{"gene":"SP100","run_date":"2026-06-10T07:46:38","timeline":{"discoveries":[{"year":1997,"finding":"Sp100 is covalently modified by PIC1/SUMO-1 (SUMO-1) in vivo. Cell fractionation and immunoblotting demonstrated that SUMO-1-modified Sp100 isoforms are exclusively nuclear, whereas non-modified Sp100 is also found in the cytoplasm, indicating that SUMOylation restricts Sp100 to the nucleus.","method":"Immunoblotting with SUMO-1-specific monoclonal antibody on cell extracts from stably transfected and untransfected cells; cell fractionation; immunofluorescence colocalization","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal immunoblot and fractionation across multiple cell lines, replicated in later studies","pmids":["9412458"],"is_preprint":false},{"year":1999,"finding":"Domains of Sp100 required for SUMOylation, nuclear localization, homodimerization, and ND targeting were mapped. A functional nuclear localization signal is required for SUMOylation, but SUMOylation is not necessary for nuclear import or ND targeting. The specific lysine residue to which SUMO-1 is conjugated was identified and lies within the HP1-binding site, suggesting SUMO modification may modulate HP1 interaction.","method":"Deletion and point mutant analysis of Sp100 domains with immunofluorescence and immunoblotting; SUMOylation site mutagenesis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — systematic mutagenesis with multiple orthogonal readouts (localization, dimerization, SUMOylation), single rigorous study","pmids":["10212234"],"is_preprint":false},{"year":1998,"finding":"Sp100 binds directly to members of the heterochromatin protein 1 (HP1) family of non-histone chromosomal proteins. A naturally occurring splice variant, SP100-HMG, contains an HMG-1 domain with potential DNA-binding activity. Both HP1 and SP100-HMG concentrate in PML/SP100 nuclear bodies, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When tethered to a promoter, SP100, SP100-HMG, and HP1 all behave as transcriptional repressors.","method":"Co-immunoprecipitation, immunofluorescence colocalization, transfection-based transcriptional reporter assays in mammalian cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus functional transcriptional repressor assays plus localization, single lab with multiple orthogonal methods; later replicated","pmids":["9636146"],"is_preprint":false},{"year":1999,"finding":"The HSV-1 ICP0 protein and the CMV IE1 protein specifically abrogate the SUMO-1 modification of PML and Sp100, correlating with complete disruption of nuclear body structure. Adenovirus E4 ORF3, which causes only moderate redistribution of PML, does not affect SUMO-1 modification of Sp100 or PML. These data support a model in which SUMO-1 conjugation is required for structural integrity of nuclear bodies.","method":"Immunoblotting for SUMO-modified Sp100 in virus-infected cells; immunofluorescence of nuclear body disruption; comparison across viral proteins","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — immunoblot and immunofluorescence across multiple viral infection conditions; findings replicated independently","pmids":["10233977"],"is_preprint":false},{"year":1999,"finding":"HSV-1 infection induces proteasome-dependent degradation of Sp100 protein without altering its mRNA level. The proteasome inhibitor MG132 abrogates HSV-1-induced Sp100 degradation and partially restores NB localization. RanGAP1 (another SUMO-1 conjugate) and PKR are not degraded, indicating specificity.","method":"Immunoblotting of Sp100 protein levels in HSV-1-infected cells with/without MG132; RT-PCR for mRNA levels; immunofluorescence","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — proteasome inhibitor rescue experiment plus protein/mRNA dissociation, replicated in subsequent studies","pmids":["10023669"],"is_preprint":false},{"year":2003,"finding":"Degradation of PML and Sp100 by HSV-1 ICP0 is mediated specifically by the E2 ubiquitin-conjugating enzyme UbcH5a. Dominant-negative UbcH5a (C85A) blocked PML and Sp100 degradation and ND10 dispersal, whereas dominant-negative UbcH6 and UbcH7, or wild-type UbcH5a, had no inhibitory effect. ICP0 site 2 (exon 2, RING finger) polyubiquitylates UbcH5a and UbcH6 in vitro.","method":"In vitro ubiquitylation assay; ectopic expression of dominant-negative E2 enzymes; immunofluorescence and immunoblotting for PML and Sp100","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro ubiquitylation reconstitution plus dominant-negative rescue experiments, mechanistically rigorous","pmids":["12855769"],"is_preprint":false},{"year":2001,"finding":"SUMO modification of SP100 enhances the stability of SP100-HP1 complexes in vitro. An additional SP100 splice variant, SP100C, was identified containing PHD-bromodomain motifs characteristic of chromatin proteins. TIF1alpha, a chromatin factor homologous to PML and SP100C, is also modified by SUMO-1.","method":"In vitro binding assay comparing SUMOylated vs non-SUMOylated SP100 interactions with HP1; cDNA cloning; immunofluorescence","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro binding assay with SUMOylated protein; single lab but biochemically defined mechanism","pmids":["11313457"],"is_preprint":false},{"year":2001,"finding":"Sp100 contains separate ND10-binding and homo-oligomerization domains. Amino acids 29–152 are sufficient for ND10 targeting and self-aggregation in the absence of endogenous Sp100. The ND10-binding domain and the self-aggregation (homo-oligomerization) domain are separable entities, as demonstrated by fusion of a p53-binding peptide that blocked self-aggregation while retaining ND10 binding.","method":"GFP-fusion deletion variant transfection, immunofluorescence localization relative to ND10 markers with and without endogenous Sp100","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic deletion analysis with functional readout, single lab","pmids":["11112690"],"is_preprint":false},{"year":2002,"finding":"SP100 physically interacts with the ETS-1 transcription factor both in vitro and in vivo. SP100 negatively modulates ETS-1 transcriptional activation of MMP1 and uPA promoters in a dose-dependent manner, decreases endogenous MMP1 and uPA expression, reduces ETS-1 DNA binding, and inhibits invasion of breast cancer cells.","method":"Yeast two-hybrid screen; in vitro pulldown; co-immunoprecipitation; transcriptional reporter assays; endogenous gene expression analysis; invasion assay","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — yeast two-hybrid plus Co-IP plus in vitro binding plus functional transcription/invasion assays, replicated by independent lab","pmids":["11909962"],"is_preprint":false},{"year":2004,"finding":"SP100 interacts with ETS1 in vitro and in vivo, reduces ETS1 DNA binding, and acts as a dose-dependent inhibitor of ETS1 transcriptional activation. SP100 expression inhibits invasion of breast cancer cells.","method":"Yeast two-hybrid; in vitro and in vivo co-immunoprecipitation; EMSA (ETS1 DNA binding); transcriptional reporter assay; Matrigel invasion assay","journal":"Oncogene","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, EMSA, reporter, invasion), independently confirmed by Wasylyk et al.","pmids":["15247905"],"is_preprint":false},{"year":2005,"finding":"SP100 negatively modulates ETS1-dependent angiogenic activity in primary endothelial cells, antagonizing ETS1-mediated network formation, migration, and invasion. Interferon upregulates SP100 in endothelial cells, and microarray analysis revealed SP100 reciprocally regulates a subset of ETS1 target genes including angiogenesis-related factors.","method":"HUVEC network formation assay; migration/invasion assays; cDNA microarray; real-time PCR","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional cellular assays with gene expression validation, single lab","pmids":["15592518"],"is_preprint":false},{"year":2005,"finding":"Overexpression of Sp100 sequesters the MRE11/RAD50/NBS1 (MRN) recombination complex away from ALT-associated PML bodies (APBs), resulting in suppression of the alternative lengthening of telomeres (ALT) mechanism. This caused progressive telomere shortening (~121 bp/population doubling), suppression of rapid telomere length changes, and suppression of APB formation. A C-terminally truncated Sp100 that did not sequester MRN failed to inhibit ALT.","method":"Sp100 overexpression in ALT cells; immunofluorescence colocalization; telomere length measurement by Southern blot; FISH; APB quantification; truncation mutant analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function mutant analysis plus multiple orthogonal telomere assays, mechanistically defined","pmids":["15767676"],"is_preprint":false},{"year":2002,"finding":"NBS1 interacts with SP100 via a BRCA carboxyl terminus (BRCT) domain-containing region of NBS1. Co-transfection of PML and SP100 into NT2 cells (lacking endogenous PML/SP100) recruited NBS1 into ectopically formed PODs, indicating SP100 mediates NBS1 recruitment to PML oncogenic domains.","method":"Co-immunoprecipitation; immunofluorescence colocalization in normal fibroblasts and ALT cells; ectopic PML/SP100 expression in NT2 cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — single Co-IP plus colocalization plus ectopic expression rescue, single lab","pmids":["12470659"],"is_preprint":false},{"year":2005,"finding":"The EBV EBNA-LP protein interacts with Sp100 and displaces Sp100 and HP1alpha from PML nuclear bodies. This interaction is mediated through conserved region 3 of EBNA-LP and the PML NB-targeting domain of Sp100. Overexpression of Sp100 lacking the PML NB-targeting domain (but not a mutant lacking the HP1alpha interaction domain) was sufficient to coactivate EBNA2 transcription independently of EBNA-LP, indicating Sp100 mediates EBNA-LP coactivation.","method":"Co-immunoprecipitation; immunofluorescence; domain deletion mutant analysis; transcriptional reporter assay","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — Co-IP plus domain mapping plus functional reporter assay with multiple mutants","pmids":["16177824"],"is_preprint":false},{"year":2007,"finding":"Both PML and Sp100 individually restrict ICP0-null HSV-1 replication. Depletion of Sp100 alone from human fibroblasts increases ICP0-null HSV-1 gene expression comparably to PML depletion alone. Simultaneous depletion of both proteins complements the mutant virus to a greater degree than either alone, demonstrating additive/independent antiviral roles. Inhibition of HDAC activity with trichostatin A could not complement ICP0-null virus in normal or PML-depleted cells.","method":"siRNA knockdown of PML and/or Sp100; quantitative RT-PCR and immunofluorescence of viral gene expression; plaque assay","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — double knockdown epistasis experiment with quantitative viral gene expression readout","pmids":["18160441"],"is_preprint":false},{"year":2009,"finding":"The three Sp100 isoforms containing a SAND domain (Sp100B, Sp100C, Sp100HMG) block transcription of HSV-1 ICP0 and ICP4 at the promoter level, but IFN-upregulated Sp100 is not degraded by ICP0 (negative result: ICP0 does not hydrolyze any Sp100 isoform). IFN alters differential splicing of Sp100 transcript in favor of the inhibitory Sp100C isoform. All four Sp100 isoforms stabilize ND10 and protect PML from ICP0-mediated degradation. Loss of all Sp100 isoforms reduces PML levels, and vice versa.","method":"Isoform-selective siRNA knockdown; IFN treatment; HSV-1 infection; promoter-luciferase reporter assay; immunoblotting; immunofluorescence","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — isoform-selective knockdowns with multiple orthogonal readouts (reporter, immunoblot, IF)","pmids":["19279115"],"is_preprint":false},{"year":2010,"finding":"The RING finger domain of VZV ORF61p has E3 ubiquitin ligase activity essential for autoubiquitination and for dispersion of Sp100-containing nuclear bodies. Disruption of the RING finger (Cys19Gly substitution) abolishes ORF61p-mediated dispersion of Sp100-containing NBs and eliminates E3 ubiquitin ligase activity.","method":"Site-directed mutagenesis of RING finger; in vitro ubiquitin ligase assay; immunofluorescence of NB dispersal","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro E3 ligase reconstitution plus mutagenesis plus cellular functional assay","pmids":["20392849"],"is_preprint":false},{"year":2011,"finding":"IE1 of HCMV directly targets Sp100 independently of PML: IE1 expression alone is sufficient to downregulate endogenous Sp100 and abrogates SUMO modification of all Sp100 isoforms. Sp100 knockdown results in more cells initiating HCMV gene expression program. Sp100 is also degraded in a proteasome-dependent manner at late times post-infection, and Sp100 knockdown results in augmented release of progeny virions and increased viral late gene products independently of enhanced IE gene expression.","method":"IE1 transfection/cotransfection; siRNA knockdown of Sp100 in HFFs; proteasome inhibitor treatment; immunoblotting; immunofluorescence; viral titer measurement","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple loss-of-function approaches with quantitative viral readouts across infection stages","pmids":["21734036"],"is_preprint":false},{"year":2011,"finding":"Cdc20, a co-activator of the APC/C, mediates proteasome-dependent degradation of Sp100 via a D-box motif. Overexpression of Cdc20 decreases endogenous Sp100 levels; knockdown of Cdc20 increases Sp100 levels. A D-box deletion mutant of Sp100 is resistant to Cdc20-mediated degradation. Despite being a Cdc20 substrate, Sp100 protein levels do not oscillate through the cell cycle, and overexpression or knockdown of Sp100 has no effect on cell-cycle progression.","method":"Cdc20 overexpression and siRNA knockdown; D-box deletion mutant analysis; MG132 proteasome inhibitor treatment; immunoblotting; flow cytometry cell cycle analysis","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — D-box mutagenesis plus Cdc20 overexpression/knockdown, single lab","pmids":["22086178"],"is_preprint":false},{"year":2012,"finding":"Herpesvirus saimiri (HVS) specifically degrades Sp100 while leaving PML and hDaxx intact. The HVS ORF3 tegument protein (which shares homology with cellular FGARAT) is the viral factor responsible for proteasomal degradation of Sp100. An ORF3-deletion virus is efficiently complemented in Sp100-depleted cells, demonstrating Sp100 restricts HVS in the absence of ORF3.","method":"Infection of cells with wild-type and ORF3-deletion HVS; immunoblotting; immunofluorescence; Sp100 knockdown complementation assay; proteasome inhibitor experiments","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — viral deletion mutant complementation by Sp100 depletion; mechanistic identification of viral effector protein","pmids":["22278248"],"is_preprint":false},{"year":2013,"finding":"Sp100 depletion in primary human keratinocytes results in substantially increased HPV18 immortalization efficiency, viral transcription, and DNA replication, establishing Sp100 as an intrinsic restriction factor against incoming HPV DNA during the initial establishment phase.","method":"siRNA depletion of Sp100 in primary keratinocytes; HPV18 quasivirus/DNA introduction; quantitative immortalization assay; viral transcription and DNA replication measurements","journal":"mBio","confidence":"High","confidence_rationale":"Tier 2 / Strong — siRNA knockdown with quantitative viral establishment assay, multiple readouts","pmids":["24194542"],"is_preprint":false},{"year":2013,"finding":"Two overlapping regions within the N-terminal half of HSV-1 ICP0 (residues 212–311 for PML degradation/dissociation; residues 212–427 for Sp100 dissociation) promote the dissociation and degradation of PML and dissociation of Sp100 from ND10.","method":"Series of ICP0 truncation mutants expressed in human embryonic lung cells; immunofluorescence of PML/Sp100 localization; immunoblotting for PML protein levels","journal":"Journal of virology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — systematic truncation mutant mapping with cellular readouts, single lab","pmids":["24089549"],"is_preprint":false},{"year":2015,"finding":"IFN-κ inhibits HPV31 transcription primarily by inducing Sp100 proteins. RNAi knockdown of Sp100 reversed IFN-κ-mediated inhibition of viral transcription in CIN612-9E cells maintaining extrachromosomal HPV31 genomes.","method":"Doxycycline-inducible IFN-κ expression; siRNA knockdown of Sp100; viral transcription measurement; RNA-seq","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — inducible expression system plus RNAi rescue, multiple readouts","pmids":["26491169"],"is_preprint":false},{"year":2016,"finding":"The rhesus monkey rhadinovirus (RRV) FGARAT homolog ORF75 is sufficient to cause proteasomal degradation of SP100 (and subsequently PML). CRISPR-Cas9 knockout of SP100 or PML in target cells increases RRV infection, confirming antiviral roles. RRV targets SP100 for degradation before PML, a distinct kinetic strategy from other gammaherpesviruses.","method":"CRISPR-Cas9 knockout of ND10 components; RRV infection of knockout cells; ORF75 expression in transfected/transduced cells; immunoblotting; immunofluorescence; proteasome inhibitor experiments","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — CRISPR-Cas9 KO plus ectopic expression of viral effector plus proteasome inhibition, multiple orthogonal approaches","pmids":["27356898"],"is_preprint":false},{"year":2017,"finding":"SP100 suppresses HSV replication and assembly of viral replication compartments. In SP100-/- HEp-2 cells, the number of viral replication compartments and wild-type virus yields (at low MOI) are significantly higher than in parental cells. Unlike PML, SP100 loss does not alter sensitivity to IFN-β and SP100-/- cells support replication of ΔICP0 virus. SP100 also enhances PML accumulation.","method":"CRISPR/gene-knockout SP100-/- cells; immunofluorescence counting of viral replication compartments; viral yield assays at low MOI; IFN-β sensitivity assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — gene KO with multiple quantitative viral readouts and IFN sensitivity comparison","pmids":["28439026"],"is_preprint":false},{"year":2017,"finding":"Sp100 localizes to HPV16 and HPV31 replication foci in primary keratinocytes and in CIN1 cervical biopsies. Sp100 binds across the HPV viral genome (by ChIP), with binding increasing at late stages of infection. siRNA depletion of Sp100 in differentiating HPV31-containing cells increases viral transcription and replication, demonstrating Sp100 represses HPV at late stages of the productive cycle.","method":"Immunofluorescence colocalization; ChIP of Sp100 on viral chromatin; siRNA knockdown in differentiating keratinocytes; viral transcription and replication measurements; analysis of patient CIN1 biopsies","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 2 / Strong — ChIP plus siRNA knockdown plus patient tissue validation, multiple orthogonal methods","pmids":["28968443"],"is_preprint":false},{"year":2010,"finding":"Selective knockdown of Sp100 isoforms in normal human diploid fibroblasts causes rapid cellular senescence followed by emergence of highly tumorigenic Sp100-minus cells that lack ND10/PML bodies, display genomic instability, cytoplasmic p53 sequestration, and activated MYC/RAS/TERT pathways. Re-introduction of Sp100A alone is sufficient to maintain senescence and prevent emergence of tumorigenic cells, identifying Sp100 as a tumor suppressor acting through chromatin-mediated gene regulation.","method":"Isoform-selective shRNA knockdown; nude mouse tumorigenicity assay; microsatellite analysis; immunofluorescence; global transcriptome analysis; Sp100A re-expression rescue","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 2 / Strong — isoform-specific KD with rescue experiment plus in vivo tumorigenicity, multiple orthogonal readouts","pmids":["21118961"],"is_preprint":false},{"year":2011,"finding":"Sp100 interacts with phage ΦC31 integrase and inhibits ΦC31 integrase-mediated recombination. The interaction maps to residues 81-84 (RILN) of ΦC31 integrase. Knockdown of endogenous Sp100 increases ΦC31 integrase-mediated recombination without affecting reporter gene expression.","method":"Yeast two-hybrid screen; co-immunoprecipitation; peptide array hybridization; siRNA knockdown with recombination reporter assay","journal":"Acta biochimica Polonica","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — Co-IP plus siRNA functional rescue, single lab","pmids":["21383994"],"is_preprint":false},{"year":1999,"finding":"Sp100 and PML are sequestered in viral translucent patches (finely granular material at the nuclear border) during prolonged HSV-1 infection, as visualized by immunoelectron microscopy. Both proteins are components not only of nuclear bodies but also of interchromatin granule-associated zones in uninfected cells, suggesting a role in splicing events.","method":"Immunoelectron microscopy of ultrathin sections of HSV-1-infected HeLa cells","journal":"Experimental cell research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — high-resolution immunoelectron microscopy, single lab, structural localization without functional manipulation","pmids":["7493645"],"is_preprint":false},{"year":1999,"finding":"Sp100 splice variants were identified: one class contains an HMG-box domain (SP100-HMG) and another contains an HNPP-box domain related to interferon-inducible nuclear phosphoproteins. When transiently expressed, both classes of variants localize in part to nuclear dots/PML bodies and additionally to other nuclear domains distinct from full-length Sp100.","method":"cDNA cloning; Sp100 gene 3'-end analysis; transient transfection with immunofluorescence localization","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — splice variant identification with cellular localization, single lab","pmids":["9973607"],"is_preprint":false},{"year":1996,"finding":"IFN-beta induces rapid, protein-synthesis-independent transcriptional upregulation of both Sp100 and PML genes. Nuclear run-on analysis revealed synergistic activation by type-I and type-II IFNs. The Sp100 promoter contains an imperfect IFN-stimulated response element (ISRE) and an IFN-gamma activation site (GAS), with the ISRE binding ISGF2 strongly and ISGF3 weakly.","method":"Nuclear run-on transcription assay; promoter cloning and functional analysis; transient transfection reporter assay; electrophoretic mobility shift assay (EMSA) with IFN-induced factors","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 / Strong — nuclear run-on plus promoter deletion analysis plus EMSA, multiple orthogonal mechanistic approaches","pmids":["8810287"],"is_preprint":false},{"year":2025,"finding":"Sp100-HMG autonomously nucleates into liquid-like condensates via its multimerization domain and intrinsically disordered region (IDR), initiating an 'inside-out' assembly of PML nuclear bodies. Sp100-HMG forms a liquid core that recruits PML (via SUMOylation-directed recruitment) and client proteins DAXX and ATRX. This hierarchical assembly is required for proper transcriptional programs and cell-cycle dynamics in HEp-2 cells.","method":"Live-cell imaging of condensate formation; cryo-EM and AlphaFold modeling (from paper 65 cross-reference); LLPS assays; domain mutant analysis; CRISPR-based perturbations; transcriptome analysis","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — preprint, single lab, novel mechanistic model not yet peer-reviewed or independently replicated","pmids":[],"is_preprint":true},{"year":2025,"finding":"SP110 directly interacts with SP100 via their CARD domains and dissolves toxic SP100 oligomers. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, causing micronucleus formation, DNA damage, and genotoxic cell death. SP100 oligomers are thus toxic during mitosis unless dissolved by SP110.","method":"Genome-wide CRISPR screen; cryo-electron microscopy; AlphaFold modeling; co-immunoprecipitation; live-cell imaging of mitosis; micronucleus quantification; DNA damage markers","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus CRISPR screen plus Co-IP plus live-cell imaging, multiple orthogonal methods in peer-reviewed study","pmids":["41826696"],"is_preprint":false},{"year":2025,"finding":"Sp100A isoform is the primary driver of HIRA histone chaperone localization to PML nuclear bodies. In Sp100 CRISPR knockout keratinocytes, HIRA and its complex members UBN1 and ASF1a fail to localize to PML-NBs even after IFN stimulation. The SUMO-interacting motif (SIM) of Sp100A plays an important role in this function. Sp100 KO does not affect keratinocyte differentiation or PML-NB formation itself.","method":"CRISPR-Cas9 Sp100 knockout; exogenous expression of individual Sp100 isoforms; immunofluorescence colocalization; IFN stimulation","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Weak — CRISPR KO plus isoform rescue with clean readout, preprint not yet peer-reviewed","pmids":[],"is_preprint":true}],"current_model":"SP100 is an IFN-inducible, SUMO-modified scaffold protein of PML nuclear bodies (ND10) that restricts viral replication (HSV, CMV, HPV, HVS, VZV, RRV) by repressing viral chromatin transcription through its SAND, PHD-bromo, and HMG domain-containing isoforms; it interacts with HP1 family proteins (enhanced by SUMO modification), recruits NBS1/MRE11/RAD50 to sequester recombination machinery (suppressing ALT), acts as a dose-dependent transcriptional co-regulator of ETS1, is degraded by viral E3 ubiquitin ligases (ICP0/UbcH5a, ORF3/FGARAT homologs) via the proteasome, undergoes cell-cycle-regulated degradation via APC/C-Cdc20, and—during mitosis—forms toxic oligomers whose dissolution by SP110 via direct CARD-domain interaction is required to prevent chromosomal segregation errors and genotoxic cell death."},"narrative":{"mechanistic_narrative":"SP100 is an interferon-inducible nuclear scaffold protein and a core constituent of PML nuclear bodies (ND10) that functions in intrinsic antiviral defense and chromatin-mediated transcriptional regulation [PMID:8810287, PMID:18160441]. Its expression is driven by type-I and type-II IFN through an ISRE and GAS element in its promoter [PMID:8810287], and it exists as multiple alternatively spliced isoforms, several carrying SAND, PHD-bromodomain, or HMG chromatin-associated motifs [PMID:9973607, PMID:11313457, PMID:19279115]. SP100 is covalently modified by SUMO-1 on a lysine within its HP1-binding region, a modification that confines it to the nucleus and enhances the stability of its complexes with HP1 family heterochromatin proteins; when tethered to DNA, SP100 and its HMG variant act as transcriptional repressors [PMID:9412458, PMID:10212234, PMID:9636146, PMID:11313457]. Distinct domains mediate ND10 targeting and homo-oligomerization, allowing SP100 (notably the HMG isoform) to nucleate nuclear body assembly and recruit partners [PMID:11112690]. SP100 restricts a broad panel of viruses — HSV-1, HCMV, HPV, EBV, HVS, VZV, and RRV — primarily by repressing viral chromatin transcription, acting independently of and additively with PML, with the SAND-domain isoforms blocking HSV-1 immediate-early promoter activity [PMID:18160441, PMID:19279115, PMID:21734036, PMID:24194542, PMID:28968443, PMID:28439026]. Viruses counteract SP100 through SUMO de-modification and proteasome-dependent degradation mediated by viral E3 ubiquitin ligases, including HSV-1 ICP0 acting via the E2 enzyme UbcH5a, VZV ORF61p, and the FGARAT-homolog tegument proteins of HVS (ORF3) and RRV (ORF75) [PMID:10233977, PMID:10023669, PMID:12855769, PMID:20392849, PMID:22278248, PMID:27356898]. Beyond viral defense, SP100 is a dose-dependent negative regulator of the ETS1 transcription factor, reducing ETS1 DNA binding and target-gene activation to suppress invasion and angiogenesis [PMID:11909962, PMID:15247905, PMID:15592518], recruits the MRN complex via NBS1 to sequester recombination machinery and suppress alternative lengthening of telomeres [PMID:15767676, PMID:12470659], and acts as a tumor suppressor whose loss triggers senescence escape and genomic instability [PMID:21118961]. SP100 is also a substrate of APC/C-Cdc20-mediated proteasomal degradation through a D-box motif [PMID:22086178], and during mitosis its toxic oligomers must be dissolved by SP110 through a direct CARD-CARD interaction to prevent chromosome segregation errors and genotoxic death [PMID:41826696].","teleology":[{"year":1996,"claim":"Established that SP100 is a bona fide interferon-response gene, defining the regulatory logic by which it is deployed in innate defense.","evidence":"Nuclear run-on, promoter deletion analysis, and EMSA identifying ISRE/GAS elements and IFN factor binding","pmids":["8810287"],"confidence":"High","gaps":["Did not connect IFN induction to a specific downstream antiviral or chromatin function","Isoform-specific transcriptional/splicing responses not resolved"]},{"year":1997,"claim":"Answered how SP100 subcellular distribution is controlled by showing SUMO-1 conjugation restricts it to the nucleus, linking a post-translational mark to localization.","evidence":"SUMO-1-specific immunoblotting plus cell fractionation and immunofluorescence across cell lines","pmids":["9412458"],"confidence":"High","gaps":["Functional consequence of SUMOylation beyond localization not yet defined","SUMO conjugation site not mapped at this stage"]},{"year":1998,"claim":"Identified SP100 as a partner of HP1 heterochromatin proteins and a transcriptional repressor, establishing a chromatin-silencing function.","evidence":"Co-IP, colocalization, and promoter-tethering reporter assays; identification of the SP100-HMG splice variant","pmids":["9636146"],"confidence":"High","gaps":["Endogenous genomic targets of repression not identified","Direct DNA binding of HMG variant not functionally demonstrated"]},{"year":1999,"claim":"Mapped the SP100 domains for SUMOylation, nuclear localization, dimerization and ND targeting and located the SUMO site within the HP1-binding region, suggesting SUMO tunes HP1 interaction.","evidence":"Deletion/point-mutant analysis with localization, dimerization and SUMOylation readouts; splice variant cloning","pmids":["10212234","9973607"],"confidence":"High","gaps":["Did not directly measure SUMO effect on HP1 binding affinity","Functional roles of individual isoforms not assigned"]},{"year":1999,"claim":"Revealed that herpesviruses dismantle SP100 by abrogating SUMO modification and triggering proteasomal degradation, defining viral antagonism of ND10 integrity.","evidence":"Immunoblotting of SUMO-modified SP100 and protein levels in HSV-1/CMV-infected cells with MG132 rescue and viral-protein comparison; immuno-EM localization","pmids":["10233977","10023669","7493645"],"confidence":"High","gaps":["Viral E3/E2 machinery not yet identified","Whether SP100 loss causally enhances viral replication not tested here"]},{"year":2001,"claim":"Provided biochemical mechanism for SUMO function by showing SUMOylation stabilizes SP100-HP1 complexes, and expanded the isoform repertoire to chromatin-reader (PHD-bromo) forms.","evidence":"In vitro binding assays comparing SUMOylated vs unmodified SP100; cDNA cloning of SP100C","pmids":["11313457"],"confidence":"High","gaps":["In vitro stabilization not validated for endogenous complexes","Chromatin substrates read by the PHD-bromo isoform unknown"]},{"year":2001,"claim":"Separated the ND10-targeting and self-oligomerization functions of SP100 into distinct domains, clarifying how it docks to and seeds nuclear bodies.","evidence":"GFP-fusion deletion variant transfection and immunofluorescence with/without endogenous SP100","pmids":["11112690"],"confidence":"Medium","gaps":["Structural basis of oligomerization not resolved","Functional importance of oligomerization in vivo not addressed"]},{"year":2002,"claim":"Defined a chromatin-independent transcriptional role by showing SP100 binds and dose-dependently inhibits ETS1, repressing invasion-associated gene programs.","evidence":"Yeast two-hybrid, in vitro pulldown, Co-IP, reporter, endogenous gene expression and invasion assays; NBS1 BRCT-mediated SP100 interaction and ectopic POD recruitment","pmids":["11909962","12470659"],"confidence":"High","gaps":["Mechanism by which SP100 reduces ETS1 DNA binding unresolved","Physiological context of NBS1 recruitment not yet linked to a pathway"]},{"year":2005,"claim":"Connected SP100 to genome maintenance and angiogenesis control: it sequesters the MRN complex to suppress ALT and antagonizes ETS1-driven endothelial behavior.","evidence":"SP100 overexpression and truncation analysis with telomere length, APB and FISH assays in ALT cells; HUVEC functional assays, microarray and EMSA; EBNA-LP displacement domain mapping","pmids":["15767676","15592518","15247905","16177824"],"confidence":"High","gaps":["Whether endogenous SP100 levels regulate ALT physiologically untested","ETS1 target-gene selectivity mechanism unknown"]},{"year":2007,"claim":"Demonstrated that SP100 is an independent intrinsic antiviral restriction factor, acting additively with but separately from PML against HSV-1.","evidence":"siRNA single and double knockdown of SP100/PML with quantitative viral gene expression and plaque assays","pmids":["18160441"],"confidence":"High","gaps":["Molecular step of restriction not defined in this study","Isoform responsible for restriction not identified"]},{"year":2009,"claim":"Pinpointed the SAND-domain isoforms as the active repressors of HSV-1 immediate-early promoters and showed IFN biases splicing toward the inhibitory SP100C isoform.","evidence":"Isoform-selective siRNA, IFN treatment, promoter-luciferase reporters and immunoblotting in infected cells","pmids":["19279115"],"confidence":"High","gaps":["Mechanism by which SAND domain represses viral chromatin not biochemically resolved","Splicing regulator directing IFN-induced isoform switch unknown"]},{"year":2011,"claim":"Identified two parallel degradation routes for SP100 — viral RING E3 ligases and host APC/C-Cdc20 — defining how its levels are controlled by infection and by the cell-cycle machinery.","evidence":"VZV ORF61p RING mutagenesis and in vitro ligase assays; HCMV IE1 transfection/knockdown with viral titers; Cdc20 overexpression/knockdown and D-box mutant analysis with MG132","pmids":["20392849","21734036","22086178"],"confidence":"High","gaps":["Cdc20-mediated degradation does not produce cell-cycle oscillation of SP100, leaving its physiological purpose unclear","Direct ubiquitination of SP100 by these ligases not all reconstituted"]},{"year":2010,"claim":"Established SP100 as a tumor suppressor whose isoform-specific loss causes senescence escape, genomic instability and tumorigenicity.","evidence":"Isoform-selective shRNA knockdown, nude mouse tumorigenicity, microsatellite and transcriptome analysis with Sp100A rescue","pmids":["21118961"],"confidence":"High","gaps":["Causal chromatin targets driving tumor suppression not defined","Relationship between tumor suppression and antiviral repression unresolved"]},{"year":2013,"claim":"Extended SP100 restriction to papillomaviruses, showing it limits HPV establishment and that ICP0 N-terminal regions mediate its ND10 dissociation.","evidence":"siRNA depletion in primary keratinocytes with HPV18 immortalization/transcription/replication readouts; ICP0 truncation mapping with localization readouts","pmids":["24194542","24089549"],"confidence":"High","gaps":["Direct engagement of incoming HPV DNA by SP100 not yet shown at this stage","ICP0 domains mapped without identifying degradation cofactors"]},{"year":2016,"claim":"Generalized viral antagonism of SP100 across gammaherpesviruses by identifying FGARAT-homolog effectors (HVS ORF3, RRV ORF75) and confirming antiviral roles by knockout.","evidence":"Wild-type vs ORF-deletion virus complementation in SP100-depleted cells; CRISPR-Cas9 knockout; ectopic effector expression and proteasome inhibition","pmids":["22278248","27356898"],"confidence":"High","gaps":["Biochemical mechanism of FGARAT-homolog-directed SP100 degradation undefined","Why RRV degrades SP100 before PML kinetically unexplained"]},{"year":2017,"claim":"Resolved the cellular step of restriction by showing SP100 suppresses formation of viral replication compartments and binds viral chromatin directly across the HPV genome.","evidence":"SP100-/- CRISPR cells with replication-compartment counting, viral yields and IFN-beta sensitivity; ChIP of SP100 on HPV chromatin, colocalization and knockdown in differentiating keratinocytes and patient biopsies","pmids":["28439026","28968443"],"confidence":"High","gaps":["Mechanism of SP100 recruitment to viral chromatin not defined","Distinct contributions of SP100 isoforms to chromatin binding unresolved"]},{"year":2025,"claim":"Defined a mitotic safety mechanism in which SP110 dissolves toxic SP100 oligomers via direct CARD-CARD interaction to prevent chromosome segregation errors and genotoxicity.","evidence":"Genome-wide CRISPR screen, cryo-EM, AlphaFold modeling, Co-IP, live-cell mitosis imaging and micronucleus/DNA-damage quantification","pmids":["41826696"],"confidence":"High","gaps":["Trigger that converts SP100 into toxic oligomers in mitosis not identified","Whether oligomerization links to other SP100 functions (antiviral/chromatin) unknown"]},{"year":2025,"claim":"Proposed an 'inside-out' phase-separation model of nuclear body assembly with SP100-HMG nucleating a liquid core, and assigned Sp100A as the driver of HIRA chaperone recruitment.","evidence":"Live-cell imaging, LLPS assays, cryo-EM/AlphaFold and CRISPR perturbations (preprint); CRISPR Sp100 KO with isoform rescue and IFN stimulation (preprint)","pmids":[],"confidence":"Low","gaps":["Preprints not yet peer-reviewed or independently replicated","LLPS-based assembly model not validated against the established SUMO-driven recruitment scheme","Functional consequence of HIRA recruitment to PML-NBs not established"]},{"year":null,"claim":"How SP100's chromatin-reader/repressor domains mechanistically silence viral and cellular promoters, and how its multiple functions (antiviral repression, ETS1 regulation, ALT suppression, mitotic oligomer toxicity) are coordinated through a unified molecular activity, remains unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of SP100 engaging chromatin","No defined catalytic or enzymatic activity for SP100 itself","Integration of distinct functional outputs by isoform/PTM state not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[2,8,9,15,26]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[2,25]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[2,6]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[11,12,32]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[2,7,28]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[25,32]}],"pathway":[{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[14,15,17,20,24,30]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[19,23,26]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[2,8,9,15]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[18,32]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[0,3,5,18]}],"complexes":["PML nuclear body / ND10"],"partners":["PML","HP1","ETS1","NBS1","SP110","DAXX","ATRX","HIRA"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P23497","full_name":"Nuclear autoantigen Sp-100","aliases":["Nuclear dot-associated Sp100 protein","Speckled 100 kDa"],"length_aa":879,"mass_kda":100.4,"function":"Together with PML, this tumor suppressor is a major constituent of the PML bodies, a subnuclear organelle involved in a large number of physiological processes including cell growth, differentiation and apoptosis. Functions as a transcriptional coactivator of ETS1 and ETS2 according to PubMed:11909962. Under certain conditions, it may also act as a corepressor of ETS1 preventing its binding to DNA according to PubMed:15247905. Through the regulation of ETS1 it may play a role in angiogenesis, controlling endothelial cell motility and invasion. Through interaction with the MRN complex it may be involved in the regulation of telomeres lengthening. May also regulate TP53-mediated transcription and through CASP8AP2, regulate FAS-mediated apoptosis. Also plays a role in infection by viruses, including human cytomegalovirus and Epstein-Barr virus, through mechanisms that may involve chromatin and/or transcriptional regulation","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P23497/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SP100","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SP100","total_profiled":1310},"omim":[{"mim_id":"619675","title":"U-BOX DOMAIN-CONTAINING PROTEIN 5; UBOX5","url":"https://www.omim.org/entry/619675"},{"mim_id":"617747","title":"NUCLEAR BODY PROTEIN SP140-LIKE; SP140L","url":"https://www.omim.org/entry/617747"},{"mim_id":"608602","title":"NUCLEAR BODY PROTEIN SP140; SP140","url":"https://www.omim.org/entry/608602"},{"mim_id":"604587","title":"CALCIUM BINDING AND COILED-COIL DOMAIN PROTEIN 2; CALCOCO2","url":"https://www.omim.org/entry/604587"},{"mim_id":"604585","title":"NUCLEAR BODY PROTEIN SP100; SP100","url":"https://www.omim.org/entry/604585"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nuclear bodies","reliability":"Supported"},{"location":"Nucleoplasm","reliability":"Additional"}],"tissue_specificity":"Tissue enhanced","tissue_distribution":"Detected in all","driving_tissues":[{"tissue":"bone marrow","ntpm":177.8}],"url":"https://www.proteinatlas.org/search/SP100"},"hgnc":{"alias_symbol":[],"prev_symbol":[]},"alphafold":{"accession":"P23497","domains":[{"cath_id":"3.10.390.10","chopping":"605-673","consensus_level":"high","plddt":86.2216,"start":605,"end":673},{"cath_id":"1.10.30.10","chopping":"770-841","consensus_level":"medium","plddt":89.0258,"start":770,"end":841},{"cath_id":"1.10.533","chopping":"46-150","consensus_level":"high","plddt":88.7698,"start":46,"end":150}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P23497","model_url":"https://alphafold.ebi.ac.uk/files/AF-P23497-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P23497-F1-predicted_aligned_error_v6.png","plddt_mean":56.94},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SP100","jax_strain_url":"https://www.jax.org/strain/search?query=SP100"},"sequence":{"accession":"P23497","fasta_url":"https://rest.uniprot.org/uniprotkb/P23497.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P23497/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P23497"}},"corpus_meta":[{"pmid":"9412458","id":"PMC_9412458","title":"Evidence 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within the N-terminal half of the herpes simplex virus 1 E3 ubiquitin ligase ICP0 facilitate the degradation and dissociation of PML and dissociation of Sp100 from ND10.","date":"2013","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/24089549","citation_count":15,"is_preprint":false},{"pmid":"27356898","id":"PMC_27356898","title":"Viral FGARAT Homolog ORF75 of Rhesus Monkey Rhadinovirus Effects Proteasomal Degradation of the ND10 Components SP100 and PML.","date":"2016","source":"Journal of virology","url":"https://pubmed.ncbi.nlm.nih.gov/27356898","citation_count":15,"is_preprint":false},{"pmid":"9268632","id":"PMC_9268632","title":"Structure and expression of the murine Sp100 nuclear dot gene.","date":"1997","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9268632","citation_count":13,"is_preprint":false},{"pmid":"21118961","id":"PMC_21118961","title":"Sp100 as a potent tumor suppressor: accelerated senescence and rapid malignant transformation of human 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Comparison of ELISA and immunofluorescence.","date":"2013","source":"Journal of immunoassay & immunochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/23859785","citation_count":11,"is_preprint":false},{"pmid":"31272209","id":"PMC_31272209","title":"Expression of Sp100 Protein in Human Papillomavirus-Associated Sinonasal Inverted Papilloma.","date":"2019","source":"Ear, nose, & throat journal","url":"https://pubmed.ncbi.nlm.nih.gov/31272209","citation_count":8,"is_preprint":false},{"pmid":"17785307","id":"PMC_17785307","title":"Comparison of two ELISA assays for anti-Sp100 determination.","date":"2007","source":"Annals of the New York Academy of Sciences","url":"https://pubmed.ncbi.nlm.nih.gov/17785307","citation_count":8,"is_preprint":false},{"pmid":"20512085","id":"PMC_20512085","title":"Low expression of Sp100 in laryngeal cancer: correlation with cell differentiation.","date":"2010","source":"Medical science monitor : international medical journal of experimental and clinical 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analysis","url":"https://pubmed.ncbi.nlm.nih.gov/34623692","citation_count":3,"is_preprint":false},{"pmid":"21383994","id":"PMC_21383994","title":"Sp100 interacts with phage ΦC31 integrase to inhibit its recombination activity.","date":"2011","source":"Acta biochimica Polonica","url":"https://pubmed.ncbi.nlm.nih.gov/21383994","citation_count":2,"is_preprint":false},{"pmid":"41188771","id":"PMC_41188771","title":"Clinical and mechanistic insights into the expression of SP100 family proteins in various cancers: a systematic review.","date":"2025","source":"BMC cancer","url":"https://pubmed.ncbi.nlm.nih.gov/41188771","citation_count":1,"is_preprint":false},{"pmid":"22419217","id":"PMC_22419217","title":"Role and fate of SP100 protein in response to Rep-dependent nonviral integration system.","date":"2012","source":"Applied microbiology and 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medicine","url":"https://pubmed.ncbi.nlm.nih.gov/40973111","citation_count":0,"is_preprint":false},{"pmid":"11574059","id":"PMC_11574059","title":"Molecular archeology of an SP100 splice variant revisited: dating the retrotranscription and Alu insertion events.","date":"2001","source":"Genome biology","url":"https://pubmed.ncbi.nlm.nih.gov/11574059","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.07.28.667104","title":"Sp100-HMG drives ‘inside-out’ PML-NB assembly to modulate transcription and cell-cycle dynamics","date":"2025-07-31","source":"bioRxiv","url":"https://doi.org/10.1101/2025.07.28.667104","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2025.09.09.675097","title":"The limited effects of AAV2 vectors on host chromatin accessibility and nuclear architecture are consistent with a favorable safety 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Factor","date":"2024-12-14","source":"bioRxiv","url":"https://doi.org/10.1101/2024.12.13.628399","citation_count":0,"is_preprint":true},{"pmid":null,"id":"bio_10.1101_2024.07.13.24310153","title":"Proteome-wide Mendelian randomization study implicates inflammaging biomarkers in retinal vasculature, cardiometabolic diseases and longevity","date":"2024-07-14","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.13.24310153","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":38789,"output_tokens":8952,"usd":0.125324,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":18808,"output_tokens":6031,"usd":0.122408,"stage2_stop_reason":"end_turn"},"total_usd":0.247732,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"Sp100 is covalently modified by PIC1/SUMO-1 (SUMO-1) in vivo. Cell fractionation and immunoblotting demonstrated that SUMO-1-modified Sp100 isoforms are exclusively nuclear, whereas non-modified Sp100 is also found in the cytoplasm, indicating that SUMOylation restricts Sp100 to the nucleus.\",\n      \"method\": \"Immunoblotting with SUMO-1-specific monoclonal antibody on cell extracts from stably transfected and untransfected cells; cell fractionation; immunofluorescence colocalization\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal immunoblot and fractionation across multiple cell lines, replicated in later studies\",\n      \"pmids\": [\"9412458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Domains of Sp100 required for SUMOylation, nuclear localization, homodimerization, and ND targeting were mapped. A functional nuclear localization signal is required for SUMOylation, but SUMOylation is not necessary for nuclear import or ND targeting. The specific lysine residue to which SUMO-1 is conjugated was identified and lies within the HP1-binding site, suggesting SUMO modification may modulate HP1 interaction.\",\n      \"method\": \"Deletion and point mutant analysis of Sp100 domains with immunofluorescence and immunoblotting; SUMOylation site mutagenesis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — systematic mutagenesis with multiple orthogonal readouts (localization, dimerization, SUMOylation), single rigorous study\",\n      \"pmids\": [\"10212234\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Sp100 binds directly to members of the heterochromatin protein 1 (HP1) family of non-histone chromosomal proteins. A naturally occurring splice variant, SP100-HMG, contains an HMG-1 domain with potential DNA-binding activity. Both HP1 and SP100-HMG concentrate in PML/SP100 nuclear bodies, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When tethered to a promoter, SP100, SP100-HMG, and HP1 all behave as transcriptional repressors.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence colocalization, transfection-based transcriptional reporter assays in mammalian cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus functional transcriptional repressor assays plus localization, single lab with multiple orthogonal methods; later replicated\",\n      \"pmids\": [\"9636146\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"The HSV-1 ICP0 protein and the CMV IE1 protein specifically abrogate the SUMO-1 modification of PML and Sp100, correlating with complete disruption of nuclear body structure. Adenovirus E4 ORF3, which causes only moderate redistribution of PML, does not affect SUMO-1 modification of Sp100 or PML. These data support a model in which SUMO-1 conjugation is required for structural integrity of nuclear bodies.\",\n      \"method\": \"Immunoblotting for SUMO-modified Sp100 in virus-infected cells; immunofluorescence of nuclear body disruption; comparison across viral proteins\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — immunoblot and immunofluorescence across multiple viral infection conditions; findings replicated independently\",\n      \"pmids\": [\"10233977\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"HSV-1 infection induces proteasome-dependent degradation of Sp100 protein without altering its mRNA level. The proteasome inhibitor MG132 abrogates HSV-1-induced Sp100 degradation and partially restores NB localization. RanGAP1 (another SUMO-1 conjugate) and PKR are not degraded, indicating specificity.\",\n      \"method\": \"Immunoblotting of Sp100 protein levels in HSV-1-infected cells with/without MG132; RT-PCR for mRNA levels; immunofluorescence\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — proteasome inhibitor rescue experiment plus protein/mRNA dissociation, replicated in subsequent studies\",\n      \"pmids\": [\"10023669\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"Degradation of PML and Sp100 by HSV-1 ICP0 is mediated specifically by the E2 ubiquitin-conjugating enzyme UbcH5a. Dominant-negative UbcH5a (C85A) blocked PML and Sp100 degradation and ND10 dispersal, whereas dominant-negative UbcH6 and UbcH7, or wild-type UbcH5a, had no inhibitory effect. ICP0 site 2 (exon 2, RING finger) polyubiquitylates UbcH5a and UbcH6 in vitro.\",\n      \"method\": \"In vitro ubiquitylation assay; ectopic expression of dominant-negative E2 enzymes; immunofluorescence and immunoblotting for PML and Sp100\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro ubiquitylation reconstitution plus dominant-negative rescue experiments, mechanistically rigorous\",\n      \"pmids\": [\"12855769\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"SUMO modification of SP100 enhances the stability of SP100-HP1 complexes in vitro. An additional SP100 splice variant, SP100C, was identified containing PHD-bromodomain motifs characteristic of chromatin proteins. TIF1alpha, a chromatin factor homologous to PML and SP100C, is also modified by SUMO-1.\",\n      \"method\": \"In vitro binding assay comparing SUMOylated vs non-SUMOylated SP100 interactions with HP1; cDNA cloning; immunofluorescence\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro binding assay with SUMOylated protein; single lab but biochemically defined mechanism\",\n      \"pmids\": [\"11313457\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Sp100 contains separate ND10-binding and homo-oligomerization domains. Amino acids 29–152 are sufficient for ND10 targeting and self-aggregation in the absence of endogenous Sp100. The ND10-binding domain and the self-aggregation (homo-oligomerization) domain are separable entities, as demonstrated by fusion of a p53-binding peptide that blocked self-aggregation while retaining ND10 binding.\",\n      \"method\": \"GFP-fusion deletion variant transfection, immunofluorescence localization relative to ND10 markers with and without endogenous Sp100\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic deletion analysis with functional readout, single lab\",\n      \"pmids\": [\"11112690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"SP100 physically interacts with the ETS-1 transcription factor both in vitro and in vivo. SP100 negatively modulates ETS-1 transcriptional activation of MMP1 and uPA promoters in a dose-dependent manner, decreases endogenous MMP1 and uPA expression, reduces ETS-1 DNA binding, and inhibits invasion of breast cancer cells.\",\n      \"method\": \"Yeast two-hybrid screen; in vitro pulldown; co-immunoprecipitation; transcriptional reporter assays; endogenous gene expression analysis; invasion assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — yeast two-hybrid plus Co-IP plus in vitro binding plus functional transcription/invasion assays, replicated by independent lab\",\n      \"pmids\": [\"11909962\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SP100 interacts with ETS1 in vitro and in vivo, reduces ETS1 DNA binding, and acts as a dose-dependent inhibitor of ETS1 transcriptional activation. SP100 expression inhibits invasion of breast cancer cells.\",\n      \"method\": \"Yeast two-hybrid; in vitro and in vivo co-immunoprecipitation; EMSA (ETS1 DNA binding); transcriptional reporter assay; Matrigel invasion assay\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, EMSA, reporter, invasion), independently confirmed by Wasylyk et al.\",\n      \"pmids\": [\"15247905\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"SP100 negatively modulates ETS1-dependent angiogenic activity in primary endothelial cells, antagonizing ETS1-mediated network formation, migration, and invasion. Interferon upregulates SP100 in endothelial cells, and microarray analysis revealed SP100 reciprocally regulates a subset of ETS1 target genes including angiogenesis-related factors.\",\n      \"method\": \"HUVEC network formation assay; migration/invasion assays; cDNA microarray; real-time PCR\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional cellular assays with gene expression validation, single lab\",\n      \"pmids\": [\"15592518\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Overexpression of Sp100 sequesters the MRE11/RAD50/NBS1 (MRN) recombination complex away from ALT-associated PML bodies (APBs), resulting in suppression of the alternative lengthening of telomeres (ALT) mechanism. This caused progressive telomere shortening (~121 bp/population doubling), suppression of rapid telomere length changes, and suppression of APB formation. A C-terminally truncated Sp100 that did not sequester MRN failed to inhibit ALT.\",\n      \"method\": \"Sp100 overexpression in ALT cells; immunofluorescence colocalization; telomere length measurement by Southern blot; FISH; APB quantification; truncation mutant analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function mutant analysis plus multiple orthogonal telomere assays, mechanistically defined\",\n      \"pmids\": [\"15767676\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"NBS1 interacts with SP100 via a BRCA carboxyl terminus (BRCT) domain-containing region of NBS1. Co-transfection of PML and SP100 into NT2 cells (lacking endogenous PML/SP100) recruited NBS1 into ectopically formed PODs, indicating SP100 mediates NBS1 recruitment to PML oncogenic domains.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence colocalization in normal fibroblasts and ALT cells; ectopic PML/SP100 expression in NT2 cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — single Co-IP plus colocalization plus ectopic expression rescue, single lab\",\n      \"pmids\": [\"12470659\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"The EBV EBNA-LP protein interacts with Sp100 and displaces Sp100 and HP1alpha from PML nuclear bodies. This interaction is mediated through conserved region 3 of EBNA-LP and the PML NB-targeting domain of Sp100. Overexpression of Sp100 lacking the PML NB-targeting domain (but not a mutant lacking the HP1alpha interaction domain) was sufficient to coactivate EBNA2 transcription independently of EBNA-LP, indicating Sp100 mediates EBNA-LP coactivation.\",\n      \"method\": \"Co-immunoprecipitation; immunofluorescence; domain deletion mutant analysis; transcriptional reporter assay\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — Co-IP plus domain mapping plus functional reporter assay with multiple mutants\",\n      \"pmids\": [\"16177824\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Both PML and Sp100 individually restrict ICP0-null HSV-1 replication. Depletion of Sp100 alone from human fibroblasts increases ICP0-null HSV-1 gene expression comparably to PML depletion alone. Simultaneous depletion of both proteins complements the mutant virus to a greater degree than either alone, demonstrating additive/independent antiviral roles. Inhibition of HDAC activity with trichostatin A could not complement ICP0-null virus in normal or PML-depleted cells.\",\n      \"method\": \"siRNA knockdown of PML and/or Sp100; quantitative RT-PCR and immunofluorescence of viral gene expression; plaque assay\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — double knockdown epistasis experiment with quantitative viral gene expression readout\",\n      \"pmids\": [\"18160441\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The three Sp100 isoforms containing a SAND domain (Sp100B, Sp100C, Sp100HMG) block transcription of HSV-1 ICP0 and ICP4 at the promoter level, but IFN-upregulated Sp100 is not degraded by ICP0 (negative result: ICP0 does not hydrolyze any Sp100 isoform). IFN alters differential splicing of Sp100 transcript in favor of the inhibitory Sp100C isoform. All four Sp100 isoforms stabilize ND10 and protect PML from ICP0-mediated degradation. Loss of all Sp100 isoforms reduces PML levels, and vice versa.\",\n      \"method\": \"Isoform-selective siRNA knockdown; IFN treatment; HSV-1 infection; promoter-luciferase reporter assay; immunoblotting; immunofluorescence\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — isoform-selective knockdowns with multiple orthogonal readouts (reporter, immunoblot, IF)\",\n      \"pmids\": [\"19279115\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"The RING finger domain of VZV ORF61p has E3 ubiquitin ligase activity essential for autoubiquitination and for dispersion of Sp100-containing nuclear bodies. Disruption of the RING finger (Cys19Gly substitution) abolishes ORF61p-mediated dispersion of Sp100-containing NBs and eliminates E3 ubiquitin ligase activity.\",\n      \"method\": \"Site-directed mutagenesis of RING finger; in vitro ubiquitin ligase assay; immunofluorescence of NB dispersal\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro E3 ligase reconstitution plus mutagenesis plus cellular functional assay\",\n      \"pmids\": [\"20392849\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"IE1 of HCMV directly targets Sp100 independently of PML: IE1 expression alone is sufficient to downregulate endogenous Sp100 and abrogates SUMO modification of all Sp100 isoforms. Sp100 knockdown results in more cells initiating HCMV gene expression program. Sp100 is also degraded in a proteasome-dependent manner at late times post-infection, and Sp100 knockdown results in augmented release of progeny virions and increased viral late gene products independently of enhanced IE gene expression.\",\n      \"method\": \"IE1 transfection/cotransfection; siRNA knockdown of Sp100 in HFFs; proteasome inhibitor treatment; immunoblotting; immunofluorescence; viral titer measurement\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple loss-of-function approaches with quantitative viral readouts across infection stages\",\n      \"pmids\": [\"21734036\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Cdc20, a co-activator of the APC/C, mediates proteasome-dependent degradation of Sp100 via a D-box motif. Overexpression of Cdc20 decreases endogenous Sp100 levels; knockdown of Cdc20 increases Sp100 levels. A D-box deletion mutant of Sp100 is resistant to Cdc20-mediated degradation. Despite being a Cdc20 substrate, Sp100 protein levels do not oscillate through the cell cycle, and overexpression or knockdown of Sp100 has no effect on cell-cycle progression.\",\n      \"method\": \"Cdc20 overexpression and siRNA knockdown; D-box deletion mutant analysis; MG132 proteasome inhibitor treatment; immunoblotting; flow cytometry cell cycle analysis\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — D-box mutagenesis plus Cdc20 overexpression/knockdown, single lab\",\n      \"pmids\": [\"22086178\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"Herpesvirus saimiri (HVS) specifically degrades Sp100 while leaving PML and hDaxx intact. The HVS ORF3 tegument protein (which shares homology with cellular FGARAT) is the viral factor responsible for proteasomal degradation of Sp100. An ORF3-deletion virus is efficiently complemented in Sp100-depleted cells, demonstrating Sp100 restricts HVS in the absence of ORF3.\",\n      \"method\": \"Infection of cells with wild-type and ORF3-deletion HVS; immunoblotting; immunofluorescence; Sp100 knockdown complementation assay; proteasome inhibitor experiments\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — viral deletion mutant complementation by Sp100 depletion; mechanistic identification of viral effector protein\",\n      \"pmids\": [\"22278248\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Sp100 depletion in primary human keratinocytes results in substantially increased HPV18 immortalization efficiency, viral transcription, and DNA replication, establishing Sp100 as an intrinsic restriction factor against incoming HPV DNA during the initial establishment phase.\",\n      \"method\": \"siRNA depletion of Sp100 in primary keratinocytes; HPV18 quasivirus/DNA introduction; quantitative immortalization assay; viral transcription and DNA replication measurements\",\n      \"journal\": \"mBio\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — siRNA knockdown with quantitative viral establishment assay, multiple readouts\",\n      \"pmids\": [\"24194542\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Two overlapping regions within the N-terminal half of HSV-1 ICP0 (residues 212–311 for PML degradation/dissociation; residues 212–427 for Sp100 dissociation) promote the dissociation and degradation of PML and dissociation of Sp100 from ND10.\",\n      \"method\": \"Series of ICP0 truncation mutants expressed in human embryonic lung cells; immunofluorescence of PML/Sp100 localization; immunoblotting for PML protein levels\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — systematic truncation mutant mapping with cellular readouts, single lab\",\n      \"pmids\": [\"24089549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"IFN-κ inhibits HPV31 transcription primarily by inducing Sp100 proteins. RNAi knockdown of Sp100 reversed IFN-κ-mediated inhibition of viral transcription in CIN612-9E cells maintaining extrachromosomal HPV31 genomes.\",\n      \"method\": \"Doxycycline-inducible IFN-κ expression; siRNA knockdown of Sp100; viral transcription measurement; RNA-seq\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — inducible expression system plus RNAi rescue, multiple readouts\",\n      \"pmids\": [\"26491169\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"The rhesus monkey rhadinovirus (RRV) FGARAT homolog ORF75 is sufficient to cause proteasomal degradation of SP100 (and subsequently PML). CRISPR-Cas9 knockout of SP100 or PML in target cells increases RRV infection, confirming antiviral roles. RRV targets SP100 for degradation before PML, a distinct kinetic strategy from other gammaherpesviruses.\",\n      \"method\": \"CRISPR-Cas9 knockout of ND10 components; RRV infection of knockout cells; ORF75 expression in transfected/transduced cells; immunoblotting; immunofluorescence; proteasome inhibitor experiments\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — CRISPR-Cas9 KO plus ectopic expression of viral effector plus proteasome inhibition, multiple orthogonal approaches\",\n      \"pmids\": [\"27356898\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"SP100 suppresses HSV replication and assembly of viral replication compartments. In SP100-/- HEp-2 cells, the number of viral replication compartments and wild-type virus yields (at low MOI) are significantly higher than in parental cells. Unlike PML, SP100 loss does not alter sensitivity to IFN-β and SP100-/- cells support replication of ΔICP0 virus. SP100 also enhances PML accumulation.\",\n      \"method\": \"CRISPR/gene-knockout SP100-/- cells; immunofluorescence counting of viral replication compartments; viral yield assays at low MOI; IFN-β sensitivity assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — gene KO with multiple quantitative viral readouts and IFN sensitivity comparison\",\n      \"pmids\": [\"28439026\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Sp100 localizes to HPV16 and HPV31 replication foci in primary keratinocytes and in CIN1 cervical biopsies. Sp100 binds across the HPV viral genome (by ChIP), with binding increasing at late stages of infection. siRNA depletion of Sp100 in differentiating HPV31-containing cells increases viral transcription and replication, demonstrating Sp100 represses HPV at late stages of the productive cycle.\",\n      \"method\": \"Immunofluorescence colocalization; ChIP of Sp100 on viral chromatin; siRNA knockdown in differentiating keratinocytes; viral transcription and replication measurements; analysis of patient CIN1 biopsies\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — ChIP plus siRNA knockdown plus patient tissue validation, multiple orthogonal methods\",\n      \"pmids\": [\"28968443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Selective knockdown of Sp100 isoforms in normal human diploid fibroblasts causes rapid cellular senescence followed by emergence of highly tumorigenic Sp100-minus cells that lack ND10/PML bodies, display genomic instability, cytoplasmic p53 sequestration, and activated MYC/RAS/TERT pathways. Re-introduction of Sp100A alone is sufficient to maintain senescence and prevent emergence of tumorigenic cells, identifying Sp100 as a tumor suppressor acting through chromatin-mediated gene regulation.\",\n      \"method\": \"Isoform-selective shRNA knockdown; nude mouse tumorigenicity assay; microsatellite analysis; immunofluorescence; global transcriptome analysis; Sp100A re-expression rescue\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — isoform-specific KD with rescue experiment plus in vivo tumorigenicity, multiple orthogonal readouts\",\n      \"pmids\": [\"21118961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Sp100 interacts with phage ΦC31 integrase and inhibits ΦC31 integrase-mediated recombination. The interaction maps to residues 81-84 (RILN) of ΦC31 integrase. Knockdown of endogenous Sp100 increases ΦC31 integrase-mediated recombination without affecting reporter gene expression.\",\n      \"method\": \"Yeast two-hybrid screen; co-immunoprecipitation; peptide array hybridization; siRNA knockdown with recombination reporter assay\",\n      \"journal\": \"Acta biochimica Polonica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — Co-IP plus siRNA functional rescue, single lab\",\n      \"pmids\": [\"21383994\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Sp100 and PML are sequestered in viral translucent patches (finely granular material at the nuclear border) during prolonged HSV-1 infection, as visualized by immunoelectron microscopy. Both proteins are components not only of nuclear bodies but also of interchromatin granule-associated zones in uninfected cells, suggesting a role in splicing events.\",\n      \"method\": \"Immunoelectron microscopy of ultrathin sections of HSV-1-infected HeLa cells\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — high-resolution immunoelectron microscopy, single lab, structural localization without functional manipulation\",\n      \"pmids\": [\"7493645\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Sp100 splice variants were identified: one class contains an HMG-box domain (SP100-HMG) and another contains an HNPP-box domain related to interferon-inducible nuclear phosphoproteins. When transiently expressed, both classes of variants localize in part to nuclear dots/PML bodies and additionally to other nuclear domains distinct from full-length Sp100.\",\n      \"method\": \"cDNA cloning; Sp100 gene 3'-end analysis; transient transfection with immunofluorescence localization\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — splice variant identification with cellular localization, single lab\",\n      \"pmids\": [\"9973607\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"IFN-beta induces rapid, protein-synthesis-independent transcriptional upregulation of both Sp100 and PML genes. Nuclear run-on analysis revealed synergistic activation by type-I and type-II IFNs. The Sp100 promoter contains an imperfect IFN-stimulated response element (ISRE) and an IFN-gamma activation site (GAS), with the ISRE binding ISGF2 strongly and ISGF3 weakly.\",\n      \"method\": \"Nuclear run-on transcription assay; promoter cloning and functional analysis; transient transfection reporter assay; electrophoretic mobility shift assay (EMSA) with IFN-induced factors\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — nuclear run-on plus promoter deletion analysis plus EMSA, multiple orthogonal mechanistic approaches\",\n      \"pmids\": [\"8810287\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Sp100-HMG autonomously nucleates into liquid-like condensates via its multimerization domain and intrinsically disordered region (IDR), initiating an 'inside-out' assembly of PML nuclear bodies. Sp100-HMG forms a liquid core that recruits PML (via SUMOylation-directed recruitment) and client proteins DAXX and ATRX. This hierarchical assembly is required for proper transcriptional programs and cell-cycle dynamics in HEp-2 cells.\",\n      \"method\": \"Live-cell imaging of condensate formation; cryo-EM and AlphaFold modeling (from paper 65 cross-reference); LLPS assays; domain mutant analysis; CRISPR-based perturbations; transcriptome analysis\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — preprint, single lab, novel mechanistic model not yet peer-reviewed or independently replicated\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SP110 directly interacts with SP100 via their CARD domains and dissolves toxic SP100 oligomers. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, causing micronucleus formation, DNA damage, and genotoxic cell death. SP100 oligomers are thus toxic during mitosis unless dissolved by SP110.\",\n      \"method\": \"Genome-wide CRISPR screen; cryo-electron microscopy; AlphaFold modeling; co-immunoprecipitation; live-cell imaging of mitosis; micronucleus quantification; DNA damage markers\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus CRISPR screen plus Co-IP plus live-cell imaging, multiple orthogonal methods in peer-reviewed study\",\n      \"pmids\": [\"41826696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Sp100A isoform is the primary driver of HIRA histone chaperone localization to PML nuclear bodies. In Sp100 CRISPR knockout keratinocytes, HIRA and its complex members UBN1 and ASF1a fail to localize to PML-NBs even after IFN stimulation. The SUMO-interacting motif (SIM) of Sp100A plays an important role in this function. Sp100 KO does not affect keratinocyte differentiation or PML-NB formation itself.\",\n      \"method\": \"CRISPR-Cas9 Sp100 knockout; exogenous expression of individual Sp100 isoforms; immunofluorescence colocalization; IFN stimulation\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Weak — CRISPR KO plus isoform rescue with clean readout, preprint not yet peer-reviewed\",\n      \"pmids\": [],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"SP100 is an IFN-inducible, SUMO-modified scaffold protein of PML nuclear bodies (ND10) that restricts viral replication (HSV, CMV, HPV, HVS, VZV, RRV) by repressing viral chromatin transcription through its SAND, PHD-bromo, and HMG domain-containing isoforms; it interacts with HP1 family proteins (enhanced by SUMO modification), recruits NBS1/MRE11/RAD50 to sequester recombination machinery (suppressing ALT), acts as a dose-dependent transcriptional co-regulator of ETS1, is degraded by viral E3 ubiquitin ligases (ICP0/UbcH5a, ORF3/FGARAT homologs) via the proteasome, undergoes cell-cycle-regulated degradation via APC/C-Cdc20, and—during mitosis—forms toxic oligomers whose dissolution by SP110 via direct CARD-domain interaction is required to prevent chromosomal segregation errors and genotoxic cell death.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SP100 is an interferon-inducible nuclear scaffold protein and a core constituent of PML nuclear bodies (ND10) that functions in intrinsic antiviral defense and chromatin-mediated transcriptional regulation [#30, #14]. Its expression is driven by type-I and type-II IFN through an ISRE and GAS element in its promoter [#30], and it exists as multiple alternatively spliced isoforms, several carrying SAND, PHD-bromodomain, or HMG chromatin-associated motifs [#29, #6, #15]. SP100 is covalently modified by SUMO-1 on a lysine within its HP1-binding region, a modification that confines it to the nucleus and enhances the stability of its complexes with HP1 family heterochromatin proteins; when tethered to DNA, SP100 and its HMG variant act as transcriptional repressors [#0, #1, #2, #6]. Distinct domains mediate ND10 targeting and homo-oligomerization, allowing SP100 (notably the HMG isoform) to nucleate nuclear body assembly and recruit partners [#7, #31]. SP100 restricts a broad panel of viruses — HSV-1, HCMV, HPV, EBV, HVS, VZV, and RRV — primarily by repressing viral chromatin transcription, acting independently of and additively with PML, with the SAND-domain isoforms blocking HSV-1 immediate-early promoter activity [#14, #15, #17, #20, #25, #24]. Viruses counteract SP100 through SUMO de-modification and proteasome-dependent degradation mediated by viral E3 ubiquitin ligases, including HSV-1 ICP0 acting via the E2 enzyme UbcH5a, VZV ORF61p, and the FGARAT-homolog tegument proteins of HVS (ORF3) and RRV (ORF75) [#3, #4, #5, #16, #19, #23]. Beyond viral defense, SP100 is a dose-dependent negative regulator of the ETS1 transcription factor, reducing ETS1 DNA binding and target-gene activation to suppress invasion and angiogenesis [#8, #9, #10], recruits the MRN complex via NBS1 to sequester recombination machinery and suppress alternative lengthening of telomeres [#11, #12], and acts as a tumor suppressor whose loss triggers senescence escape and genomic instability [#26]. SP100 is also a substrate of APC/C-Cdc20-mediated proteasomal degradation through a D-box motif [#18], and during mitosis its toxic oligomers must be dissolved by SP110 through a direct CARD-CARD interaction to prevent chromosome segregation errors and genotoxic death [#32].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Established that SP100 is a bona fide interferon-response gene, defining the regulatory logic by which it is deployed in innate defense.\",\n      \"evidence\": \"Nuclear run-on, promoter deletion analysis, and EMSA identifying ISRE/GAS elements and IFN factor binding\",\n      \"pmids\": [\"8810287\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not connect IFN induction to a specific downstream antiviral or chromatin function\", \"Isoform-specific transcriptional/splicing responses not resolved\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Answered how SP100 subcellular distribution is controlled by showing SUMO-1 conjugation restricts it to the nucleus, linking a post-translational mark to localization.\",\n      \"evidence\": \"SUMO-1-specific immunoblotting plus cell fractionation and immunofluorescence across cell lines\",\n      \"pmids\": [\"9412458\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional consequence of SUMOylation beyond localization not yet defined\", \"SUMO conjugation site not mapped at this stage\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Identified SP100 as a partner of HP1 heterochromatin proteins and a transcriptional repressor, establishing a chromatin-silencing function.\",\n      \"evidence\": \"Co-IP, colocalization, and promoter-tethering reporter assays; identification of the SP100-HMG splice variant\",\n      \"pmids\": [\"9636146\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous genomic targets of repression not identified\", \"Direct DNA binding of HMG variant not functionally demonstrated\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Mapped the SP100 domains for SUMOylation, nuclear localization, dimerization and ND targeting and located the SUMO site within the HP1-binding region, suggesting SUMO tunes HP1 interaction.\",\n      \"evidence\": \"Deletion/point-mutant analysis with localization, dimerization and SUMOylation readouts; splice variant cloning\",\n      \"pmids\": [\"10212234\", \"9973607\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not directly measure SUMO effect on HP1 binding affinity\", \"Functional roles of individual isoforms not assigned\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Revealed that herpesviruses dismantle SP100 by abrogating SUMO modification and triggering proteasomal degradation, defining viral antagonism of ND10 integrity.\",\n      \"evidence\": \"Immunoblotting of SUMO-modified SP100 and protein levels in HSV-1/CMV-infected cells with MG132 rescue and viral-protein comparison; immuno-EM localization\",\n      \"pmids\": [\"10233977\", \"10023669\", \"7493645\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Viral E3/E2 machinery not yet identified\", \"Whether SP100 loss causally enhances viral replication not tested here\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Provided biochemical mechanism for SUMO function by showing SUMOylation stabilizes SP100-HP1 complexes, and expanded the isoform repertoire to chromatin-reader (PHD-bromo) forms.\",\n      \"evidence\": \"In vitro binding assays comparing SUMOylated vs unmodified SP100; cDNA cloning of SP100C\",\n      \"pmids\": [\"11313457\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vitro stabilization not validated for endogenous complexes\", \"Chromatin substrates read by the PHD-bromo isoform unknown\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Separated the ND10-targeting and self-oligomerization functions of SP100 into distinct domains, clarifying how it docks to and seeds nuclear bodies.\",\n      \"evidence\": \"GFP-fusion deletion variant transfection and immunofluorescence with/without endogenous SP100\",\n      \"pmids\": [\"11112690\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of oligomerization not resolved\", \"Functional importance of oligomerization in vivo not addressed\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Defined a chromatin-independent transcriptional role by showing SP100 binds and dose-dependently inhibits ETS1, repressing invasion-associated gene programs.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro pulldown, Co-IP, reporter, endogenous gene expression and invasion assays; NBS1 BRCT-mediated SP100 interaction and ectopic POD recruitment\",\n      \"pmids\": [\"11909962\", \"12470659\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which SP100 reduces ETS1 DNA binding unresolved\", \"Physiological context of NBS1 recruitment not yet linked to a pathway\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected SP100 to genome maintenance and angiogenesis control: it sequesters the MRN complex to suppress ALT and antagonizes ETS1-driven endothelial behavior.\",\n      \"evidence\": \"SP100 overexpression and truncation analysis with telomere length, APB and FISH assays in ALT cells; HUVEC functional assays, microarray and EMSA; EBNA-LP displacement domain mapping\",\n      \"pmids\": [\"15767676\", \"15592518\", \"15247905\", \"16177824\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether endogenous SP100 levels regulate ALT physiologically untested\", \"ETS1 target-gene selectivity mechanism unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrated that SP100 is an independent intrinsic antiviral restriction factor, acting additively with but separately from PML against HSV-1.\",\n      \"evidence\": \"siRNA single and double knockdown of SP100/PML with quantitative viral gene expression and plaque assays\",\n      \"pmids\": [\"18160441\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular step of restriction not defined in this study\", \"Isoform responsible for restriction not identified\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Pinpointed the SAND-domain isoforms as the active repressors of HSV-1 immediate-early promoters and showed IFN biases splicing toward the inhibitory SP100C isoform.\",\n      \"evidence\": \"Isoform-selective siRNA, IFN treatment, promoter-luciferase reporters and immunoblotting in infected cells\",\n      \"pmids\": [\"19279115\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which SAND domain represses viral chromatin not biochemically resolved\", \"Splicing regulator directing IFN-induced isoform switch unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identified two parallel degradation routes for SP100 — viral RING E3 ligases and host APC/C-Cdc20 — defining how its levels are controlled by infection and by the cell-cycle machinery.\",\n      \"evidence\": \"VZV ORF61p RING mutagenesis and in vitro ligase assays; HCMV IE1 transfection/knockdown with viral titers; Cdc20 overexpression/knockdown and D-box mutant analysis with MG132\",\n      \"pmids\": [\"20392849\", \"21734036\", \"22086178\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cdc20-mediated degradation does not produce cell-cycle oscillation of SP100, leaving its physiological purpose unclear\", \"Direct ubiquitination of SP100 by these ligases not all reconstituted\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established SP100 as a tumor suppressor whose isoform-specific loss causes senescence escape, genomic instability and tumorigenicity.\",\n      \"evidence\": \"Isoform-selective shRNA knockdown, nude mouse tumorigenicity, microsatellite and transcriptome analysis with Sp100A rescue\",\n      \"pmids\": [\"21118961\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Causal chromatin targets driving tumor suppression not defined\", \"Relationship between tumor suppression and antiviral repression unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Extended SP100 restriction to papillomaviruses, showing it limits HPV establishment and that ICP0 N-terminal regions mediate its ND10 dissociation.\",\n      \"evidence\": \"siRNA depletion in primary keratinocytes with HPV18 immortalization/transcription/replication readouts; ICP0 truncation mapping with localization readouts\",\n      \"pmids\": [\"24194542\", \"24089549\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct engagement of incoming HPV DNA by SP100 not yet shown at this stage\", \"ICP0 domains mapped without identifying degradation cofactors\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Generalized viral antagonism of SP100 across gammaherpesviruses by identifying FGARAT-homolog effectors (HVS ORF3, RRV ORF75) and confirming antiviral roles by knockout.\",\n      \"evidence\": \"Wild-type vs ORF-deletion virus complementation in SP100-depleted cells; CRISPR-Cas9 knockout; ectopic effector expression and proteasome inhibition\",\n      \"pmids\": [\"22278248\", \"27356898\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Biochemical mechanism of FGARAT-homolog-directed SP100 degradation undefined\", \"Why RRV degrades SP100 before PML kinetically unexplained\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the cellular step of restriction by showing SP100 suppresses formation of viral replication compartments and binds viral chromatin directly across the HPV genome.\",\n      \"evidence\": \"SP100-/- CRISPR cells with replication-compartment counting, viral yields and IFN-beta sensitivity; ChIP of SP100 on HPV chromatin, colocalization and knockdown in differentiating keratinocytes and patient biopsies\",\n      \"pmids\": [\"28439026\", \"28968443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of SP100 recruitment to viral chromatin not defined\", \"Distinct contributions of SP100 isoforms to chromatin binding unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined a mitotic safety mechanism in which SP110 dissolves toxic SP100 oligomers via direct CARD-CARD interaction to prevent chromosome segregation errors and genotoxicity.\",\n      \"evidence\": \"Genome-wide CRISPR screen, cryo-EM, AlphaFold modeling, Co-IP, live-cell mitosis imaging and micronucleus/DNA-damage quantification\",\n      \"pmids\": [\"41826696\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Trigger that converts SP100 into toxic oligomers in mitosis not identified\", \"Whether oligomerization links to other SP100 functions (antiviral/chromatin) unknown\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Proposed an 'inside-out' phase-separation model of nuclear body assembly with SP100-HMG nucleating a liquid core, and assigned Sp100A as the driver of HIRA chaperone recruitment.\",\n      \"evidence\": \"Live-cell imaging, LLPS assays, cryo-EM/AlphaFold and CRISPR perturbations (preprint); CRISPR Sp100 KO with isoform rescue and IFN stimulation (preprint)\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprints not yet peer-reviewed or independently replicated\", \"LLPS-based assembly model not validated against the established SUMO-driven recruitment scheme\", \"Functional consequence of HIRA recruitment to PML-NBs not established\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SP100's chromatin-reader/repressor domains mechanistically silence viral and cellular promoters, and how its multiple functions (antiviral repression, ETS1 regulation, ALT suppression, mitotic oligomer toxicity) are coordinated through a unified molecular activity, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of SP100 engaging chromatin\", \"No defined catalytic or enzymatic activity for SP100 itself\", \"Integration of distinct functional outputs by isoform/PTM state not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [2, 8, 9, 15, 26]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [2, 25]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [2, 6]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [11, 12, 32]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [2, 7, 28]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [25, 32]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [14, 15, 17, 20, 24, 30]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [19, 23, 26]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [2, 8, 9, 15]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [18, 32]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [0, 3, 5, 18]}\n    ],\n    \"complexes\": [\"PML nuclear body / ND10\"],\n    \"partners\": [\"PML\", \"HP1\", \"ETS1\", \"NBS1\", \"SP110\", \"DAXX\", \"ATRX\", \"HIRA\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}