab","pmids":["37572669"],"is_preprint":false},{"year":2021,"finding":"HDAC8 inhibition in cardiac hypertrophy promotes p38 MAPK dephosphorylation; HDAC8 overexpression promoted p38 MAPK phosphorylation and cardiac hypertrophic marker expression, while HDAC8 knockdown or selective inhibition (PCI34051) reversed isoproterenol-induced hypertrophy via reduction of p38 MAPK activity.","method":"HDAC8 selective inhibitor (PCI34051), HDAC8 knockdown/overexpression, Western blot (p38 MAPK phosphorylation), mouse isoproterenol model, echocardiography","journal":"Frontiers in pharmacology","confidence":"Medium","confidence_rationale":"Tier 2/3 — gain and loss of function with defined signaling readout, single lab","pmids":["33959033"],"is_preprint":false},{"year":2020,"finding":"HDAC8 interacts with galectin-3 (Gal-3) as shown by co-immunoprecipitation; selective HDAC8 inhibition (PCI-34051) synchronously reduces HDAC8-Gal-3 complex levels and M2 macrophage polarization, attenuating airway hyperresponsiveness.","method":"Co-immunoprecipitation, immunofluorescence, selective inhibitor (PCI-34051), shRNA knockdown","journal":"Respiratory research","confidence":"Low","confidence_rationale":"Tier 3 — single co-IP, single lab","pmids":["32111211"],"is_preprint":false}],"current_model":"HDAC8 is a class I, Zn2+-dependent histone deacetylase whose core catalytic function—requiring active-site histidine residues—removes acetyl groups from lysine residues on both histone substrates (particularly H3K27 and H4) and non-histone proteins including SMC3 (cohesin, required for cohesin recycling and cell cycle progression), p53 (regulating apoptosis in HSCs and AML), PKM2 (promoting nuclear localization and β-catenin/CCND1 signaling), c-Jun (increasing AP-1 transcriptional activity in melanoma), and EP300 (inactivating this acetyltransferase to alter chromatin accessibility); HDAC8 is regulated by AMPK-mediated phosphorylation that drives its nuclear-to-cytoplasmic translocation, operates in complex with partners such as EZH2, STAT3, DEC1, ADRM1, Gal-3, and SSRP1/SUPT16H-FACT, and its loss-of-function mutations underlie Cornelia de Lange Syndrome by impairing SMC3 deacetylation and cohesin recycling."},"narrative":{"teleology":[{"year":2000,"claim":"Establishing HDAC8 as a bona fide class I histone deacetylase answered the question of whether additional HDACs with distinct expression patterns existed; mutagenesis of active-site histidines proved these residues are essential for catalysis.","evidence":"Cloning, immunopurified in vitro HDAC assays on H4 peptide/core histones, active-site His mutagenesis","pmids":["10926844"],"confidence":"High","gaps":["Physiological substrates beyond bulk histones unknown","In vivo function not addressed","Regulatory mechanisms not explored"]},{"year":2008,"claim":"Nuclear localization and interaction with CREB/PP1 suggested HDAC8 could modulate signaling-responsive transcription, expanding its role beyond histone deacetylation.","evidence":"Co-immunoprecipitation and overexpression with luciferase reporter in HEK293 cells","pmids":["19070599"],"confidence":"Low","gaps":["Single co-IP without reciprocal validation or endogenous confirmation","No direct deacetylation of CREB shown","Relevance to physiological signaling unclear"]},{"year":2014,"claim":"Identification of SMC3 as the key HDAC8 substrate for cohesin recycling, and structural characterization of Cornelia de Lange Syndrome mutations, established the molecular basis by which HDAC8 loss-of-function causes a developmental disorder.","evidence":"HDAC8 inhibitor/siRNA with ac-SMC3 Western blot and cell cycle analysis; X-ray crystallography of five CdLS mutants with enzymatic and thermostability assays plus activator rescue","pmids":["27072133","25075551","26463496"],"confidence":"High","gaps":["Whether CdLS phenotypes are entirely explained by impaired SMC3 deacetylation versus other substrates","No patient-derived cell rescue data shown"]},{"year":2014,"claim":"Discovery that HDAC8 partners with EZH2 to co-repress Wnt antagonists, and with STAT3 to repress BMF, revealed that HDAC8 operates in transcriptional repressor complexes with distinct co-factors at specific promoters.","evidence":"Co-IP and ChIP in HCC and leukemia cells, with RNAi/overexpression functional validation","pmids":["26383163","25321483","24404147"],"confidence":"Medium","gaps":["Genome-wide extent of HDAC8–EZH2 co-occupancy not mapped","DEC1 and STAT3 interactions each from single labs"]},{"year":2017,"claim":"Demonstration that HDAC8 deacetylates p53 to suppress apoptosis in hematopoietic stem cells, with genetic epistasis rescue by p53 deletion, established a non-histone substrate axis controlling stem cell maintenance.","evidence":"Hdac8 conditional KO mice, serial repopulation assays, co-IP, p53 double KO rescue","pmids":["29084772"],"confidence":"High","gaps":["Specific p53 lysine residue(s) deacetylated by HDAC8 not mapped","Whether this axis operates in non-hematopoietic stem cells unknown"]},{"year":2019,"claim":"Identification of c-Jun K273 as an HDAC8 deacetylation site driving AP-1 activity and BRAF inhibitor resistance in melanoma demonstrated a direct role for HDAC8 in drug resistance signaling through non-histone substrate modification.","evidence":"MS-based phosphoproteomics, K273 site mutagenesis, in vivo xenograft studies","pmids":["30987999"],"confidence":"High","gaps":["Whether HDAC8 deacetylates additional AP-1 family members","Clinical relevance of HDAC8 inhibition in BRAF-resistant melanoma not established"]},{"year":2020,"claim":"AMPK phosphorylation of HDAC8 was shown to drive its nuclear-to-cytoplasmic translocation under glucose deprivation, establishing a metabolic signaling axis that dynamically regulates HDAC8 chromatin occupancy.","evidence":"AMPK activation/inhibition, nuclear-cytoplasmic fractionation, Western blot in lung cancer cells","pmids":["32171858"],"confidence":"Medium","gaps":["Specific phosphorylation site on HDAC8 not identified","Whether AMPK-mediated translocation alters non-histone substrate deacetylation unknown","Single-lab observation"]},{"year":2020,"claim":"PKM2 K62 deacetylation by HDAC8 was shown to promote PKM2 nuclear translocation and β-catenin/CCND1 signaling, extending HDAC8's non-histone substrate repertoire to metabolic enzymes with moonlighting transcriptional roles.","evidence":"Co-IP, K62 site-directed mutagenesis, subcellular fractionation, ChIP, enzymatic assays in HCC cells","pmids":["33279948"],"confidence":"High","gaps":["Whether PKM2 deacetylation by HDAC8 occurs in non-cancer contexts","Structural basis for HDAC8–PKM2 recognition not defined"]},{"year":2021,"claim":"Genome-wide chromatin profiling established HDAC8 as an H3K27-specific deacetylase in HCC, and its interaction with the FACT complex at the MAP2K3 promoter in keratinocytes revealed tissue-specific chromatin complexes maintaining immune tolerance.","evidence":"ChIP-Seq (H3K27ac), selective HDAC8 inhibitor in humanized mouse models; proteomic identification of FACT complex, keratinocyte-specific KO mice","pmids":["33827976","34021025"],"confidence":"High","gaps":["Whether H3K27 specificity holds across all cell types or is context-dependent","Precise mechanism of HDAC8–FACT cooperation at chromatin not structurally resolved"]},{"year":2023,"claim":"HDAC8 deacetylation of the acetyltransferase EP300 was shown to inactivate EP300 and redirect its chromatin occupancy, establishing a direct enzymatic crosstalk between a deacetylase and an acetyltransferase that drives melanoma cell-state switching and brain metastasis.","evidence":"Mass spectrometry, ATAC-Seq, ChIP-Seq (H3K27ac), HDAC8 inhibition, in vivo brain metastasis model","pmids":["38030596"],"confidence":"High","gaps":["Specific EP300 lysine(s) deacetylated by HDAC8 not fully mapped","Whether EP300 inactivation is a general HDAC8 mechanism or melanoma-specific"]},{"year":2023,"claim":"PROTAC-mediated degradation of HDAC8 (and HDAC3) showed that loss of these proteins does not globally alter histone acetylation, raising the question of whether HDAC8's primary physiological substrates are non-histone proteins and whether inhibitor-induced hyperacetylation reflects off-target effects.","evidence":"PROTAC-induced degradation, quantitative proteomics, histone acetylation Western blot, RNA-Seq","pmids":["37572669"],"confidence":"Medium","gaps":["Whether locus-specific histone acetylation changes were missed in bulk analysis","Relative contribution of catalytic versus scaffolding functions of HDAC8 not resolved","Single-lab observation"]},{"year":null,"claim":"The full catalog of physiological HDAC8 substrates, the structural basis for its context-dependent histone versus non-histone substrate selectivity, and how AMPK-mediated translocation integrates with its chromatin and cytoplasmic functions remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No systematic in vivo substrate identification (e.g., acetylomics in HDAC8-null cells)","No structure of HDAC8 bound to a full-length non-histone substrate","Tissue-specific regulatory mechanisms largely uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,1,7,8,13,14,20]},{"term_id":"GO:0016787","term_label":"hydrolase activity","supporting_discovery_ids":[0,1,6,8,13,14]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[0,14,15,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,3,10,15]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,12,14,18]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[12]},{"term_id":"GO:0005694","term_label":"chromosome","supporting_discovery_ids":[1,14,15]}],"pathway":[{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[0,2,14,15,16]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,13]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[7,11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[8,13,22]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,5]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[4,5,8,11,20]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,3,10,15]}],"complexes":["HDAC8–EZH2 repressive complex","HDAC8–FACT complex (SSRP1/SUPT16H)"],"partners":["EZH2","STAT3","SMC3","TP53","SSRP1","SUPT16H","EP300","DEC1"],"other_free_text":[]},"mechanistic_narrative":"HDAC8 is a class I, Zn²⁺-dependent lysine deacetylase that removes acetyl groups from both histone and non-histone substrates to regulate chromatin state, cohesin recycling, and cell signaling. Its catalytic activity, dependent on conserved active-site histidine residues, targets histone H3K27 and H4 to repress transcription at specific loci—often in concert with EZH2 or the FACT complex (SSRP1/SUPT16H)—and deacetylates non-histone substrates including SMC3 (enabling cohesin recycling during the cell cycle), p53 (modulating apoptosis in hematopoietic stem cells and AML), c-Jun (increasing AP-1 transcriptional activity), PKM2 (promoting its nuclear translocation and β-catenin/CCND1 signaling), and EP300 (inactivating this acetyltransferase to redirect chromatin accessibility) [PMID:10926844, PMID:27072133, PMID:33827976, PMID:29084772, PMID:30987999, PMID:33279948, PMID:38030596, PMID:34021025]. Loss-of-function mutations in HDAC8 cause Cornelia de Lange Syndrome by disrupting active-site geometry and impairing SMC3 deacetylation, and these catalytic defects can be partially rescued by small-molecule HDAC8 activators [PMID:25075551, PMID:26463496]. AMPK-mediated phosphorylation drives HDAC8 nuclear-to-cytoplasmic translocation, uncoupling it from histone substrates and derepressing target genes such as PGM1 [PMID:32171858]."},"prefetch_data":{"uniprot":{"accession":"Q9BY41","full_name":"Histone deacetylase 8","aliases":["Protein deacetylase HDAC8","Protein decrotonylase HDAC8"],"length_aa":377,"mass_kda":41.8,"function":"Histone deacetylase that catalyzes the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4) (PubMed:10748112, PubMed:10922473, PubMed:10926844, PubMed:14701748, PubMed:28497810). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events (PubMed:10748112, PubMed:10922473, PubMed:10926844, PubMed:14701748). Histone deacetylases act via the formation of large multiprotein complexes (PubMed:10748112, PubMed:10922473, PubMed:10926844, PubMed:14701748). Also involved in the deacetylation of cohesin complex protein SMC3 regulating release of cohesin complexes from chromatin (PubMed:22885700). May play a role in smooth muscle cell contractility (PubMed:15772115). In addition to protein deacetylase activity, also has protein-lysine deacylase activity: acts as a protein decrotonylase by mediating decrotonylation ((2E)-butenoyl) of histones (PubMed:28497810)","subcellular_location":"Nucleus; Chromosome; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9BY41/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HDAC8","classification":"Not Classified","n_dependent_lines":74,"n_total_lines":1208,"dependency_fraction":0.061258278145695365},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/HDAC8","total_profiled":1310},"omim":[{"mim_id":"610962","title":"SMG5 NONSENSE-MEDIATED mRNA DECAY FACTOR; SMG5","url":"https://www.omim.org/entry/610962"},{"mim_id":"601844","title":"PROTEIN KINASE, LYSINE-DEFICIENT 4; WNK4","url":"https://www.omim.org/entry/601844"},{"mim_id":"300882","title":"CORNELIA DE LANGE SYNDROME 5; CDLS5","url":"https://www.omim.org/entry/300882"},{"mim_id":"300269","title":"HISTONE DEACETYLASE 8; HDAC8","url":"https://www.omim.org/entry/300269"},{"mim_id":"122470","title":"CORNELIA DE LANGE SYNDROME 1; CDLS1","url":"https://www.omim.org/entry/122470"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/HDAC8"},"hgnc":{"alias_symbol":["RPD3","KDAC8"],"prev_symbol":["HDACL1","WTS","MRXS6"]},"alphafold":{"accession":"Q9BY41","domains":[{"cath_id":"3.40.800.20","chopping":"16-57_116-373","consensus_level":"high","plddt":97.9628,"start":16,"end":373}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BY41","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BY41-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q9BY41-F1-predicted_aligned_error_v6.png","plddt_mean":95.31},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=HDAC8","jax_strain_url":"https://www.jax.org/strain/search?query=HDAC8"},"sequence":{"accession":"Q9BY41","fasta_url":"https://rest.uniprot.org/uniprotkb/Q9BY41.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q9BY41/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q9BY41"}},"corpus_meta":[{"pmid":"18292778","id":"PMC_18292778","title":"The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men.","date":"2008","source":"Nature reviews. 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1950)","url":"https://pubmed.ncbi.nlm.nih.gov/2117034","citation_count":27,"is_preprint":false},{"pmid":"23719282","id":"PMC_23719282","title":"Synthesis and biological evaluation of a targeted DNA-binding transcriptional activator with HDAC8 inhibitory activity.","date":"2013","source":"Bioorganic & medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23719282","citation_count":27,"is_preprint":false},{"pmid":"37098288","id":"PMC_37098288","title":"Discovery of highly potent HDAC8 PROTACs with anti-tumor activity.","date":"2023","source":"Bioorganic chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37098288","citation_count":26,"is_preprint":false},{"pmid":"35330681","id":"PMC_35330681","title":"Long noncoding RNA LINC01435 impedes diabetic wound healing by facilitating YY1-mediated HDAC8 expression.","date":"2022","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/35330681","citation_count":26,"is_preprint":false},{"pmid":"32111211","id":"PMC_32111211","title":"HDAC8 inhibitor attenuates airway responses to antigen stimulus through synchronously suppressing galectin-3 expression and reducing macrophage-2 polarization.","date":"2020","source":"Respiratory research","url":"https://pubmed.ncbi.nlm.nih.gov/32111211","citation_count":25,"is_preprint":false},{"pmid":"31421254","id":"PMC_31421254","title":"Design, synthesis, biological evaluation and molecular docking study of arylcarboxamido piperidine and piperazine-based hydroxamates as potential HDAC8 inhibitors with promising anticancer activity.","date":"2019","source":"European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31421254","citation_count":25,"is_preprint":false},{"pmid":"38030596","id":"PMC_38030596","title":"HDAC8-mediated inhibition of EP300 drives a transcriptional state that increases melanoma brain metastasis.","date":"2023","source":"Nature communications","url":"https://pubmed.ncbi.nlm.nih.gov/38030596","citation_count":24,"is_preprint":false},{"pmid":"26625202","id":"PMC_26625202","title":"Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.","date":"2016","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/26625202","citation_count":24,"is_preprint":false},{"pmid":"30632447","id":"PMC_30632447","title":"Selective and nonselective HDAC8 inhibitors: a therapeutic patent review.","date":"2019","source":"Pharmaceutical patent analyst","url":"https://pubmed.ncbi.nlm.nih.gov/30632447","citation_count":23,"is_preprint":false},{"pmid":"37429084","id":"PMC_37429084","title":"Are inhibitors of histone deacetylase 8 (HDAC8) effective in hematological cancers especially acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)?","date":"2023","source":"European journal of medicinal chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/37429084","citation_count":23,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":47506,"output_tokens":5493,"usd":0.112457},"stage2":{"model":"claude-opus-4-6","input_tokens":9095,"output_tokens":3378,"usd":0.194887},"total_usd":0.307344,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2000,\n      \"finding\": \"HDAC8 was cloned and characterized as a new class I HDAC with trichostatin A- and sodium butyrate-inhibitable deacetylase activity on histone H4 peptide substrates and core histones; mutation of two adjacent histidine residues in the predicted active site severely decreased activity, confirming these residues as essential for catalysis.\",\n      \"method\": \"Expression in cell systems, immunopurification, in vitro HDAC activity assay, active-site histidine mutagenesis\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro enzymatic assay plus mutagenesis in the original characterization paper\",\n      \"pmids\": [\"10926844\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HDAC8 is the Zn2+-dependent SMC3 (cohesin subunit) lysine deacetylase required for cohesin recycling during the cell cycle; HDAC8 inhibition leads to accumulation of acetylated SMC3, delays cell cycle progression, and suppresses proliferation without affecting cohesin-dependent transcription of estrogen-responsive genes.\",\n      \"method\": \"HDAC8-specific inhibitor (PCI-34051), Western blot for ac-SMC3, ChIP, siRNA knockdown, cell cycle analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (inhibitor, siRNA, ChIP) in a single rigorous study, consistent with structural studies on CdLS mutations\",\n      \"pmids\": [\"27072133\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HDAC8 physically interacts with EZH2 to concordantly repress Wnt antagonists via histone H4 deacetylation and H3K27 trimethylation in NAFLD-associated hepatocellular carcinoma cells.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, lentiviral knockdown in vivo, Western blot\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — reciprocal co-IP plus in vivo knockdown, single lab\",\n      \"pmids\": [\"26383163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HDAC8 and STAT3 directly repress the BMF gene promoter; HDAC8 occupancy at the BMF promoter was identified as a direct transcriptional target, and overexpression of HDAC8 interfered with BMF induction whereas HDAC8 RNAi activated it.\",\n      \"method\": \"ChIP, RNAi knockdown, transient overexpression, p300 inhibitor treatment, reporter assay\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — ChIP plus functional RNAi/overexpression, single lab\",\n      \"pmids\": [\"25321483\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"X-ray crystal structures of five CdLS-associated HDAC8 missense mutants (C153F, A188T, I243N, T311M, H334R) revealed local structural changes that compromise catalysis and/or thermostability; the catalytic activity of these mutants could be partially or fully rescued in vitro by the HDAC8 activator N-(phenylcarbamothioyl)benzamide.\",\n      \"method\": \"X-ray crystallography, enzymatic activity assays, thermostability assays, activator rescue experiments\",\n      \"journal\": \"ACS chemical biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structures plus in vitro enzymatic characterization with mutagenesis and activator rescue\",\n      \"pmids\": [\"25075551\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"X-ray crystal structures of additional CdLS HDAC8 mutants (G117E, P91L-Y306F, D233G-Y306F) showed that CdLS mutations disrupt key hydrogen bond networks and active-site residue positioning, compromising catalysis and thermostability; molecular dynamics simulations of H180R and G304R showed bulky arginine side chains protruding into the substrate binding site.\",\n      \"method\": \"X-ray crystallography, molecular dynamics simulations, enzymatic activity assays, thermostability assays\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple crystal structures plus MD simulations and enzymatic assays\",\n      \"pmids\": [\"26463496\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Structure-based identification of novel HDAC8 non-histone substrates using Rosetta FlexPepBind revealed that SMC3, ERRα, and ARID1A are among known non-histone HDAC8 substrates, and the in silico approach identified many additional acetyl-lysine-containing peptides as HDAC8 substrates in vitro.\",\n      \"method\": \"Structure-based computational docking (Rosetta FlexPepBind), in vitro peptide deacetylation assays\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/3 — in vitro substrate assays validated computationally, single lab\",\n      \"pmids\": [\"26933971\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HDAC8 protein interacts with p53 and modulates p53 activity via deacetylation; HDAC8-deficient LT-HSCs displayed hyperactivation of p53, increased apoptosis under stress, and compromised long-term repopulating activity, all of which were rescued by genetic inactivation of p53.\",\n      \"method\": \"Co-immunoprecipitation, Hdac8 conditional knockout, serial repopulation assays, genetic epistasis (p53 double KO), Western blot\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal co-IP, clean conditional KO with defined phenotype, and genetic epistasis rescue\",\n      \"pmids\": [\"29084772\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HDAC8 deacetylates c-Jun at lysine 273, decreasing c-Jun acetylation and increasing its transcriptional activity; HDAC8-mediated BRAF inhibitor resistance in melanoma is mediated through receptor tyrosine kinase activation and MAPK/AP-1 signaling.\",\n      \"method\": \"Mass spectrometry-based phosphoproteomics, HDAC8 introduction into drug-naïve cells, in vitro and in vivo xenograft studies, c-Jun K273 acetylation site mutagenesis\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — MS-based substrate identification, site mutagenesis, in vivo validation\",\n      \"pmids\": [\"30987999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HDAC8 interacts with the proteasome receptor ADRM1 to regulate MGMT protein levels independent of MGMT promoter methylation in glioblastoma cells; HDAC8 inhibition or shRNA decreased MGMT levels and increased DNA damage and cell cycle arrest.\",\n      \"method\": \"Co-immunoprecipitation, HDAC8-specific inhibitor (PCI34051), shRNA knockdown, Western blot\",\n      \"journal\": \"Genes & cancer\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP and pharmacological inhibition, single lab\",\n      \"pmids\": [\"31798765\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HDAC8 binds the ID2 enhancer and represses ID2 transcription; lncRNA ID2-AS1 suppresses HCC metastasis by blocking HDAC8 binding to the ID2 enhancer, thereby increasing H3K27 acetylation and ID2 expression.\",\n      \"method\": \"ChIP, RNAi knockdown, overexpression, migration/invasion assays in vitro and in vivo\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — ChIP demonstrating HDAC8 occupancy and functional KD, single lab\",\n      \"pmids\": [\"31730902\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"FLT3 inhibition induces HDAC8 upregulation through FOXO1- and FOXO3-mediated transactivation; upregulated HDAC8 deacetylates and inactivates p53, promoting FLT3-ITD+ AML maintenance and TKI resistance.\",\n      \"method\": \"Genetic/pharmacological HDAC8 inhibition, FOXO1/3 ChIP, p53 acetylation Western blot, patient-derived xenograft models\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — ChIP for transcriptional regulation, p53 deacetylation shown, in vivo PDX validation\",\n      \"pmids\": [\"32315388\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"AMPK phosphorylates HDAC8 in response to glucose deprivation, stimulating HDAC8 translocation from the nucleus to the cytoplasm and disrupting HDAC8 binding to histone H3, thereby derepressing PGM1 expression in lung cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, nuclear/cytoplasmic fractionation, Western blot, AMPK inhibition/activation\",\n      \"journal\": \"Cancer letters\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — fractionation with functional consequence, single lab\",\n      \"pmids\": [\"32171858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HDAC8 binds and deacetylates PKM2 at the K62 residue; K62 deacetylation facilitates PKM2 nuclear translocation where it binds β-catenin to promote CCND1 transcription and cell cycle progression in hepatocellular carcinoma.\",\n      \"method\": \"Co-immunoprecipitation, site-directed mutagenesis (K62), subcellular fractionation, ChIP, enzymatic activity assays\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — in vitro deacetylation assay, mutagenesis, ChIP, and nuclear localization with functional readout\",\n      \"pmids\": [\"33279948\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HDAC8 is an H3K27-specific deacetylase in HCC; pharmacological inhibition of HDAC8 increases global and enhancer H3K27 acetylation, reactivating T cell-trafficking chemokines and relieving T cell exclusion.\",\n      \"method\": \"Chromatin profiling (ChIP-Seq), selective HDAC8 inhibitor treatment, humanized and immunodeficient mouse models\",\n      \"journal\": \"Science translational medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — genome-wide chromatin profiling plus multiple in vivo models demonstrating H3K27 substrate specificity\",\n      \"pmids\": [\"33827976\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HDAC8 interacts with HDAC9 and the FACT complex (SSRP1 and SUPT16H) to regulate H3K9 and H3K27 acetylation at the MAP2K3 promoter, thereby suppressing MAP2K3 expression and maintaining cutaneous innate immune tolerance in keratinocytes.\",\n      \"method\": \"Proteomic analysis (HDAC8/9 interactors), ChIP for histone marks, HDAC8/9 siRNA, keratinocyte-specific KO mice, cytokine assays\",\n      \"journal\": \"Science immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — proteomics identified complex, ChIP confirmed histone marks, KO mouse with in vivo phenotype\",\n      \"pmids\": [\"34021025\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"HDAC8 associates with α-smooth muscle actin (α-SMA) in TGFβ1-treated lung fibroblasts as shown by co-immunoprecipitation; HDAC8 inhibition represses TGFβ1-induced fibroblast contraction, α-SMA expression, and fibrotic gene expression, and increases H3K27ac at the PPARγ gene enhancer.\",\n      \"method\": \"Co-immunoprecipitation, HDAC8-selective inhibitor (NCC170), siRNA knockdown, ChIP-qPCR (H3K27ac), collagen gel contraction assay, bleomycin mouse model\",\n      \"journal\": \"American journal of physiology. Lung cellular and molecular physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP, ChIP, selective inhibitor, siRNA, and in vivo mouse model with multiple orthogonal methods\",\n      \"pmids\": [\"30358439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"DEC1 interacts with HDAC8 and recruits HDAC8 to the TAp73 promoter to enhance TAp73 expression; HDAC8 is required for DEC1-mediated transcriptional activation of TAp73 but not ΔNp73.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, RNAi knockdown, reporter assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — co-IP and ChIP demonstrating recruitment, single lab\",\n      \"pmids\": [\"24404147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"HDAC8 localizes in the nucleus of HEK293 cells and binds both CREB and PP1; expression of HDAC8 decreases CREB phosphorylation at S133 and CREB-mediated gene transcription in response to forskolin.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization, overexpression, luciferase reporter assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP/overexpression, single lab, single method\",\n      \"pmids\": [\"19070599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HDAC8 maintains Notch1 protein stability in breast cancer cells through a non-epigenetic mechanism; HDAC8 inhibition promotes Notch1 proteasomal degradation via Fbxw7, although HDAC8 does not form a complex with Notch1 and inhibition does not affect Notch1 acetylation.\",\n      \"method\": \"Co-immunoprecipitation, siRNA/shRNA knockdown, proteasome inhibitor (MG132), Fbxw7 siRNA, xenograft model\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — multiple genetic approaches and proteasome rescue, but mechanism linking HDAC8 to Notch1 stability remains indirect\",\n      \"pmids\": [\"26625202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"HDAC8 deacetylates the histone acetyltransferase EP300, causing its enzymatic inactivation; increased HDAC8 activity leads to EP300 binding at c-Jun transcriptional sites (increasing chromatin accessibility) and away from MITF sites, driving a neural crest-stem cell state that promotes melanoma brain metastasis.\",\n      \"method\": \"ATAC-Seq, ChIP-Seq (H3K27ac), mass spectrometry, HDAC8 inhibition, in vivo brain metastasis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1/2 — MS-based substrate identification (EP300 deacetylation), ATAC-Seq, ChIP-Seq, in vivo model with multiple orthogonal methods\",\n      \"pmids\": [\"38030596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Simultaneous PROTAC-mediated degradation of HDAC3 and HDAC8 (YX968) does not induce global histone hyperacetylation or broad transcriptomic perturbation, suggesting that histone hyperacetylation is a major factor driving transcriptional changes induced by HDAC inhibitors rather than loss of HDAC3/8 catalytic activity per se.\",\n      \"method\": \"PROTAC-induced protein degradation, quantitative proteomics, histone acetylation Western blot, RNA-Seq, cell viability assays\",\n      \"journal\": \"Cell chemical biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1/2 — selective protein degradation with unbiased quantitative proteomics confirming selectivity, single lab\",\n      \"pmids\": [\"37572669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HDAC8 inhibition in cardiac hypertrophy promotes p38 MAPK dephosphorylation; HDAC8 overexpression promoted p38 MAPK phosphorylation and cardiac hypertrophic marker expression, while HDAC8 knockdown or selective inhibition (PCI34051) reversed isoproterenol-induced hypertrophy via reduction of p38 MAPK activity.\",\n      \"method\": \"HDAC8 selective inhibitor (PCI34051), HDAC8 knockdown/overexpression, Western blot (p38 MAPK phosphorylation), mouse isoproterenol model, echocardiography\",\n      \"journal\": \"Frontiers in pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — gain and loss of function with defined signaling readout, single lab\",\n      \"pmids\": [\"33959033\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HDAC8 interacts with galectin-3 (Gal-3) as shown by co-immunoprecipitation; selective HDAC8 inhibition (PCI-34051) synchronously reduces HDAC8-Gal-3 complex levels and M2 macrophage polarization, attenuating airway hyperresponsiveness.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence, selective inhibitor (PCI-34051), shRNA knockdown\",\n      \"journal\": \"Respiratory research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single co-IP, single lab\",\n      \"pmids\": [\"32111211\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HDAC8 is a class I, Zn2+-dependent histone deacetylase whose core catalytic function—requiring active-site histidine residues—removes acetyl groups from lysine residues on both histone substrates (particularly H3K27 and H4) and non-histone proteins including SMC3 (cohesin, required for cohesin recycling and cell cycle progression), p53 (regulating apoptosis in HSCs and AML), PKM2 (promoting nuclear localization and β-catenin/CCND1 signaling), c-Jun (increasing AP-1 transcriptional activity in melanoma), and EP300 (inactivating this acetyltransferase to alter chromatin accessibility); HDAC8 is regulated by AMPK-mediated phosphorylation that drives its nuclear-to-cytoplasmic translocation, operates in complex with partners such as EZH2, STAT3, DEC1, ADRM1, Gal-3, and SSRP1/SUPT16H-FACT, and its loss-of-function mutations underlie Cornelia de Lange Syndrome by impairing SMC3 deacetylation and cohesin recycling.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HDAC8 is a class I, Zn²⁺-dependent lysine deacetylase that removes acetyl groups from both histone and non-histone substrates to regulate chromatin state, cohesin recycling, and cell signaling. Its catalytic activity, dependent on conserved active-site histidine residues, targets histone H3K27 and H4 to repress transcription at specific loci—often in concert with EZH2 or the FACT complex (SSRP1/SUPT16H)—and deacetylates non-histone substrates including SMC3 (enabling cohesin recycling during the cell cycle), p53 (modulating apoptosis in hematopoietic stem cells and AML), c-Jun (increasing AP-1 transcriptional activity), PKM2 (promoting its nuclear translocation and β-catenin/CCND1 signaling), and EP300 (inactivating this acetyltransferase to redirect chromatin accessibility) [PMID:10926844, PMID:27072133, PMID:33827976, PMID:29084772, PMID:30987999, PMID:33279948, PMID:38030596, PMID:34021025]. Loss-of-function mutations in HDAC8 cause Cornelia de Lange Syndrome by disrupting active-site geometry and impairing SMC3 deacetylation, and these catalytic defects can be partially rescued by small-molecule HDAC8 activators [PMID:25075551, PMID:26463496]. AMPK-mediated phosphorylation drives HDAC8 nuclear-to-cytoplasmic translocation, uncoupling it from histone substrates and derepressing target genes such as PGM1 [PMID:32171858].\",\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing HDAC8 as a bona fide class I histone deacetylase answered the question of whether additional HDACs with distinct expression patterns existed; mutagenesis of active-site histidines proved these residues are essential for catalysis.\",\n      \"evidence\": \"Cloning, immunopurified in vitro HDAC assays on H4 peptide/core histones, active-site His mutagenesis\",\n      \"pmids\": [\"10926844\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological substrates beyond bulk histones unknown\", \"In vivo function not addressed\", \"Regulatory mechanisms not explored\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Nuclear localization and interaction with CREB/PP1 suggested HDAC8 could modulate signaling-responsive transcription, expanding its role beyond histone deacetylation.\",\n      \"evidence\": \"Co-immunoprecipitation and overexpression with luciferase reporter in HEK293 cells\",\n      \"pmids\": [\"19070599\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single co-IP without reciprocal validation or endogenous confirmation\", \"No direct deacetylation of CREB shown\", \"Relevance to physiological signaling unclear\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of SMC3 as the key HDAC8 substrate for cohesin recycling, and structural characterization of Cornelia de Lange Syndrome mutations, established the molecular basis by which HDAC8 loss-of-function causes a developmental disorder.\",\n      \"evidence\": \"HDAC8 inhibitor/siRNA with ac-SMC3 Western blot and cell cycle analysis; X-ray crystallography of five CdLS mutants with enzymatic and thermostability assays plus activator rescue\",\n      \"pmids\": [\"27072133\", \"25075551\", \"26463496\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether CdLS phenotypes are entirely explained by impaired SMC3 deacetylation versus other substrates\", \"No patient-derived cell rescue data shown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Discovery that HDAC8 partners with EZH2 to co-repress Wnt antagonists, and with STAT3 to repress BMF, revealed that HDAC8 operates in transcriptional repressor complexes with distinct co-factors at specific promoters.\",\n      \"evidence\": \"Co-IP and ChIP in HCC and leukemia cells, with RNAi/overexpression functional validation\",\n      \"pmids\": [\"26383163\", \"25321483\", \"24404147\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genome-wide extent of HDAC8–EZH2 co-occupancy not mapped\", \"DEC1 and STAT3 interactions each from single labs\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Demonstration that HDAC8 deacetylates p53 to suppress apoptosis in hematopoietic stem cells, with genetic epistasis rescue by p53 deletion, established a non-histone substrate axis controlling stem cell maintenance.\",\n      \"evidence\": \"Hdac8 conditional KO mice, serial repopulation assays, co-IP, p53 double KO rescue\",\n      \"pmids\": [\"29084772\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific p53 lysine residue(s) deacetylated by HDAC8 not mapped\", \"Whether this axis operates in non-hematopoietic stem cells unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identification of c-Jun K273 as an HDAC8 deacetylation site driving AP-1 activity and BRAF inhibitor resistance in melanoma demonstrated a direct role for HDAC8 in drug resistance signaling through non-histone substrate modification.\",\n      \"evidence\": \"MS-based phosphoproteomics, K273 site mutagenesis, in vivo xenograft studies\",\n      \"pmids\": [\"30987999\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HDAC8 deacetylates additional AP-1 family members\", \"Clinical relevance of HDAC8 inhibition in BRAF-resistant melanoma not established\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"AMPK phosphorylation of HDAC8 was shown to drive its nuclear-to-cytoplasmic translocation under glucose deprivation, establishing a metabolic signaling axis that dynamically regulates HDAC8 chromatin occupancy.\",\n      \"evidence\": \"AMPK activation/inhibition, nuclear-cytoplasmic fractionation, Western blot in lung cancer cells\",\n      \"pmids\": [\"32171858\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific phosphorylation site on HDAC8 not identified\", \"Whether AMPK-mediated translocation alters non-histone substrate deacetylation unknown\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"PKM2 K62 deacetylation by HDAC8 was shown to promote PKM2 nuclear translocation and β-catenin/CCND1 signaling, extending HDAC8's non-histone substrate repertoire to metabolic enzymes with moonlighting transcriptional roles.\",\n      \"evidence\": \"Co-IP, K62 site-directed mutagenesis, subcellular fractionation, ChIP, enzymatic assays in HCC cells\",\n      \"pmids\": [\"33279948\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PKM2 deacetylation by HDAC8 occurs in non-cancer contexts\", \"Structural basis for HDAC8–PKM2 recognition not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Genome-wide chromatin profiling established HDAC8 as an H3K27-specific deacetylase in HCC, and its interaction with the FACT complex at the MAP2K3 promoter in keratinocytes revealed tissue-specific chromatin complexes maintaining immune tolerance.\",\n      \"evidence\": \"ChIP-Seq (H3K27ac), selective HDAC8 inhibitor in humanized mouse models; proteomic identification of FACT complex, keratinocyte-specific KO mice\",\n      \"pmids\": [\"33827976\", \"34021025\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether H3K27 specificity holds across all cell types or is context-dependent\", \"Precise mechanism of HDAC8–FACT cooperation at chromatin not structurally resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"HDAC8 deacetylation of the acetyltransferase EP300 was shown to inactivate EP300 and redirect its chromatin occupancy, establishing a direct enzymatic crosstalk between a deacetylase and an acetyltransferase that drives melanoma cell-state switching and brain metastasis.\",\n      \"evidence\": \"Mass spectrometry, ATAC-Seq, ChIP-Seq (H3K27ac), HDAC8 inhibition, in vivo brain metastasis model\",\n      \"pmids\": [\"38030596\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific EP300 lysine(s) deacetylated by HDAC8 not fully mapped\", \"Whether EP300 inactivation is a general HDAC8 mechanism or melanoma-specific\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"PROTAC-mediated degradation of HDAC8 (and HDAC3) showed that loss of these proteins does not globally alter histone acetylation, raising the question of whether HDAC8's primary physiological substrates are non-histone proteins and whether inhibitor-induced hyperacetylation reflects off-target effects.\",\n      \"evidence\": \"PROTAC-induced degradation, quantitative proteomics, histone acetylation Western blot, RNA-Seq\",\n      \"pmids\": [\"37572669\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether locus-specific histone acetylation changes were missed in bulk analysis\", \"Relative contribution of catalytic versus scaffolding functions of HDAC8 not resolved\", \"Single-lab observation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The full catalog of physiological HDAC8 substrates, the structural basis for its context-dependent histone versus non-histone substrate selectivity, and how AMPK-mediated translocation integrates with its chromatin and cytoplasmic functions remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No systematic in vivo substrate identification (e.g., acetylomics in HDAC8-null cells)\", \"No structure of HDAC8 bound to a full-length non-histone substrate\", \"Tissue-specific regulatory mechanisms largely uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 1, 7, 8, 13, 14, 20]},\n      {\"term_id\": \"GO:0016787\", \"supporting_discovery_ids\": [0, 1, 6, 8, 13, 14]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [0, 14, 15, 16]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 3, 10, 15]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 12, 14, 18]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [12]},\n      {\"term_id\": \"GO:0005694\", \"supporting_discovery_ids\": [1, 14, 15]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [0, 2, 14, 15, 16]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 13]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [7, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [8, 13, 22]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [4, 5, 8, 11, 20]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 3, 10, 15]}\n    ],\n    \"complexes\": [\n      \"HDAC8–EZH2 repressive complex\",\n      \"HDAC8–FACT complex (SSRP1/SUPT16H)\"\n    ],\n    \"partners\": [\n      \"EZH2\",\n      \"STAT3\",\n      \"SMC3\",\n      \"TP53\",\n      \"SSRP1\",\n      \"SUPT16H\",\n      \"EP300\",\n      \"DEC1\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}