{"gene":"PSIP1","run_date":"2026-06-10T06:43:36","timeline":{"discoveries":[{"year":2002,"finding":"HIV-1 integrase (IN) forms stable tetramers in human cell nuclei and associates with LEDGF/p75 to form a larger ~61 Å complex; recombinant LEDGF/p75 robustly enhanced strand transfer activity of HIV-1 IN in vitro, and IN co-localizes with LEDGF/p75 in the nucleus.","method":"FLAG-tag co-immunoprecipitation from nuclear extracts, gel filtration, in vitro strand transfer assay, co-localization by fluorescence microscopy","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (Co-IP, in vitro enzymatic assay, co-localization), foundational paper replicated by many subsequent studies","pmids":["12407101"],"is_preprint":false},{"year":2003,"finding":"LEDGF/p75 is essential for nuclear localization and chromosomal targeting of HIV-1 integrase; knockdown of endogenous LEDGF/p75 by RNAi abolished nuclear/chromosomal localization of IN. The alternative splice variant p52 did not interact with HIV-1 IN in vitro or in living cells.","method":"RNAi knockdown, EGFP/HcRed1 live-cell fluorescence imaging, in vitro pull-down, domain deletion/mutant analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — RNAi loss-of-function with clear phenotypic readout, live-cell imaging, and in vitro binding, replicated across multiple studies","pmids":["12796494"],"is_preprint":false},{"year":2000,"finding":"LEDGF/p75 and p52 are derived from a single gene (PSIP1) by alternative splicing; exons 1–15 encode p75 and exons 1–9 plus part of intron 9 encode p52; the gene is located on chromosome 9p22.2.","method":"Gene cloning, sequencing, exon/intron mapping, RT-PCR expression analysis","journal":"Gene","confidence":"High","confidence_rationale":"Tier 1 / Strong — definitive molecular characterization of gene structure, replicated in subsequent literature","pmids":["10721720"],"is_preprint":false},{"year":2001,"finding":"LEDGF binds to heat shock element (HSE; nGAAn) and stress-related regulatory element (STRE; A/TGGGGA/T) in the promoters of stress-related genes (Hsp27 and αB-crystallin) to activate their transcription.","method":"DNA-binding assay, transcriptional reporter assay, promoter element deletion/mutation","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and transactivation assays, single lab but two orthogonal methods","pmids":["11350077"],"is_preprint":false},{"year":2001,"finding":"LEDGF/p75 is distributed diffusely in the nucleoplasm during G1 and attaches to condensed chromatin during G2/M (mitosis), whereas p52 localizes to the nuclear periphery in G1 and forms speckles at S-phase, indicating distinct nuclear compartments and functions.","method":"Live-cell fluorescence imaging of GFP-tagged proteins across cell cycle stages in CHO-K1, MCDK, and NRK cells","journal":"Cell and tissue research","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — direct localization by live imaging in multiple cell lines, single lab","pmids":["11512661"],"is_preprint":false},{"year":2004,"finding":"LEDGF/p75 nuclear import is GTP-, Ran-, importin-α/β-, and energy-dependent; the functional NLS was mapped to residues 148–156 (GRKRKAEKQ); a single amino acid change in the NLS excluded LEDGF/p75 from the nucleus and abolished nuclear import of HIV-1 integrase.","method":"Deletion analysis, site-directed mutagenesis, semipermeabilized cell nuclear import assay, GFP fusion constructs in live cells, β-galactosidase NLS transfer assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis with functional nuclear import assay and multiple orthogonal validations","pmids":["15163664"],"is_preprint":false},{"year":2005,"finding":"LEDGF/p75 interacts with HIV-1, HIV-2, and feline immunodeficiency virus integrase but not with non-lentiviral integrases (HTLV-2, MoMLV, RSV); LEDGF/p75 strongly promoted binding of HIV-1 and HIV-2 IN to DNA, an effect specific to the p75 isoform and not seen with p52.","method":"Pull-down binding assay, fluorescence correlation spectroscopy for DNA-binding measurements, isoform-specific comparisons","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro binding with biophysical quantification, lentiviral-specificity shown across multiple integrases","pmids":["15749713"],"is_preprint":false},{"year":2005,"finding":"Interaction of HIV-1 integrase with LEDGF/p75 is essential for viral replication and chromosomal tethering of IN; a single integrase mutation that disrupts LEDGF/p75 interaction (without reducing enzymatic activity) results in defective HIV-1 replication.","method":"Two-hybrid assays, random and directed mutagenesis, viral replication assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic mutation + viral replication assay, multiple orthogonal approaches, replicated in other studies","pmids":["15855167"],"is_preprint":false},{"year":2005,"finding":"Both the IN-binding and DNA-binding activities of LEDGF/p75 contribute to functional reconstitution of HIV-1 preintegration complex (PIC) in vitro; recombinant LEDGF/p75 efficiently reconstitutes high-salt-disrupted PIC activity.","method":"In vitro PIC reconstitution assay with recombinant proteins, mutational analysis of LEDGF/p75 domains","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — reconstitution in vitro shown, single lab, not independently replicated in this form","pmids":["16337983"],"is_preprint":false},{"year":2006,"finding":"LEDGF/p75 chromatin association in living cells is mediated by a tripartite element comprising the NLS and two AT-hook motifs (residues 146–197); the PWWP domain is not required for binding condensed mitotic chromosomes but subtly affects interphase nucleoplasmic distribution; neither DNA-binding-deficient LEDGF/p75 mutants nor the AT-hook mutant lost chromatin binding unless combined with NLS mutations or PWWP deletion.","method":"SPR and EMSA for DNA binding, EGFP fusion localization in interphase and mitotic cells, domain deletion and AT-hook mutagenesis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (SPR, EMSA, live-cell imaging, systematic mutagenesis) in single rigorous study","pmids":["16549878"],"is_preprint":false},{"year":2006,"finding":"LEDGF/p75 interacts with and stimulates strand transfer activity of all lentiviral integrases tested (BIV, MVV, EIAV) but not non-lentiviral integrases; mutation D366N in LEDGF ablates interaction with all lentiviral INs, suggesting a conserved recognition mechanism; in the presence of LEDGF, EIAV IN almost exclusively catalyzes concerted integration.","method":"Pull-down, yeast two-hybrid, in vitro strand transfer assay with divergent lentiviral INs, site-directed mutagenesis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro enzymatic assay with mutagenesis across multiple integrases, demonstrates conserved mechanism","pmids":["17158150"],"is_preprint":false},{"year":2006,"finding":"Psip1/Ledgf knockout mice exhibit perinatal mortality, homeotic skeletal transformations, motor/behavioral defects, and craniofacial abnormalities, implicating PSIP1 in Hox gene expression regulation in vivo.","method":"Gene trap mutagenesis in mouse ES cells, homozygous knockout mouse phenotypic analysis","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic knockout mouse model with multiple well-defined phenotypic readouts","pmids":["16980622"],"is_preprint":false},{"year":2006,"finding":"The LEDGF/p75-binding site on HIV-1 integrase maps to two regions: residues around W131/W132 and residues I161–E170; mutations at W131, I161, R166, Q168, and E170 impair LEDGF/p75 interaction but retain IN enzymatic activity; the W131A mutation reduces HIV-1 replication.","method":"Yeast two-hybrid, in vitro binding/pull-down, IN enzymatic activity assays, HIV-1 replication assays, structural comparison","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — systematic mutagenesis with multiple functional assays and structural comparison","pmids":["17137594"],"is_preprint":false},{"year":2007,"finding":"LEDGF/p75 functions downstream from HIV-1 preintegration complex formation to direct integration into transcription units; in LEDGF-null cells, HIV-1 loses its strong bias toward integrating into transcription units and instead shows increased affinity for promoter regions and CpG islands, while 3'-end processing and local target DNA sequence preference are unaffected.","method":"Genetic knockout of LEDGF, HIV-1 vector integration site mapping, quantitative PCR, in vitro strand transfer assays with PICs from knockout cells","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic KO with integration site mapping and in vitro PIC assays, replicated in subsequent studies","pmids":["17639082"],"is_preprint":false},{"year":2007,"finding":"JPO2 binds the C-terminal integrase-binding domain (IBD) of LEDGF/p75 in a mutually exclusive manner with HIV-1 integrase; the binding mechanism differs (JPO2 continues to interact with some IN-defective LEDGF/p75 mutants like I365A, D366A, F406A); JPO2 overexpression modestly inhibits HIV-1 replication.","method":"Yeast two-hybrid, pull-down, AlphaScreen, co-immunoprecipitation, competition assays with recombinant proteins, HIV-1 replication assay","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, AlphaScreen, and competition assays with multiple orthogonal methods in single study","pmids":["17669426"],"is_preprint":false},{"year":2008,"finding":"LEDGF/p75 strongly stabilizes IN-IN subunit interactions and promotes IN tetramerization; mass spectrometric protein footprinting revealed novel intra- and inter-protein contacts in the full-length IN-LEDGF complex beyond the IBD-CCD co-crystal structure; the IN tetramer interface is important for enzymatic activities and high-affinity LEDGF binding.","method":"Mass spectrometric protein footprinting, molecular modeling, biochemical characterization of full-length IN-LEDGF interactions, enzymatic assays","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — MS footprinting combined with enzymatic assays, multiple orthogonal methods","pmids":["18801737"],"is_preprint":false},{"year":2008,"finding":"The PWWP domain of LEDGF/p75 is required for chromatin binding and HIV-1 infectivity; mutations at W21 or A51 (which line a hydrophobic cavity conserved among Tudor clan members) disrupt chromatin binding and virus infectivity; W21A recombinant protein is preferentially defective for enhancing integration into chromatinized (but not naked) DNA in vitro.","method":"Systematic mutagenesis of 24 PWWP residues, chromatin binding assays, HIV-1 infectivity assays, in vitro integration into chromatinized templates","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — systematic mutagenesis with multiple functional readouts (chromatin binding, infectivity, in vitro integration)","pmids":["18799576"],"is_preprint":false},{"year":2008,"finding":"The PWWP domain of LEDGF/p75 is required for stimulation of HIV-1 integration into reconstituted polynucleosome (chromatinized) templates; the PWWP domain binds directly to nucleosomes in GST pull-down assays; with naked DNA, full removal of N-terminal chromatin-binding elements is needed to abate cofactor function, but with polynucleosomes, PWWP domain is the primary determinant.","method":"In vitro integration assay with reconstituted polynucleosome templates, GST pull-down, domain deletion analysis","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution with defined substrates, multiple domain mutants, and direct nucleosome-binding assay","pmids":["18174227"],"is_preprint":false},{"year":2009,"finding":"LEDGF/p75 interacts with the Cdc7-ASK (activator of S-phase kinase) heterodimer through its integrase-binding domain (IBD); the interaction requires autophosphorylation of Cdc7 and the C-terminal 50 residues of ASK; Cdc7-ASK phosphorylates LEDGF at Ser-206 (primarily during S phase); LEDGF potently stimulates Cdc7-ASK kinase activity, increasing MCM2 phosphorylation >10-fold in vitro, by relieving ASK C-terminus-mediated autoinhibition.","method":"Co-immunoprecipitation of endogenous proteins, truncation analysis, in vitro kinase assay with MCM2 substrate, phosphorylation site mapping","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reciprocal Co-IP of endogenous proteins, in vitro kinase assay, phosphorylation site mapping, mechanistic relief-of-autoinhibition demonstrated","pmids":["19864417"],"is_preprint":false},{"year":2009,"finding":"LEDGF/p75 crystal structure (HIV-2 IN NTD+CCD in complex with LEDGF IBD) revealed charge-charge interactions between the IN N-terminal domain (NTD) and the IBD; a constellation of acidic residues on the NTD is characteristic of lentiviral INs; mutations of positively charged IBD residues severely impaired interaction with all lentiviral INs and abrogated stimulation of concerted integration and HIV-1 replication.","method":"X-ray crystallography, site-directed mutagenesis, in vitro concerted integration assay, HIV-1 infectivity assays","journal":"PLoS pathogens","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and functional assays in single study","pmids":["19132083"],"is_preprint":false},{"year":2010,"finding":"LEDGF/p75 is primarily bound downstream of transcription start sites of active transcription units in chromatin, co-localizing with active chromatin markers (H3/H4 acetylation, H3K4me1, RNA Pol II); not all LEDGF/p75-bound chromosomal complexes are amenable to HIV-1 integration.","method":"DamID chromatin profiling in ENCODE regions, correlation analysis with >200 genomic features including HIV-1 integration sites","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide chromatin profiling (DamID), single lab study with bioinformatic correlation","pmids":["20484370"],"is_preprint":false},{"year":2010,"finding":"LEDGF/p75 chromatin binding in living cells follows a 'scan-and-lock' mechanism: the protein moves in a chromatin hopping/scanning mode; the PWWP domain is necessary but not sufficient for in vivo chromatin binding; upon binding HIV-1 integrase via its IBD, LEDGF/p75 kinetics shift to 75-fold larger chromatin affinity, with PWWP domain crucial for locking the complex on chromatin.","method":"FRAP (spot- and half-nucleus), continuous photobleaching, fluorescence correlation spectroscopy, tunable focus FCS in living HeLa cells","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple quantitative live-cell fluorescence methods, mechanistic scan-and-lock model with quantitative parameters","pmids":["20974633"],"is_preprint":false},{"year":2010,"finding":"Replacement of the LEDGF/p75 chromatin interaction domain with CBX1 (which binds H3K9me2/3 at pericentric heterochromatin) redirects lentiviral vector integration away from genes and toward heterochromatin, demonstrating that LEDGF/p75 chromatin-tethering domain determines integration site selection.","method":"Chimeric protein engineering, lentiviral vector transduction, integration site sequencing","journal":"Molecular therapy","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic domain swap with integration site mapping provides causal evidence for tethering mechanism","pmids":["20195265"],"is_preprint":false},{"year":2011,"finding":"FRET analysis revealed that HIV-1 IN tetramers adopt distinct conformations in the presence of viral DNA versus LEDGF/p75; pre-formed IN-viral DNA complex conformation is unchanged upon subsequent LEDGF/p75 binding, but pre-incubation with LEDGF followed by viral DNA yields IN conformation similar to the IN-LEDGF complex.","method":"FRET monitoring of IN subunit assembly in the presence of viral DNA and LEDGF/p75, order-of-addition experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — quantitative FRET with systematic order-of-addition experiments reveals structural mechanistic insight","pmids":["21771857"],"is_preprint":false},{"year":2011,"finding":"LEDGF/p75 tethers HIV-1 IN to DNA in vitro: LEDGF/p75 stimulates IN binding to DNA 10–30-fold in a manner requiring direct IN-LEDGF C-terminus interaction; overexpression of LEDGF C-terminus or NLS/AT-hook mutant LEDGF inhibits IN-DNA interaction.","method":"AlphaScreen assay for IN-DNA interaction, domain-deletion analysis, in vitro DNA-binding","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — quantitative in vitro tethering assay, single lab, two orthogonal approaches","pmids":["21763490"],"is_preprint":false},{"year":2011,"finding":"LEDGF/p75 recognizes supercoiled DNA preferentially over unconstrained DNA via a novel 'supercoiled DNA-recognition domain' (SRD) containing a K/E/D cluster; GFP-SRD in cells localizes to transcriptionally active regions (H3K4me3, Br-UTP incorporation); deletion of SRD abolishes this localization.","method":"Recombinant protein fragments with in vitro supercoiled DNA-binding assays, EGFP-tagged localization with immunostaining in live cells","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro binding assay combined with cellular localization experiment, single lab","pmids":["21345933"],"is_preprint":false},{"year":2012,"finding":"LEDGF/p75 promotes DNA double-strand break repair by homologous recombination; LEDGF depletion impairs recruitment of CtIP to DNA DSBs and CtIP-dependent DNA-end resection; LEDGF binds CtIP in a DNA damage-dependent manner through its PWWP domain interaction with chromatin, tethering CtIP to active chromatin near DSBs.","method":"RNAi depletion, CtIP recruitment assay at DSBs, DNA-end resection assay, co-immunoprecipitation (damage-dependent), chromatin binding","journal":"Nature structural & molecular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — loss-of-function with specific molecular readout (CtIP recruitment, resection), damage-dependent Co-IP, published in high-impact journal","pmids":["22773103"],"is_preprint":false},{"year":2012,"finding":"The PWWP domain of Psip1/Ledgf specifically recognizes trimethylated H3K36; the p52 (short) isoform, enriched at active genes like H3K36me3, co-localizes and interacts with Srsf1 and other mRNA processing proteins; in Psip1 mutant cells, H3K36me3-associated Srsf1 is reduced and alternative splicing of specific genes is altered.","method":"PWWP domain H3K36me3 binding assay, co-immunoprecipitation of p52 with splicing factors, ChIP, splicing analysis in Psip1 mutant cells","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding assay, Co-IP, ChIP, RNA splicing analysis) with genetic knockout cells","pmids":["22615581"],"is_preprint":false},{"year":2013,"finding":"The LEDGF PWWP domain binds H3K36me3-containing mononucleosomes with nanomolar affinity via two distinct functional interfaces: a hydrophobic cavity that selectively contacts H3K36me3 peptide, and an adjacent basic surface that non-specifically binds DNA; cooperative binding to both methylated histone tail and nucleosomal DNA is essential for high-affinity chromatin binding.","method":"NMR solution structure of PWWP domain, binding to H3K36me3 peptide and DNA monitored by NMR, affinity measurements to native and reconstituted mononucleosomes, mutational analysis, proteomic experiments","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR structure with functional validation, mutagenesis, and quantitative binding to reconstituted mononucleosomes","pmids":["23396443"],"is_preprint":false},{"year":2013,"finding":"PSIP1-PWWP binds H3K36-methylated nucleosomes through simultaneous bipartite interactions with the methylated histone tail and nucleosomal DNA; binding is enhanced ~10,000-fold compared to methylated peptide alone; this bipartite mechanism was structurally characterized by NMR and mutational analysis.","method":"NMR spectroscopy, extensive mutational analysis, computational approaches, thermodynamic binding measurements","journal":"Epigenetics & chromatin","confidence":"High","confidence_rationale":"Tier 1 / Strong — NMR-derived structure with mutational analysis and quantitative thermodynamic measurements","pmids":["23656834"],"is_preprint":false},{"year":2013,"finding":"In the HIV-1 preintegration complex, LEDGF/p75 organizes and stabilizes an active integrase tetramer (stoichiometry IN4:LEDGF2:INI1-IBD2:DNA2); INI1-IBD occupies the cellular DNA binding site of the IN-LEDGF complex and constrains integrase in a stable conformation.","method":"Stable complex reconstitution, mass spectrometry stoichiometry determination, fluorescence correlation spectroscopy, functional integration assay, cryo-electron microscopy","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro reconstitution, cryo-EM structure, mass spectrometry stoichiometry, and functional assays","pmids":["23593299"],"is_preprint":false},{"year":2014,"finding":"NMR spectroscopy identified an additional LEDGF/p75-MLL interface overlapping with the HIV-1 integrase, PogZ, and JPO2 binding site on the IBD; binding of MLL, HIV-1 IN, PogZ, and JPO2 to LEDGF/p75 is mutually exclusive; the MLL interaction is primarily sustained by two aromatic residues (F148 and F151 of MLL); mutation of this interface impairs MLL-AF9+ leukemic cell transformation.","method":"NMR spectroscopy for interface mapping, mutational analysis, AlphaScreen competition assays, colony-forming assays","journal":"Cancer research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — NMR structure with mutational analysis and functional transformation assay","pmids":["25082813"],"is_preprint":false},{"year":2014,"finding":"Complete TALEN-mediated knockout of PSIP1 in human cells severely impairs HIV-1 spreading replication and single-cycle integration; HIV-1 particle assembly and the main ALLINI (allosteric integrase inhibitor) mechanism are LEDGF/p75-independent.","method":"TALEN-mediated gene knockout (whole-gene deletion and IBD exon deletion), HIV-1 replication and single-cycle infectivity assays, virus assembly assays","journal":"Journal of virology","confidence":"High","confidence_rationale":"Tier 2 / Strong — definitive gene knockout with specific mechanistic readouts for integration, assembly, and inhibitor mechanism","pmids":["24942577"],"is_preprint":false},{"year":2015,"finding":"LEDGF/p75 is associated with multiple mRNA splicing factors by affinity purification; LEDGF/p75 (or its IBD) contributes to splicing patterns in approximately half of transcription units with alternative isoforms; HIV-1 integration density in transcription units correlates with intron number in a LEDGF-dependent manner.","method":"Affinity purification of LEDGF/p75-associated splicing factors, RNA-seq analysis of LEDGF/p75 or IBD knockout HEK293T cells, HIV-1 integration site sequencing (>1 million sites), multivariate analysis","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — affinity purification + RNA-seq in KO cells + integration site mapping, multiple orthogonal methods","pmids":["26545813"],"is_preprint":false},{"year":2017,"finding":"Conditional Psip1 knockout in hematopoietic cells decreases Hox gene expression, reduces MLL occupancy at MLL target genes, and impairs MLL-fusion-mediated leukemia initiation in vitro and in vivo, but is dispensable for steady-state hematopoiesis.","method":"Conditional knockout mouse model, Hox gene expression analysis, ChIP for MLL occupancy, in vitro colony-forming assays, in vivo bone marrow transplant leukemia model","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — conditional KO mouse model with molecular readouts (ChIP, gene expression) and both in vitro and in vivo functional assays","pmids":["29084774"],"is_preprint":false},{"year":2017,"finding":"PSIP1/p75 interacts with RNA polymerase II and facilitates its association with promoters of cell cycle genes, thereby regulating their transcription and promoting triple-negative breast cancer tumorigenicity.","method":"Co-immunoprecipitation of PSIP1 with RNA Pol II, ChIP for RNA Pol II at promoters in PSIP1-depleted cells, gene expression analysis, tumorigenicity assays","journal":"Carcinogenesis","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and ChIP showing PSIP1-Pol II interaction and promoter association, single lab","pmids":["28633434"],"is_preprint":false},{"year":2017,"finding":"Psip1/p52 (short isoform) specifically regulates expression of lncRNA Hottip at the Hoxa locus; Hottip is required for activation of posterior Hoxa genes (Hoxa13, Hoxa10/11) and for retaining Mll1 at the 5' end of Hoxa; the Hottip RNA molecule itself (not just its transcription) is required for Hox gene activation.","method":"Knockdown and knockout approaches, Hottip overexpression, premature transcription termination engineering, Mll1 ChIP","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO/KD with multiple orthogonal functional approaches, single lab","pmids":["28384324"],"is_preprint":false},{"year":2019,"finding":"LEDGF/p75 (and HDGF2) bind H3K36me2 and H3K36me3 via their PWWP domains and, like FACT, facilitate RNA Pol II transcription elongation past nucleosome barriers; in differentiated myotubes, HDGF2 depletion causes paused RNAPII accumulation within transcribed regions of target genes, indicating a defect in early elongation.","method":"Genome-wide co-localization with H3K36me2/3, HDGF2 knockout in myoblasts/myotubes, RNAPII ChIP-seq in KO cells","journal":"Science advances","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO with RNAPII ChIP-seq readout; primary mechanistic focus is on HDGF2, but LEDGF role is defined by co-localization and parallel domain architecture","pmids":["31616795"],"is_preprint":false},{"year":2020,"finding":"LEDGF/p75 differentially regulates HIV transcription: during latency it suppresses proviral transcription by recruiting PAF1 complex to promote RNAPII promoter-proximal pausing; upon latency reversal, casein kinase II (CKII)-dependent MLL1 complex competitively displaces PAF1 from LEDGF/p75 at the provirus, enabling Super Elongation Complex recruitment and transcriptional activation.","method":"Co-immunoprecipitation, ChIP for PAF1/MLL1/SEC at provirus, CKII pharmacological inhibition and depletion, HIV latency reversal assays","journal":"Science advances","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, ChIP at provirus, pharmacological inhibition with clear mechanistic model supported by multiple orthogonal experiments","pmids":["32426500"],"is_preprint":false},{"year":2024,"finding":"PSIP1 interacts with R-loops and proteins involved in R-loop homeostasis including PARP1; depletion of PSIP1 leads to genome-wide accumulation of R-loops and DNA damage (γ-H2AX) at gene promoters, local transcriptional arrest, transcription-replication conflicts, increased 53BP1 foci, and reduced RAD51 foci (indicating altered DNA repair pathway choice toward NHEJ); PSIP1 depletion sensitizes cancer cells to PARP1 inhibitors.","method":"Co-immunoprecipitation of PSIP1 with PARP1 and R-loop-associated proteins, genome-wide R-loop mapping (DRIP-seq), γ-H2AX ChIP-seq, 53BP1 and RAD51 foci analysis, cell viability assays with PARP inhibitors in PSIP1-depleted cells","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal genome-wide and cellular assays (DRIP-seq, ChIP-seq, foci analysis, Co-IP) with functional consequence (PARP inhibitor sensitivity)","pmids":["38191578"],"is_preprint":false},{"year":2009,"finding":"LEDGF/p75 binds the VEGF-C promoter (demonstrated by ChIP); FSH augments this binding and increases LEDGF/p75 mRNA and protein levels; siRNA suppression of LEDGF/p75 reduces hormonally induced VEGF-C expression, linking LEDGF/p75 to gonadotropin-regulated lymphangiogenesis in ovarian cancer cells.","method":"ChIP, siRNA knockdown, promoter-luciferase reporter assay, RT-PCR","journal":"Cancer research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and siRNA with functional reporter assay, single lab","pmids":["19934313"],"is_preprint":false},{"year":2012,"finding":"LEDGF/p75 and p52 both interact with MeCP2 in vitro and in human cancer cells; the interaction maps to the N-terminal PWWP-CR1 domain shared by both isoforms; LEDGF/p75 modulates MeCP2-induced Hsp27 promoter transactivation in a cell-context-dependent manner.","method":"Transcription factor protein arrays, pull-down, AlphaScreen, co-immunoprecipitation, nuclear co-localization by confocal microscopy, Hsp27 promoter reporter assays","journal":"Molecular cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — multiple orthogonal binding methods and functional reporter assay, single lab","pmids":["22275515"],"is_preprint":false},{"year":2003,"finding":"LEDGF/p75 is cleaved by caspases at three sites during apoptosis, generating 65 kDa and 58 kDa fragments; caspase cleavage abolishes the survival function of LEDGF/p75.","method":"Immunoblotting of apoptotic cell extracts, caspase cleavage site mapping","journal":"Autoimmunity reviews","confidence":"Medium","confidence_rationale":"Tier 3 / Moderate — biochemical detection of cleavage products with functional inference, multiple cell systems","pmids":["12965181"],"is_preprint":false},{"year":2004,"finding":"LEDGF/p75 binds to and transactivates heat shock (nGAAn) and stress response elements (A/TGGGGA/T) in promoters of ADH1, ADH4, and RALDH2 genes; overexpression in lens epithelial cells elevates RA production and protects against ethanol stress.","method":"EMSA, supershift assay, CAT reporter transfection, RT-PCR in overexpressing cells","journal":"American journal of physiology. Cell physiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA showing direct DNA binding and reporter assay, two orthogonal methods, single lab","pmids":["15238362"],"is_preprint":false}],"current_model":"PSIP1/LEDGF/p75 is a multifunctional chromatin-associated adaptor protein that tethers protein cargoes to actively transcribed genomic regions via two key domains: an N-terminal PWWP domain that reads H3K36me2/3 marks on nucleosomes through cooperative binding to both the methylated histone tail and nucleosomal DNA, and a C-terminal integrase-binding domain (IBD) that engages diverse partners (HIV-1 integrase, MLL, JPO2, PogZ, Cdc7-ASK) through a shared IBD-binding motif in a mutually exclusive manner; during HIV-1 infection, LEDGF/p75 tethers lentiviral preintegration complexes to active transcription units by forming a stable IN tetramer–LEDGF dimer complex, stimulating integrase strand transfer activity and directing integration site selection, while at the cellular level it also recruits CtIP to DNA double-strand breaks to promote homologous recombination, associates with splicing factors via the p52 isoform to regulate alternative splicing, facilitates RNA Pol II elongation through nucleosome barriers, suppresses R-loop accumulation at gene promoters through interaction with PARP1 and other R-loop homeostasis factors, and transcriptionally activates stress-response genes (Hsp27, αB-crystallin, ADH1, RALDH2, VEGF-C) by binding HSE and STRE promoter elements."},"narrative":{"mechanistic_narrative":"PSIP1 (LEDGF/p75) is a chromatin-associated adaptor that tethers diverse protein cargoes to transcriptionally active genomic regions, reading the H3K36me2/3 mark through its N-terminal PWWP domain and engaging partners through its C-terminal integrase-binding domain (IBD) [PMID:22615581, PMID:23396443, PMID:25082813]. The PWWP domain binds H3K36me3-containing nucleosomes with nanomolar affinity via a bipartite mechanism, simultaneously contacting the methylated histone tail through a hydrophobic cavity and nucleosomal DNA through an adjacent basic surface, an interaction enhanced ~10,000-fold over peptide alone [PMID:23396443, PMID:23656834]; in living cells this supports a 'scan-and-lock' mode of chromatin engagement in which the PWWP domain locks complexes onto chromatin [PMID:20974633]. Through its IBD, PSIP1 binds HIV-1 integrase, MLL, JPO2, PogZ, and the Cdc7-ASK kinase in a mutually exclusive manner using a shared interface [PMID:17669426, PMID:19864417, PMID:25082813]. PSIP1 is the principal cellular cofactor directing HIV-1 integration: it stabilizes and organizes an active integrase tetramer, stimulates strand transfer activity, tethers preintegration complexes to transcription units, and dictates integration site selection — a function shown to be portable, since swapping its chromatin-binding domain for the heterochromatin reader CBX1 redirects integration toward heterochromatin [PMID:12407101, PMID:17639082, PMID:18801737, PMID:20195265, PMID:23593299]. Beyond viral integration, PSIP1 functions broadly in genome regulation: it tethers CtIP to DNA double-strand breaks to promote homologous-recombination repair [PMID:22773103], suppresses genome-wide R-loop accumulation at promoters in concert with PARP1 such that its loss sensitizes cancer cells to PARP inhibitors [PMID:38191578], and facilitates RNA Pol II transcription elongation past nucleosomes [PMID:31616795]. Its short p52 isoform associates with SRSF1 and other splicing factors at H3K36me3-marked active genes to regulate alternative splicing [PMID:22615581, PMID:26545813]. Through the IBD it recruits MLL to Hox target genes, and PSIP1 loss reduces MLL occupancy and impairs MLL-fusion leukemogenesis; correspondingly, Psip1-null mice show homeotic skeletal transformations consistent with disrupted Hox regulation [PMID:16980622, PMID:25082813, PMID:29084774]. PSIP1 also binds heat-shock and stress-response promoter elements to transactivate stress and survival genes [PMID:11350077, PMID:15238362].","teleology":[{"year":2000,"claim":"Establishing that the PSIP1 locus generates two isoforms by alternative splicing defined the molecular basis for the distinct activities later assigned to p75 and p52.","evidence":"gene cloning, exon/intron mapping, and RT-PCR of the chromosome 9p22.2 locus","pmids":["10721720"],"confidence":"High","gaps":["Did not assign function to either isoform","No protein structure or domain function defined"]},{"year":2002,"claim":"Identifying LEDGF/p75 as a stable interactor of HIV-1 integrase that enhances strand transfer activity defined the protein's role as a candidate cellular cofactor for integration.","evidence":"Co-IP from nuclear extracts, gel filtration, in vitro strand transfer assay, and co-localization microscopy","pmids":["12407101"],"confidence":"High","gaps":["Did not establish requirement for viral replication","Domain responsible for IN binding not yet mapped"]},{"year":2003,"claim":"RNAi depletion showed LEDGF/p75 is required for nuclear and chromosomal targeting of integrase and that p52 does not bind IN, establishing isoform-specific cofactor function.","evidence":"RNAi knockdown with live-cell imaging and in vitro pull-down of isoforms","pmids":["12796494"],"confidence":"High","gaps":["Did not distinguish tethering from import requirement","Functional consequence for replication not yet shown"]},{"year":2004,"claim":"Mapping the NLS and demonstrating Ran/importin-dependent nuclear import explained how LEDGF/p75 carries integrase into the nucleus.","evidence":"deletion/mutagenesis with semipermeabilized-cell nuclear import assay and GFP fusions","pmids":["15163664"],"confidence":"High","gaps":["Did not separate import role from chromatin-tethering role"]},{"year":2005,"claim":"Showing the IN-LEDGF interaction is lentivirus-specific and essential for viral replication, separable from IN enzymatic activity, defined the interaction as a discrete druggable cofactor function.","evidence":"isoform/integrase-specific pull-downs, FCS DNA-binding, two-hybrid mutagenesis, and viral replication assays","pmids":["15749713","15855167"],"confidence":"High","gaps":["Atomic basis of the interface not yet resolved","Chromatin-tethering step not yet directly demonstrated"]},{"year":2006,"claim":"Mapping the IN-binding residues on integrase and the conserved D366 contact on LEDGF, plus showing in vivo Hox/developmental phenotypes in knockout mice, jointly defined both the viral interface and an endogenous developmental role.","evidence":"systematic mutagenesis across lentiviral integrases, in vitro strand transfer assays, and gene-trap knockout mouse phenotyping","pmids":["17158150","17137594","16980622"],"confidence":"High","gaps":["Mechanistic link between PSIP1 and Hox gene regulation not yet defined","Chromatin-reading domain function still unresolved"]},{"year":2006,"claim":"Live-cell mutagenesis showed chromatin association is driven by a tripartite NLS/AT-hook element with a modulatory role for the PWWP domain, refining how LEDGF docks on chromatin.","evidence":"SPR/EMSA DNA binding with EGFP-fusion localization in interphase and mitotic cells","pmids":["16549878"],"confidence":"High","gaps":["Did not identify the histone mark recognized by PWWP","Relative contribution of each element to integration not yet quantified"]},{"year":2007,"claim":"Genetic knockout showed LEDGF acts downstream of PIC formation to bias integration into transcription units, establishing the tethering model of integration site selection.","evidence":"LEDGF-null cells with HIV-1 integration site mapping and PIC strand transfer assays","pmids":["17639082"],"confidence":"High","gaps":["Chromatin feature read by LEDGF to define targets not yet identified"]},{"year":2007,"claim":"Defining JPO2 as a cellular IBD ligand competing with integrase introduced the principle that the IBD engages multiple partners through one mutually exclusive site.","evidence":"two-hybrid, AlphaScreen, reciprocal Co-IP, and competition assays","pmids":["17669426"],"confidence":"High","gaps":["Cellular function of the JPO2 interaction unresolved"]},{"year":2008,"claim":"Demonstrating that the PWWP domain binds nucleosomes directly and is the primary determinant for stimulating integration into chromatinized templates connected chromatin reading to cofactor function.","evidence":"systematic PWWP mutagenesis, GST pull-down of nucleosomes, and in vitro integration into polynucleosome templates","pmids":["18799576","18174227"],"confidence":"High","gaps":["Specific histone modification recognized not yet identified"]},{"year":2008,"claim":"Showing LEDGF stabilizes IN-IN contacts and promotes integrase tetramerization revealed it functions as an allosteric organizer of the active integrase multimer, not merely a tether.","evidence":"MS protein footprinting, molecular modeling, and enzymatic assays of full-length IN-LEDGF","pmids":["18801737"],"confidence":"High","gaps":["Stoichiometry of the assembled complex not yet defined"]},{"year":2009,"claim":"Discovery of the Cdc7-ASK interaction and LEDGF-stimulated MCM2 phosphorylation revealed a cellular role in DNA replication kinase activation via the IBD.","evidence":"endogenous reciprocal Co-IP, in vitro kinase assays with MCM2, and phosphorylation-site mapping","pmids":["19864417"],"confidence":"High","gaps":["In vivo importance for replication timing not established"]},{"year":2009,"claim":"The IN NTD+CCD-IBD crystal structure defined the charge-charge basis of lentiviral specificity, providing the structural template for understanding the interaction and its inhibition.","evidence":"X-ray crystallography with mutagenesis and concerted integration/infectivity assays","pmids":["19132083"],"confidence":"High","gaps":["Structure of the full-length IN-LEDGF-DNA assembly not yet resolved"]},{"year":2009,"claim":"ChIP linking LEDGF/p75 to the VEGF-C promoter under hormonal control extended its function to transcriptional regulation of lymphangiogenesis genes.","evidence":"ChIP, siRNA knockdown, and promoter-reporter assays in ovarian cancer cells","pmids":["19934313"],"confidence":"Medium","gaps":["Direct DNA-binding versus tethered recruitment at this promoter not distinguished","Single lab"]},{"year":2010,"claim":"Genome-wide chromatin profiling and the scan-and-lock kinetic model defined where and how LEDGF engages active chromatin, and that integrase binding markedly increases its chromatin affinity.","evidence":"DamID profiling and FRAP/FCS live-cell kinetics with domain mutants","pmids":["20484370","20974633"],"confidence":"High","gaps":["Why only a subset of LEDGF-bound sites support integration unresolved"]},{"year":2010,"claim":"Replacing the chromatin-binding domain with CBX1 to redirect integration to heterochromatin provided causal proof that the tethering domain dictates integration site selection.","evidence":"chimeric protein engineering with lentiviral integration site sequencing","pmids":["20195265"],"confidence":"High","gaps":["Did not address endogenous cellular consequences of retargeting"]},{"year":2011,"claim":"FRET order-of-addition and in vitro tethering assays defined how LEDGF shapes integrase conformation and stimulates IN-DNA binding, sharpening the mechanistic model of complex assembly.","evidence":"FRET of IN assembly and AlphaScreen IN-DNA tethering assays with domain mutants","pmids":["21771857","21763490"],"confidence":"High","gaps":["Medium-confidence quantitation for the tethering assay; single lab"]},{"year":2011,"claim":"Identifying a supercoiled-DNA recognition domain that localizes to transcriptionally active regions added a DNA-topology preference to LEDGF chromatin targeting.","evidence":"recombinant fragment supercoiled-DNA binding and EGFP-SRD localization with immunostaining","pmids":["21345933"],"confidence":"Medium","gaps":["Relationship of SRD to PWWP-based targeting not integrated","Single lab"]},{"year":2012,"claim":"Demonstrating damage-dependent recruitment of CtIP to DSBs via the PWWP domain established a direct role for PSIP1 in homologous-recombination repair.","evidence":"RNAi depletion, CtIP recruitment and DNA-end resection assays, and damage-dependent Co-IP","pmids":["22773103"],"confidence":"High","gaps":["Whether the same PWWP-chromatin contacts mediate both integration and repair tethering not directly compared"]},{"year":2012,"claim":"Identifying MeCP2 as an N-terminal-domain partner shared by both isoforms expanded the transcriptional-regulator interactome of PSIP1.","evidence":"protein arrays, AlphaScreen, Co-IP, confocal co-localization, and Hsp27 reporter assays","pmids":["22275515"],"confidence":"Medium","gaps":["Cell-context dependence of the functional effect unresolved","Single lab"]},{"year":2012,"claim":"Establishing that the PWWP domain specifically reads H3K36me3 and that p52 couples this mark to SRSF1 connected chromatin reading to alternative splicing regulation.","evidence":"PWWP H3K36me3 binding, p52-splicing-factor Co-IP, ChIP, and splicing analysis in Psip1 mutant cells","pmids":["22615581"],"confidence":"High","gaps":["Genome-wide breadth of splicing control not yet quantified"]},{"year":2013,"claim":"NMR structures resolved the bipartite PWWP mechanism — cooperative histone-tail and nucleosomal-DNA contacts producing nanomolar, ~10,000-fold-enhanced nucleosome binding — explaining high-affinity targeting to H3K36me3 chromatin.","evidence":"NMR solution structure, mutagenesis, and thermodynamic binding to reconstituted mononucleosomes","pmids":["23396443","23656834"],"confidence":"High","gaps":["How partner binding at the IBD couples to this PWWP affinity not structurally resolved"]},{"year":2013,"claim":"Defining the IN4:LEDGF2:INI1-IBD2:DNA2 stoichiometry by cryo-EM and MS established the architecture of the LEDGF-stabilized active integration intasome.","evidence":"reconstitution, MS stoichiometry, FCS, integration assays, and cryo-EM","pmids":["23593299"],"confidence":"High","gaps":["High-resolution atomic model of the full assembly not obtained"]},{"year":2014,"claim":"Mapping the MLL interface to the same mutually exclusive IBD site and showing its mutation impairs MLL-AF9 transformation linked the cofactor function to leukemogenesis.","evidence":"NMR interface mapping, mutagenesis, AlphaScreen competition, and colony-forming assays","pmids":["25082813"],"confidence":"High","gaps":["In vivo leukemia dependence not yet tested in this study"]},{"year":2014,"claim":"Complete TALEN knockout confirmed PSIP1's requirement for HIV integration while showing particle assembly and ALLINI action are LEDGF-independent, delimiting the cofactor's mechanistic scope.","evidence":"whole-gene and IBD-exon TALEN knockouts with replication, infectivity, and assembly assays","pmids":["24942577"],"confidence":"High","gaps":["Residual integration in knockout cells not fully explained"]},{"year":2015,"claim":"Affinity purification and RNA-seq in knockout cells established PSIP1 as a broad regulator of alternative splicing and linked intron density to LEDGF-dependent integration patterns.","evidence":"affinity purification of splicing factors and RNA-seq plus >1M integration-site mapping in KO cells","pmids":["26545813"],"confidence":"High","gaps":["Direct versus indirect contribution to each splicing event not resolved"]},{"year":2017,"claim":"Conditional knockout and ChIP showed PSIP1 sustains MLL occupancy at Hox genes and is required for MLL-fusion leukemia initiation but dispensable for normal hematopoiesis, defining a context-specific oncogenic dependency.","evidence":"conditional KO mouse, Hox expression, MLL ChIP, and in vitro/in vivo leukemia models","pmids":["29084774"],"confidence":"High","gaps":["Molecular basis of leukemia-specific dependence not fully defined"]},{"year":2017,"claim":"Additional studies tied p52 to lncRNA Hottip-dependent Hoxa activation and p75 to RNA Pol II recruitment at cell-cycle gene promoters in breast cancer, broadening PSIP1's transcriptional roles.","evidence":"Hottip knockdown/knockout and Mll1 ChIP; PSIP1-Pol II Co-IP, promoter ChIP, and tumorigenicity assays","pmids":["28384324","28633434"],"confidence":"Medium","gaps":["Single-lab findings","Mechanistic link between Pol II recruitment and tumorigenicity not fully resolved"]},{"year":2019,"claim":"Showing PWWP-mediated H3K36me2/3 binding supports RNA Pol II elongation past nucleosomes defined a FACT-like role in transcription elongation.","evidence":"genome-wide co-localization with H3K36me2/3 and RNAPII ChIP-seq (primary focus on paralog HDGF2)","pmids":["31616795"],"confidence":"Medium","gaps":["Direct elongation defect upon LEDGF/p75 loss not separately demonstrated","Mechanism inferred largely from HDGF2"]},{"year":2020,"claim":"Dissecting PSIP1-dependent competition between PAF1 and the CKII-activated MLL1/SEC at the provirus explained how LEDGF toggles HIV transcription between latency and activation.","evidence":"reciprocal Co-IP, ChIP at the provirus, and CKII inhibition in latency-reversal assays","pmids":["32426500"],"confidence":"High","gaps":["Whether the same switch operates at host genes not addressed"]},{"year":2024,"claim":"Defining PSIP1's interaction with PARP1 and R-loop homeostasis factors revealed it suppresses promoter R-loops and transcription-replication conflicts, with loss imposing PARP-inhibitor sensitivity.","evidence":"Co-IP, DRIP-seq, γ-H2AX ChIP-seq, 53BP1/RAD51 foci, and PARP-inhibitor viability assays in PSIP1-depleted cells","pmids":["38191578"],"confidence":"High","gaps":["Mechanism by which PSIP1 resolves R-loops not established","Relationship to its HR/CtIP function not integrated"]},{"year":null,"claim":"How PSIP1 coordinates its single chromatin-reading domain and one mutually exclusive IBD across its many roles — integration, HR repair, splicing, elongation, R-loop control, and MLL-driven transcription — into context-specific outcomes remains unresolved.","evidence":"","pmids":[],"confidence":"High","gaps":["No unified model of how partner selection at the IBD is regulated in cells","Whether PWWP chromatin reading is rate-limiting for each function is untested","High-resolution structures of most non-viral complexes are lacking"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[13,22,26,31]},{"term_id":"GO:0042393","term_label":"histone binding","supporting_discovery_ids":[27,28,29]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[6,9,25,28]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[3,35,43]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[15,18,30]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[4,5]},{"term_id":"GO:0000228","term_label":"nuclear chromosome","supporting_discovery_ids":[9,20,21]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[0,13,32,38]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[26,39]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[27,33,35,37]},{"term_id":"R-HSA-8953854","term_label":"Metabolism of RNA","supporting_discovery_ids":[27,33]},{"term_id":"R-HSA-4839726","term_label":"Chromatin organization","supporting_discovery_ids":[20,28,31,34]}],"complexes":["HIV-1 preintegration complex (IN tetramer–LEDGF dimer)","MLL complex","PAF1 complex (at HIV provirus)"],"partners":["HIV-1 INTEGRASE","MLL","JPO2","POGZ","CDC7","CTIP","PARP1","SRSF1"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"O75475","full_name":"PC4 and SFRS1-interacting protein","aliases":["CLL-associated antigen KW-7","Dense fine speckles 70 kDa protein","DFS 70","Lens epithelium-derived growth factor","Transcriptional coactivator p75/p52"],"length_aa":530,"mass_kda":60.1,"function":"Transcriptional coactivator involved in neuroepithelial stem cell differentiation and neurogenesis. Involved in particular in lens epithelial cell gene regulation and stress responses. May play an important role in lens epithelial to fiber cell terminal differentiation. May play a protective role during stress-induced apoptosis. Isoform 2 is a more general and stronger transcriptional coactivator. Isoform 2 may also act as an adapter to coordinate pre-mRNA splicing. Cellular cofactor for lentiviral integration","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/O75475/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PSIP1","classification":"Not Classified","n_dependent_lines":16,"n_total_lines":1208,"dependency_fraction":0.013245033112582781},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"NUCKS1","stoichiometry":4.0},{"gene":"SLC7A6","stoichiometry":4.0},{"gene":"CBX1","stoichiometry":0.2},{"gene":"H2AFZ","stoichiometry":0.2},{"gene":"HIST2H2BE","stoichiometry":0.2},{"gene":"HMGA1","stoichiometry":0.2},{"gene":"NUMA1","stoichiometry":0.2},{"gene":"PARP1","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/PSIP1","total_profiled":1310},"omim":[{"mim_id":"603620","title":"PC4- AND SFRS1-INTERACTING PROTEIN 1; PSIP1","url":"https://www.omim.org/entry/603620"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Enhanced","locations":[{"location":"Nucleoplasm","reliability":"Enhanced"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PSIP1"},"hgnc":{"alias_symbol":["p52","LEDGF","p75","DFS70"],"prev_symbol":["PSIP2"]},"alphafold":{"accession":"O75475","domains":[{"cath_id":"2.30.30.140","chopping":"10-88","consensus_level":"high","plddt":91.9628,"start":10,"end":88},{"cath_id":"1.20.930.10","chopping":"343-431","consensus_level":"high","plddt":93.6458,"start":343,"end":431}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/O75475","model_url":"https://alphafold.ebi.ac.uk/files/AF-O75475-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-O75475-F1-predicted_aligned_error_v6.png","plddt_mean":62.62},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PSIP1","jax_strain_url":"https://www.jax.org/strain/search?query=PSIP1"},"sequence":{"accession":"O75475","fasta_url":"https://rest.uniprot.org/uniprotkb/O75475.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/O75475/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/O75475"}},"corpus_meta":[{"pmid":"12407101","id":"PMC_12407101","title":"HIV-1 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The alternative splice variant p52 did not interact with HIV-1 IN in vitro or in living cells.\",\n      \"method\": \"RNAi knockdown, EGFP/HcRed1 live-cell fluorescence imaging, in vitro pull-down, domain deletion/mutant analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — RNAi loss-of-function with clear phenotypic readout, live-cell imaging, and in vitro binding, replicated across multiple studies\",\n      \"pmids\": [\"12796494\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"LEDGF/p75 and p52 are derived from a single gene (PSIP1) by alternative splicing; exons 1–15 encode p75 and exons 1–9 plus part of intron 9 encode p52; the gene is located on chromosome 9p22.2.\",\n      \"method\": \"Gene cloning, sequencing, exon/intron mapping, RT-PCR expression analysis\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — definitive molecular characterization of gene structure, replicated in subsequent literature\",\n      \"pmids\": [\"10721720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"LEDGF binds to heat shock element (HSE; nGAAn) and stress-related regulatory element (STRE; A/TGGGGA/T) in the promoters of stress-related genes (Hsp27 and αB-crystallin) to activate their transcription.\",\n      \"method\": \"DNA-binding assay, transcriptional reporter assay, promoter element deletion/mutation\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and transactivation assays, single lab but two orthogonal methods\",\n      \"pmids\": [\"11350077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"LEDGF/p75 is distributed diffusely in the nucleoplasm during G1 and attaches to condensed chromatin during G2/M (mitosis), whereas p52 localizes to the nuclear periphery in G1 and forms speckles at S-phase, indicating distinct nuclear compartments and functions.\",\n      \"method\": \"Live-cell fluorescence imaging of GFP-tagged proteins across cell cycle stages in CHO-K1, MCDK, and NRK cells\",\n      \"journal\": \"Cell and tissue research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — direct localization by live imaging in multiple cell lines, single lab\",\n      \"pmids\": [\"11512661\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"LEDGF/p75 nuclear import is GTP-, Ran-, importin-α/β-, and energy-dependent; the functional NLS was mapped to residues 148–156 (GRKRKAEKQ); a single amino acid change in the NLS excluded LEDGF/p75 from the nucleus and abolished nuclear import of HIV-1 integrase.\",\n      \"method\": \"Deletion analysis, site-directed mutagenesis, semipermeabilized cell nuclear import assay, GFP fusion constructs in live cells, β-galactosidase NLS transfer assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis with functional nuclear import assay and multiple orthogonal validations\",\n      \"pmids\": [\"15163664\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"LEDGF/p75 interacts with HIV-1, HIV-2, and feline immunodeficiency virus integrase but not with non-lentiviral integrases (HTLV-2, MoMLV, RSV); LEDGF/p75 strongly promoted binding of HIV-1 and HIV-2 IN to DNA, an effect specific to the p75 isoform and not seen with p52.\",\n      \"method\": \"Pull-down binding assay, fluorescence correlation spectroscopy for DNA-binding measurements, isoform-specific comparisons\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro binding with biophysical quantification, lentiviral-specificity shown across multiple integrases\",\n      \"pmids\": [\"15749713\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Interaction of HIV-1 integrase with LEDGF/p75 is essential for viral replication and chromosomal tethering of IN; a single integrase mutation that disrupts LEDGF/p75 interaction (without reducing enzymatic activity) results in defective HIV-1 replication.\",\n      \"method\": \"Two-hybrid assays, random and directed mutagenesis, viral replication assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic mutation + viral replication assay, multiple orthogonal approaches, replicated in other studies\",\n      \"pmids\": [\"15855167\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Both the IN-binding and DNA-binding activities of LEDGF/p75 contribute to functional reconstitution of HIV-1 preintegration complex (PIC) in vitro; recombinant LEDGF/p75 efficiently reconstitutes high-salt-disrupted PIC activity.\",\n      \"method\": \"In vitro PIC reconstitution assay with recombinant proteins, mutational analysis of LEDGF/p75 domains\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — reconstitution in vitro shown, single lab, not independently replicated in this form\",\n      \"pmids\": [\"16337983\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"LEDGF/p75 chromatin association in living cells is mediated by a tripartite element comprising the NLS and two AT-hook motifs (residues 146–197); the PWWP domain is not required for binding condensed mitotic chromosomes but subtly affects interphase nucleoplasmic distribution; neither DNA-binding-deficient LEDGF/p75 mutants nor the AT-hook mutant lost chromatin binding unless combined with NLS mutations or PWWP deletion.\",\n      \"method\": \"SPR and EMSA for DNA binding, EGFP fusion localization in interphase and mitotic cells, domain deletion and AT-hook mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (SPR, EMSA, live-cell imaging, systematic mutagenesis) in single rigorous study\",\n      \"pmids\": [\"16549878\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"LEDGF/p75 interacts with and stimulates strand transfer activity of all lentiviral integrases tested (BIV, MVV, EIAV) but not non-lentiviral integrases; mutation D366N in LEDGF ablates interaction with all lentiviral INs, suggesting a conserved recognition mechanism; in the presence of LEDGF, EIAV IN almost exclusively catalyzes concerted integration.\",\n      \"method\": \"Pull-down, yeast two-hybrid, in vitro strand transfer assay with divergent lentiviral INs, site-directed mutagenesis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro enzymatic assay with mutagenesis across multiple integrases, demonstrates conserved mechanism\",\n      \"pmids\": [\"17158150\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Psip1/Ledgf knockout mice exhibit perinatal mortality, homeotic skeletal transformations, motor/behavioral defects, and craniofacial abnormalities, implicating PSIP1 in Hox gene expression regulation in vivo.\",\n      \"method\": \"Gene trap mutagenesis in mouse ES cells, homozygous knockout mouse phenotypic analysis\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic knockout mouse model with multiple well-defined phenotypic readouts\",\n      \"pmids\": [\"16980622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The LEDGF/p75-binding site on HIV-1 integrase maps to two regions: residues around W131/W132 and residues I161–E170; mutations at W131, I161, R166, Q168, and E170 impair LEDGF/p75 interaction but retain IN enzymatic activity; the W131A mutation reduces HIV-1 replication.\",\n      \"method\": \"Yeast two-hybrid, in vitro binding/pull-down, IN enzymatic activity assays, HIV-1 replication assays, structural comparison\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — systematic mutagenesis with multiple functional assays and structural comparison\",\n      \"pmids\": [\"17137594\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"LEDGF/p75 functions downstream from HIV-1 preintegration complex formation to direct integration into transcription units; in LEDGF-null cells, HIV-1 loses its strong bias toward integrating into transcription units and instead shows increased affinity for promoter regions and CpG islands, while 3'-end processing and local target DNA sequence preference are unaffected.\",\n      \"method\": \"Genetic knockout of LEDGF, HIV-1 vector integration site mapping, quantitative PCR, in vitro strand transfer assays with PICs from knockout cells\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic KO with integration site mapping and in vitro PIC assays, replicated in subsequent studies\",\n      \"pmids\": [\"17639082\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"JPO2 binds the C-terminal integrase-binding domain (IBD) of LEDGF/p75 in a mutually exclusive manner with HIV-1 integrase; the binding mechanism differs (JPO2 continues to interact with some IN-defective LEDGF/p75 mutants like I365A, D366A, F406A); JPO2 overexpression modestly inhibits HIV-1 replication.\",\n      \"method\": \"Yeast two-hybrid, pull-down, AlphaScreen, co-immunoprecipitation, competition assays with recombinant proteins, HIV-1 replication assay\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, AlphaScreen, and competition assays with multiple orthogonal methods in single study\",\n      \"pmids\": [\"17669426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"LEDGF/p75 strongly stabilizes IN-IN subunit interactions and promotes IN tetramerization; mass spectrometric protein footprinting revealed novel intra- and inter-protein contacts in the full-length IN-LEDGF complex beyond the IBD-CCD co-crystal structure; the IN tetramer interface is important for enzymatic activities and high-affinity LEDGF binding.\",\n      \"method\": \"Mass spectrometric protein footprinting, molecular modeling, biochemical characterization of full-length IN-LEDGF interactions, enzymatic assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — MS footprinting combined with enzymatic assays, multiple orthogonal methods\",\n      \"pmids\": [\"18801737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The PWWP domain of LEDGF/p75 is required for chromatin binding and HIV-1 infectivity; mutations at W21 or A51 (which line a hydrophobic cavity conserved among Tudor clan members) disrupt chromatin binding and virus infectivity; W21A recombinant protein is preferentially defective for enhancing integration into chromatinized (but not naked) DNA in vitro.\",\n      \"method\": \"Systematic mutagenesis of 24 PWWP residues, chromatin binding assays, HIV-1 infectivity assays, in vitro integration into chromatinized templates\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — systematic mutagenesis with multiple functional readouts (chromatin binding, infectivity, in vitro integration)\",\n      \"pmids\": [\"18799576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The PWWP domain of LEDGF/p75 is required for stimulation of HIV-1 integration into reconstituted polynucleosome (chromatinized) templates; the PWWP domain binds directly to nucleosomes in GST pull-down assays; with naked DNA, full removal of N-terminal chromatin-binding elements is needed to abate cofactor function, but with polynucleosomes, PWWP domain is the primary determinant.\",\n      \"method\": \"In vitro integration assay with reconstituted polynucleosome templates, GST pull-down, domain deletion analysis\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution with defined substrates, multiple domain mutants, and direct nucleosome-binding assay\",\n      \"pmids\": [\"18174227\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"LEDGF/p75 interacts with the Cdc7-ASK (activator of S-phase kinase) heterodimer through its integrase-binding domain (IBD); the interaction requires autophosphorylation of Cdc7 and the C-terminal 50 residues of ASK; Cdc7-ASK phosphorylates LEDGF at Ser-206 (primarily during S phase); LEDGF potently stimulates Cdc7-ASK kinase activity, increasing MCM2 phosphorylation >10-fold in vitro, by relieving ASK C-terminus-mediated autoinhibition.\",\n      \"method\": \"Co-immunoprecipitation of endogenous proteins, truncation analysis, in vitro kinase assay with MCM2 substrate, phosphorylation site mapping\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reciprocal Co-IP of endogenous proteins, in vitro kinase assay, phosphorylation site mapping, mechanistic relief-of-autoinhibition demonstrated\",\n      \"pmids\": [\"19864417\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"LEDGF/p75 crystal structure (HIV-2 IN NTD+CCD in complex with LEDGF IBD) revealed charge-charge interactions between the IN N-terminal domain (NTD) and the IBD; a constellation of acidic residues on the NTD is characteristic of lentiviral INs; mutations of positively charged IBD residues severely impaired interaction with all lentiviral INs and abrogated stimulation of concerted integration and HIV-1 replication.\",\n      \"method\": \"X-ray crystallography, site-directed mutagenesis, in vitro concerted integration assay, HIV-1 infectivity assays\",\n      \"journal\": \"PLoS pathogens\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and functional assays in single study\",\n      \"pmids\": [\"19132083\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"LEDGF/p75 is primarily bound downstream of transcription start sites of active transcription units in chromatin, co-localizing with active chromatin markers (H3/H4 acetylation, H3K4me1, RNA Pol II); not all LEDGF/p75-bound chromosomal complexes are amenable to HIV-1 integration.\",\n      \"method\": \"DamID chromatin profiling in ENCODE regions, correlation analysis with >200 genomic features including HIV-1 integration sites\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide chromatin profiling (DamID), single lab study with bioinformatic correlation\",\n      \"pmids\": [\"20484370\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"LEDGF/p75 chromatin binding in living cells follows a 'scan-and-lock' mechanism: the protein moves in a chromatin hopping/scanning mode; the PWWP domain is necessary but not sufficient for in vivo chromatin binding; upon binding HIV-1 integrase via its IBD, LEDGF/p75 kinetics shift to 75-fold larger chromatin affinity, with PWWP domain crucial for locking the complex on chromatin.\",\n      \"method\": \"FRAP (spot- and half-nucleus), continuous photobleaching, fluorescence correlation spectroscopy, tunable focus FCS in living HeLa cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple quantitative live-cell fluorescence methods, mechanistic scan-and-lock model with quantitative parameters\",\n      \"pmids\": [\"20974633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Replacement of the LEDGF/p75 chromatin interaction domain with CBX1 (which binds H3K9me2/3 at pericentric heterochromatin) redirects lentiviral vector integration away from genes and toward heterochromatin, demonstrating that LEDGF/p75 chromatin-tethering domain determines integration site selection.\",\n      \"method\": \"Chimeric protein engineering, lentiviral vector transduction, integration site sequencing\",\n      \"journal\": \"Molecular therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic domain swap with integration site mapping provides causal evidence for tethering mechanism\",\n      \"pmids\": [\"20195265\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"FRET analysis revealed that HIV-1 IN tetramers adopt distinct conformations in the presence of viral DNA versus LEDGF/p75; pre-formed IN-viral DNA complex conformation is unchanged upon subsequent LEDGF/p75 binding, but pre-incubation with LEDGF followed by viral DNA yields IN conformation similar to the IN-LEDGF complex.\",\n      \"method\": \"FRET monitoring of IN subunit assembly in the presence of viral DNA and LEDGF/p75, order-of-addition experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — quantitative FRET with systematic order-of-addition experiments reveals structural mechanistic insight\",\n      \"pmids\": [\"21771857\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"LEDGF/p75 tethers HIV-1 IN to DNA in vitro: LEDGF/p75 stimulates IN binding to DNA 10–30-fold in a manner requiring direct IN-LEDGF C-terminus interaction; overexpression of LEDGF C-terminus or NLS/AT-hook mutant LEDGF inhibits IN-DNA interaction.\",\n      \"method\": \"AlphaScreen assay for IN-DNA interaction, domain-deletion analysis, in vitro DNA-binding\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — quantitative in vitro tethering assay, single lab, two orthogonal approaches\",\n      \"pmids\": [\"21763490\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"LEDGF/p75 recognizes supercoiled DNA preferentially over unconstrained DNA via a novel 'supercoiled DNA-recognition domain' (SRD) containing a K/E/D cluster; GFP-SRD in cells localizes to transcriptionally active regions (H3K4me3, Br-UTP incorporation); deletion of SRD abolishes this localization.\",\n      \"method\": \"Recombinant protein fragments with in vitro supercoiled DNA-binding assays, EGFP-tagged localization with immunostaining in live cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro binding assay combined with cellular localization experiment, single lab\",\n      \"pmids\": [\"21345933\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"LEDGF/p75 promotes DNA double-strand break repair by homologous recombination; LEDGF depletion impairs recruitment of CtIP to DNA DSBs and CtIP-dependent DNA-end resection; LEDGF binds CtIP in a DNA damage-dependent manner through its PWWP domain interaction with chromatin, tethering CtIP to active chromatin near DSBs.\",\n      \"method\": \"RNAi depletion, CtIP recruitment assay at DSBs, DNA-end resection assay, co-immunoprecipitation (damage-dependent), chromatin binding\",\n      \"journal\": \"Nature structural & molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — loss-of-function with specific molecular readout (CtIP recruitment, resection), damage-dependent Co-IP, published in high-impact journal\",\n      \"pmids\": [\"22773103\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"The PWWP domain of Psip1/Ledgf specifically recognizes trimethylated H3K36; the p52 (short) isoform, enriched at active genes like H3K36me3, co-localizes and interacts with Srsf1 and other mRNA processing proteins; in Psip1 mutant cells, H3K36me3-associated Srsf1 is reduced and alternative splicing of specific genes is altered.\",\n      \"method\": \"PWWP domain H3K36me3 binding assay, co-immunoprecipitation of p52 with splicing factors, ChIP, splicing analysis in Psip1 mutant cells\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding assay, Co-IP, ChIP, RNA splicing analysis) with genetic knockout cells\",\n      \"pmids\": [\"22615581\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"The LEDGF PWWP domain binds H3K36me3-containing mononucleosomes with nanomolar affinity via two distinct functional interfaces: a hydrophobic cavity that selectively contacts H3K36me3 peptide, and an adjacent basic surface that non-specifically binds DNA; cooperative binding to both methylated histone tail and nucleosomal DNA is essential for high-affinity chromatin binding.\",\n      \"method\": \"NMR solution structure of PWWP domain, binding to H3K36me3 peptide and DNA monitored by NMR, affinity measurements to native and reconstituted mononucleosomes, mutational analysis, proteomic experiments\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR structure with functional validation, mutagenesis, and quantitative binding to reconstituted mononucleosomes\",\n      \"pmids\": [\"23396443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PSIP1-PWWP binds H3K36-methylated nucleosomes through simultaneous bipartite interactions with the methylated histone tail and nucleosomal DNA; binding is enhanced ~10,000-fold compared to methylated peptide alone; this bipartite mechanism was structurally characterized by NMR and mutational analysis.\",\n      \"method\": \"NMR spectroscopy, extensive mutational analysis, computational approaches, thermodynamic binding measurements\",\n      \"journal\": \"Epigenetics & chromatin\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — NMR-derived structure with mutational analysis and quantitative thermodynamic measurements\",\n      \"pmids\": [\"23656834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In the HIV-1 preintegration complex, LEDGF/p75 organizes and stabilizes an active integrase tetramer (stoichiometry IN4:LEDGF2:INI1-IBD2:DNA2); INI1-IBD occupies the cellular DNA binding site of the IN-LEDGF complex and constrains integrase in a stable conformation.\",\n      \"method\": \"Stable complex reconstitution, mass spectrometry stoichiometry determination, fluorescence correlation spectroscopy, functional integration assay, cryo-electron microscopy\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro reconstitution, cryo-EM structure, mass spectrometry stoichiometry, and functional assays\",\n      \"pmids\": [\"23593299\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"NMR spectroscopy identified an additional LEDGF/p75-MLL interface overlapping with the HIV-1 integrase, PogZ, and JPO2 binding site on the IBD; binding of MLL, HIV-1 IN, PogZ, and JPO2 to LEDGF/p75 is mutually exclusive; the MLL interaction is primarily sustained by two aromatic residues (F148 and F151 of MLL); mutation of this interface impairs MLL-AF9+ leukemic cell transformation.\",\n      \"method\": \"NMR spectroscopy for interface mapping, mutational analysis, AlphaScreen competition assays, colony-forming assays\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — NMR structure with mutational analysis and functional transformation assay\",\n      \"pmids\": [\"25082813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Complete TALEN-mediated knockout of PSIP1 in human cells severely impairs HIV-1 spreading replication and single-cycle integration; HIV-1 particle assembly and the main ALLINI (allosteric integrase inhibitor) mechanism are LEDGF/p75-independent.\",\n      \"method\": \"TALEN-mediated gene knockout (whole-gene deletion and IBD exon deletion), HIV-1 replication and single-cycle infectivity assays, virus assembly assays\",\n      \"journal\": \"Journal of virology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — definitive gene knockout with specific mechanistic readouts for integration, assembly, and inhibitor mechanism\",\n      \"pmids\": [\"24942577\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"LEDGF/p75 is associated with multiple mRNA splicing factors by affinity purification; LEDGF/p75 (or its IBD) contributes to splicing patterns in approximately half of transcription units with alternative isoforms; HIV-1 integration density in transcription units correlates with intron number in a LEDGF-dependent manner.\",\n      \"method\": \"Affinity purification of LEDGF/p75-associated splicing factors, RNA-seq analysis of LEDGF/p75 or IBD knockout HEK293T cells, HIV-1 integration site sequencing (>1 million sites), multivariate analysis\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — affinity purification + RNA-seq in KO cells + integration site mapping, multiple orthogonal methods\",\n      \"pmids\": [\"26545813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Conditional Psip1 knockout in hematopoietic cells decreases Hox gene expression, reduces MLL occupancy at MLL target genes, and impairs MLL-fusion-mediated leukemia initiation in vitro and in vivo, but is dispensable for steady-state hematopoiesis.\",\n      \"method\": \"Conditional knockout mouse model, Hox gene expression analysis, ChIP for MLL occupancy, in vitro colony-forming assays, in vivo bone marrow transplant leukemia model\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — conditional KO mouse model with molecular readouts (ChIP, gene expression) and both in vitro and in vivo functional assays\",\n      \"pmids\": [\"29084774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PSIP1/p75 interacts with RNA polymerase II and facilitates its association with promoters of cell cycle genes, thereby regulating their transcription and promoting triple-negative breast cancer tumorigenicity.\",\n      \"method\": \"Co-immunoprecipitation of PSIP1 with RNA Pol II, ChIP for RNA Pol II at promoters in PSIP1-depleted cells, gene expression analysis, tumorigenicity assays\",\n      \"journal\": \"Carcinogenesis\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and ChIP showing PSIP1-Pol II interaction and promoter association, single lab\",\n      \"pmids\": [\"28633434\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Psip1/p52 (short isoform) specifically regulates expression of lncRNA Hottip at the Hoxa locus; Hottip is required for activation of posterior Hoxa genes (Hoxa13, Hoxa10/11) and for retaining Mll1 at the 5' end of Hoxa; the Hottip RNA molecule itself (not just its transcription) is required for Hox gene activation.\",\n      \"method\": \"Knockdown and knockout approaches, Hottip overexpression, premature transcription termination engineering, Mll1 ChIP\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO/KD with multiple orthogonal functional approaches, single lab\",\n      \"pmids\": [\"28384324\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"LEDGF/p75 (and HDGF2) bind H3K36me2 and H3K36me3 via their PWWP domains and, like FACT, facilitate RNA Pol II transcription elongation past nucleosome barriers; in differentiated myotubes, HDGF2 depletion causes paused RNAPII accumulation within transcribed regions of target genes, indicating a defect in early elongation.\",\n      \"method\": \"Genome-wide co-localization with H3K36me2/3, HDGF2 knockout in myoblasts/myotubes, RNAPII ChIP-seq in KO cells\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO with RNAPII ChIP-seq readout; primary mechanistic focus is on HDGF2, but LEDGF role is defined by co-localization and parallel domain architecture\",\n      \"pmids\": [\"31616795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"LEDGF/p75 differentially regulates HIV transcription: during latency it suppresses proviral transcription by recruiting PAF1 complex to promote RNAPII promoter-proximal pausing; upon latency reversal, casein kinase II (CKII)-dependent MLL1 complex competitively displaces PAF1 from LEDGF/p75 at the provirus, enabling Super Elongation Complex recruitment and transcriptional activation.\",\n      \"method\": \"Co-immunoprecipitation, ChIP for PAF1/MLL1/SEC at provirus, CKII pharmacological inhibition and depletion, HIV latency reversal assays\",\n      \"journal\": \"Science advances\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, ChIP at provirus, pharmacological inhibition with clear mechanistic model supported by multiple orthogonal experiments\",\n      \"pmids\": [\"32426500\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"PSIP1 interacts with R-loops and proteins involved in R-loop homeostasis including PARP1; depletion of PSIP1 leads to genome-wide accumulation of R-loops and DNA damage (γ-H2AX) at gene promoters, local transcriptional arrest, transcription-replication conflicts, increased 53BP1 foci, and reduced RAD51 foci (indicating altered DNA repair pathway choice toward NHEJ); PSIP1 depletion sensitizes cancer cells to PARP1 inhibitors.\",\n      \"method\": \"Co-immunoprecipitation of PSIP1 with PARP1 and R-loop-associated proteins, genome-wide R-loop mapping (DRIP-seq), γ-H2AX ChIP-seq, 53BP1 and RAD51 foci analysis, cell viability assays with PARP inhibitors in PSIP1-depleted cells\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal genome-wide and cellular assays (DRIP-seq, ChIP-seq, foci analysis, Co-IP) with functional consequence (PARP inhibitor sensitivity)\",\n      \"pmids\": [\"38191578\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"LEDGF/p75 binds the VEGF-C promoter (demonstrated by ChIP); FSH augments this binding and increases LEDGF/p75 mRNA and protein levels; siRNA suppression of LEDGF/p75 reduces hormonally induced VEGF-C expression, linking LEDGF/p75 to gonadotropin-regulated lymphangiogenesis in ovarian cancer cells.\",\n      \"method\": \"ChIP, siRNA knockdown, promoter-luciferase reporter assay, RT-PCR\",\n      \"journal\": \"Cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and siRNA with functional reporter assay, single lab\",\n      \"pmids\": [\"19934313\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"LEDGF/p75 and p52 both interact with MeCP2 in vitro and in human cancer cells; the interaction maps to the N-terminal PWWP-CR1 domain shared by both isoforms; LEDGF/p75 modulates MeCP2-induced Hsp27 promoter transactivation in a cell-context-dependent manner.\",\n      \"method\": \"Transcription factor protein arrays, pull-down, AlphaScreen, co-immunoprecipitation, nuclear co-localization by confocal microscopy, Hsp27 promoter reporter assays\",\n      \"journal\": \"Molecular cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — multiple orthogonal binding methods and functional reporter assay, single lab\",\n      \"pmids\": [\"22275515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"LEDGF/p75 is cleaved by caspases at three sites during apoptosis, generating 65 kDa and 58 kDa fragments; caspase cleavage abolishes the survival function of LEDGF/p75.\",\n      \"method\": \"Immunoblotting of apoptotic cell extracts, caspase cleavage site mapping\",\n      \"journal\": \"Autoimmunity reviews\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 / Moderate — biochemical detection of cleavage products with functional inference, multiple cell systems\",\n      \"pmids\": [\"12965181\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"LEDGF/p75 binds to and transactivates heat shock (nGAAn) and stress response elements (A/TGGGGA/T) in promoters of ADH1, ADH4, and RALDH2 genes; overexpression in lens epithelial cells elevates RA production and protects against ethanol stress.\",\n      \"method\": \"EMSA, supershift assay, CAT reporter transfection, RT-PCR in overexpressing cells\",\n      \"journal\": \"American journal of physiology. Cell physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA showing direct DNA binding and reporter assay, two orthogonal methods, single lab\",\n      \"pmids\": [\"15238362\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PSIP1/LEDGF/p75 is a multifunctional chromatin-associated adaptor protein that tethers protein cargoes to actively transcribed genomic regions via two key domains: an N-terminal PWWP domain that reads H3K36me2/3 marks on nucleosomes through cooperative binding to both the methylated histone tail and nucleosomal DNA, and a C-terminal integrase-binding domain (IBD) that engages diverse partners (HIV-1 integrase, MLL, JPO2, PogZ, Cdc7-ASK) through a shared IBD-binding motif in a mutually exclusive manner; during HIV-1 infection, LEDGF/p75 tethers lentiviral preintegration complexes to active transcription units by forming a stable IN tetramer–LEDGF dimer complex, stimulating integrase strand transfer activity and directing integration site selection, while at the cellular level it also recruits CtIP to DNA double-strand breaks to promote homologous recombination, associates with splicing factors via the p52 isoform to regulate alternative splicing, facilitates RNA Pol II elongation through nucleosome barriers, suppresses R-loop accumulation at gene promoters through interaction with PARP1 and other R-loop homeostasis factors, and transcriptionally activates stress-response genes (Hsp27, αB-crystallin, ADH1, RALDH2, VEGF-C) by binding HSE and STRE promoter elements.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PSIP1 (LEDGF/p75) is a chromatin-associated adaptor that tethers diverse protein cargoes to transcriptionally active genomic regions, reading the H3K36me2/3 mark through its N-terminal PWWP domain and engaging partners through its C-terminal integrase-binding domain (IBD) [#27, #28, #31]. The PWWP domain binds H3K36me3-containing nucleosomes with nanomolar affinity via a bipartite mechanism, simultaneously contacting the methylated histone tail through a hydrophobic cavity and nucleosomal DNA through an adjacent basic surface, an interaction enhanced ~10,000-fold over peptide alone [#28, #29]; in living cells this supports a 'scan-and-lock' mode of chromatin engagement in which the PWWP domain locks complexes onto chromatin [#21]. Through its IBD, PSIP1 binds HIV-1 integrase, MLL, JPO2, PogZ, and the Cdc7-ASK kinase in a mutually exclusive manner using a shared interface [#14, #18, #31]. PSIP1 is the principal cellular cofactor directing HIV-1 integration: it stabilizes and organizes an active integrase tetramer, stimulates strand transfer activity, tethers preintegration complexes to transcription units, and dictates integration site selection — a function shown to be portable, since swapping its chromatin-binding domain for the heterochromatin reader CBX1 redirects integration toward heterochromatin [#0, #13, #15, #22, #30]. Beyond viral integration, PSIP1 functions broadly in genome regulation: it tethers CtIP to DNA double-strand breaks to promote homologous-recombination repair [#26], suppresses genome-wide R-loop accumulation at promoters in concert with PARP1 such that its loss sensitizes cancer cells to PARP inhibitors [#39], and facilitates RNA Pol II transcription elongation past nucleosomes [#37]. Its short p52 isoform associates with SRSF1 and other splicing factors at H3K36me3-marked active genes to regulate alternative splicing [#27, #33]. Through the IBD it recruits MLL to Hox target genes, and PSIP1 loss reduces MLL occupancy and impairs MLL-fusion leukemogenesis; correspondingly, Psip1-null mice show homeotic skeletal transformations consistent with disrupted Hox regulation [#11, #31, #34]. PSIP1 also binds heat-shock and stress-response promoter elements to transactivate stress and survival genes [#3, #43].\"\n  ,\n  \"teleology\": [\n    {\n      \"year\": 2000,\n      \"claim\": \"Establishing that the PSIP1 locus generates two isoforms by alternative splicing defined the molecular basis for the distinct activities later assigned to p75 and p52.\",\n      \"evidence\": \"gene cloning, exon/intron mapping, and RT-PCR of the chromosome 9p22.2 locus\",\n      \"pmids\": [\"10721720\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not assign function to either isoform\", \"No protein structure or domain function defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identifying LEDGF/p75 as a stable interactor of HIV-1 integrase that enhances strand transfer activity defined the protein's role as a candidate cellular cofactor for integration.\",\n      \"evidence\": \"Co-IP from nuclear extracts, gel filtration, in vitro strand transfer assay, and co-localization microscopy\",\n      \"pmids\": [\"12407101\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish requirement for viral replication\", \"Domain responsible for IN binding not yet mapped\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"RNAi depletion showed LEDGF/p75 is required for nuclear and chromosomal targeting of integrase and that p52 does not bind IN, establishing isoform-specific cofactor function.\",\n      \"evidence\": \"RNAi knockdown with live-cell imaging and in vitro pull-down of isoforms\",\n      \"pmids\": [\"12796494\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not distinguish tethering from import requirement\", \"Functional consequence for replication not yet shown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Mapping the NLS and demonstrating Ran/importin-dependent nuclear import explained how LEDGF/p75 carries integrase into the nucleus.\",\n      \"evidence\": \"deletion/mutagenesis with semipermeabilized-cell nuclear import assay and GFP fusions\",\n      \"pmids\": [\"15163664\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not separate import role from chromatin-tethering role\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showing the IN-LEDGF interaction is lentivirus-specific and essential for viral replication, separable from IN enzymatic activity, defined the interaction as a discrete druggable cofactor function.\",\n      \"evidence\": \"isoform/integrase-specific pull-downs, FCS DNA-binding, two-hybrid mutagenesis, and viral replication assays\",\n      \"pmids\": [\"15749713\", \"15855167\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic basis of the interface not yet resolved\", \"Chromatin-tethering step not yet directly demonstrated\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Mapping the IN-binding residues on integrase and the conserved D366 contact on LEDGF, plus showing in vivo Hox/developmental phenotypes in knockout mice, jointly defined both the viral interface and an endogenous developmental role.\",\n      \"evidence\": \"systematic mutagenesis across lentiviral integrases, in vitro strand transfer assays, and gene-trap knockout mouse phenotyping\",\n      \"pmids\": [\"17158150\", \"17137594\", \"16980622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link between PSIP1 and Hox gene regulation not yet defined\", \"Chromatin-reading domain function still unresolved\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Live-cell mutagenesis showed chromatin association is driven by a tripartite NLS/AT-hook element with a modulatory role for the PWWP domain, refining how LEDGF docks on chromatin.\",\n      \"evidence\": \"SPR/EMSA DNA binding with EGFP-fusion localization in interphase and mitotic cells\",\n      \"pmids\": [\"16549878\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not identify the histone mark recognized by PWWP\", \"Relative contribution of each element to integration not yet quantified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Genetic knockout showed LEDGF acts downstream of PIC formation to bias integration into transcription units, establishing the tethering model of integration site selection.\",\n      \"evidence\": \"LEDGF-null cells with HIV-1 integration site mapping and PIC strand transfer assays\",\n      \"pmids\": [\"17639082\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Chromatin feature read by LEDGF to define targets not yet identified\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Defining JPO2 as a cellular IBD ligand competing with integrase introduced the principle that the IBD engages multiple partners through one mutually exclusive site.\",\n      \"evidence\": \"two-hybrid, AlphaScreen, reciprocal Co-IP, and competition assays\",\n      \"pmids\": [\"17669426\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Cellular function of the JPO2 interaction unresolved\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Demonstrating that the PWWP domain binds nucleosomes directly and is the primary determinant for stimulating integration into chromatinized templates connected chromatin reading to cofactor function.\",\n      \"evidence\": \"systematic PWWP mutagenesis, GST pull-down of nucleosomes, and in vitro integration into polynucleosome templates\",\n      \"pmids\": [\"18799576\", \"18174227\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Specific histone modification recognized not yet identified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Showing LEDGF stabilizes IN-IN contacts and promotes integrase tetramerization revealed it functions as an allosteric organizer of the active integrase multimer, not merely a tether.\",\n      \"evidence\": \"MS protein footprinting, molecular modeling, and enzymatic assays of full-length IN-LEDGF\",\n      \"pmids\": [\"18801737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry of the assembled complex not yet defined\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Discovery of the Cdc7-ASK interaction and LEDGF-stimulated MCM2 phosphorylation revealed a cellular role in DNA replication kinase activation via the IBD.\",\n      \"evidence\": \"endogenous reciprocal Co-IP, in vitro kinase assays with MCM2, and phosphorylation-site mapping\",\n      \"pmids\": [\"19864417\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo importance for replication timing not established\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"The IN NTD+CCD-IBD crystal structure defined the charge-charge basis of lentiviral specificity, providing the structural template for understanding the interaction and its inhibition.\",\n      \"evidence\": \"X-ray crystallography with mutagenesis and concerted integration/infectivity assays\",\n      \"pmids\": [\"19132083\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure of the full-length IN-LEDGF-DNA assembly not yet resolved\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"ChIP linking LEDGF/p75 to the VEGF-C promoter under hormonal control extended its function to transcriptional regulation of lymphangiogenesis genes.\",\n      \"evidence\": \"ChIP, siRNA knockdown, and promoter-reporter assays in ovarian cancer cells\",\n      \"pmids\": [\"19934313\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct DNA-binding versus tethered recruitment at this promoter not distinguished\", \"Single lab\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Genome-wide chromatin profiling and the scan-and-lock kinetic model defined where and how LEDGF engages active chromatin, and that integrase binding markedly increases its chromatin affinity.\",\n      \"evidence\": \"DamID profiling and FRAP/FCS live-cell kinetics with domain mutants\",\n      \"pmids\": [\"20484370\", \"20974633\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Why only a subset of LEDGF-bound sites support integration unresolved\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Replacing the chromatin-binding domain with CBX1 to redirect integration to heterochromatin provided causal proof that the tethering domain dictates integration site selection.\",\n      \"evidence\": \"chimeric protein engineering with lentiviral integration site sequencing\",\n      \"pmids\": [\"20195265\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address endogenous cellular consequences of retargeting\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"FRET order-of-addition and in vitro tethering assays defined how LEDGF shapes integrase conformation and stimulates IN-DNA binding, sharpening the mechanistic model of complex assembly.\",\n      \"evidence\": \"FRET of IN assembly and AlphaScreen IN-DNA tethering assays with domain mutants\",\n      \"pmids\": [\"21771857\", \"21763490\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Medium-confidence quantitation for the tethering assay; single lab\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Identifying a supercoiled-DNA recognition domain that localizes to transcriptionally active regions added a DNA-topology preference to LEDGF chromatin targeting.\",\n      \"evidence\": \"recombinant fragment supercoiled-DNA binding and EGFP-SRD localization with immunostaining\",\n      \"pmids\": [\"21345933\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relationship of SRD to PWWP-based targeting not integrated\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Demonstrating damage-dependent recruitment of CtIP to DSBs via the PWWP domain established a direct role for PSIP1 in homologous-recombination repair.\",\n      \"evidence\": \"RNAi depletion, CtIP recruitment and DNA-end resection assays, and damage-dependent Co-IP\",\n      \"pmids\": [\"22773103\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same PWWP-chromatin contacts mediate both integration and repair tethering not directly compared\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Identifying MeCP2 as an N-terminal-domain partner shared by both isoforms expanded the transcriptional-regulator interactome of PSIP1.\",\n      \"evidence\": \"protein arrays, AlphaScreen, Co-IP, confocal co-localization, and Hsp27 reporter assays\",\n      \"pmids\": [\"22275515\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-context dependence of the functional effect unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Establishing that the PWWP domain specifically reads H3K36me3 and that p52 couples this mark to SRSF1 connected chromatin reading to alternative splicing regulation.\",\n      \"evidence\": \"PWWP H3K36me3 binding, p52-splicing-factor Co-IP, ChIP, and splicing analysis in Psip1 mutant cells\",\n      \"pmids\": [\"22615581\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Genome-wide breadth of splicing control not yet quantified\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"NMR structures resolved the bipartite PWWP mechanism — cooperative histone-tail and nucleosomal-DNA contacts producing nanomolar, ~10,000-fold-enhanced nucleosome binding — explaining high-affinity targeting to H3K36me3 chromatin.\",\n      \"evidence\": \"NMR solution structure, mutagenesis, and thermodynamic binding to reconstituted mononucleosomes\",\n      \"pmids\": [\"23396443\", \"23656834\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How partner binding at the IBD couples to this PWWP affinity not structurally resolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining the IN4:LEDGF2:INI1-IBD2:DNA2 stoichiometry by cryo-EM and MS established the architecture of the LEDGF-stabilized active integration intasome.\",\n      \"evidence\": \"reconstitution, MS stoichiometry, FCS, integration assays, and cryo-EM\",\n      \"pmids\": [\"23593299\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution atomic model of the full assembly not obtained\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Mapping the MLL interface to the same mutually exclusive IBD site and showing its mutation impairs MLL-AF9 transformation linked the cofactor function to leukemogenesis.\",\n      \"evidence\": \"NMR interface mapping, mutagenesis, AlphaScreen competition, and colony-forming assays\",\n      \"pmids\": [\"25082813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo leukemia dependence not yet tested in this study\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Complete TALEN knockout confirmed PSIP1's requirement for HIV integration while showing particle assembly and ALLINI action are LEDGF-independent, delimiting the cofactor's mechanistic scope.\",\n      \"evidence\": \"whole-gene and IBD-exon TALEN knockouts with replication, infectivity, and assembly assays\",\n      \"pmids\": [\"24942577\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Residual integration in knockout cells not fully explained\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Affinity purification and RNA-seq in knockout cells established PSIP1 as a broad regulator of alternative splicing and linked intron density to LEDGF-dependent integration patterns.\",\n      \"evidence\": \"affinity purification of splicing factors and RNA-seq plus >1M integration-site mapping in KO cells\",\n      \"pmids\": [\"26545813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct versus indirect contribution to each splicing event not resolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Conditional knockout and ChIP showed PSIP1 sustains MLL occupancy at Hox genes and is required for MLL-fusion leukemia initiation but dispensable for normal hematopoiesis, defining a context-specific oncogenic dependency.\",\n      \"evidence\": \"conditional KO mouse, Hox expression, MLL ChIP, and in vitro/in vivo leukemia models\",\n      \"pmids\": [\"29084774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of leukemia-specific dependence not fully defined\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Additional studies tied p52 to lncRNA Hottip-dependent Hoxa activation and p75 to RNA Pol II recruitment at cell-cycle gene promoters in breast cancer, broadening PSIP1's transcriptional roles.\",\n      \"evidence\": \"Hottip knockdown/knockout and Mll1 ChIP; PSIP1-Pol II Co-IP, promoter ChIP, and tumorigenicity assays\",\n      \"pmids\": [\"28384324\", \"28633434\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab findings\", \"Mechanistic link between Pol II recruitment and tumorigenicity not fully resolved\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showing PWWP-mediated H3K36me2/3 binding supports RNA Pol II elongation past nucleosomes defined a FACT-like role in transcription elongation.\",\n      \"evidence\": \"genome-wide co-localization with H3K36me2/3 and RNAPII ChIP-seq (primary focus on paralog HDGF2)\",\n      \"pmids\": [\"31616795\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct elongation defect upon LEDGF/p75 loss not separately demonstrated\", \"Mechanism inferred largely from HDGF2\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Dissecting PSIP1-dependent competition between PAF1 and the CKII-activated MLL1/SEC at the provirus explained how LEDGF toggles HIV transcription between latency and activation.\",\n      \"evidence\": \"reciprocal Co-IP, ChIP at the provirus, and CKII inhibition in latency-reversal assays\",\n      \"pmids\": [\"32426500\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether the same switch operates at host genes not addressed\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defining PSIP1's interaction with PARP1 and R-loop homeostasis factors revealed it suppresses promoter R-loops and transcription-replication conflicts, with loss imposing PARP-inhibitor sensitivity.\",\n      \"evidence\": \"Co-IP, DRIP-seq, γ-H2AX ChIP-seq, 53BP1/RAD51 foci, and PARP-inhibitor viability assays in PSIP1-depleted cells\",\n      \"pmids\": [\"38191578\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which PSIP1 resolves R-loops not established\", \"Relationship to its HR/CtIP function not integrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PSIP1 coordinates its single chromatin-reading domain and one mutually exclusive IBD across its many roles — integration, HR repair, splicing, elongation, R-loop control, and MLL-driven transcription — into context-specific outcomes remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No unified model of how partner selection at the IBD is regulated in cells\", \"Whether PWWP chromatin reading is rate-limiting for each function is untested\", \"High-resolution structures of most non-viral complexes are lacking\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [13, 22, 26, 31]},\n      {\"term_id\": \"GO:0042393\", \"supporting_discovery_ids\": [27, 28, 29]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [6, 9, 25, 28]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [3, 35, 43]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [15, 18, 30]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [4, 5]},\n      {\"term_id\": \"GO:0000228\", \"supporting_discovery_ids\": [9, 20, 21]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [0, 13, 32, 38]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [26, 39]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [27, 33, 35, 37]},\n      {\"term_id\": \"R-HSA-8953854\", \"supporting_discovery_ids\": [27, 33]},\n      {\"term_id\": \"R-HSA-4839726\", \"supporting_discovery_ids\": [20, 28, 31, 34]}\n    ],\n    \"complexes\": [\n      \"HIV-1 preintegration complex (IN tetramer–LEDGF dimer)\",\n      \"MLL complex\",\n      \"PAF1 complex (at HIV provirus)\"\n    ],\n    \"partners\": [\n      \"HIV-1 integrase\",\n      \"MLL\",\n      \"JPO2\",\n      \"PogZ\",\n      \"CDC7\",\n      \"CtIP\",\n      \"PARP1\",\n      \"SRSF1\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}