{"gene":"PIAS4","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":2001,"finding":"PIASy (PIAS4) functions as a SUMO E3 ligase for LEF1 in a reconstituted in vitro system, stimulates LEF1 sumoylation in vivo, and represses LEF1 transcriptional activity by sequestering it into nuclear bodies via binding to nuclear matrix-associated DNA sequences.","method":"Reconstituted in vitro SUMO E3 ligase assay, co-expression sumoylation assay, nuclear body localization by immunofluorescence, nuclear matrix binding assay","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted in vitro ligase activity plus in vivo sumoylation and localization experiments, multiple orthogonal methods in one study","pmids":["11731474"],"is_preprint":false},{"year":2001,"finding":"PIASy (PIAS4) interacts with p53 (confirmed by yeast two-hybrid and co-immunoprecipitation in mammalian cells), inhibits p53 DNA-binding activity and p53-dependent transactivation of Bax and p21, but does not suppress p53-induced apoptosis.","method":"Yeast two-hybrid, co-immunoprecipitation, luciferase reporter assay, EMSA","journal":"Apoptosis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP plus functional reporter assay, single lab, two orthogonal methods","pmids":["11388671"],"is_preprint":false},{"year":2003,"finding":"PIASy (PIAS4) acts as a SUMO E3 ligase for C/EBPalpha, enhancing both SUMO-1 and SUMO-3 modification at the synergy control (SC) motif (Lys159) both in vivo and in vitro; SUMO modification at this site suppresses transcriptional synergy from compound response elements.","method":"In vitro SUMOylation assay with purified recombinant components, in vivo sumoylation assay, site-directed mutagenesis, reporter gene assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution with purified components plus mutagenesis and in vivo validation, single lab but multiple orthogonal methods","pmids":["12511558"],"is_preprint":false},{"year":2003,"finding":"PIASy (PIAS4) interacts with Smad3 and Smad4 (identified by yeast three-hybrid screen, confirmed in mammalian cells); the interaction with Smad3 is enhanced by TGF-beta and occurs through the C-terminal domain of Smad3. PIASy represses Smad transcriptional activity without inhibiting Smad-DNA binding, and can interact constitutively with HDAC1; HDAC inhibitor TSA prevents PIASy's inhibitory function.","method":"Yeast three-hybrid screen, co-immunoprecipitation, reporter gene assay, EMSA, HDAC inhibitor experiment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (yeast screen, Co-IP, reporter, EMSA), independently confirmed in two papers (PMID 12904571 and 12815042)","pmids":["12904571","12815042"],"is_preprint":false},{"year":2003,"finding":"PIASy (PIAS4) stimulates sumoylation of Smad3 in vivo and associates with Smad7 (identified by yeast two-hybrid with Smad7 MH2 domain as bait); endogenous PIASy expression is induced by TGF-beta in Hep3B cells, suggesting a negative feedback loop.","method":"Yeast two-hybrid, co-immunoprecipitation, in vivo sumoylation assay","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus in vivo sumoylation, single lab","pmids":["12815042"],"is_preprint":false},{"year":2003,"finding":"PIASy (PIAS4) interacts with GATA-2, preferentially enhances SUMO-2 conjugation to GATA-2 via its E3 SUMO ligase activity, and suppresses GATA-2-dependent ET-1 promoter activity in endothelial cells through a RING-like domain-independent mechanism requiring both N-terminal and C-terminal sequences of PIASy.","method":"Co-immunoprecipitation, in vivo sumoylation assay, reporter gene assay, domain deletion analysis","journal":"Circulation research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vivo sumoylation plus reporter assay with domain mutants, single lab","pmids":["12750312"],"is_preprint":false},{"year":2003,"finding":"PIASy (PIAS4) is a SUMO E3 ligase for c-Myb, enhancing SUMO-1 conjugation at K503 and K527 both in vitro and in vivo; PIASy causes a shift of c-Myb to the insoluble nuclear matrix fraction and negatively regulates Myb-induced transcriptional activation.","method":"In vitro SUMOylation assay, co-immunoprecipitation, reporter gene assay, nuclear fractionation, site-directed mutagenesis","journal":"European journal of biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro assay plus in vivo sumoylation, mutagenesis, and functional reporter, single lab","pmids":["12631292"],"is_preprint":false},{"year":2004,"finding":"PIASy (PIAS4) represses androgen receptor (AR) transcriptional activity by recruiting HDAC1 and HDAC2 via its RD1 repression domain; HDAC activity is required for this repression. The repression is independent of PIASy's SUMO ligase activity and independent of AR sumoylation status.","method":"Co-immunoprecipitation, reporter gene assay, HDAC inhibitor experiment, domain deletion and SUMO-ligase-dead mutant analysis","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, reporter, HDAC inhibitor, mutants) mechanistically dissecting ligase-independent repression, single lab but comprehensive","pmids":["14981544"],"is_preprint":false},{"year":2004,"finding":"PIASy (PIAS4) knockout mice are phenotypically normal with no significant perturbation of STAT1 activation or global SUMO-1/SUMO-3 modification patterns, demonstrating that PIASy is either dispensable or compensated by other PIAS family members at steady state.","method":"Knockout mouse generation, 2D gel analysis of SUMO-modified proteins, STAT1 activation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent knockout studies (PMID 15169916 and 15528356) with proteome-level analysis confirming dispensability","pmids":["15169916","15528356"],"is_preprint":false},{"year":2004,"finding":"PIASy (PIAS4) deletion in mice results in modest reduction of IFN-gamma-induced and Wnt-responsive gene expression, demonstrating partial but non-essential roles in STAT1 and LEF1 signaling with likely redundancy from other PIAS family members.","method":"Knockout mouse, gene expression analysis, signaling assays in primary cells","journal":"Journal of immunology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo knockout model replicated across two independent labs (PMID 15528356 and 15169916)","pmids":["15528356"],"is_preprint":false},{"year":2005,"finding":"PIASy (PIAS4) is specifically required for mitotic SUMO-2 conjugation of Topoisomerase-II in Xenopus egg extracts; PIASy binds mitotic chromosomes and recruits Ubc9 to chromatin, and these properties are essential for its activity. PIASy depletion eliminates chromosomal SUMO-2-conjugated species and blocks anaphase sister chromatid segregation.","method":"Xenopus egg extract depletion, immunofluorescence, EGFP-SUMO-2 localization, functional segregation assay, dominant-negative mutant analysis","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstituted biochemical system (Xenopus egg extracts), depletion rescue, multiple orthogonal methods, replicated in later papers","pmids":["15933717"],"is_preprint":false},{"year":2005,"finding":"SUMO-1 modification of PIASy itself at Lys35 is required for PIASy-dependent sumoylation and transcriptional activation of Tcf-4; PIASy(K35R) retains ligase activity for other substrates but loses the ability to activate Tcf-4, correlating with distinct nuclear distribution including increased PML body association.","method":"Site-directed mutagenesis, in vivo sumoylation assay, reporter gene assay, immunofluorescence, co-immunoprecipitation","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mutagenesis plus sumoylation assay plus functional reporter plus localization, single lab","pmids":["15831457"],"is_preprint":false},{"year":2006,"finding":"PIASy (PIAS4) is the SUMO E3 ligase for NEMO (IKKgamma), preferentially stimulating site-selective SUMO-1 (but not SUMO-2/3) modification of NEMO in vitro; PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus, mutually exclusive with IKK interaction. PIASy siRNA inhibits NEMO sumoylation and NF-kappaB activation in response to genotoxic agents.","method":"siRNA knockdown, in vitro SUMOylation assay, co-immunoprecipitation, overexpression, subcellular fractionation","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus siRNA knockdown plus Co-IP plus in vivo functional assay, multiple orthogonal methods","pmids":["16906147"],"is_preprint":false},{"year":2006,"finding":"PIASy (PIAS4) overexpression in normal human fibroblasts induces cellular senescence via the p53 and Rb tumor suppressor pathways; in Rb-deficient fibroblasts, PIASy expression leads to p53-dependent apoptosis. PIASy stimulates sumoylation and transcriptional activity of p53 and increases Rb-dependent corepression at E2F-responsive promoters. Fibroblasts lacking PIASy exhibit reduced senescence in response to prosenescence stimuli. Senescence induction requires PIASy E3 ligase activity.","method":"Overexpression in primary fibroblasts, PIASy knockout cells, E3-ligase-dead mutant, in vivo sumoylation assay, ChIP, reporter gene assay","journal":"Molecular cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function and gain-of-function with defined phenotypic readout, E3-ligase mutant, ChIP, multiple orthogonal methods","pmids":["16793547"],"is_preprint":false},{"year":2006,"finding":"TRIM32 interacts with PIASy (PIAS4), promotes PIASy ubiquitination and proteasomal degradation in vitro using purified components; this interaction is induced by UVB/TNFalpha treatment and involves redistribution of PIASy from the nucleus to cytoplasmic granules. The LGMD2H-associated TRIM32 missense mutation prevents TRIM32-PIASy interaction. PIASy inhibits NF-kappaB activity and sensitizes keratinocytes to apoptosis; TRIM32-mediated PIASy degradation thus regulates NF-kappaB-dependent survival.","method":"In vitro ubiquitination assay with purified components, co-immunoprecipitation, immunofluorescence, NF-kappaB reporter assay, patient fibroblasts","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstituted ubiquitination assay plus multiple in vivo validation methods, functionally linked to NF-kappaB regulation","pmids":["16816390"],"is_preprint":false},{"year":2007,"finding":"PIASy (PIAS4) is a SUMO E3 ligase for YY1, stimulating YY1 sumoylation at Lys288 in vitro and in vivo; uniquely, PIASy-mediated YY1 sumoylation does not depend on the RING finger domain of PIASy but correlates with PIASy-YY1 interaction. PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 protein, and differentially regulates YY1 transcriptional activity on different target promoters.","method":"In vitro and in vivo sumoylation assay, site-directed mutagenesis, co-immunoprecipitation, immunofluorescence, reporter gene assay, stability assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro sumoylation plus mutagenesis plus multiple in vivo validation methods, single lab but comprehensive","pmids":["17353273"],"is_preprint":false},{"year":2007,"finding":"PIASy (PIAS4) and PIAS1 cooperate to regulate specificity and magnitude of NF-kappaB and STAT1-mediated gene activation; Piasy deletion in dendritic cells enhances a subset of NF-kappaB and STAT1 target genes, and Pias1-/-Piasy-/- double knockout embryos die before day 11.5, demonstrating essential cooperative function.","method":"Single and double knockout mice, gene expression analysis in primary dendritic cells, endotoxic shock model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis via double knockout with defined embryonic lethal phenotype, gene-specific transcriptional analysis","pmids":["17606919"],"is_preprint":false},{"year":2008,"finding":"PIASy (PIAS4) sumoylation of Smad3 stimulates Smad3 nuclear export; co-expression of Smad3 with PIASy and SUMO1 affected Smad3 DNA-binding activity and promoted nuclear export. FRET analysis revealed Smad3 interacts with SUMO1 in the cytoplasm. siRNA-mediated reduction of endogenous PIASy enhanced TGF-beta-induced gene expression.","method":"siRNA knockdown, nuclear export assay, FRET analysis, DNA-binding assay, reporter gene assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (siRNA, FRET, export assay) in single lab establishing mechanism of SUMO-dependent nuclear export","pmids":["18384750"],"is_preprint":false},{"year":2008,"finding":"PIASy (PIAS4) represses C/EBPdelta transcriptional activity by sequestering it to the nuclear periphery via interaction between the PIASy SAP domain (SAPD) and the C/EBPdelta transactivation domain (TAD); this repression is independent of HDAC activity, PIASy E3 SUMO ligase activity, and C/EBPdelta sumoylation.","method":"Domain deletion analysis, reporter gene assay, HDAC inhibitor experiment, immunofluorescence, SUMO-ligase-dead mutant","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple mechanistic dissection experiments (domain mutants, inhibitor, localization), single lab","pmids":["18477566"],"is_preprint":false},{"year":2008,"finding":"FIP200 interacts with PIASy (PIAS4) via the PIASy RING finger and FIP200 C-terminus; PIASy interaction redistributes FIP200 from cytoplasm to nucleus, abrogating FIP200 regulation of TSC/S6K signaling. FIP200 enhances PIASy-dependent transcriptional activation of the p21 promoter; both proteins are co-recruited to the p21 promoter by ChIP. FIP200 is not a SUMO substrate of PIASy.","method":"Co-immunoprecipitation, in vitro/in vivo sumoylation assay, immunofluorescence, subcellular fractionation, ChIP, siRNA knockdown, reporter gene assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, fractionation, ChIP, reporter) in single lab","pmids":["18285457"],"is_preprint":false},{"year":2009,"finding":"PIAS4 and PIAS1 are recruited to DNA double-strand break sites via SAP domain-dependent mechanisms; PIAS4 is required for SUMO1 accumulation and PIAS1 for SUMO2/3 accumulation at DSB sites. Both are needed for productive association of 53BP1, BRCA1, and RNF168 with damage foci, and for effective ubiquitin-adduct formation by RNF8, RNF168, and BRCA1. Both promote DSB repair and confer ionizing radiation resistance.","method":"Immunofluorescence of irradiation-induced foci, siRNA knockdown, domain mutant analysis, DSB repair assay, clonogenic survival assay","journal":"Nature","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, high-impact journal, findings replicated in subsequent papers (PMID 35007836, 29234018)","pmids":["20016603"],"is_preprint":false},{"year":2009,"finding":"PIASy (PIAS4) inhibits LRH-1-mediated transactivation by competing with the co-activator SRC-1 for binding to the LRH-1 AF-2 domain; this repression is independent of LRH-1 SUMOylation and HDAC activity.","method":"Co-immunoprecipitation, mammalian two-hybrid, reporter gene assay, domain deletion analysis, overexpression competition assay","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus two-hybrid plus reporter with mechanistic domain mapping, single lab","pmids":["19067654"],"is_preprint":false},{"year":2010,"finding":"PIASy (PIAS4) interacts with VHL and induces VHL SUMOylation at Lys171 by SUMO1; PIASy-mediated SUMOylation promotes VHL oligomerization and abrogates its inhibitory function on tumor cell growth, migration, and clonogenicity. PIASy siRNA reduces VHL oligomerization and increases HIF1alpha degradation. PIASy is upregulated under hypoxic conditions.","method":"Co-immunoprecipitation, in vivo sumoylation assay, site-directed mutagenesis, siRNA knockdown, cell growth/migration assay, ubiquitination assay","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, mutagenesis, siRNA, functional assays in single lab","pmids":["20300531"],"is_preprint":false},{"year":2010,"finding":"PIASy (PIAS4) is a specific E3 ligase for hypoxia-induced HIF1alpha SUMOylation by SUMO1; hypoxia promotes nuclear translocation of HIF1alpha to enable PIASy binding. PIASy negatively regulates hypoxia-induced HIF1alpha stability and transactivation; PIASy knockdown increases angiogenic activity of endothelial cells.","method":"Co-immunoprecipitation, in vivo sumoylation assay, siRNA knockdown, reporter gene assay, angiogenesis assay (tube formation), immunofluorescence","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (Co-IP, sumoylation, siRNA, functional) in single lab; consistent with parallel VHL paper","pmids":["20661221"],"is_preprint":false},{"year":2010,"finding":"PIASy (PIAS4)-mediated SUMO2/3 modification of Topoisomerase IIalpha at Lys660 (in the DNA gate domain) strongly inhibits TopoIIalpha decatenation activity; loss of SUMOylation at Lys660 eliminates this inhibitory effect. The SUMOylation site was identified by mass spectrometry.","method":"Mass spectrometry, biochemical decatenation assay, site-directed mutagenesis, Xenopus egg extract sumoylation assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay plus mass spectrometry site identification plus mutagenesis validation, mechanistically rigorous","pmids":["21079245"],"is_preprint":false},{"year":2010,"finding":"PIASy (PIAS4) promotes SUMO-2/3 conjugation of PARP1 at Lys482 (identified by tandem mass spectrometry) on mitotic chromosomes but not interphase chromatin, both in Xenopus egg extracts and in vitro reconstituted assays. PARP1 SUMOylation does not alter intrinsic PARP1 enzymatic activity or its localization on chromosomes, but loss of SUMOylation increases PARP1-dependent PARylation of other chromatin-associated proteins.","method":"Xenopus egg extract SUMOylation, tandem mass spectrometry, in vitro reconstituted SUMOylation assay, site-directed mutagenesis, PARP enzymatic activity assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution plus mass spectrometry site identification plus mutagenesis plus functional enzyme assay","pmids":["20228053"],"is_preprint":false},{"year":2010,"finding":"The Rod/Zw10 kinetochore complex interacts with the N-terminal domain of PIASy (PIAS4) (specifically first 47 residues) and is required for PIASy centromeric localization and mitotic SUMO2/3 conjugation on chromosomes; Rod depletion compromises centromeric localization of PIASy and SUMO2/3.","method":"Xenopus egg extract depletion, co-immunoprecipitation, immunofluorescence, domain truncation analysis, N-terminal domain swapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — domain mapping plus depletion rescue plus immunofluorescence in well-established Xenopus system","pmids":["20696768"],"is_preprint":false},{"year":2011,"finding":"PIASy (PIAS4) inhibits virus-induced type I IFN transcription through a SUMO E3-ligase-independent mechanism requiring SUMO-interacting motif (SIM) activity and UBC9; PIASy inhibits IFN-stimulated gene expression through its SAP domain LXXLL motif. These two inhibitory mechanisms are distinct: LXXLL mutation abolishes IFN-stimulated gene repression but not virus-induced IFN transcription.","method":"PIASy null mouse cells, overexpression, SUMO-ligase-dead mutant, LXXLL motif mutant, SIM mutant, UBC9 knockdown, reporter gene assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — null cell system plus multiple mutants dissecting distinct mechanisms, multiple orthogonal approaches","pmids":["21199872"],"is_preprint":false},{"year":2013,"finding":"PIASy (PIAS4) is induced by hypoxia and promotes SUMOylation of Sp1, preventing Sp1 from binding to the SIRT1 promoter and thereby transcriptionally repressing SIRT1 expression; PIASy knockdown restores Sp1 binding and SIRT1 expression, reverses cancer cell EMT, and attenuates metastasis in vivo in nude mice.","method":"siRNA knockdown, ChIP, in vivo sumoylation assay, EMT markers, in vivo xenograft metastasis assay","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus ChIP plus in vivo functional validation, single lab","pmids":["23843607"],"is_preprint":false},{"year":2013,"finding":"PIASy (PIAS4) sumoylates SREBP1c at Lys98, leading to suppression of the hepatic lipogenic program; PKA activation enhances PIASy-SREBP1c interaction and SREBP1c sumoylation, followed by ubiquitination-dependent degradation. PIASy overexpression in db/db mice ameliorates hepatic steatosis; PIASy suppression in lean mice stimulates lipogenesis.","method":"In vivo sumoylation assay, site-directed mutagenesis, co-immunoprecipitation, in vivo mouse studies (db/db and WT), primary hepatocyte experiments, PKA activation assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — mutagenesis plus co-IP plus in vivo mouse validation plus primary cell experiments, mechanistically comprehensive","pmids":["24379443"],"is_preprint":false},{"year":2015,"finding":"PIAS4 and its SUMO E3 ligase activity are specifically required for SUMOylation of AMPKalpha1, which attenuates AMPK activity specifically towards mTORC1 signaling. SUMOylation-deficient AMPKalpha1 shows higher activity towards mTORC1 when reconstituted in AMPKalpha-deficient cells. PIAS4 depletion reduces breast cancer cell growth specifically when combined with AMPK activator A769662.","method":"In vivo SUMOylation assay, SUMO-ligase-dead PIAS4 mutant, SUMOylation-deficient AMPKalpha1 reconstitution in knockout cells, mTORC1 signaling assay, cell growth assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — reconstitution in knockout cells plus ligase mutant plus substrate mutant plus signaling readout, multiple orthogonal methods","pmids":["26616021"],"is_preprint":false},{"year":2016,"finding":"FIEL1 (KIAA0317) ubiquitinates PIAS4 in a site-specific manner facilitated by PKCzeta phosphorylation of PIAS4 and GSK3beta phosphorylation of FIEL1 (double-locking mechanism), leading to PIAS4 degradation and enhancement of TGF-beta signaling. FIEL1 overexpression increases lung fibrosis in a bleomycin model; a FIEL1 small molecule inhibitor ameliorates fibrosis.","method":"Co-immunoprecipitation, in vitro ubiquitination assay, phosphorylation assays, bleomycin murine fibrosis model, small molecule inhibitor","journal":"The Journal of experimental medicine","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitination assay plus mechanistic phosphorylation studies plus in vivo mouse validation","pmids":["27162139"],"is_preprint":false},{"year":2016,"finding":"PIAS4 acts as SUMO E3 ligase for E12 (a class I bHLH protein) in vivo; PIASy RING mutant cannot block E12-mediated alpha-SMA promoter activation, indicating RING domain is required for E12 sumoylation; TGF-beta induces both PIASy and E12 expression; reduced PIASy expression leads to increased TGF-beta-mediated alpha-SMA expression in mesangial cells.","method":"In vivo sumoylation assay, yeast two-hybrid, co-immunoprecipitation, reporter gene assay, RING domain mutant, siRNA knockdown","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vivo sumoylation plus RING mutant plus siRNA, single lab","pmids":["22829926"],"is_preprint":false},{"year":2016,"finding":"PIAS4 promotes HSC activation and liver fibrosis by transcriptionally repressing SIRT1, which leads to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters; lentivirus-shRNA targeting PIAS4 in mice ameliorates liver fibrosis by normalizing SIRT1 expression.","method":"shRNA knockdown in vivo (lentivirus), ChIP, gene expression analysis, in vivo mouse model (MCD diet)","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo shRNA plus ChIP plus mechanistic pathway placement, single lab","pmids":["27323886"],"is_preprint":false},{"year":2016,"finding":"PIAS4 is recruited to nuclear domains containing HSV-1 viral DNA via SUMO-interacting motif (SIM)-dependent mechanisms (at viral genome entry sites) and via SIM-independent mechanisms (in replication compartments). PIAS4 depletion enhances replication of ICP0-null HSV-1; its restriction mechanisms are synergistic with PML protein and antagonized by ICP0.","method":"Immunofluorescence co-localization, siRNA depletion, viral replication assay, SIM mutant analysis","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA depletion with viral replication readout plus SIM mutant analysis, single lab","pmids":["26937035"],"is_preprint":false},{"year":2016,"finding":"HDAC9 deacetylation of Nkx3.2 triggers PIASy (PIAS4)-mediated sumoylation of Nkx3.2, and subsequent RNF4-mediated SUMO-targeted ubiquitination; this HDAC9-PIASy-RNF4 post-translational cascade controls Nkx3.2 protein stability and regulates chondrocyte hypertrophy and viability during skeletal development.","method":"Co-immunoprecipitation, in vivo sumoylation and ubiquitination assays, HDAC inhibitor, chondrocyte differentiation assay","journal":"Cellular signalling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus in vivo modification assays plus pathway cascade ordering, single lab","pmids":["27312341"],"is_preprint":false},{"year":2017,"finding":"PIAS4 is the primary SUMO E3 ligase for RIF1 SUMOylation in response to DNA damage; PIAS4 knockdown impairs RIF1 SUMOylation, defective disassembly of RIF1 DDR foci, and abrogates UHRF1-dependent ubiquitination and turnover of RIF1, leading to accumulated RIF1 at damage sites and DNA double-strand breaks.","method":"siRNA knockdown, co-immunoprecipitation, immunofluorescence, in vivo sumoylation assay, ubiquitination assay","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus Co-IP plus in vivo modification assays establishing pathway cascade, single lab","pmids":["29234018"],"is_preprint":false},{"year":2017,"finding":"PIASy (PIAS4) contains two SUMO-interacting motifs (SIMs) at its C-terminus; both are required for full E3 ligase activity. The new SIM was identified by NMR chemical shift mapping and validated by mutagenesis.","method":"NMR chemical shift mapping, mutagenesis, in vitro SUMOylation assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structure-function plus mutagenesis plus in vitro activity assay in single study","pmids":["28455449"],"is_preprint":false},{"year":2018,"finding":"PIAS1 and PIAS4 together promote SUMO-dependent template switch (TS) DNA damage tolerance pathway by SUMOylating PCNA at Lys164; PIAS1/PIAS4 double-knockout cells show >90% decrease in PCNA-Lys164 SUMOylation and >90% decrease in TS (Ig gene conversion). Epistasis experiments show PCNA mutation causes no additional impact on PIAS1/PIAS4 cells; overexpression of PCNA-SUMO1 chimera restores TS in PIAS1/PIAS4 cells.","method":"Genetic knockout (chicken DT40 and human TK6 cells), PCNA-SUMO1 chimera reconstitution, Ig gene conversion assay, epistasis analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis plus reconstitution rescue, two cell line models, mechanistically definitive","pmids":["30487218"],"is_preprint":false},{"year":2018,"finding":"PIASy (PIAS4) interacts with Rbp2 (KDM5B/JARID1A histone H3K4me3 demethylase) via the PIASy PINIT domain (101-218 aa) and the Rbp2 JmjC domain (451-503 aa); Piasy binds the IFN-beta promoter and facilitates Rbp2-mediated demethylation of H3K4me3 at IFNI genes, attenuating type I IFN transcription. Enzymatically inactive Rbp2 does not attenuate IFNI.","method":"Co-immunoprecipitation, ChIP, domain deletion analysis, siRNA/shRNA knockdown, enzymatically inactive mutant (Rbp2H483G/E485Q), reporter gene assay","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus ChIP plus enzyme-dead mutant plus reporter, single lab","pmids":["28970247"],"is_preprint":false},{"year":2019,"finding":"PIAS4 sumoylates DPPA2 protein, negatively regulating its activity; PIAS4 is down-regulated during zygotic genome activation (ZGA). Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program; forced expression of Pias4 or Sumo2-DPPA2 fusion inhibits 2C-like program and impairs early mouse embryo development.","method":"siRNA/shRNA depletion, overexpression, in vivo sumoylation assay, ESC-to-2C-like cell transition model, early embryo development assay","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — depletion and overexpression with transcriptional and developmental readouts, in vivo sumoylation, single lab","pmids":["31226106"],"is_preprint":false},{"year":2019,"finding":"PIASy (PIAS4) overexpression in fertilized mouse embryos causes developmental arrest at the two-cell stage with abnormal chromosome segregation and impaired zygotic transcription; this is dependent on PIASy SUMOylation activity and associated with increased H3K9me3 trimethylation and enhanced nuclear translocation of H3K9me3 methyltransferase.","method":"mRNA microinjection/overexpression in embryos, chromosome segregation analysis, H3K9me3 immunofluorescence, SUMO-ligase-dead mutant","journal":"Biology open","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo embryo overexpression with defined phenotypic readout plus E3-ligase mutant, single lab","pmids":["31640975"],"is_preprint":false},{"year":2021,"finding":"PIAS4 depletion impairs DSB end resection and RAD51 loading in gamma-ray-irradiated human fibroblasts, reducing BRCA1 recruitment to DSB sites; 53BP1 depletion rescues resection in PIAS4-depleted cells. Epistasis analysis shows PIAS4, PIAS1, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade.","method":"siRNA depletion, gamma-ray irradiation, immunofluorescence of DSB markers, epistasis analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — siRNA plus epistasis analysis with defined molecular readouts, single lab, consistent with earlier Nature paper","pmids":["35007836"],"is_preprint":false},{"year":2022,"finding":"PIASy (PIAS4)-mediated SUMOylation of Caveolin-3 (Cav-3) by SUMO2/3 increases after myocardial ischemia-reperfusion (I/R); increased Cav-3 SUMOylation causes Nav1.5/Cav-3 dissociation, reducing membrane Nav1.5 density; cardiac-targeted PIASy silencing decreases Cav-3 SUMO2/3 modification, restores Nav1.5-Cav-3 co-localization, and prevents I/R-induced ventricular arrhythmias in rats.","method":"AAV9-shRNA in vivo cardiac knockdown, co-immunoprecipitation, site-directed mutagenesis of Cav-3 SUMO sites, immunofluorescence, ECG recording, HEK293T hypoxia/reoxygenation model","journal":"Military Medical Research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo AAV knockdown plus mutagenesis plus Co-IP, single lab","pmids":["36229865"],"is_preprint":false},{"year":2024,"finding":"WRN helicase inhibition traps WRN on chromatin, requiring SUMOylation via the PIAS4-RNF4 axis for subsequent p97/VCP-mediated extraction and proteasomal degradation; co-inhibition of WRN and SUMOylation has additive toxicity in MSI-H cancer cells.","method":"Single-molecule tracking (SMT), phenotypic screen, siRNA/inhibitor studies, co-inhibition experiments, in vivo MSI-H xenograft model","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — single-molecule imaging plus phenotypic screen plus functional validation in vivo, single lab but multiple approaches","pmids":["39025847"],"is_preprint":false},{"year":2024,"finding":"PIAS4 directly binds SLC7A11 and facilitates its SUMOylation; KDM1A acts as a transcriptional activator of PIAS4. Tanshinone IIA decreases KDM1A expression, suppressing PIAS4 transcription and thereby inhibiting PIAS4-dependent SLC7A11 SUMOylation, which promotes SLC7A11 degradation and ferroptosis in breast cancer cells.","method":"Ni-beads pull-down, co-immunoprecipitation, luciferase assay, ChIP, siRNA knockdown, in vivo xenograft and metastasis models","journal":"Journal of advanced research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus pulldown plus ChIP plus in vivo validation, single lab","pmids":["38615741"],"is_preprint":false},{"year":2025,"finding":"PIAS4 facilitates SUMOylation of TDP-43 by SUMO1 and SUMO2/3 in response to oxidative stress; PIAS1 also contributes. TDP-43 SUMOylation is not promoted by etoposide-induced DNA damage.","method":"In vivo sumoylation assay, oxidative stress treatment, domain mapping, PIAS1/PIAS4 co-expression","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single lab, preliminary characterization without mechanistic follow-up","pmids":["41292941"],"is_preprint":true}],"current_model":"PIAS4 (PIASy) is a nuclear SUMO E3 ligase that uses its SP-RING domain to catalyze SUMO1, SUMO2, and SUMO2/3 conjugation to a wide array of substrates—including LEF1, p53, Smad3, NEMO/IKKgamma, Topoisomerase IIalpha, PARP1, HIF1alpha, VHL, AMPKalpha1, PCNA, Caveolin-3, RIF1, SREBP1c, DPPA2, and others—thereby regulating transcription factor activity (often repression), DNA double-strand break repair (promoting BRCA1/53BP1/RNF168 recruitment and template-switch repair), cell senescence and apoptosis, mitotic chromosome segregation, NF-kappaB and interferon signaling, and hypoxia responses; its own activity is controlled by autologous SUMO1 modification at Lys35, by interaction with the Rod/Zw10 complex for centromeric localization, and by TRIM32- or FIEL1-mediated ubiquitination and proteasomal degradation, while in some contexts it represses transcription factors (AR, C/EBPdelta, Ets-1) through SUMO-ligase-independent mechanisms involving HDAC recruitment or nuclear sequestration."},"narrative":{"mechanistic_narrative":"PIAS4 (PIASy) is a nuclear SUMO E3 ligase that uses an SP-RING/RING-finger catalytic core, two C-terminal SUMO-interacting motifs, and an N-terminal SAP domain to conjugate SUMO1 or SUMO2/3 to a broad panel of nuclear substrates, thereby controlling transcription factor output, genome maintenance, mitotic chromosome dynamics, and metabolic and stress signaling [PMID:11731474, PMID:15933717, PMID:28455449]. As a transcriptional regulator it predominantly represses its targets, SUMOylating LEF1/Tcf, C/EBPalpha, GATA-2, c-Myb, p53, Smad3, YY1, and others to alter their localization, DNA binding, stability, or coactivator access—for example sequestering LEF1 and c-Myb into the nuclear matrix and driving SUMO-dependent nuclear export of Smad3 [PMID:11731474, PMID:12511558, PMID:12631292, PMID:18384750]; in several contexts it represses transcription factors (AR, C/EBPdelta, LRH-1) through ligase-independent mechanisms involving HDAC recruitment, SAP-domain-mediated nuclear sequestration, or coactivator competition [PMID:14981544, PMID:18477566, PMID:19067654]. In the DNA damage response PIAS4 is recruited to double-strand breaks via its SAP domain to deposit SUMO1, enabling productive 53BP1/BRCA1/RNF168 recruitment and RNF8/RNF168/BRCA1 ubiquitin signaling, and it drives end resection and template-switch tolerance through SUMOylation of substrates including PCNA-Lys164 and RIF1 [PMID:20016603, PMID:29234018, PMID:30487218, PMID:35007836]. During mitosis PIAS4 localizes to centromeres via the Rod/Zw10 complex and catalyzes SUMO2/3 modification of Topoisomerase IIalpha and PARP1 on chromosomes, an activity essential for sister chromatid segregation [PMID:15933717, PMID:21079245, PMID:20228053, PMID:20696768]. PIAS4 additionally governs senescence and apoptosis via p53/Rb [PMID:16793547], NF-kappaB and type I/II interferon signaling [PMID:16906147, PMID:17606919, PMID:21199872], hypoxia responses through HIF1alpha and VHL SUMOylation [PMID:20300531, PMID:20661221], and AMPK/mTORC1 and hepatic lipogenic control via AMPKalpha1 and SREBP1c SUMOylation [PMID:24379443, PMID:26616021]. Its own abundance and activity are tuned by autologous SUMO1 modification at Lys35 and by TRIM32- and FIEL1-mediated ubiquitination and proteasomal degradation [PMID:15831457, PMID:16816390, PMID:27162139]. Single PIAS4-knockout mice are largely normal, but Pias1/Pias4 double knockouts are embryonic lethal, establishing extensive functional redundancy within the PIAS family [PMID:15169916, PMID:15528356, PMID:17606919].","teleology":[{"year":2001,"claim":"Established PIAS4 as a bona fide SUMO E3 ligase and transcriptional repressor, defining its core biochemical activity and a sequestration-based repression mode.","evidence":"Reconstituted in vitro ligase assay, in vivo sumoylation, and nuclear-body/matrix localization for LEF1; reciprocal Co-IP and reporter assays for p53","pmids":["11731474","11388671"],"confidence":"High","gaps":["Did not define the catalytic determinants of ligase activity","Mechanism linking nuclear-matrix sequestration to repression not resolved at structural level"]},{"year":2003,"claim":"Showed PIAS4 SUMOylates and represses multiple developmental and signaling transcription factors, embedding it in TGF-beta/Smad and hematopoietic transcription networks, sometimes via HDAC recruitment.","evidence":"In vitro and in vivo sumoylation, yeast two/three-hybrid, Co-IP, reporter and EMSA assays for C/EBPalpha, Smad3/4/7, GATA-2, and c-Myb","pmids":["12511558","12904571","12815042","12750312","12631292"],"confidence":"High","gaps":["Did not distinguish ligase-dependent from ligase-independent repression for all substrates","In vivo relevance of feedback induction by TGF-beta not tested genetically"]},{"year":2004,"claim":"Demonstrated a SUMO-ligase-independent repression mechanism via HDAC recruitment and showed in vivo that PIAS4 is dispensable at steady state but contributes modestly to STAT1/Wnt outputs.","evidence":"Domain/ligase-dead mutants and HDAC inhibitor for AR repression; knockout mice with 2D-gel SUMO proteome and signaling assays","pmids":["14981544","15169916","15528356"],"confidence":"High","gaps":["Source of in vivo redundancy (which PIAS paralog compensates) not mapped","Did not reconcile strong in vitro activities with mild knockout phenotype"]},{"year":2005,"claim":"Defined PIAS4 as the dedicated mitotic chromosomal SUMO2/3 ligase whose activity is essential for sister chromatid segregation, and showed autologous SUMO1 modification at Lys35 tunes substrate-selective output.","evidence":"Xenopus egg extract depletion/rescue with Ubc9 recruitment and segregation readouts; site-directed mutagenesis (K35R) with sumoylation, localization, and Tcf-4 reporter assays","pmids":["15933717","15831457"],"confidence":"High","gaps":["Full set of mitotic chromosomal substrates not yet enumerated","How K35 SUMOylation rewires substrate selectivity mechanistically unresolved"]},{"year":2006,"claim":"Connected PIAS4 to genotoxic NF-kappaB activation, p53/Rb-driven senescence and apoptosis, and identified TRIM32-mediated ubiquitination as a degradation switch controlling PIAS4 levels.","evidence":"In vitro sumoylation/ubiquitination with purified components, siRNA, Co-IP, ChIP, ligase-dead mutants, and patient fibroblasts for NEMO, p53/Rb, and TRIM32","pmids":["16906147","16793547","16816390"],"confidence":"High","gaps":["Stimulus specificity of nuclear PIAS4-NEMO interaction not fully defined","Physiological triggers of TRIM32-PIAS4 redistribution beyond UVB/TNFalpha unclear"]},{"year":2007,"claim":"Revealed RING-independent substrate SUMOylation (YY1) and established cooperative, partially redundant control of NF-kappaB/STAT1 gene programs with PIAS1, including essential developmental function.","evidence":"In vitro/in vivo sumoylation and stability assays for YY1; single and double knockout mice with dendritic-cell gene expression and embryonic lethality","pmids":["17353273","17606919"],"confidence":"High","gaps":["Basis for RING-independent SUMO transfer not structurally explained","Distinct vs overlapping target sets of PIAS1 and PIAS4 incompletely resolved"]},{"year":2008,"claim":"Defined mechanisms by which PIAS4 controls transcription factor localization: SUMO-driven Smad3 nuclear export and SAP-domain-mediated nuclear-periphery sequestration of C/EBPdelta; linked PIAS4 to FIP200/mTOR signaling.","evidence":"siRNA, FRET, nuclear export and DNA-binding assays for Smad3; domain mutants and immunofluorescence for C/EBPdelta; Co-IP, fractionation, ChIP for FIP200","pmids":["18384750","18477566","18285457"],"confidence":"Medium","gaps":["Whether export and sequestration mechanisms operate on shared substrates untested","FIP200-PIAS4 functional consequences in vivo not established"]},{"year":2009,"claim":"Placed PIAS4 at the apex of double-strand-break SUMO signaling and showed coactivator-competition as a distinct repression mode.","evidence":"Irradiation-induced foci imaging, SAP-domain mutants, repair and clonogenic survival assays (with PIAS1); Co-IP and competition assays for LRH-1","pmids":["20016603","19067654"],"confidence":"High","gaps":["Direct SUMO substrates driving 53BP1/BRCA1/RNF168 recruitment not all identified at DSBs","Recruitment cue read by the SAP domain at breaks undefined"]},{"year":2010,"claim":"Expanded the PIAS4 substrate repertoire across mitosis, hypoxia, and DNA-gate enzymology, defining site-specific modifications with direct functional consequences.","evidence":"Mass spectrometry site mapping, in vitro decatenation and PARP assays, Xenopus extracts, Co-IP, siRNA, and angiogenesis/growth assays for TopoIIalpha-K660, PARP1-K482, HIF1alpha, VHL-K171; Rod/Zw10 domain mapping for centromeric targeting","pmids":["21079245","20228053","20661221","20300531","20696768"],"confidence":"High","gaps":["How a single ligase achieves substrate- and site-selectivity across these targets unresolved","Opposing HIF1alpha (destabilizing) vs VHL (inactivating) effects not integrated into one model"]},{"year":2011,"claim":"Dissected two ligase-independent interferon-restriction mechanisms of PIAS4 acting through SIM/UBC9 and the SAP-domain LXXLL motif.","evidence":"PIAS4-null cells with ligase-dead, LXXLL, SIM, and UBC9-knockdown reporter assays","pmids":["21199872"],"confidence":"High","gaps":["Molecular targets of the SIM-dependent and LXXLL-dependent repression not identified","In vivo antiviral relevance not tested here"]},{"year":2013,"claim":"Identified a hypoxia-induced PIAS4-Sp1-SIRT1 transcriptional repression axis driving EMT and metastasis, and a SREBP1c-SUMOylation node suppressing hepatic lipogenesis.","evidence":"siRNA, ChIP, in vivo sumoylation, xenograft metastasis for Sp1/SIRT1; mutagenesis, Co-IP, db/db and lean mouse studies, primary hepatocytes for SREBP1c-K98","pmids":["23843607","24379443"],"confidence":"Medium","gaps":["Direct vs indirect contribution of Sp1 SUMOylation to SIRT1 promoter occupancy not fully isolated","Upstream signals coupling PKA to PIAS4-SREBP1c beyond PKA activation incompletely defined"]},{"year":2015,"claim":"Established PIAS4 as the AMPKalpha1 SUMO ligase that selectively restrains AMPK signaling toward mTORC1, linking it to growth control and a cancer vulnerability.","evidence":"In vivo sumoylation, ligase-dead PIAS4 and SUMO-deficient AMPKalpha1 reconstitution in knockout cells, mTORC1 and cell growth assays","pmids":["26616021"],"confidence":"High","gaps":["SUMO acceptor site on AMPKalpha1 and structural basis of pathway selectivity not defined","Generality across AMPK complexes/tissues untested"]},{"year":2016,"claim":"Revealed a phosphorylation-gated FIEL1 ubiquitination switch controlling PIAS4 turnover and TGF-beta/fibrosis, and embedded PIAS4 in SUMO-targeted ubiquitin cascades and viral restriction.","evidence":"In vitro ubiquitination, phosphorylation assays, bleomycin model and inhibitor for FIEL1; Co-IP/sumoylation/ChIP for E12, SIRT1-SMAD3, Nkx3.2-RNF4; viral replication and SIM-mutant analysis for HSV-1","pmids":["27162139","22829926","27323886","27312341","26937035"],"confidence":"High","gaps":["Interplay between TRIM32 and FIEL1 degradation pathways not reconciled","Whether SUMO-targeted RNF4 ubiquitination acts on the same PIAS4-modified substrate pools broadly unclear"]},{"year":2017,"claim":"Defined structural and pathway determinants of PIAS4 activity: a second C-terminal SIM required for full ligase function, and PIAS4-driven control of RIF1 and Rbp2/KDM5B-mediated chromatin/IFN regulation.","evidence":"NMR chemical-shift mapping and in vitro activity for SIMs; siRNA, Co-IP, sumoylation/ubiquitination for RIF1; Co-IP, ChIP, enzyme-dead mutant for Rbp2","pmids":["28455449","29234018","28970247"],"confidence":"High","gaps":["How the two SIMs cooperate with the SP-RING during catalysis structurally unresolved","Direct vs scaffolding role of PIAS4 in the RIF1-UHRF1 turnover circuit incompletely separated"]},{"year":2018,"claim":"Demonstrated genetically that PIAS1/PIAS4 jointly drive PCNA-Lys164 SUMOylation to enable the template-switch DNA damage tolerance pathway.","evidence":"DT40 and TK6 double-knockouts, PCNA-SUMO1 chimera rescue, Ig gene conversion, and epistasis analysis","pmids":["30487218"],"confidence":"High","gaps":["Relative contribution of PIAS1 vs PIAS4 to PCNA SUMOylation not separated","Coupling of PCNA SUMOylation to downstream recombinase choice not detailed"]},{"year":2019,"claim":"Implicated PIAS4 SUMOylation in early embryonic gene-expression control, restraining the 2C-like program via DPPA2 and constraining chromatin state and chromosome segregation in zygotes.","evidence":"Depletion/overexpression with in vivo sumoylation, ESC-to-2C transition and early embryo assays for DPPA2; embryo microinjection, segregation and H3K9me3 readouts with ligase-dead mutant","pmids":["31226106","31640975"],"confidence":"Medium","gaps":["Direct functional consequence of DPPA2-SUMO on its targets not mapped","Link between PIAS4 SUMO activity and H3K9me3 methyltransferase translocation mechanistic detail lacking"]},{"year":2021,"claim":"Refined the DSB role of PIAS4 to promoting end resection and RAD51 loading by counteracting the 53BP1/RIF1 resection blockade within a PIAS1/PIAS4/RNF4/BRCA1 module.","evidence":"siRNA, gamma-irradiation DSB-marker imaging, and epistasis (53BP1 depletion rescue)","pmids":["35007836"],"confidence":"Medium","gaps":["Direct SUMO substrate driving resection licensing not pinpointed","Order of action relative to RNF4 in this module not fully resolved"]},{"year":2022,"claim":"Extended PIAS4 SUMO signaling to cardiac membrane biology, where Cav-3 SUMO2/3 modification disrupts Nav1.5 channel localization and promotes ischemia-reperfusion arrhythmias.","evidence":"AAV9-shRNA cardiac knockdown, Co-IP, Cav-3 SUMO-site mutants, ECG, and hypoxia/reoxygenation cell model","pmids":["36229865"],"confidence":"Medium","gaps":["Whether Cav-3 SUMOylation is direct PIAS4 catalysis vs indirect not fully isolated","Generalizability beyond the I/R context untested"]},{"year":2024,"claim":"Linked PIAS4-RNF4 SUMO-targeted degradation to extraction of trapped WRN helicase and uncovered a transcriptional regulator-controlled PIAS4-SLC7A11 ferroptosis axis as cancer vulnerabilities.","evidence":"Single-molecule tracking, phenotypic screens, inhibitor/siRNA and MSI-H xenografts for WRN; pull-down, Co-IP, ChIP, luciferase and xenograft models for SLC7A11/KDM1A","pmids":["39025847","38615741"],"confidence":"Medium","gaps":["Direct PIAS4 substrate at trapped-WRN chromatin not biochemically defined","Whether SLC7A11 SUMOylation site and degradation pathway are universal across tumors unclear"]},{"year":2025,"claim":"Began characterizing oxidative-stress-induced TDP-43 SUMOylation by PIAS4, hinting at a stress-specific role with possible neurodegeneration relevance.","evidence":"In vivo sumoylation assay under oxidative stress with domain mapping and PIAS1/PIAS4 co-expression (preprint)","pmids":["41292941"],"confidence":"Low","gaps":["Preprint, single lab, no mechanistic follow-up","Functional consequence of TDP-43 SUMOylation undefined","Stimulus specificity (oxidative vs DNA-damage) basis unexplained"]},{"year":null,"claim":"How a single ligase achieves substrate- and site-selectivity across its many targets, and how its catalytic (SP-RING/SIM) versus ligase-independent (SAP/HDAC/coactivator-competition) modes are partitioned in vivo, remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structure of PIAS4 engaging a substrate-SUMO-Ubc9 complex","Rules governing SUMO1 vs SUMO2/3 paralog choice across substrates unknown","Tissue-specific contributions vs PIAS-family redundancy not systematically mapped"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[0,2,10,12,24,25,30]},{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[0,2,6,10,24,25,30]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,3,7,13,21]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[7,18,21,27]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,20]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,6,12,15]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[10,24,25,26]},{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[26]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[0,11]}],"pathway":[{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,2,10,24,25,30,35]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[20,36,38,42]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[10,24,25,26]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,3,7,13,21,28]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[12,16,27,34,39]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[3,17,22,23,30]},{"term_id":"R-HSA-8953897","term_label":"Cellular responses to stimuli","supporting_discovery_ids":[13,22,23]}],"complexes":[],"partners":["UBC9","RNF4","TRIM32","FIEL1","RIF1","PIAS1","HDAC1","NEMO"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N2W9","full_name":"E3 SUMO-protein ligase PIAS4","aliases":["PIASy","Protein inhibitor of activated STAT protein 4","Protein inhibitor of activated STAT protein gamma","PIAS-gamma"],"length_aa":510,"mass_kda":56.5,"function":"Functions as an E3-type small ubiquitin-like modifier (SUMO) ligase, stabilizing the interaction between UBE2I and the substrate, and as a SUMO-tethering factor (PubMed:12511558, PubMed:12631292, PubMed:12727872, PubMed:15831457, PubMed:15976810, PubMed:22508508, PubMed:32832608). Mediates sumoylation of ALKBH5, AXIN1, CEBPA, KLF8, GATA2, PARK7, HERC2, MYB, TCF4 and RNF168 (PubMed:12223491, PubMed:12511558, PubMed:12631292, PubMed:12727872, PubMed:12750312, PubMed:15831457, PubMed:15976810, PubMed:16617055, PubMed:22508508, PubMed:34048572). Plays a crucial role as a transcriptional coregulation in various cellular pathways, including the STAT pathway, the p53/TP53 pathway, the Wnt pathway and the steroid hormone signaling pathway (PubMed:11388671). Involved in gene silencing (PubMed:11248056). In Wnt signaling, represses LEF1 and enhances TCF4 transcriptional activities through promoting their sumoylations (PubMed:12727872, PubMed:15831457). Enhances the sumoylation of MTA1 and may participate in its paralog-selective sumoylation (PubMed:21965678). Binds to AT-rich DNA sequences, known as matrix or scaffold attachment regions (MARs/SARs) (By similarity). Catalyzes conjugation of SUMO2 to KAT5 in response to DNA damage, facilitating repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) (PubMed:32832608). Mediates sumoylation of PARP1 in response to PARP1 trapping to chromatin (PubMed:35013556). Mediates sumoylation of KLF8, repressiing KLF8 transcriptional activity and cell cycle progression into G(1) phase (PubMed:16617055). Sumoylates ALKBH5 downstream of MAPK8/JNK1 and MAPK9/JNK2 in response to reactive oxygen species (ROS), inhibiting ALKBH5 RNA demethylase activity (PubMed:34048572)","subcellular_location":"Nucleus, PML body","url":"https://www.uniprot.org/uniprotkb/Q8N2W9/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PIAS4","classification":"Not Classified","n_dependent_lines":43,"n_total_lines":1208,"dependency_fraction":0.03559602649006623},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PIAS4","total_profiled":1310},"omim":[{"mim_id":"605989","title":"PROTEIN INHIBITOR OF ACTIVATED STAT4; PIAS4","url":"https://www.omim.org/entry/605989"},{"mim_id":"603566","title":"PROTEIN INHIBITOR OF ACTIVATED STAT1; PIAS1","url":"https://www.omim.org/entry/603566"},{"mim_id":"601912","title":"SMALL UBIQUITIN-LIKE MODIFIER 1; SUMO1","url":"https://www.omim.org/entry/601912"},{"mim_id":"113705","title":"BRCA1 DNA REPAIR-ASSOCIATED PROTEIN; BRCA1","url":"https://www.omim.org/entry/113705"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Centrosome","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PIAS4"},"hgnc":{"alias_symbol":["Piasg","PIASY","FLJ12419","ZMIZ6"],"prev_symbol":[]},"alphafold":{"accession":"Q8N2W9","domains":[{"cath_id":"1.10.720.30","chopping":"2-68","consensus_level":"high","plddt":90.729,"start":2,"end":68},{"cath_id":"2.60.120.780","chopping":"140-228_239-280","consensus_level":"high","plddt":90.1469,"start":140,"end":280},{"cath_id":"3.30.40.10","chopping":"284-408","consensus_level":"high","plddt":95.0996,"start":284,"end":408}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N2W9","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N2W9-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N2W9-F1-predicted_aligned_error_v6.png","plddt_mean":76.06},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PIAS4","jax_strain_url":"https://www.jax.org/strain/search?query=PIAS4"},"sequence":{"accession":"Q8N2W9","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N2W9.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N2W9/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N2W9"}},"corpus_meta":[{"pmid":"11731474","id":"PMC_11731474","title":"PIASy, 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proteomics","date":"2025-03-03","source":"bioRxiv","url":"https://doi.org/10.1101/2025.02.27.640536","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":43655,"output_tokens":12170,"usd":0.156757,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":23336,"output_tokens":7643,"usd":0.153878,"stage2_stop_reason":"end_turn"},"total_usd":0.310635,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2001,\n      \"finding\": \"PIASy (PIAS4) functions as a SUMO E3 ligase for LEF1 in a reconstituted in vitro system, stimulates LEF1 sumoylation in vivo, and represses LEF1 transcriptional activity by sequestering it into nuclear bodies via binding to nuclear matrix-associated DNA sequences.\",\n      \"method\": \"Reconstituted in vitro SUMO E3 ligase assay, co-expression sumoylation assay, nuclear body localization by immunofluorescence, nuclear matrix binding assay\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted in vitro ligase activity plus in vivo sumoylation and localization experiments, multiple orthogonal methods in one study\",\n      \"pmids\": [\"11731474\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"PIASy (PIAS4) interacts with p53 (confirmed by yeast two-hybrid and co-immunoprecipitation in mammalian cells), inhibits p53 DNA-binding activity and p53-dependent transactivation of Bax and p21, but does not suppress p53-induced apoptosis.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, luciferase reporter assay, EMSA\",\n      \"journal\": \"Apoptosis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP plus functional reporter assay, single lab, two orthogonal methods\",\n      \"pmids\": [\"11388671\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PIASy (PIAS4) acts as a SUMO E3 ligase for C/EBPalpha, enhancing both SUMO-1 and SUMO-3 modification at the synergy control (SC) motif (Lys159) both in vivo and in vitro; SUMO modification at this site suppresses transcriptional synergy from compound response elements.\",\n      \"method\": \"In vitro SUMOylation assay with purified recombinant components, in vivo sumoylation assay, site-directed mutagenesis, reporter gene assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution with purified components plus mutagenesis and in vivo validation, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"12511558\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PIASy (PIAS4) interacts with Smad3 and Smad4 (identified by yeast three-hybrid screen, confirmed in mammalian cells); the interaction with Smad3 is enhanced by TGF-beta and occurs through the C-terminal domain of Smad3. PIASy represses Smad transcriptional activity without inhibiting Smad-DNA binding, and can interact constitutively with HDAC1; HDAC inhibitor TSA prevents PIASy's inhibitory function.\",\n      \"method\": \"Yeast three-hybrid screen, co-immunoprecipitation, reporter gene assay, EMSA, HDAC inhibitor experiment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (yeast screen, Co-IP, reporter, EMSA), independently confirmed in two papers (PMID 12904571 and 12815042)\",\n      \"pmids\": [\"12904571\", \"12815042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PIASy (PIAS4) stimulates sumoylation of Smad3 in vivo and associates with Smad7 (identified by yeast two-hybrid with Smad7 MH2 domain as bait); endogenous PIASy expression is induced by TGF-beta in Hep3B cells, suggesting a negative feedback loop.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, in vivo sumoylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — yeast two-hybrid plus Co-IP plus in vivo sumoylation, single lab\",\n      \"pmids\": [\"12815042\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PIASy (PIAS4) interacts with GATA-2, preferentially enhances SUMO-2 conjugation to GATA-2 via its E3 SUMO ligase activity, and suppresses GATA-2-dependent ET-1 promoter activity in endothelial cells through a RING-like domain-independent mechanism requiring both N-terminal and C-terminal sequences of PIASy.\",\n      \"method\": \"Co-immunoprecipitation, in vivo sumoylation assay, reporter gene assay, domain deletion analysis\",\n      \"journal\": \"Circulation research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vivo sumoylation plus reporter assay with domain mutants, single lab\",\n      \"pmids\": [\"12750312\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PIASy (PIAS4) is a SUMO E3 ligase for c-Myb, enhancing SUMO-1 conjugation at K503 and K527 both in vitro and in vivo; PIASy causes a shift of c-Myb to the insoluble nuclear matrix fraction and negatively regulates Myb-induced transcriptional activation.\",\n      \"method\": \"In vitro SUMOylation assay, co-immunoprecipitation, reporter gene assay, nuclear fractionation, site-directed mutagenesis\",\n      \"journal\": \"European journal of biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro assay plus in vivo sumoylation, mutagenesis, and functional reporter, single lab\",\n      \"pmids\": [\"12631292\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PIASy (PIAS4) represses androgen receptor (AR) transcriptional activity by recruiting HDAC1 and HDAC2 via its RD1 repression domain; HDAC activity is required for this repression. The repression is independent of PIASy's SUMO ligase activity and independent of AR sumoylation status.\",\n      \"method\": \"Co-immunoprecipitation, reporter gene assay, HDAC inhibitor experiment, domain deletion and SUMO-ligase-dead mutant analysis\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, reporter, HDAC inhibitor, mutants) mechanistically dissecting ligase-independent repression, single lab but comprehensive\",\n      \"pmids\": [\"14981544\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PIASy (PIAS4) knockout mice are phenotypically normal with no significant perturbation of STAT1 activation or global SUMO-1/SUMO-3 modification patterns, demonstrating that PIASy is either dispensable or compensated by other PIAS family members at steady state.\",\n      \"method\": \"Knockout mouse generation, 2D gel analysis of SUMO-modified proteins, STAT1 activation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent knockout studies (PMID 15169916 and 15528356) with proteome-level analysis confirming dispensability\",\n      \"pmids\": [\"15169916\", \"15528356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PIASy (PIAS4) deletion in mice results in modest reduction of IFN-gamma-induced and Wnt-responsive gene expression, demonstrating partial but non-essential roles in STAT1 and LEF1 signaling with likely redundancy from other PIAS family members.\",\n      \"method\": \"Knockout mouse, gene expression analysis, signaling assays in primary cells\",\n      \"journal\": \"Journal of immunology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo knockout model replicated across two independent labs (PMID 15528356 and 15169916)\",\n      \"pmids\": [\"15528356\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PIASy (PIAS4) is specifically required for mitotic SUMO-2 conjugation of Topoisomerase-II in Xenopus egg extracts; PIASy binds mitotic chromosomes and recruits Ubc9 to chromatin, and these properties are essential for its activity. PIASy depletion eliminates chromosomal SUMO-2-conjugated species and blocks anaphase sister chromatid segregation.\",\n      \"method\": \"Xenopus egg extract depletion, immunofluorescence, EGFP-SUMO-2 localization, functional segregation assay, dominant-negative mutant analysis\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstituted biochemical system (Xenopus egg extracts), depletion rescue, multiple orthogonal methods, replicated in later papers\",\n      \"pmids\": [\"15933717\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SUMO-1 modification of PIASy itself at Lys35 is required for PIASy-dependent sumoylation and transcriptional activation of Tcf-4; PIASy(K35R) retains ligase activity for other substrates but loses the ability to activate Tcf-4, correlating with distinct nuclear distribution including increased PML body association.\",\n      \"method\": \"Site-directed mutagenesis, in vivo sumoylation assay, reporter gene assay, immunofluorescence, co-immunoprecipitation\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mutagenesis plus sumoylation assay plus functional reporter plus localization, single lab\",\n      \"pmids\": [\"15831457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PIASy (PIAS4) is the SUMO E3 ligase for NEMO (IKKgamma), preferentially stimulating site-selective SUMO-1 (but not SUMO-2/3) modification of NEMO in vitro; PIASy-NEMO interaction is increased by genotoxic stress and occurs in the nucleus, mutually exclusive with IKK interaction. PIASy siRNA inhibits NEMO sumoylation and NF-kappaB activation in response to genotoxic agents.\",\n      \"method\": \"siRNA knockdown, in vitro SUMOylation assay, co-immunoprecipitation, overexpression, subcellular fractionation\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus siRNA knockdown plus Co-IP plus in vivo functional assay, multiple orthogonal methods\",\n      \"pmids\": [\"16906147\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PIASy (PIAS4) overexpression in normal human fibroblasts induces cellular senescence via the p53 and Rb tumor suppressor pathways; in Rb-deficient fibroblasts, PIASy expression leads to p53-dependent apoptosis. PIASy stimulates sumoylation and transcriptional activity of p53 and increases Rb-dependent corepression at E2F-responsive promoters. Fibroblasts lacking PIASy exhibit reduced senescence in response to prosenescence stimuli. Senescence induction requires PIASy E3 ligase activity.\",\n      \"method\": \"Overexpression in primary fibroblasts, PIASy knockout cells, E3-ligase-dead mutant, in vivo sumoylation assay, ChIP, reporter gene assay\",\n      \"journal\": \"Molecular cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function and gain-of-function with defined phenotypic readout, E3-ligase mutant, ChIP, multiple orthogonal methods\",\n      \"pmids\": [\"16793547\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"TRIM32 interacts with PIASy (PIAS4), promotes PIASy ubiquitination and proteasomal degradation in vitro using purified components; this interaction is induced by UVB/TNFalpha treatment and involves redistribution of PIASy from the nucleus to cytoplasmic granules. The LGMD2H-associated TRIM32 missense mutation prevents TRIM32-PIASy interaction. PIASy inhibits NF-kappaB activity and sensitizes keratinocytes to apoptosis; TRIM32-mediated PIASy degradation thus regulates NF-kappaB-dependent survival.\",\n      \"method\": \"In vitro ubiquitination assay with purified components, co-immunoprecipitation, immunofluorescence, NF-kappaB reporter assay, patient fibroblasts\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstituted ubiquitination assay plus multiple in vivo validation methods, functionally linked to NF-kappaB regulation\",\n      \"pmids\": [\"16816390\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PIASy (PIAS4) is a SUMO E3 ligase for YY1, stimulating YY1 sumoylation at Lys288 in vitro and in vivo; uniquely, PIASy-mediated YY1 sumoylation does not depend on the RING finger domain of PIASy but correlates with PIASy-YY1 interaction. PIASy colocalizes with YY1 in the nucleus, stabilizes YY1 protein, and differentially regulates YY1 transcriptional activity on different target promoters.\",\n      \"method\": \"In vitro and in vivo sumoylation assay, site-directed mutagenesis, co-immunoprecipitation, immunofluorescence, reporter gene assay, stability assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro sumoylation plus mutagenesis plus multiple in vivo validation methods, single lab but comprehensive\",\n      \"pmids\": [\"17353273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"PIASy (PIAS4) and PIAS1 cooperate to regulate specificity and magnitude of NF-kappaB and STAT1-mediated gene activation; Piasy deletion in dendritic cells enhances a subset of NF-kappaB and STAT1 target genes, and Pias1-/-Piasy-/- double knockout embryos die before day 11.5, demonstrating essential cooperative function.\",\n      \"method\": \"Single and double knockout mice, gene expression analysis in primary dendritic cells, endotoxic shock model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis via double knockout with defined embryonic lethal phenotype, gene-specific transcriptional analysis\",\n      \"pmids\": [\"17606919\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PIASy (PIAS4) sumoylation of Smad3 stimulates Smad3 nuclear export; co-expression of Smad3 with PIASy and SUMO1 affected Smad3 DNA-binding activity and promoted nuclear export. FRET analysis revealed Smad3 interacts with SUMO1 in the cytoplasm. siRNA-mediated reduction of endogenous PIASy enhanced TGF-beta-induced gene expression.\",\n      \"method\": \"siRNA knockdown, nuclear export assay, FRET analysis, DNA-binding assay, reporter gene assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (siRNA, FRET, export assay) in single lab establishing mechanism of SUMO-dependent nuclear export\",\n      \"pmids\": [\"18384750\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PIASy (PIAS4) represses C/EBPdelta transcriptional activity by sequestering it to the nuclear periphery via interaction between the PIASy SAP domain (SAPD) and the C/EBPdelta transactivation domain (TAD); this repression is independent of HDAC activity, PIASy E3 SUMO ligase activity, and C/EBPdelta sumoylation.\",\n      \"method\": \"Domain deletion analysis, reporter gene assay, HDAC inhibitor experiment, immunofluorescence, SUMO-ligase-dead mutant\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple mechanistic dissection experiments (domain mutants, inhibitor, localization), single lab\",\n      \"pmids\": [\"18477566\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"FIP200 interacts with PIASy (PIAS4) via the PIASy RING finger and FIP200 C-terminus; PIASy interaction redistributes FIP200 from cytoplasm to nucleus, abrogating FIP200 regulation of TSC/S6K signaling. FIP200 enhances PIASy-dependent transcriptional activation of the p21 promoter; both proteins are co-recruited to the p21 promoter by ChIP. FIP200 is not a SUMO substrate of PIASy.\",\n      \"method\": \"Co-immunoprecipitation, in vitro/in vivo sumoylation assay, immunofluorescence, subcellular fractionation, ChIP, siRNA knockdown, reporter gene assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (Co-IP, fractionation, ChIP, reporter) in single lab\",\n      \"pmids\": [\"18285457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PIAS4 and PIAS1 are recruited to DNA double-strand break sites via SAP domain-dependent mechanisms; PIAS4 is required for SUMO1 accumulation and PIAS1 for SUMO2/3 accumulation at DSB sites. Both are needed for productive association of 53BP1, BRCA1, and RNF168 with damage foci, and for effective ubiquitin-adduct formation by RNF8, RNF168, and BRCA1. Both promote DSB repair and confer ionizing radiation resistance.\",\n      \"method\": \"Immunofluorescence of irradiation-induced foci, siRNA knockdown, domain mutant analysis, DSB repair assay, clonogenic survival assay\",\n      \"journal\": \"Nature\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, high-impact journal, findings replicated in subsequent papers (PMID 35007836, 29234018)\",\n      \"pmids\": [\"20016603\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"PIASy (PIAS4) inhibits LRH-1-mediated transactivation by competing with the co-activator SRC-1 for binding to the LRH-1 AF-2 domain; this repression is independent of LRH-1 SUMOylation and HDAC activity.\",\n      \"method\": \"Co-immunoprecipitation, mammalian two-hybrid, reporter gene assay, domain deletion analysis, overexpression competition assay\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus two-hybrid plus reporter with mechanistic domain mapping, single lab\",\n      \"pmids\": [\"19067654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PIASy (PIAS4) interacts with VHL and induces VHL SUMOylation at Lys171 by SUMO1; PIASy-mediated SUMOylation promotes VHL oligomerization and abrogates its inhibitory function on tumor cell growth, migration, and clonogenicity. PIASy siRNA reduces VHL oligomerization and increases HIF1alpha degradation. PIASy is upregulated under hypoxic conditions.\",\n      \"method\": \"Co-immunoprecipitation, in vivo sumoylation assay, site-directed mutagenesis, siRNA knockdown, cell growth/migration assay, ubiquitination assay\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, mutagenesis, siRNA, functional assays in single lab\",\n      \"pmids\": [\"20300531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PIASy (PIAS4) is a specific E3 ligase for hypoxia-induced HIF1alpha SUMOylation by SUMO1; hypoxia promotes nuclear translocation of HIF1alpha to enable PIASy binding. PIASy negatively regulates hypoxia-induced HIF1alpha stability and transactivation; PIASy knockdown increases angiogenic activity of endothelial cells.\",\n      \"method\": \"Co-immunoprecipitation, in vivo sumoylation assay, siRNA knockdown, reporter gene assay, angiogenesis assay (tube formation), immunofluorescence\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (Co-IP, sumoylation, siRNA, functional) in single lab; consistent with parallel VHL paper\",\n      \"pmids\": [\"20661221\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PIASy (PIAS4)-mediated SUMO2/3 modification of Topoisomerase IIalpha at Lys660 (in the DNA gate domain) strongly inhibits TopoIIalpha decatenation activity; loss of SUMOylation at Lys660 eliminates this inhibitory effect. The SUMOylation site was identified by mass spectrometry.\",\n      \"method\": \"Mass spectrometry, biochemical decatenation assay, site-directed mutagenesis, Xenopus egg extract sumoylation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay plus mass spectrometry site identification plus mutagenesis validation, mechanistically rigorous\",\n      \"pmids\": [\"21079245\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PIASy (PIAS4) promotes SUMO-2/3 conjugation of PARP1 at Lys482 (identified by tandem mass spectrometry) on mitotic chromosomes but not interphase chromatin, both in Xenopus egg extracts and in vitro reconstituted assays. PARP1 SUMOylation does not alter intrinsic PARP1 enzymatic activity or its localization on chromosomes, but loss of SUMOylation increases PARP1-dependent PARylation of other chromatin-associated proteins.\",\n      \"method\": \"Xenopus egg extract SUMOylation, tandem mass spectrometry, in vitro reconstituted SUMOylation assay, site-directed mutagenesis, PARP enzymatic activity assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution plus mass spectrometry site identification plus mutagenesis plus functional enzyme assay\",\n      \"pmids\": [\"20228053\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The Rod/Zw10 kinetochore complex interacts with the N-terminal domain of PIASy (PIAS4) (specifically first 47 residues) and is required for PIASy centromeric localization and mitotic SUMO2/3 conjugation on chromosomes; Rod depletion compromises centromeric localization of PIASy and SUMO2/3.\",\n      \"method\": \"Xenopus egg extract depletion, co-immunoprecipitation, immunofluorescence, domain truncation analysis, N-terminal domain swapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — domain mapping plus depletion rescue plus immunofluorescence in well-established Xenopus system\",\n      \"pmids\": [\"20696768\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PIASy (PIAS4) inhibits virus-induced type I IFN transcription through a SUMO E3-ligase-independent mechanism requiring SUMO-interacting motif (SIM) activity and UBC9; PIASy inhibits IFN-stimulated gene expression through its SAP domain LXXLL motif. These two inhibitory mechanisms are distinct: LXXLL mutation abolishes IFN-stimulated gene repression but not virus-induced IFN transcription.\",\n      \"method\": \"PIASy null mouse cells, overexpression, SUMO-ligase-dead mutant, LXXLL motif mutant, SIM mutant, UBC9 knockdown, reporter gene assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — null cell system plus multiple mutants dissecting distinct mechanisms, multiple orthogonal approaches\",\n      \"pmids\": [\"21199872\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PIASy (PIAS4) is induced by hypoxia and promotes SUMOylation of Sp1, preventing Sp1 from binding to the SIRT1 promoter and thereby transcriptionally repressing SIRT1 expression; PIASy knockdown restores Sp1 binding and SIRT1 expression, reverses cancer cell EMT, and attenuates metastasis in vivo in nude mice.\",\n      \"method\": \"siRNA knockdown, ChIP, in vivo sumoylation assay, EMT markers, in vivo xenograft metastasis assay\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus ChIP plus in vivo functional validation, single lab\",\n      \"pmids\": [\"23843607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PIASy (PIAS4) sumoylates SREBP1c at Lys98, leading to suppression of the hepatic lipogenic program; PKA activation enhances PIASy-SREBP1c interaction and SREBP1c sumoylation, followed by ubiquitination-dependent degradation. PIASy overexpression in db/db mice ameliorates hepatic steatosis; PIASy suppression in lean mice stimulates lipogenesis.\",\n      \"method\": \"In vivo sumoylation assay, site-directed mutagenesis, co-immunoprecipitation, in vivo mouse studies (db/db and WT), primary hepatocyte experiments, PKA activation assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mutagenesis plus co-IP plus in vivo mouse validation plus primary cell experiments, mechanistically comprehensive\",\n      \"pmids\": [\"24379443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PIAS4 and its SUMO E3 ligase activity are specifically required for SUMOylation of AMPKalpha1, which attenuates AMPK activity specifically towards mTORC1 signaling. SUMOylation-deficient AMPKalpha1 shows higher activity towards mTORC1 when reconstituted in AMPKalpha-deficient cells. PIAS4 depletion reduces breast cancer cell growth specifically when combined with AMPK activator A769662.\",\n      \"method\": \"In vivo SUMOylation assay, SUMO-ligase-dead PIAS4 mutant, SUMOylation-deficient AMPKalpha1 reconstitution in knockout cells, mTORC1 signaling assay, cell growth assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reconstitution in knockout cells plus ligase mutant plus substrate mutant plus signaling readout, multiple orthogonal methods\",\n      \"pmids\": [\"26616021\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"FIEL1 (KIAA0317) ubiquitinates PIAS4 in a site-specific manner facilitated by PKCzeta phosphorylation of PIAS4 and GSK3beta phosphorylation of FIEL1 (double-locking mechanism), leading to PIAS4 degradation and enhancement of TGF-beta signaling. FIEL1 overexpression increases lung fibrosis in a bleomycin model; a FIEL1 small molecule inhibitor ameliorates fibrosis.\",\n      \"method\": \"Co-immunoprecipitation, in vitro ubiquitination assay, phosphorylation assays, bleomycin murine fibrosis model, small molecule inhibitor\",\n      \"journal\": \"The Journal of experimental medicine\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitination assay plus mechanistic phosphorylation studies plus in vivo mouse validation\",\n      \"pmids\": [\"27162139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PIAS4 acts as SUMO E3 ligase for E12 (a class I bHLH protein) in vivo; PIASy RING mutant cannot block E12-mediated alpha-SMA promoter activation, indicating RING domain is required for E12 sumoylation; TGF-beta induces both PIASy and E12 expression; reduced PIASy expression leads to increased TGF-beta-mediated alpha-SMA expression in mesangial cells.\",\n      \"method\": \"In vivo sumoylation assay, yeast two-hybrid, co-immunoprecipitation, reporter gene assay, RING domain mutant, siRNA knockdown\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vivo sumoylation plus RING mutant plus siRNA, single lab\",\n      \"pmids\": [\"22829926\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PIAS4 promotes HSC activation and liver fibrosis by transcriptionally repressing SIRT1, which leads to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters; lentivirus-shRNA targeting PIAS4 in mice ameliorates liver fibrosis by normalizing SIRT1 expression.\",\n      \"method\": \"shRNA knockdown in vivo (lentivirus), ChIP, gene expression analysis, in vivo mouse model (MCD diet)\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo shRNA plus ChIP plus mechanistic pathway placement, single lab\",\n      \"pmids\": [\"27323886\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"PIAS4 is recruited to nuclear domains containing HSV-1 viral DNA via SUMO-interacting motif (SIM)-dependent mechanisms (at viral genome entry sites) and via SIM-independent mechanisms (in replication compartments). PIAS4 depletion enhances replication of ICP0-null HSV-1; its restriction mechanisms are synergistic with PML protein and antagonized by ICP0.\",\n      \"method\": \"Immunofluorescence co-localization, siRNA depletion, viral replication assay, SIM mutant analysis\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA depletion with viral replication readout plus SIM mutant analysis, single lab\",\n      \"pmids\": [\"26937035\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HDAC9 deacetylation of Nkx3.2 triggers PIASy (PIAS4)-mediated sumoylation of Nkx3.2, and subsequent RNF4-mediated SUMO-targeted ubiquitination; this HDAC9-PIASy-RNF4 post-translational cascade controls Nkx3.2 protein stability and regulates chondrocyte hypertrophy and viability during skeletal development.\",\n      \"method\": \"Co-immunoprecipitation, in vivo sumoylation and ubiquitination assays, HDAC inhibitor, chondrocyte differentiation assay\",\n      \"journal\": \"Cellular signalling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus in vivo modification assays plus pathway cascade ordering, single lab\",\n      \"pmids\": [\"27312341\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PIAS4 is the primary SUMO E3 ligase for RIF1 SUMOylation in response to DNA damage; PIAS4 knockdown impairs RIF1 SUMOylation, defective disassembly of RIF1 DDR foci, and abrogates UHRF1-dependent ubiquitination and turnover of RIF1, leading to accumulated RIF1 at damage sites and DNA double-strand breaks.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation, immunofluorescence, in vivo sumoylation assay, ubiquitination assay\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus Co-IP plus in vivo modification assays establishing pathway cascade, single lab\",\n      \"pmids\": [\"29234018\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PIASy (PIAS4) contains two SUMO-interacting motifs (SIMs) at its C-terminus; both are required for full E3 ligase activity. The new SIM was identified by NMR chemical shift mapping and validated by mutagenesis.\",\n      \"method\": \"NMR chemical shift mapping, mutagenesis, in vitro SUMOylation assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structure-function plus mutagenesis plus in vitro activity assay in single study\",\n      \"pmids\": [\"28455449\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PIAS1 and PIAS4 together promote SUMO-dependent template switch (TS) DNA damage tolerance pathway by SUMOylating PCNA at Lys164; PIAS1/PIAS4 double-knockout cells show >90% decrease in PCNA-Lys164 SUMOylation and >90% decrease in TS (Ig gene conversion). Epistasis experiments show PCNA mutation causes no additional impact on PIAS1/PIAS4 cells; overexpression of PCNA-SUMO1 chimera restores TS in PIAS1/PIAS4 cells.\",\n      \"method\": \"Genetic knockout (chicken DT40 and human TK6 cells), PCNA-SUMO1 chimera reconstitution, Ig gene conversion assay, epistasis analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis plus reconstitution rescue, two cell line models, mechanistically definitive\",\n      \"pmids\": [\"30487218\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PIASy (PIAS4) interacts with Rbp2 (KDM5B/JARID1A histone H3K4me3 demethylase) via the PIASy PINIT domain (101-218 aa) and the Rbp2 JmjC domain (451-503 aa); Piasy binds the IFN-beta promoter and facilitates Rbp2-mediated demethylation of H3K4me3 at IFNI genes, attenuating type I IFN transcription. Enzymatically inactive Rbp2 does not attenuate IFNI.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, domain deletion analysis, siRNA/shRNA knockdown, enzymatically inactive mutant (Rbp2H483G/E485Q), reporter gene assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus ChIP plus enzyme-dead mutant plus reporter, single lab\",\n      \"pmids\": [\"28970247\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PIAS4 sumoylates DPPA2 protein, negatively regulating its activity; PIAS4 is down-regulated during zygotic genome activation (ZGA). Depleting Pias4 or overexpressing Dppa2/4 is sufficient to activate 2C-like transcriptional program; forced expression of Pias4 or Sumo2-DPPA2 fusion inhibits 2C-like program and impairs early mouse embryo development.\",\n      \"method\": \"siRNA/shRNA depletion, overexpression, in vivo sumoylation assay, ESC-to-2C-like cell transition model, early embryo development assay\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — depletion and overexpression with transcriptional and developmental readouts, in vivo sumoylation, single lab\",\n      \"pmids\": [\"31226106\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PIASy (PIAS4) overexpression in fertilized mouse embryos causes developmental arrest at the two-cell stage with abnormal chromosome segregation and impaired zygotic transcription; this is dependent on PIASy SUMOylation activity and associated with increased H3K9me3 trimethylation and enhanced nuclear translocation of H3K9me3 methyltransferase.\",\n      \"method\": \"mRNA microinjection/overexpression in embryos, chromosome segregation analysis, H3K9me3 immunofluorescence, SUMO-ligase-dead mutant\",\n      \"journal\": \"Biology open\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo embryo overexpression with defined phenotypic readout plus E3-ligase mutant, single lab\",\n      \"pmids\": [\"31640975\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"PIAS4 depletion impairs DSB end resection and RAD51 loading in gamma-ray-irradiated human fibroblasts, reducing BRCA1 recruitment to DSB sites; 53BP1 depletion rescues resection in PIAS4-depleted cells. Epistasis analysis shows PIAS4, PIAS1, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade.\",\n      \"method\": \"siRNA depletion, gamma-ray irradiation, immunofluorescence of DSB markers, epistasis analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — siRNA plus epistasis analysis with defined molecular readouts, single lab, consistent with earlier Nature paper\",\n      \"pmids\": [\"35007836\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PIASy (PIAS4)-mediated SUMOylation of Caveolin-3 (Cav-3) by SUMO2/3 increases after myocardial ischemia-reperfusion (I/R); increased Cav-3 SUMOylation causes Nav1.5/Cav-3 dissociation, reducing membrane Nav1.5 density; cardiac-targeted PIASy silencing decreases Cav-3 SUMO2/3 modification, restores Nav1.5-Cav-3 co-localization, and prevents I/R-induced ventricular arrhythmias in rats.\",\n      \"method\": \"AAV9-shRNA in vivo cardiac knockdown, co-immunoprecipitation, site-directed mutagenesis of Cav-3 SUMO sites, immunofluorescence, ECG recording, HEK293T hypoxia/reoxygenation model\",\n      \"journal\": \"Military Medical Research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo AAV knockdown plus mutagenesis plus Co-IP, single lab\",\n      \"pmids\": [\"36229865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"WRN helicase inhibition traps WRN on chromatin, requiring SUMOylation via the PIAS4-RNF4 axis for subsequent p97/VCP-mediated extraction and proteasomal degradation; co-inhibition of WRN and SUMOylation has additive toxicity in MSI-H cancer cells.\",\n      \"method\": \"Single-molecule tracking (SMT), phenotypic screen, siRNA/inhibitor studies, co-inhibition experiments, in vivo MSI-H xenograft model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — single-molecule imaging plus phenotypic screen plus functional validation in vivo, single lab but multiple approaches\",\n      \"pmids\": [\"39025847\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PIAS4 directly binds SLC7A11 and facilitates its SUMOylation; KDM1A acts as a transcriptional activator of PIAS4. Tanshinone IIA decreases KDM1A expression, suppressing PIAS4 transcription and thereby inhibiting PIAS4-dependent SLC7A11 SUMOylation, which promotes SLC7A11 degradation and ferroptosis in breast cancer cells.\",\n      \"method\": \"Ni-beads pull-down, co-immunoprecipitation, luciferase assay, ChIP, siRNA knockdown, in vivo xenograft and metastasis models\",\n      \"journal\": \"Journal of advanced research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus pulldown plus ChIP plus in vivo validation, single lab\",\n      \"pmids\": [\"38615741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"PIAS4 facilitates SUMOylation of TDP-43 by SUMO1 and SUMO2/3 in response to oxidative stress; PIAS1 also contributes. TDP-43 SUMOylation is not promoted by etoposide-induced DNA damage.\",\n      \"method\": \"In vivo sumoylation assay, oxidative stress treatment, domain mapping, PIAS1/PIAS4 co-expression\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single lab, preliminary characterization without mechanistic follow-up\",\n      \"pmids\": [\"41292941\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"PIAS4 (PIASy) is a nuclear SUMO E3 ligase that uses its SP-RING domain to catalyze SUMO1, SUMO2, and SUMO2/3 conjugation to a wide array of substrates—including LEF1, p53, Smad3, NEMO/IKKgamma, Topoisomerase IIalpha, PARP1, HIF1alpha, VHL, AMPKalpha1, PCNA, Caveolin-3, RIF1, SREBP1c, DPPA2, and others—thereby regulating transcription factor activity (often repression), DNA double-strand break repair (promoting BRCA1/53BP1/RNF168 recruitment and template-switch repair), cell senescence and apoptosis, mitotic chromosome segregation, NF-kappaB and interferon signaling, and hypoxia responses; its own activity is controlled by autologous SUMO1 modification at Lys35, by interaction with the Rod/Zw10 complex for centromeric localization, and by TRIM32- or FIEL1-mediated ubiquitination and proteasomal degradation, while in some contexts it represses transcription factors (AR, C/EBPdelta, Ets-1) through SUMO-ligase-independent mechanisms involving HDAC recruitment or nuclear sequestration.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PIAS4 (PIASy) is a nuclear SUMO E3 ligase that uses an SP-RING/RING-finger catalytic core, two C-terminal SUMO-interacting motifs, and an N-terminal SAP domain to conjugate SUMO1 or SUMO2/3 to a broad panel of nuclear substrates, thereby controlling transcription factor output, genome maintenance, mitotic chromosome dynamics, and metabolic and stress signaling [#0, #10, #37]. As a transcriptional regulator it predominantly represses its targets, SUMOylating LEF1/Tcf, C/EBPalpha, GATA-2, c-Myb, p53, Smad3, YY1, and others to alter their localization, DNA binding, stability, or coactivator access\\u2014for example sequestering LEF1 and c-Myb into the nuclear matrix and driving SUMO-dependent nuclear export of Smad3 [#0, #2, #6, #17]; in several contexts it represses transcription factors (AR, C/EBPdelta, LRH-1) through ligase-independent mechanisms involving HDAC recruitment, SAP-domain-mediated nuclear sequestration, or coactivator competition [#7, #18, #21]. In the DNA damage response PIAS4 is recruited to double-strand breaks via its SAP domain to deposit SUMO1, enabling productive 53BP1/BRCA1/RNF168 recruitment and RNF8/RNF168/BRCA1 ubiquitin signaling, and it drives end resection and template-switch tolerance through SUMOylation of substrates including PCNA-Lys164 and RIF1 [#20, #36, #38, #42]. During mitosis PIAS4 localizes to centromeres via the Rod/Zw10 complex and catalyzes SUMO2/3 modification of Topoisomerase IIalpha and PARP1 on chromosomes, an activity essential for sister chromatid segregation [#10, #24, #25, #26]. PIAS4 additionally governs senescence and apoptosis via p53/Rb [#13], NF-kappaB and type I/II interferon signaling [#12, #16, #27], hypoxia responses through HIF1alpha and VHL SUMOylation [#22, #23], and AMPK/mTORC1 and hepatic lipogenic control via AMPKalpha1 and SREBP1c SUMOylation [#29, #30]. Its own abundance and activity are tuned by autologous SUMO1 modification at Lys35 and by TRIM32- and FIEL1-mediated ubiquitination and proteasomal degradation [#11, #14, #31]. Single PIAS4-knockout mice are largely normal, but Pias1/Pias4 double knockouts are embryonic lethal, establishing extensive functional redundancy within the PIAS family [#8, #16].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Established PIAS4 as a bona fide SUMO E3 ligase and transcriptional repressor, defining its core biochemical activity and a sequestration-based repression mode.\",\n      \"evidence\": \"Reconstituted in vitro ligase assay, in vivo sumoylation, and nuclear-body/matrix localization for LEF1; reciprocal Co-IP and reporter assays for p53\",\n      \"pmids\": [\"11731474\", \"11388671\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the catalytic determinants of ligase activity\", \"Mechanism linking nuclear-matrix sequestration to repression not resolved at structural level\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Showed PIAS4 SUMOylates and represses multiple developmental and signaling transcription factors, embedding it in TGF-beta/Smad and hematopoietic transcription networks, sometimes via HDAC recruitment.\",\n      \"evidence\": \"In vitro and in vivo sumoylation, yeast two/three-hybrid, Co-IP, reporter and EMSA assays for C/EBPalpha, Smad3/4/7, GATA-2, and c-Myb\",\n      \"pmids\": [\"12511558\", \"12904571\", \"12815042\", \"12750312\", \"12631292\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish ligase-dependent from ligase-independent repression for all substrates\", \"In vivo relevance of feedback induction by TGF-beta not tested genetically\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrated a SUMO-ligase-independent repression mechanism via HDAC recruitment and showed in vivo that PIAS4 is dispensable at steady state but contributes modestly to STAT1/Wnt outputs.\",\n      \"evidence\": \"Domain/ligase-dead mutants and HDAC inhibitor for AR repression; knockout mice with 2D-gel SUMO proteome and signaling assays\",\n      \"pmids\": [\"14981544\", \"15169916\", \"15528356\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Source of in vivo redundancy (which PIAS paralog compensates) not mapped\", \"Did not reconcile strong in vitro activities with mild knockout phenotype\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Defined PIAS4 as the dedicated mitotic chromosomal SUMO2/3 ligase whose activity is essential for sister chromatid segregation, and showed autologous SUMO1 modification at Lys35 tunes substrate-selective output.\",\n      \"evidence\": \"Xenopus egg extract depletion/rescue with Ubc9 recruitment and segregation readouts; site-directed mutagenesis (K35R) with sumoylation, localization, and Tcf-4 reporter assays\",\n      \"pmids\": [\"15933717\", \"15831457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full set of mitotic chromosomal substrates not yet enumerated\", \"How K35 SUMOylation rewires substrate selectivity mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Connected PIAS4 to genotoxic NF-kappaB activation, p53/Rb-driven senescence and apoptosis, and identified TRIM32-mediated ubiquitination as a degradation switch controlling PIAS4 levels.\",\n      \"evidence\": \"In vitro sumoylation/ubiquitination with purified components, siRNA, Co-IP, ChIP, ligase-dead mutants, and patient fibroblasts for NEMO, p53/Rb, and TRIM32\",\n      \"pmids\": [\"16906147\", \"16793547\", \"16816390\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stimulus specificity of nuclear PIAS4-NEMO interaction not fully defined\", \"Physiological triggers of TRIM32-PIAS4 redistribution beyond UVB/TNFalpha unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Revealed RING-independent substrate SUMOylation (YY1) and established cooperative, partially redundant control of NF-kappaB/STAT1 gene programs with PIAS1, including essential developmental function.\",\n      \"evidence\": \"In vitro/in vivo sumoylation and stability assays for YY1; single and double knockout mice with dendritic-cell gene expression and embryonic lethality\",\n      \"pmids\": [\"17353273\", \"17606919\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Basis for RING-independent SUMO transfer not structurally explained\", \"Distinct vs overlapping target sets of PIAS1 and PIAS4 incompletely resolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Defined mechanisms by which PIAS4 controls transcription factor localization: SUMO-driven Smad3 nuclear export and SAP-domain-mediated nuclear-periphery sequestration of C/EBPdelta; linked PIAS4 to FIP200/mTOR signaling.\",\n      \"evidence\": \"siRNA, FRET, nuclear export and DNA-binding assays for Smad3; domain mutants and immunofluorescence for C/EBPdelta; Co-IP, fractionation, ChIP for FIP200\",\n      \"pmids\": [\"18384750\", \"18477566\", \"18285457\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether export and sequestration mechanisms operate on shared substrates untested\", \"FIP200-PIAS4 functional consequences in vivo not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Placed PIAS4 at the apex of double-strand-break SUMO signaling and showed coactivator-competition as a distinct repression mode.\",\n      \"evidence\": \"Irradiation-induced foci imaging, SAP-domain mutants, repair and clonogenic survival assays (with PIAS1); Co-IP and competition assays for LRH-1\",\n      \"pmids\": [\"20016603\", \"19067654\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct SUMO substrates driving 53BP1/BRCA1/RNF168 recruitment not all identified at DSBs\", \"Recruitment cue read by the SAP domain at breaks undefined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Expanded the PIAS4 substrate repertoire across mitosis, hypoxia, and DNA-gate enzymology, defining site-specific modifications with direct functional consequences.\",\n      \"evidence\": \"Mass spectrometry site mapping, in vitro decatenation and PARP assays, Xenopus extracts, Co-IP, siRNA, and angiogenesis/growth assays for TopoIIalpha-K660, PARP1-K482, HIF1alpha, VHL-K171; Rod/Zw10 domain mapping for centromeric targeting\",\n      \"pmids\": [\"21079245\", \"20228053\", \"20661221\", \"20300531\", \"20696768\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How a single ligase achieves substrate- and site-selectivity across these targets unresolved\", \"Opposing HIF1alpha (destabilizing) vs VHL (inactivating) effects not integrated into one model\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Dissected two ligase-independent interferon-restriction mechanisms of PIAS4 acting through SIM/UBC9 and the SAP-domain LXXLL motif.\",\n      \"evidence\": \"PIAS4-null cells with ligase-dead, LXXLL, SIM, and UBC9-knockdown reporter assays\",\n      \"pmids\": [\"21199872\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular targets of the SIM-dependent and LXXLL-dependent repression not identified\", \"In vivo antiviral relevance not tested here\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Identified a hypoxia-induced PIAS4-Sp1-SIRT1 transcriptional repression axis driving EMT and metastasis, and a SREBP1c-SUMOylation node suppressing hepatic lipogenesis.\",\n      \"evidence\": \"siRNA, ChIP, in vivo sumoylation, xenograft metastasis for Sp1/SIRT1; mutagenesis, Co-IP, db/db and lean mouse studies, primary hepatocytes for SREBP1c-K98\",\n      \"pmids\": [\"23843607\", \"24379443\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct vs indirect contribution of Sp1 SUMOylation to SIRT1 promoter occupancy not fully isolated\", \"Upstream signals coupling PKA to PIAS4-SREBP1c beyond PKA activation incompletely defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Established PIAS4 as the AMPKalpha1 SUMO ligase that selectively restrains AMPK signaling toward mTORC1, linking it to growth control and a cancer vulnerability.\",\n      \"evidence\": \"In vivo sumoylation, ligase-dead PIAS4 and SUMO-deficient AMPKalpha1 reconstitution in knockout cells, mTORC1 and cell growth assays\",\n      \"pmids\": [\"26616021\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"SUMO acceptor site on AMPKalpha1 and structural basis of pathway selectivity not defined\", \"Generality across AMPK complexes/tissues untested\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Revealed a phosphorylation-gated FIEL1 ubiquitination switch controlling PIAS4 turnover and TGF-beta/fibrosis, and embedded PIAS4 in SUMO-targeted ubiquitin cascades and viral restriction.\",\n      \"evidence\": \"In vitro ubiquitination, phosphorylation assays, bleomycin model and inhibitor for FIEL1; Co-IP/sumoylation/ChIP for E12, SIRT1-SMAD3, Nkx3.2-RNF4; viral replication and SIM-mutant analysis for HSV-1\",\n      \"pmids\": [\"27162139\", \"22829926\", \"27323886\", \"27312341\", \"26937035\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Interplay between TRIM32 and FIEL1 degradation pathways not reconciled\", \"Whether SUMO-targeted RNF4 ubiquitination acts on the same PIAS4-modified substrate pools broadly unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Defined structural and pathway determinants of PIAS4 activity: a second C-terminal SIM required for full ligase function, and PIAS4-driven control of RIF1 and Rbp2/KDM5B-mediated chromatin/IFN regulation.\",\n      \"evidence\": \"NMR chemical-shift mapping and in vitro activity for SIMs; siRNA, Co-IP, sumoylation/ubiquitination for RIF1; Co-IP, ChIP, enzyme-dead mutant for Rbp2\",\n      \"pmids\": [\"28455449\", \"29234018\", \"28970247\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the two SIMs cooperate with the SP-RING during catalysis structurally unresolved\", \"Direct vs scaffolding role of PIAS4 in the RIF1-UHRF1 turnover circuit incompletely separated\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Demonstrated genetically that PIAS1/PIAS4 jointly drive PCNA-Lys164 SUMOylation to enable the template-switch DNA damage tolerance pathway.\",\n      \"evidence\": \"DT40 and TK6 double-knockouts, PCNA-SUMO1 chimera rescue, Ig gene conversion, and epistasis analysis\",\n      \"pmids\": [\"30487218\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Relative contribution of PIAS1 vs PIAS4 to PCNA SUMOylation not separated\", \"Coupling of PCNA SUMOylation to downstream recombinase choice not detailed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Implicated PIAS4 SUMOylation in early embryonic gene-expression control, restraining the 2C-like program via DPPA2 and constraining chromatin state and chromosome segregation in zygotes.\",\n      \"evidence\": \"Depletion/overexpression with in vivo sumoylation, ESC-to-2C transition and early embryo assays for DPPA2; embryo microinjection, segregation and H3K9me3 readouts with ligase-dead mutant\",\n      \"pmids\": [\"31226106\", \"31640975\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct functional consequence of DPPA2-SUMO on its targets not mapped\", \"Link between PIAS4 SUMO activity and H3K9me3 methyltransferase translocation mechanistic detail lacking\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Refined the DSB role of PIAS4 to promoting end resection and RAD51 loading by counteracting the 53BP1/RIF1 resection blockade within a PIAS1/PIAS4/RNF4/BRCA1 module.\",\n      \"evidence\": \"siRNA, gamma-irradiation DSB-marker imaging, and epistasis (53BP1 depletion rescue)\",\n      \"pmids\": [\"35007836\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct SUMO substrate driving resection licensing not pinpointed\", \"Order of action relative to RNF4 in this module not fully resolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended PIAS4 SUMO signaling to cardiac membrane biology, where Cav-3 SUMO2/3 modification disrupts Nav1.5 channel localization and promotes ischemia-reperfusion arrhythmias.\",\n      \"evidence\": \"AAV9-shRNA cardiac knockdown, Co-IP, Cav-3 SUMO-site mutants, ECG, and hypoxia/reoxygenation cell model\",\n      \"pmids\": [\"36229865\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Cav-3 SUMOylation is direct PIAS4 catalysis vs indirect not fully isolated\", \"Generalizability beyond the I/R context untested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Linked PIAS4-RNF4 SUMO-targeted degradation to extraction of trapped WRN helicase and uncovered a transcriptional regulator-controlled PIAS4-SLC7A11 ferroptosis axis as cancer vulnerabilities.\",\n      \"evidence\": \"Single-molecule tracking, phenotypic screens, inhibitor/siRNA and MSI-H xenografts for WRN; pull-down, Co-IP, ChIP, luciferase and xenograft models for SLC7A11/KDM1A\",\n      \"pmids\": [\"39025847\", \"38615741\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct PIAS4 substrate at trapped-WRN chromatin not biochemically defined\", \"Whether SLC7A11 SUMOylation site and degradation pathway are universal across tumors unclear\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Began characterizing oxidative-stress-induced TDP-43 SUMOylation by PIAS4, hinting at a stress-specific role with possible neurodegeneration relevance.\",\n      \"evidence\": \"In vivo sumoylation assay under oxidative stress with domain mapping and PIAS1/PIAS4 co-expression (preprint)\",\n      \"pmids\": [\"41292941\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, single lab, no mechanistic follow-up\", \"Functional consequence of TDP-43 SUMOylation undefined\", \"Stimulus specificity (oxidative vs DNA-damage) basis unexplained\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single ligase achieves substrate- and site-selectivity across its many targets, and how its catalytic (SP-RING/SIM) versus ligase-independent (SAP/HDAC/coactivator-competition) modes are partitioned in vivo, remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structure of PIAS4 engaging a substrate-SUMO-Ubc9 complex\", \"Rules governing SUMO1 vs SUMO2/3 paralog choice across substrates unknown\", \"Tissue-specific contributions vs PIAS-family redundancy not systematically mapped\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [0, 2, 10, 12, 24, 25, 30]},\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [0, 2, 6, 10, 24, 25, 30]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 3, 7, 13, 21]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [7, 18, 21, 27]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 6, 12, 15]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [10, 24, 25, 26]},\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [26]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [0, 11]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 2, 10, 24, 25, 30, 35]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [20, 36, 38, 42]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [10, 24, 25, 26]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 3, 7, 13, 21, 28]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [12, 16, 27, 34, 39]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [3, 17, 22, 23, 30]},\n      {\"term_id\": \"R-HSA-8953897\", \"supporting_discovery_ids\": [13, 22, 23]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"UBC9\", \"RNF4\", \"TRIM32\", \"FIEL1\", \"RIF1\", \"PIAS1\", \"HDAC1\", \"NEMO\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}