{"gene":"PKNOX1","run_date":"2026-06-10T06:43:35","timeline":{"discoveries":[{"year":1998,"finding":"PREP1 forms a stable heterodimer with PBX proteins independent of DNA binding, requiring sequences in the N-terminal portions of both proteins (HR1/HR2 domains). The PREP1-PBX interaction is essential for DNA-binding activity, and PREP1 enhances the ability of the HOXB1-PBX1 complex to activate transcription cooperatively, including formation of a ternary PREP1-PBX1-HOXB1 complex on a HOXB1-responsive target in vitro.","method":"Co-immunoprecipitation, in vitro protein-protein interaction assays, EMSA, transient transfection transcriptional assays, mutagenesis of homeodomain and N-terminal interaction surfaces","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (co-IP, EMSA, mutagenesis, transcriptional assays) in founding paper, replicated across multiple subsequent studies","pmids":["9482740"],"is_preprint":false},{"year":1998,"finding":"PREP1 was cloned as a subunit of the UEF3 complex (urokinase plasminogen activator enhancer factor 3). PREP1 is a TALE-class homeodomain protein that forms a stable complex with PBX proteins in solution independent of DNA, and the heterodimer binds the TGACAG motif in the uPA promoter with high affinity. The other UEF3 subunit was identified as a PBX family member.","method":"cDNA cloning, EMSA, co-immunoprecipitation, in vitro binding assays","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — founding biochemical characterization with multiple orthogonal methods; replicated by companion paper","pmids":["9482739"],"is_preprint":false},{"year":1997,"finding":"PKNOX1 (PREP1) dimerizes with PBX1 on the TGATTGAC motif and binds DNA cooperatively. The PKNOX1/MEIS1-interaction domain in PBX1 resides in a conserved N-terminal PBX domain deleted in the leukemic oncoprotein E2a-PBX1; consequently, PKNOX1 and MEIS1 fail to dimerize significantly with E2a-PBX1.","method":"EMSA, GST pull-down, nuclear extract binding assays","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reconstitution-level biochemical assays with domain mapping; replicated by multiple subsequent studies","pmids":["9405651"],"is_preprint":false},{"year":1999,"finding":"PREP1 is cytoplasmic and requires association with PBX/EXD for nuclear localization. Coexpression of EXD/PBX with PREP1/HTH causes nuclear localization of their heterodimers in both mammalian fibroblasts and Drosophila Schneider cells. Heterodimerization with PBX induces nuclear translocation of PREP1 by blocking nuclear export of PBX.","method":"Immunofluorescence microscopy, subcellular fractionation, co-expression experiments in multiple cell contexts","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct localization experiments with functional consequence, tested in multiple cell contexts, replicated independently","pmids":["10215622"],"is_preprint":false},{"year":1999,"finding":"PREP1-PBX heterodimers bind the UE-A element of the somatostatin promoter cooperatively (neither protein alone can bind), and both PBX1 and PREP1 are required for transcriptional activation from this element. In the context of the intact somatostatin mini-enhancer, PREP1-PBX1 cooperates with the pancreatic homeodomain factor PDX1 to produce drastic transcriptional activation.","method":"EMSA with recombinant proteins, transient transfection reporter assays, co-expression experiments","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution with recombinant proteins plus cellular reporter assays, multiple orthogonal methods","pmids":["9933599"],"is_preprint":false},{"year":2000,"finding":"PREP1-PBX complexes bind Pbx-Meinox (PM) sites in the Hoxb2 r4 enhancer, and the PM site is essential in vivo for r4-restricted expression. Both PM and PH (Pbx-Hox) sites are required for formation and binding of a ternary HOXB1-PBX1a-PREP1 complex in vitro. A similar ternary complex forms in nuclear extracts from retinoic acid-induced P19 cells, containing PREP1 (or MEIS1), PBX, and HOXB1.","method":"EMSA, transgenic mouse enhancer assays, nuclear extract binding assays, site-directed mutagenesis of enhancer elements","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro biochemical assays combined with in vivo transgenic validation, multiple orthogonal methods","pmids":["10654609"],"is_preprint":false},{"year":1999,"finding":"PREP1 is present in mouse embryos from at least E9.5, is ubiquitously nuclear in embryonic tissues (E7.5–17.5), and forms tissue-specific DNA-binding complexes with different PBX family members depending on cell type. PREP1 super-activates the PBX-HOXB1 autoregulated Hoxb-1 promoter, and all three proteins (PREP1, PBX, HOXB1) are co-localized in rhombomere 4 in vivo.","method":"Co-immunoprecipitation, EMSA, immunohistochemistry, Northern blot, in situ hybridization","journal":"Mechanisms of development","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods, in vivo validation, replicated across tissues","pmids":["10381567"],"is_preprint":false},{"year":1999,"finding":"PBX-MEIS1/PREP1 heterodimers cooperate with myogenic bHLH proteins (MyoD, myogenin, Mrf-4, Myf-5) for DNA binding. A conserved tryptophan motif N-terminal to the DNA-binding domain of each myogenic bHLH protein is required for cooperative DNA binding with PBX-MEIS1/PREP1. MyoD requires this tryptophan motif to evoke chromatin remodeling at the Myogenin promoter.","method":"EMSA, mutagenesis, chromatin remodeling assays, transcriptional reporter assays","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods including mutagenesis and chromatin assays; mechanistically defined interaction surface","pmids":["10471746"],"is_preprint":false},{"year":2000,"finding":"PREP1-PBX heterodimers bind the glucagon gene promoter (domain B of the G3 enhancer and the cAMP response element and G5 element) and restrict transcription in non-glucagon-producing cells. Coexpression of Pbx1a/1b-Prep1 inhibited the glucagon promoter activated by Pax6 in non-glucagon-producing cells, but had no effect in glucagon-producing islet cells.","method":"EMSA with specific antiserum and in vitro translated proteins, transient transfection reporter assays","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA with antibody supershift and functional reporter assays, single lab","pmids":["10869353"],"is_preprint":false},{"year":2003,"finding":"PREP1 overexpression increases PBX-2 protein levels by preventing its proteasomal degradation. Dimerization with PREP1 stabilizes PBX-2 protein against cycloheximide-induced decay; the proteasome inhibitor MG132 prevents PBX-2 decay in control cells, indicating PBX-2 is normally degraded by the proteasome and PREP1 binding protects it.","method":"Cycloheximide chase, proteasome inhibitor (MG132) treatment, Western blot, EMSA, stable transfection","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — biochemical mechanism established with multiple orthogonal methods (cycloheximide chase, proteasome inhibition, Western blot); replicated in subsequent studies","pmids":["12871956"],"is_preprint":false},{"year":2004,"finding":"PREP1 binds the FSHβ promoter in a complex with SMAD4 and PBX1 at the -134 activin response element; PBX1 and PREP1 interact physically with SMAD2 and SMAD3 as shown by GST pull-down. Pbx1 and Prep1 bind the mouse FSHβ gene in vivo, and mutation of the -134 site abrogates activin responsiveness.","method":"EMSA, GST interaction assays, transient transfection, chromatin immunoprecipitation (in vivo binding)","journal":"Molecular endocrinology (Baltimore, Md.)","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (GST pulldown, EMSA with antibody supershift, ChIP, reporter assays) establishing physical and functional interaction with SMADs","pmids":["14764653"],"is_preprint":false},{"year":2005,"finding":"PREP1-PBX1 (PM) binding sites in the Hoxb1 autoregulatory enhancer include three sites (PM1, PM2, and R2/PM3). PM1 and PM2 cooperate to modulate in vivo regulatory activity. R2/PM3 has high binding affinity for PREP1-PBX1 dimers and, in combination with PM1 and R3, inhibits ternary PREP1-PBX1-HOXB1 complex formation in vitro and restricts reporter expression in transgenic embryos.","method":"EMSA, site-directed mutagenesis, transgenic chicken and mouse embryo reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro biochemical assays combined with in vivo transgenic validation in two species","pmids":["16166636"],"is_preprint":false},{"year":2006,"finding":"PREP1 deficiency in hypomorphic Prep1(i/i) mice causes an overall decrease in protein levels of MEIS1, PBX1, and PBX2, strongly reducing total PREP1/MEIS-PBX DNA-binding activity. PREP1 acts upstream of a PBX-MEIS network. The levels of cMyb and Pax6 are significantly decreased in fetal liver and retina of Prep1(i/i) embryos, correlating with hematopoietic and eye phenotypes.","method":"Western blot, EMSA, immunohistochemistry, hypomorphic mouse model","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo genetic model with multiple biochemical readouts, replicated across tissues","pmids":["16847320"],"is_preprint":false},{"year":2006,"finding":"The fetal adrenal enhancer (FAdE) in intron 4 of Ad4BP/SF-1 contains binding sites for PBX-PREP1, PBX-HOX, and Ad4BP/SF-1. PBX/PREP1 and PBX/HOX sites initiate transcription prior to the establishment of the Ad4BP/SF-1 autoregulatory loop during fetal adrenal development.","method":"Transgenic mouse assays, site-directed mutagenesis of enhancer elements, EMSA","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — transgenic in vivo validation with mutagenesis, single lab","pmids":["16705164"],"is_preprint":false},{"year":2007,"finding":"p160 Myb-binding protein (p160) is a novel PREP1-interacting protein that competes with PBX1 for PREP1 binding in vitro. The N-terminal truncated form p67 binds the sequence 63-LFPLL-67 in the HR1 domain of PREP1; mutation of L63 and L66 impairs binding to both p160/p67 and PBX1. p160 inhibits PREP1-dependent HoxB2 expression.","method":"Co-immunoprecipitation, in vitro competition assay, site-directed mutagenesis, immunofluorescence colocalization, RT-PCR","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — mutagenesis identifying specific binding residues, competition assay, co-IP, and functional gene expression assay; replicated in multiple subsequent studies","pmids":["17875935"],"is_preprint":false},{"year":2007,"finding":"Tandem affinity purification of PREP1 from nuclear and cytoplasmic fractions identified its interactome, including PBX1b, PBX2, p160 Myb-binding protein (p160), beta-actin, and NMMHCIIA as co-purifying partners.","method":"Tandem affinity purification (TAP), mass spectrometry","journal":"Proteomics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — TAP/MS interactome from functional tagged protein, single lab","pmids":["17623278"],"is_preprint":false},{"year":2007,"finding":"IL-10 production by macrophages in response to apoptotic cells is regulated at transcription via the apoptotic-cell-response element (ACRE) in the human IL10 promoter; PREP1 (Pbx-regulating protein 1) and PBX1b bind ACRE and mediate transcriptional activation of IL-10 in response to apoptotic cells in a p38 MAPK-dependent manner.","method":"Reporter assay (ACRE mapping), EMSA, ChIP, gene knockdown, p38 inhibitor treatment","journal":"Immunity","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — multiple orthogonal methods (reporter assay, EMSA, ChIP, functional knockdown) in peer-reviewed high-impact journal","pmids":["18093541"],"is_preprint":false},{"year":2008,"finding":"PREP1 directly controls the intrinsic apoptotic pathway by regulating Bcl-x gene expression. Prep1(i/i) MEFs have decreased Bcl-X(L) mRNA and protein. Chromatin immunoprecipitation and transient transfection identified the Bcl-x promoter as a direct transcriptional target of PREP1. Overexpression of Bcl-x rescues the apoptotic defect of Prep1(i/i) MEFs.","method":"ChIP, transient transfection reporter assay, Western blot, annexin V assay, caspase activity assay, Bcl-x overexpression rescue","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP plus reporter assay plus genetic rescue, multiple orthogonal methods in single rigorous study","pmids":["19103748"],"is_preprint":false},{"year":2008,"finding":"PREP1 deficiency in skeletal muscle increases insulin sensitivity through a p160-GLUT4 pathway. Reduced Prep1 decreases p160 levels, leading to increased PGC-1α and GLUT4 expression. PREP1 controls the stability of the p160 protein. Overexpression of Prep1 or p160 (but not PBX1) reduces GLUT4 and insulin sensitivity; in vivo delivery of p160 cDNA reverses the molecular phenotype of Prep1(i/i) muscle.","method":"Hypomorphic mouse model, L6 cell overexpression/knockdown, Western blot, glucose uptake assay, in vivo muscle cDNA delivery","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro and in vivo experiments, genetic rescue, pathway placement established","pmids":["18644868"],"is_preprint":false},{"year":2009,"finding":"Cytoplasmic PREP1 in mouse oocytes interacts with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Hoxb4 mRNA. PREP1 binds the Hoxb4 3'UTR (RNA EMSA), inhibits Hoxb4 mRNA translation >95% in vitro in the presence of 4EHP, and a functional 4EHP-binding motif in PREP1 was identified by mutagenesis. Prep1(i/i) oocytes show oocyte growth failure and increased Hoxb4 protein.","method":"Confocal microscopy, co-immunoprecipitation, GST pull-down, mutagenesis of 4EHP-binding motif, RNA EMSA, in vitro translation assay, immunohistochemistry","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — in vitro reconstitution of translational repression, mutagenesis of functional motif, multiple orthogonal methods, in vivo genetic validation","pmids":["19365557"],"is_preprint":false},{"year":2010,"finding":"PREP1 transcriptionally activates the SHP1 tyrosine phosphatase gene via a promoter element between -2113 and -1778. Both PREP1 and PBX1 bind the SHP1 promoter in vivo and cooperatively induce luciferase activity 30-fold. This leads to attenuation of insulin receptor/IRS1/2 phosphorylation and reduced glycogen storage in liver. Antisense silencing of SHP1 (but not SYP) rescues insulin-dependent IR phosphorylation in PREP1-overexpressing cells.","method":"ChIP, luciferase reporter assay, Western blot, euglycemic hyperinsulinemic clamp, antisense knockdown, HepG2 overexpression","journal":"Diabetes","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP plus reporter assay plus functional rescue, in vivo and in vitro orthogonal methods","pmids":["20864515"],"is_preprint":false},{"year":2010,"finding":"Prep1 is required for genomic stability. Prep1(i/i) fetal liver cells and MEFs exhibit increased basal DNA damage and chromosomal aberrations/aneuploidy. Acute PREP1 knockdown by siRNA in human fibroblasts induces DNA damage response and increases heterochromatin-associated histone methylation with decreased satellite DNA transcription. Ectopic PREP1 expression rescues DNA damage and heterochromatin methylation. PREP1 deficiency facilitates immortalization and H-Ras(V12)-dependent transformation, partially rescued by Prep1 restoration.","method":"γH2AX immunofluorescence, cytogenetic analysis, siRNA knockdown, ChIP, satellite DNA transcription assay, transformation assay, rescue experiment","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vivo and in vitro methods with genetic rescue; establishes genomic stability function","pmids":["21715654"],"is_preprint":false},{"year":2010,"finding":"Absence of Prep1 in null embryos causes p53-dependent apoptosis of epiblast cells. The embryonic lethality of Prep1(-/-) embryos is partially rescued in a p53(-/-) background but not in a p16(-/-) background, placing PREP1 upstream of p53-dependent apoptosis. Atm downregulation exacerbates the Prep1(-/-) phenotype, suggesting DNA damage as the cause of apoptosis.","method":"Genetic epistasis (Prep1(-/-)/p53(-/-) double mutants), TUNEL staining, cleaved caspase 3 immunostaining, immunohistochemistry","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with double mutant rescue, multiple mutant backgrounds tested","pmids":["20826531"],"is_preprint":false},{"year":2010,"finding":"Excess PREP1 increases sensitivity of cells to genotoxic stress via p53. PREP1 overexpression induces p53, and p53 depletion reverts the apoptotic phenotype of PREP1-overexpressing cells. p53 is identified as a direct transcriptional target of PREP1. PREP1 overexpression also increases anti-apoptotic Bcl-X(L) levels, but pro-apoptotic p53 induction dominates.","method":"Apoptosis assays (genotoxic stress), p53 siRNA rescue, Western blot, transient transfection reporter assay (implied by 'direct target')","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic rescue experiments and functional assays; abstract implies direct target relationship but does not explicitly describe ChIP for p53","pmids":["20110257"],"is_preprint":false},{"year":2011,"finding":"PREP1 deficiency results in premature cycling and exhaustion of fetal hematopoietic stem cells (HSCs) through induction of interferon signaling (Stat1-Sca1 IFN-dependent pathway). Prep1 controls the self-renewal ability of FL HSCs, as shown by serial transplantation experiments.","method":"Hypomorphic mouse model, serial transplantation, flow cytometry, gene expression analysis","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo serial transplantation with pathway identification, single lab","pmids":["25233378"],"is_preprint":false},{"year":2013,"finding":"PREP1 regulates mitochondrial oxidative phosphorylation components in skeletal muscle through direct and indirect mechanisms. Muscle-specific ablation of Prep1 increases expression of respiratory chain subunits and mitochondrial enzyme activity. PREP1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1α activity; Prep1 ablation reduces p160, elevating PGC-1α. ChIP-seq identifies direct PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2).","method":"Muscle-specific conditional knockout, electron microscopy, treadmill exercise testing, Western blot, ChIP-seq","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — tissue-specific KO, genome-wide ChIP-seq, multiple phenotypic and biochemical readouts; establishes both direct and indirect mechanisms","pmids":["24216763"],"is_preprint":false},{"year":2013,"finding":"PREP1/PBX1 complex transcriptionally activates the SHIP2 (Inppl1) promoter, and the PREP1-HR mutant (unable to bind PBX1) has no effect on Ship2, demonstrating that PREP1-PBX1 dimerization is required for this transcriptional activation. PREP1 deficiency reduces SHIP2, leading to increased PKCζ/LKB1/AMPK/ACC phosphorylation and reduced hepatic lipogenesis.","method":"ChIP, luciferase reporter assay, PBX1-binding mutant (HR mutant), Western blot, hypomorphic mouse model","journal":"Diabetologia","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP plus reporter assay plus dimerization-deficient mutant establishing PBX1-dependence; multiple orthogonal methods","pmids":["24052111"],"is_preprint":false},{"year":2014,"finding":"PREP1 and MEIS1 compete for PBX1 binding in mouse embryonic fibroblasts. PREP1 posttranslationally controls MEIS1 levels by decreasing its stability through sequestering PBX1. MEIS1 alone transforms Prep1-deficient fibroblasts; PREP1 overexpression inhibits MEIS1 tumorigenicity. PREP1 also modifies the DNA binding landscape of MEIS1 and prevents MEIS1 interaction with DDX3X and DDX5.","method":"Co-immunoprecipitation, protein stability assays, transformation assay, transcriptional profiling, MEF genetic model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods establishing competition mechanism, post-translational stability control, and functional consequences; peer-reviewed PNAS","pmids":["24578510"],"is_preprint":false},{"year":2014,"finding":"PREP1 is a novel EMT inducer and prometastatic transcription factor in non-small cell lung cancer. PREP1 overexpression triggers EMT, and PREP1 down-regulation inhibits TGF-β-induced EMT. Mechanistically, PREP1 induces SMAD3 nuclear translocation through transactivation of a regulatory element in the SMAD3 first intron. PREP1 also stabilizes and accumulates PBX1 and induces FRA-1 expression; both FRA-1 and PBX1 are required for PREP1-induced mesenchymal changes.","method":"Overexpression/knockdown, reporter assay (SMAD3 intron element), Western blot, immunofluorescence (SMAD3 localization), lung colonization assay","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — reporter assay establishing direct transcriptional target, subcellular localization experiments, functional rescue with FRA-1/PBX1 knockdown, in vivo colonization assay","pmids":["25157139"],"is_preprint":false},{"year":2015,"finding":"In embryonic stem cells, PREP1 binds ~2,000 novel genomic sites compared to embryo trunk, including a higher proportion of enhancers. RNA-seq identifies ~1800 genes down-regulated in Prep1(-/-) ES cells, including essential components of Wnt and Fgf pathways. Prep1(-/-) ES cells show deficient embryoid body formation and differentiation, rescued by re-expression of PREP1.","method":"ChIP-seq, RNA-seq, embryoid body differentiation assay, Prep1 knockout ES cells, rescue experiment","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genome-wide ChIP-seq and RNA-seq with genetic rescue; identifies Wnt/Fgf as PREP1 target pathways in ES cells","pmids":["25875616"],"is_preprint":false},{"year":2015,"finding":"PREP1 and PBX1 homeodomains (HDs) bind DNA cooperatively. The kinetics of individual PREP1 and PBX1 HDs and their combination show cooperative DNA binding. A novel PREP1 motif flanking the HD at the C-terminus is required for cooperativity.","method":"NMR spectroscopy, biophysical characterization of recombinant PREP1 HD, DNA binding kinetics","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 1 / Moderate — structural (NMR) plus biochemical characterization, identification of novel cooperativity motif; single lab but rigorous biophysical approach","pmids":["25856340"],"is_preprint":false},{"year":2015,"finding":"High glucose exposure increases NF-κB p65 binding and recruitment of SET7 histone methyltransferase and p300 acetyltransferase to the 5' region of Prep1, leading to enhanced H3K4 methylation and H3K9/14 acetylation and increased Prep1 transcription. Prep1-induced recruitment of MEF2/HDAC5 repressor complex at the Glut4 promoter represses Glut4 expression.","method":"Chromatin immunoprecipitation (ChIP), Western blot, L6 cell model, streptozotocin diabetic mouse model","journal":"Diabetologia","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP establishing histone modifications and transcription factor binding at endogenous loci, validated in vivo in diabetic mice","pmids":["26453063"],"is_preprint":false},{"year":2017,"finding":"NMR spectroscopy of the PREP1 homeodomain shows that residues flanking the HD participate in DNA binding. Isolated PREP1 and PBX1 HDs bind DNA cooperatively, and a novel PREP1 motif C-terminal to the HD is required for cooperativity with PBX1.","method":"NMR spectroscopy, surface plasmon resonance/kinetic binding assays, mutagenesis","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1 / Moderate — NMR structural characterization with functional validation by binding kinetics; first biophysical characterization of PREP1 HD","pmids":["28094776"],"is_preprint":false},{"year":2017,"finding":"The synthetic PREP1(54-72) peptide mimicking the PREP1 region involved in interaction with p160 disrupts the PREP1-p160 interaction in vitro (IC50 = 0.20 μM) and restores insulin signaling impaired by ceramide in L6 skeletal muscle cells. The PREP1-p160 interaction is induced by ceramide treatment.","method":"In vitro binding competition assay (IC50 determination), co-immunoprecipitation, insulin signaling Western blot, glucose uptake assay, glycogen assay","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — IC50 of peptide inhibitor determined in vitro, mechanistic rescue in cellular model, multiple orthogonal readouts","pmids":["29069751"],"is_preprint":false},{"year":2017,"finding":"PREP1 down-regulation in mesenchymal stromal cells and 3T3-L1 preadipocytes increases adipogenic differentiation. PREP1 restricts DNA binding of C/EBPβ to adipogenic enhancers without affecting C/EBPβ expression or activation, thereby suppressing adipogenesis.","method":"Prep1 siRNA knockdown, adipogenic differentiation assay, ChIP (C/EBPβ binding), ex vivo bone marrow-derived MSC","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP establishing mechanism of C/EBPβ binding restriction, single lab with ex vivo and in vitro models","pmids":["29138456"],"is_preprint":false},{"year":2018,"finding":"PREP1 depletion affects DNA replication timing of middle-to-late-replicating DNA. PREP1 down-regulation increases replication rate, number of simultaneously firing origins, and asymmetry of DNA replication, leading to DNA damage. Genome-wide Repli-seq shows 25% of genome (LADs, LRRs, TTRs) replicates earlier upon PREP1 depletion. PREP1 down-regulation also decreases Lamin B1 levels, suggesting DNA release from the nuclear lamina.","method":"DNA combing, Repli-seq (genome-wide replication timing), Western blot (Lamin B1), DNA damage assays","journal":"Scientific reports","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genome-wide replication timing assay plus DNA combing with quantitative analysis; establishes novel function in DNA replication regulation","pmids":["29453404"],"is_preprint":false},{"year":2018,"finding":"PKnox1/PREP1 is required in a germ cell-intrinsic manner for adult spermatogenesis. Tamoxifen-induced or germ cell-specific PKnox1 loss causes arrest at the c-Kit+ spermatogonia stage with loss of meiotic spermatocytes and compromised c-Kit+ spermatogonia differentiation.","method":"Conditional/inducible knockout (tamoxifen-Cre, germ cell-specific Cre), immunohistochemistry, flow cytometry","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific conditional KO with defined stage of arrest; germ cell-intrinsic requirement established","pmids":["29293683"],"is_preprint":false},{"year":2018,"finding":"PREP1 regulates angiogenesis through the p160/PGC-1α pathway. PREP1 overexpression in mouse aortic endothelial cells increases p160 binding and levels (~4-fold) and reduces PGC-1α (~70%), stimulating angiogenic migration, proliferation, and tube formation. The PREP1(54-72) peptide blocking PREP1-p160 interaction reverts the proangiogenic effects. PREP1 levels increase during bFGF-mediated endothelial colony activation.","method":"Endothelial cell overexpression, PREP1(54-72) peptide inhibition, migration/proliferation/tube formation assays, Western blot, co-immunoprecipitation, hypomorphic mouse Matrigel plug assay","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal in vitro and in vivo methods, pharmacological disruption of PREP1-p160 with functional readout","pmids":["31618597"],"is_preprint":false},{"year":2018,"finding":"PREP1 deficiency reduces olfactory bulb development and impairs BDNF-TrkB signaling: Prep1(i/+) mice show reduced TrkB receptor levels and decreased BDNF-induced ERK1/2 activation. Overexpression of Prep1 in N2A neuronal cells increases TrkB expression levels, BDNF-induced ERK phosphorylation, and cell viability.","method":"Immunohistochemistry, cytochrome C oxidase activity, Western blot, overexpression in N2A cells, olfactory perception behavioral test","journal":"Molecular neurobiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro overexpression and in vivo hypomorphic model, single lab, links PREP1 to TrkB pathway","pmids":["29349576"],"is_preprint":false},{"year":2018,"finding":"PREP1 and p160 domains interact with a KD of approximately 0.3–1 μM. Both PREP1 (residues 45–155) and p160 (residues 20–160) form folded, monomeric, predominantly alpha-helical domains with unusually high thermal stability.","method":"Recombinant protein expression, CD spectroscopy, thermal stability assay, binding affinity measurement (KD determination)","journal":"Molecular biotechnology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct biophysical characterization of recombinant domains; single lab, first quantification of binding affinity","pmids":["26979610"],"is_preprint":false},{"year":2018,"finding":"SP2 transcription factor forms a complex with PREP1-PBX1 and NF-Y at composite genomic motifs where TALE and NF-Y recognition sequences are separated by 11 bp. SP2 potentiates binding of PBX1:PREP1 and NF-Y to co-occupied regulatory elements; reciprocally, PBX1:PREP1 together with NF-Y recruits SP2 to these sites.","method":"ChIP-exo sequencing, co-immunoprecipitation, ChIP, motif analysis in MEFs","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — ChIP-exo at nucleotide resolution, co-IP, multiple binding/functional assays establishing novel complex and mutual potentiation","pmids":["30337366"],"is_preprint":false},{"year":2022,"finding":"PREP1 (PKNOX1) regulates nuclear stiffness, expression of LINC complex proteins, and YAP-TAZ mechanotransduction. PREP1 depletion upsets nuclear membrane protein stoichiometry, renders nuclei soft, leads to fortified actomyosin networks with larger focal adhesions and greater traction forces, but impairs YAP-TAZ nuclear translocation.","method":"PREP1 knockdown, atomic force microscopy (nuclear stiffness), traction force microscopy, YAP-TAZ immunofluorescence, Western blot of LINC complex proteins","journal":"Communications biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biophysical and cell biological methods in single lab; establishes novel PREP1 function in mechanosignaling","pmids":["35550602"],"is_preprint":false},{"year":2023,"finding":"PKNOX1 acts as a transcription factor for the DHH (desert hedgehog) gene, directly binding the DHH promoter and promoting its expression, thereby activating Hedgehog signaling in stomach adenocarcinoma. ANKRD49 physically interacts with PKNOX1 (co-IP) and uses PKNOX1-mediated transcription of TGF-β1 to activate SMAD signaling and promote EMT.","method":"Dual-luciferase reporter assay (DHH promoter), ChIP (PKNOX1 binding to TGF-β1 promoter), co-immunoprecipitation (ANKRD49-PKNOX1), Western blot, knockdown experiments","journal":"Cancer cell international","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase establishing direct transcriptional target relationship, co-IP for protein interaction; single lab, recent paper","pmids":["41821002"],"is_preprint":false},{"year":2003,"finding":"PBX1 and PREP1 bind the PBX consensus element (PCE, TGATTGAC) in the long terminal repeat of Moloney murine leukemia virus (MLV), confirmed by gel shift assay. Overexpression of PBX1 and PREP1 together enhances MLV transcription, and mutations in the PCE completely inhibit viral transcription.","method":"EMSA (gel shift), overexpression, site-directed mutagenesis of PCE, transcriptional reporter assay","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA and functional overexpression assay, mutagenesis of binding site; single lab","pmids":["12529389"],"is_preprint":false},{"year":2002,"finding":"PBX2-PREP1 heterodimer binds to the Pbx site in the UGT2B17 promoter and interferes with HNF1α binding to the adjacent HNF1 site, resulting in down-regulation of HNF1α-mediated activation of the UGT2B17 promoter.","method":"EMSA, GST interaction assays, transient transfection reporter assays","journal":"Molecular pharmacology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gel shift, GST pulldown, functional reporter assay; single lab","pmids":["12065766"],"is_preprint":false},{"year":2008,"finding":"PREP1/PBX2 complex preferentially binds the -2578 G allele in the CCL2 promoter (created by the -2578 G polymorphism) and suppresses basal CCL2 promoter activity compared to the ancestral A allele in astrocytes.","method":"EMSA, transient transfection reporter assay, supershift with specific antibodies","journal":"Genes and immunity","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA with allele-specific binding and functional reporter assay; single lab","pmids":["18480829"],"is_preprint":false},{"year":2003,"finding":"PKNOX1 (PREP1) specifically binds the Pbx/POU binding site of the FABP7 promoter in vitro, and overexpression of PKNOX1 in neuroblastoma cells trans-activates the FABP7 promoter.","method":"EMSA (in vitro binding), transient transfection reporter assay","journal":"Nucleic acids research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, two methods (EMSA and reporter assay) but limited mechanistic depth","pmids":["12771203"],"is_preprint":false},{"year":2011,"finding":"The MEIS1 C-terminal transactivating domain, when fused to full-length PKNOX1, confers oncogenic activity: the chimeric PKNOX1-MC protein accelerates onset of HOXA9-induced leukemia in a mouse bone marrow transduction/transplantation model. The absence of a transactivating domain in native PKNOX1 accounts for its lack of pro-leukemic activity despite similar DNA-binding properties.","method":"Bone marrow transduction/transplantation leukemia model, gene expression profiling, chimeric protein construction","journal":"Blood","confidence":"High","confidence_rationale":"Tier 2 / Strong — in vivo leukemia model with chimeric protein establishing structure-function relationship; clean genetic experiment","pmids":["21900201"],"is_preprint":false},{"year":2016,"finding":"miR-17-5p and miR-19a-3p of the miR-17~92 cluster target PKNOX1, validated as a direct target. PKNOX1 competitively inhibits MEIS1-PBX complex formation; PKNOX1 acts antagonistically to leukemia in an MLL-AF9 murine model.","method":"miRNA target validation (luciferase reporter), co-immunoprecipitation (competitive PBX binding), murine MLL-AF9 leukemia model, knockdown studies","journal":"Leukemia research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reporter assay for miRNA targeting, co-IP for competitive binding, in vivo leukemia model; single lab","pmids":["27123834"],"is_preprint":false},{"year":2019,"finding":"PREP1 forms complexes with SNAIL2 in mammary stem/progenitor cells and protects SNAIL2 from proteasome-mediated degradation. Prep1-deficient mammary progenitor cells show reduced Snail2, Snail1, and vimentin expression, reduced mammosphere formation and branching in 3D assay, and increased TP53-dependent apoptosis.","method":"Co-immunoprecipitation (PREP1-SNAIL2 complex), proteasome inhibitor rescue, mammosphere assay, 3D branching assay, Western blot, conditional KO mouse model","journal":"The International journal of developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — co-IP establishing complex, proteasomal stabilization mechanism; single lab with in vitro and in vivo data","pmids":["30604852"],"is_preprint":false}],"current_model":"PKNOX1/PREP1 is a ubiquitous TALE-class homeodomain transcription factor that functions primarily as a stable heterodimer with PBX proteins (requiring N-terminal interaction domains in both proteins), which are mutually stabilized against proteasomal degradation; the PREP1-PBX heterodimer binds specific DNA motifs (TGACAG/TGATTGAC) cooperatively and can form ternary complexes with HOX proteins to regulate developmental gene expression (Hox, SF-1, FSHβ, Bcl-x, SHP1, SHIP2, SMAD3, DHH promoters), while competing with MEIS1 for PBX1 binding to control MEIS1 stability and suppress tumorigenesis; additionally, cytoplasmic PREP1 represses translation of Hoxb4 mRNA via 4EHP interaction in oocytes, PREP1 regulates genomic stability and DNA replication timing of lamin-associated domains (via Lamin B1), controls nuclear stiffness and YAP-TAZ mechanotransduction through LINC complex protein expression, and modulates insulin sensitivity through the p160-PGC-1α-GLUT4 axis in skeletal muscle and SHP1/SHIP2 in liver."},"narrative":{"mechanistic_narrative":"PKNOX1/PREP1 is a ubiquitous TALE-class homeodomain transcription factor that controls developmental gene expression as an obligate, stabilizing heterodimer with PBX proteins and governs cellular fate, genomic stability, and metabolic homeostasis [PMID:9482740, PMID:9482739, PMID:21715654]. PREP1 binds PBX through N-terminal HR1/HR2 interaction surfaces independent of DNA, an association that is required for high-affinity, cooperative DNA binding at TGACAG/TGATTGAC-class motifs and that mutually stabilizes both partners against proteasomal degradation [PMID:9482740, PMID:12871956, PMID:25856340]; because PREP1 is intrinsically cytoplasmic, dimerization with PBX also drives its nuclear translocation by blocking PBX export [PMID:10215622]. The PREP1-PBX dimer nucleates higher-order complexes — ternary assemblies with HOX proteins on developmental enhancers (Hoxb1, Hoxb2, SF-1/FAdE) and composite complexes with SMADs, NF-Y/SP2, and myogenic bHLH factors — to activate or restrict transcription in a context-dependent manner [PMID:10654609, PMID:10471746, PMID:14764653, PMID:16705164, PMID:30337366]. Through this network PREP1 acts upstream of a PBX-MEIS module, and by sequestering PBX1 it competes with MEIS1, destabilizing MEIS1 and suppressing its oncogenic and pro-leukemic activity [PMID:16847320, PMID:24578510, PMID:21900201]. PREP1 directly governs cell survival and genome maintenance, transactivating Bcl-x and p53 and being required to prevent DNA damage, chromosomal instability, and aberrant replication timing of lamin-associated domains via Lamin B1 [PMID:19103748, PMID:21715654, PMID:20826531, PMID:29453404]. In metabolic and signaling tissues, PREP1 controls insulin sensitivity and mitochondrial biogenesis by stabilizing the p160/Mybbp1a inhibitor of PGC-1α and transactivating the phosphatases SHP1 and SHIP2 [PMID:18644868, PMID:20864515, PMID:24216763, PMID:24052111], and it additionally functions in the cytoplasm of oocytes by binding 4EHP to repress Hoxb4 mRNA translation [PMID:19365557]. PREP1 is required in vivo for hematopoietic stem cell self-renewal, spermatogenesis, embryonic stem cell differentiation, and epiblast survival [PMID:20826531, PMID:25233378, PMID:25875616, PMID:29293683].","teleology":[{"year":1998,"claim":"Established the founding biochemical identity of PREP1: how does this TALE homeodomain protein engage DNA and partner proteins to regulate transcription?","evidence":"cDNA cloning, co-IP, EMSA, and transcriptional assays identifying PREP1 as a UEF3 subunit forming a DNA-independent heterodimer with PBX that binds the TGACAG motif","pmids":["9482740","9482739","9405651"],"confidence":"High","gaps":["Did not resolve the structural basis of cooperative DNA binding","Physiological target genes beyond uPA/Hox reporters not yet defined"]},{"year":1999,"claim":"Explained the subcellular logic of the dimer: why does PREP1 require PBX for function?","evidence":"Immunofluorescence and fractionation in mammalian and Drosophila cells showing PBX/EXD blocks PREP1 nuclear export, driving heterodimer nuclear translocation","pmids":["10215622"],"confidence":"High","gaps":["NLS/NES sequences mediating the switch not mapped"]},{"year":2000,"claim":"Defined PREP1-PBX as a developmental enhancer organizer that nucleates ternary HOX complexes and tissue-restricted expression in vivo.","evidence":"EMSA, transgenic mouse enhancer assays, and IHC at Hoxb1/Hoxb2 r4 enhancers and the somatostatin and glucagon promoters showing PM-site-dependent ternary PREP1-PBX-HOX complexes","pmids":["10654609","10381567","9933599","10869353"],"confidence":"High","gaps":["Whether PREP1 vs MEIS1 occupancy at PM sites is regulated was not resolved","Co-activator recruitment mechanism not defined"]},{"year":2003,"claim":"Revealed mutual protein stabilization as a core mechanism: PREP1 dimerization protects PBX from proteasomal degradation.","evidence":"Cycloheximide chase plus MG132 treatment and Western blot showing PREP1 prevents PBX-2 proteasomal decay","pmids":["12871956"],"confidence":"High","gaps":["E3 ligase targeting free PBX not identified","Reciprocal stabilization of PREP1 by PBX not quantified here"]},{"year":2006,"claim":"Placed PREP1 atop a PBX-MEIS regulatory network in vivo via a hypomorphic mouse, linking its dosage to network-wide protein levels and tissue phenotypes.","evidence":"Prep1(i/i) hypomorphic mice with Western blot, EMSA, and IHC showing reduced MEIS1/PBX1/PBX2 and downstream cMyb/Pax6, plus FAdE/SF-1 enhancer transgenic assays","pmids":["16847320","16705164"],"confidence":"High","gaps":["Direct vs indirect contributions to each downstream factor not fully separated"]},{"year":2007,"claim":"Expanded the PREP1 interactome beyond PBX, identifying p160/Mybbp1a as a competitive partner and immune/cytoskeletal associates.","evidence":"Co-IP, in vitro competition with mapped HR1 residues, TAP-MS interactome, and ACRE reporter/ChIP in macrophages","pmids":["17875935","17623278","18093541"],"confidence":"High","gaps":["Functional significance of beta-actin/NMMHCIIA association not pursued","Stoichiometry of p160 vs PBX competition in vivo unknown"]},{"year":2008,"claim":"Connected PREP1 to cell survival and metabolism: direct transcriptional control of Bcl-x and a p160-GLUT4 axis governing insulin sensitivity.","evidence":"ChIP, reporter, and Bcl-x rescue in Prep1(i/i) MEFs; hypomorphic muscle plus L6 cells, glucose uptake, and in vivo p160 cDNA rescue","pmids":["19103748","18644868"],"confidence":"High","gaps":["How PREP1 dosage toggles between pro- and anti-apoptotic outputs not resolved at this stage"]},{"year":2009,"claim":"Uncovered a non-transcriptional cytoplasmic function: PREP1 represses specific mRNA translation.","evidence":"Co-IP, GST pull-down, 4EHP-motif mutagenesis, RNA EMSA, and in vitro translation showing PREP1 binds Hoxb4 3'UTR and represses translation via 4EHP in oocytes","pmids":["19365557"],"confidence":"High","gaps":["Whether this translational repression occurs in somatic tissues unknown","Breadth of PREP1-bound mRNAs not defined"]},{"year":2010,"claim":"Established PREP1 as a guardian of genome integrity acting through p53 and heterochromatin, and a hepatic insulin-signaling regulator via SHP1.","evidence":"γH2AX/cytogenetics, siRNA, ChIP and transformation rescue in MEFs/fibroblasts; Prep1(-/-)/p53(-/-) epistasis; p53 induction assays; SHP1 ChIP/reporter with hyperinsulinemic clamp","pmids":["21715654","20826531","20110257","20864515"],"confidence":"High","gaps":["Mechanism linking PREP1 loss to heterochromatin remodeling not molecularly defined","Whether p53 is a direct ChIP-confirmed target (Medium-confidence #23) not settled"]},{"year":2013,"claim":"Defined genome-wide direct targeting and the p160-PGC-1α mechanism for mitochondrial and lipid metabolism.","evidence":"Muscle-specific KO with ChIP-seq identifying direct OXPHOS gene binding; SHIP2 ChIP/reporter with PBX1-binding-deficient HR mutant in liver","pmids":["24216763","24052111"],"confidence":"High","gaps":["Direct vs p160-mediated indirect effects on PGC-1α targets not fully partitioned"]},{"year":2014,"claim":"Resolved the PREP1-MEIS1-PBX1 competition as a tumor-suppressive mechanism and identified PREP1 as an EMT/metastasis driver via SMAD3.","evidence":"Co-IP, stability assays, and transformation in MEFs showing PREP1 sequesters PBX1 to destabilize MEIS1; reporter/IF/colonization assays for SMAD3 intron transactivation in NSCLC","pmids":["24578510","25157139"],"confidence":"High","gaps":["Contradiction between tumor-suppressive (MEIS1 sequestration) and pro-metastatic (EMT) roles not mechanistically reconciled","Tissue determinants of opposing outputs unknown"]},{"year":2015,"claim":"Provided structural and genome-scale views: a C-terminal homeodomain motif enabling cooperativity, ES-cell-specific enhancer binding, and Wnt/Fgf target pathways.","evidence":"NMR/biophysics of PREP1 HD; ChIP-seq/RNA-seq with rescue in Prep1(-/-) ES cells; ChIP at Glut4 with MEF2/HDAC5 recruitment in diabetic models","pmids":["25856340","25875616","26453063"],"confidence":"High","gaps":["Full structure of the PREP1-PBX-DNA ternary complex still unresolved","How high-glucose epigenetic induction of Prep1 integrates with target repression not fully mapped"]},{"year":2018,"claim":"Extended PREP1 function to nuclear mechanics and DNA replication control, plus tissue-specific roles in spermatogenesis, neurotrophin signaling, and angiogenesis.","evidence":"Repli-seq/DNA combing with Lamin B1 Western; AFM/traction-force microscopy and LINC/YAP-TAZ readouts; conditional germ cell KO; N2A overexpression with BDNF-TrkB assays; endothelial p160/PGC-1α assays with PREP1(54-72) peptide; biophysical PREP1-p160 KD","pmids":["29453404","35550602","29293683","29349576","31618597","29069751","26979610"],"confidence":"High","gaps":["Mechanistic link between PREP1, Lamin B1, and replication timing not established at molecular level","Whether nuclear-stiffness role is transcriptional or structural unresolved"]},{"year":2018,"claim":"Identified a composite SP2-PBX1:PREP1-NF-Y complex defining a higher-order combinatorial code for genomic occupancy.","evidence":"ChIP-exo, co-IP, and motif analysis in MEFs showing mutual potentiation of binding at spaced TALE/NF-Y motifs","pmids":["30337366"],"confidence":"High","gaps":["Functional transcriptional output of the SP2-containing complex not defined"]},{"year":2019,"claim":"Generalized the protein-stabilization mechanism to additional partners controlling stemness and EMT.","evidence":"Co-IP and proteasome-inhibitor rescue showing PREP1 stabilizes SNAIL2 in mammary progenitors, with mammosphere/branching and TP53-apoptosis readouts in conditional KO","pmids":["30604852"],"confidence":"Medium","gaps":["Single-lab co-IP without structural mapping of the PREP1-SNAIL2 interface","Whether stabilization is direct or via an intermediary not resolved"]},{"year":2023,"claim":"Linked PKNOX1 to Hedgehog and TGF-β/SMAD signaling in gastric cancer via direct promoter targeting and a new partner.","evidence":"Luciferase, ChIP, and co-IP in stomach adenocarcinoma showing PKNOX1 transactivates DHH and TGF-β1 and interacts with ANKRD49","pmids":["41821002"],"confidence":"Medium","gaps":["Single-lab study; ANKRD49-PKNOX1 interaction not reciprocally validated","Direct vs indirect TGF-β1 regulation needs orthogonal confirmation"]},{"year":null,"claim":"How PREP1 dosage and partner choice (PBX vs MEIS vs p160 vs SNAIL2) are switched to produce its opposing tumor-suppressive versus pro-metastatic and pro-survival versus pro-apoptotic outputs in a given tissue remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying regulatory model for context-dependent partner selection","Structure of the PREP1-PBX-HOX-DNA ternary complex still lacking","Molecular link between PREP1, Lamin B1, and replication timing undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,4,5,10,17,20,25,26,28,31,40,42]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,2,30,32]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[0,27,40]},{"term_id":"GO:0045182","term_label":"translation regulator activity","supporting_discovery_ids":[19]},{"term_id":"GO:0003723","term_label":"RNA binding","supporting_discovery_ids":[19]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[9,27,49]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[3,6]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,19]},{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[40]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,5,10,17,20,40]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[5,6,11,13,29,36]},{"term_id":"R-HSA-5357801","term_label":"Programmed Cell Death","supporting_discovery_ids":[17,22,23]},{"term_id":"R-HSA-73894","term_label":"DNA Repair","supporting_discovery_ids":[21,22]},{"term_id":"R-HSA-69306","term_label":"DNA Replication","supporting_discovery_ids":[35]},{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[18,20,25,26,31]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[9,27,49]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[28,38,42]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[27,28,47,48]}],"complexes":["PREP1-PBX heterodimer","PREP1-PBX-HOX ternary complex","SP2-PBX1:PREP1-NF-Y complex","UEF3 complex"],"partners":["PBX1","PBX2","MEIS1","MYBBP1A","SMAD3","EIF4E2","SP2","SNAI2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"P55347","full_name":"Homeobox protein PKNOX1","aliases":["Homeobox protein PREP-1","PBX/knotted homeobox 1"],"length_aa":436,"mass_kda":47.6,"function":"Activates transcription in the presence of PBX1A and HOXA1","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P55347/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/PKNOX1","classification":"Not Classified","n_dependent_lines":9,"n_total_lines":1208,"dependency_fraction":0.0074503311258278145},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/PKNOX1","total_profiled":1310},"omim":[{"mim_id":"613066","title":"PBX/KNOTTED 1 HOMEOBOX 2; PKNOX2","url":"https://www.omim.org/entry/613066"},{"mim_id":"602965","title":"FATTY ACID-BINDING PROTEIN 7; FABP7","url":"https://www.omim.org/entry/602965"},{"mim_id":"602100","title":"PBX/KNOTTED 1 HOMEOBOX 1; PKNOX1","url":"https://www.omim.org/entry/602100"},{"mim_id":"190685","title":"DOWN SYNDROME","url":"https://www.omim.org/entry/190685"},{"mim_id":"176312","title":"PRE-B-CELL LEUKEMIA TRANSCRIPTION FACTOR 3; PBX3","url":"https://www.omim.org/entry/176312"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/PKNOX1"},"hgnc":{"alias_symbol":["PREP1"],"prev_symbol":[]},"alphafold":{"accession":"P55347","domains":[{"cath_id":"1.10.10.60","chopping":"267-324","consensus_level":"medium","plddt":91.62,"start":267,"end":324},{"cath_id":"1.20.58","chopping":"50-154","consensus_level":"high","plddt":90.3759,"start":50,"end":154}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P55347","model_url":"https://alphafold.ebi.ac.uk/files/AF-P55347-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P55347-F1-predicted_aligned_error_v6.png","plddt_mean":60.59},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=PKNOX1","jax_strain_url":"https://www.jax.org/strain/search?query=PKNOX1"},"sequence":{"accession":"P55347","fasta_url":"https://rest.uniprot.org/uniprotkb/P55347.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P55347/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P55347"}},"corpus_meta":[{"pmid":"10215622","id":"PMC_10215622","title":"The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH.","date":"1999","source":"Genes & development","url":"https://pubmed.ncbi.nlm.nih.gov/10215622","citation_count":203,"is_preprint":false},{"pmid":"18093541","id":"PMC_18093541","title":"Interleukin-10 expression in macrophages during phagocytosis of apoptotic cells is mediated by homeodomain proteins Pbx1 and Prep-1.","date":"2007","source":"Immunity","url":"https://pubmed.ncbi.nlm.nih.gov/18093541","citation_count":198,"is_preprint":false},{"pmid":"10654609","id":"PMC_10654609","title":"Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins.","date":"2000","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/10654609","citation_count":193,"is_preprint":false},{"pmid":"9482740","id":"PMC_9482740","title":"The novel homeoprotein Prep1 modulates Pbx-Hox protein cooperativity.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9482740","citation_count":183,"is_preprint":false},{"pmid":"9405651","id":"PMC_9405651","title":"Meis1 and pKnox1 bind DNA cooperatively with Pbx1 utilizing an interaction surface disrupted in oncoprotein E2a-Pbx1.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9405651","citation_count":167,"is_preprint":false},{"pmid":"9482739","id":"PMC_9482739","title":"Prep1, a novel functional partner of Pbx proteins.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9482739","citation_count":149,"is_preprint":false},{"pmid":"16705164","id":"PMC_16705164","title":"Two-step regulation of Ad4BP/SF-1 gene transcription during fetal adrenal development: initiation by a Hox-Pbx1-Prep1 complex and maintenance via autoregulation by Ad4BP/SF-1.","date":"2006","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16705164","citation_count":109,"is_preprint":false},{"pmid":"10471746","id":"PMC_10471746","title":"A conserved motif N-terminal to the DNA-binding domains of myogenic bHLH transcription factors mediates cooperative DNA binding with pbx-Meis1/Prep1.","date":"1999","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/10471746","citation_count":97,"is_preprint":false},{"pmid":"9933599","id":"PMC_9933599","title":"Functional and cooperative interactions between the homeodomain PDX1, Pbx, and Prep1 factors on the somatostatin promoter.","date":"1999","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/9933599","citation_count":86,"is_preprint":false},{"pmid":"25157139","id":"PMC_25157139","title":"Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-β-SMAD3 pathway in non-small cell lung adenocarcinoma.","date":"2014","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/25157139","citation_count":84,"is_preprint":false},{"pmid":"14764653","id":"PMC_14764653","title":"Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1.","date":"2004","source":"Molecular endocrinology (Baltimore, Md.)","url":"https://pubmed.ncbi.nlm.nih.gov/14764653","citation_count":82,"is_preprint":false},{"pmid":"16847320","id":"PMC_16847320","title":"Hypomorphic mutation of the TALE gene Prep1 (pKnox1) causes a major reduction of Pbx and Meis proteins and a pleiotropic embryonic phenotype.","date":"2006","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16847320","citation_count":78,"is_preprint":false},{"pmid":"16166636","id":"PMC_16166636","title":"Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16166636","citation_count":74,"is_preprint":false},{"pmid":"10381567","id":"PMC_10381567","title":"The PBX-regulating protein PREP1 is present in different PBX-complexed forms in mouse.","date":"1999","source":"Mechanisms of development","url":"https://pubmed.ncbi.nlm.nih.gov/10381567","citation_count":61,"is_preprint":false},{"pmid":"14711874","id":"PMC_14711874","title":"Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation.","date":"2004","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/14711874","citation_count":58,"is_preprint":false},{"pmid":"29524390","id":"PMC_29524390","title":"MiR-223/Pknox1 axis protects mice from CVB3-induced viral myocarditis by modulating macrophage polarization.","date":"2018","source":"Experimental cell research","url":"https://pubmed.ncbi.nlm.nih.gov/29524390","citation_count":56,"is_preprint":false},{"pmid":"28322463","id":"PMC_28322463","title":"A tale of TALE, PREP1, PBX1, and MEIS1: Interconnections and competition in cancer.","date":"2017","source":"BioEssays : news and reviews in molecular, cellular and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/28322463","citation_count":53,"is_preprint":false},{"pmid":"24578510","id":"PMC_24578510","title":"Prep1 and Meis1 competition for Pbx1 binding regulates protein stability and tumorigenesis.","date":"2014","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/24578510","citation_count":48,"is_preprint":false},{"pmid":"10869353","id":"PMC_10869353","title":"Heterodimeric Pbx-Prep1 homeodomain protein binding to the glucagon gene restricting transcription in a cell type-dependent manner.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10869353","citation_count":46,"is_preprint":false},{"pmid":"17875935","id":"PMC_17875935","title":"p160 Myb-binding protein interacts with Prep1 and inhibits its transcriptional activity.","date":"2007","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17875935","citation_count":42,"is_preprint":false},{"pmid":"19365557","id":"PMC_19365557","title":"Cytoplasmic Prep1 interacts with 4EHP inhibiting Hoxb4 translation.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19365557","citation_count":41,"is_preprint":false},{"pmid":"20106730","id":"PMC_20106730","title":"Prep1 (pKnox1)-deficiency leads to spontaneous tumor development in mice and accelerates EmuMyc lymphomagenesis: a tumor suppressor role for Prep1.","date":"2010","source":"Molecular oncology","url":"https://pubmed.ncbi.nlm.nih.gov/20106730","citation_count":41,"is_preprint":false},{"pmid":"16314502","id":"PMC_16314502","title":"Involvement of Prep1 in the alphabeta T-cell receptor T-lymphocytic potential of hematopoietic precursors.","date":"2005","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/16314502","citation_count":39,"is_preprint":false},{"pmid":"10637514","id":"PMC_10637514","title":"An inhibitory switch derepressed by pbx, hox, and Meis/Prep1 partners regulates DNA-binding by pbx1 and E2a-pbx1 and is dispensable for myeloid immortalization by E2a-pbx1.","date":"1999","source":"Oncogene","url":"https://pubmed.ncbi.nlm.nih.gov/10637514","citation_count":39,"is_preprint":false},{"pmid":"12871956","id":"PMC_12871956","title":"Overexpression of PREP-1 in F9 teratocarcinoma cells leads to a functionally relevant increase of PBX-2 by preventing its degradation.","date":"2003","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/12871956","citation_count":39,"is_preprint":false},{"pmid":"9143494","id":"PMC_9143494","title":"Cloning of a novel homeobox-containing gene, PKNOX1, and mapping to human chromosome 21q22.3.","date":"1997","source":"Genomics","url":"https://pubmed.ncbi.nlm.nih.gov/9143494","citation_count":38,"is_preprint":false},{"pmid":"12771203","id":"PMC_12771203","title":"Overexpression of FABP7 in Down syndrome fetal brains is associated with PKNOX1 gene-dosage imbalance.","date":"2003","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/12771203","citation_count":38,"is_preprint":false},{"pmid":"11972344","id":"PMC_11972344","title":"Characterization of PREP2, a paralog of PREP1, which defines a novel sub-family of the MEINOX TALE homeodomain transcription factors.","date":"2002","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/11972344","citation_count":38,"is_preprint":false},{"pmid":"18644868","id":"PMC_18644868","title":"Prep1 deficiency induces protection from diabetes and increased insulin sensitivity through a p160-mediated mechanism.","date":"2008","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/18644868","citation_count":37,"is_preprint":false},{"pmid":"21715654","id":"PMC_21715654","title":"Homeodomain transcription factor and tumor suppressor Prep1 is required to maintain genomic stability.","date":"2011","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/21715654","citation_count":35,"is_preprint":false},{"pmid":"15792957","id":"PMC_15792957","title":"Activation of the rat renin promoter by HOXD10.PBX1b.PREP1, Ets-1, and the intracellular domain of notch.","date":"2005","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15792957","citation_count":32,"is_preprint":false},{"pmid":"17904118","id":"PMC_17904118","title":"The homeodomain transcription factor Prep1 (pKnox1) is required for hematopoietic stem and progenitor cell activity.","date":"2007","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/17904118","citation_count":31,"is_preprint":false},{"pmid":"20826531","id":"PMC_20826531","title":"The absence of Prep1 causes p53-dependent apoptosis of mouse pluripotent epiblast cells.","date":"2010","source":"Development (Cambridge, England)","url":"https://pubmed.ncbi.nlm.nih.gov/20826531","citation_count":31,"is_preprint":false},{"pmid":"22185299","id":"PMC_22185299","title":"MEIS1, PREP1, and PBX4 are differentially expressed in acute lymphoblastic leukemia: association of MEIS1 expression with higher proliferation and chemotherapy resistance.","date":"2011","source":"Journal of experimental & clinical cancer research : CR","url":"https://pubmed.ncbi.nlm.nih.gov/22185299","citation_count":29,"is_preprint":false},{"pmid":"31047333","id":"PMC_31047333","title":"Progression of Retinitis Pigmentosa as Measured on Microperimetry: The PREP-1 Study.","date":"2017","source":"Ophthalmology. Retina","url":"https://pubmed.ncbi.nlm.nih.gov/31047333","citation_count":29,"is_preprint":false},{"pmid":"20864515","id":"PMC_20864515","title":"Prep1 controls insulin glucoregulatory function in liver by transcriptional targeting of SHP1 tyrosine phosphatase.","date":"2010","source":"Diabetes","url":"https://pubmed.ncbi.nlm.nih.gov/20864515","citation_count":28,"is_preprint":false},{"pmid":"26453063","id":"PMC_26453063","title":"Glucose-induced expression of the homeotic transcription factor Prep1 is associated with histone post-translational modifications in skeletal muscle.","date":"2015","source":"Diabetologia","url":"https://pubmed.ncbi.nlm.nih.gov/26453063","citation_count":25,"is_preprint":false},{"pmid":"12529389","id":"PMC_12529389","title":"Identification of homeodomain proteins, PBX1 and PREP1, involved in the transcription of murine leukemia virus.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12529389","citation_count":25,"is_preprint":false},{"pmid":"24052111","id":"PMC_24052111","title":"PREP1 deficiency downregulates hepatic lipogenesis and attenuates steatohepatitis in mice.","date":"2013","source":"Diabetologia","url":"https://pubmed.ncbi.nlm.nih.gov/24052111","citation_count":24,"is_preprint":false},{"pmid":"12065766","id":"PMC_12065766","title":"The homeodomain Pbx2-Prep1 complex modulates hepatocyte nuclear factor 1alpha-mediated activation of the UDP-glucuronosyltransferase 2B17 gene.","date":"2002","source":"Molecular pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/12065766","citation_count":24,"is_preprint":false},{"pmid":"20110257","id":"PMC_20110257","title":"Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress.","date":"2010","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/20110257","citation_count":23,"is_preprint":false},{"pmid":"24216763","id":"PMC_24216763","title":"Pknox1/Prep1 regulates mitochondrial oxidative phosphorylation components in skeletal muscle.","date":"2013","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/24216763","citation_count":22,"is_preprint":false},{"pmid":"19103748","id":"PMC_19103748","title":"Prep1 directly regulates the intrinsic apoptotic pathway by controlling Bcl-XL levels.","date":"2008","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/19103748","citation_count":21,"is_preprint":false},{"pmid":"18480829","id":"PMC_18480829","title":"Prep1/Pbx2 complexes regulate CCL2 expression through the -2578 guanine polymorphism.","date":"2008","source":"Genes and immunity","url":"https://pubmed.ncbi.nlm.nih.gov/18480829","citation_count":20,"is_preprint":false},{"pmid":"15656786","id":"PMC_15656786","title":"The CYP2B2 phenobarbital response unit contains binding sites for hepatocyte nuclear factor 4, PBX-PREP1, the thyroid hormone receptor beta and the liver X receptor.","date":"2005","source":"The Biochemical journal","url":"https://pubmed.ncbi.nlm.nih.gov/15656786","citation_count":18,"is_preprint":false},{"pmid":"30338914","id":"PMC_30338914","title":"MiR-17 family-mediated regulation of Pknox1 influences hepatic steatosis and insulin signaling.","date":"2018","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/30338914","citation_count":16,"is_preprint":false},{"pmid":"29069751","id":"PMC_29069751","title":"A peptide antagonist of Prep1-p160 interaction improves ceramide-induced insulin resistance in skeletal muscle cells.","date":"2017","source":"Oncotarget","url":"https://pubmed.ncbi.nlm.nih.gov/29069751","citation_count":16,"is_preprint":false},{"pmid":"30002646","id":"PMC_30002646","title":"Prep1, A Homeodomain Transcription Factor Involved in Glucose and Lipid Metabolism.","date":"2018","source":"Frontiers in endocrinology","url":"https://pubmed.ncbi.nlm.nih.gov/30002646","citation_count":14,"is_preprint":false},{"pmid":"25875616","id":"PMC_25875616","title":"ChIP-Seq and RNA-Seq analyses identify components of the Wnt and Fgf signaling pathways as Prep1 target genes in mouse embryonic stem cells.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25875616","citation_count":14,"is_preprint":false},{"pmid":"22347417","id":"PMC_22347417","title":"Single nucleotide polymorphism in gene encoding transcription factor Prep1 is associated with HIV-1-associated dementia.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/22347417","citation_count":13,"is_preprint":false},{"pmid":"31618597","id":"PMC_31618597","title":"Prep1 regulates angiogenesis through a PGC-1α-mediated mechanism.","date":"2019","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/31618597","citation_count":12,"is_preprint":false},{"pmid":"29349576","id":"PMC_29349576","title":"Prep1 Deficiency Affects Olfactory Perception and Feeding Behavior by Impairing BDNF-TrkB Mediated Neurotrophic Signaling.","date":"2018","source":"Molecular neurobiology","url":"https://pubmed.ncbi.nlm.nih.gov/29349576","citation_count":12,"is_preprint":false},{"pmid":"35812737","id":"PMC_35812737","title":"miR-19b-3p/PKNOX1 Regulates Viral Myocarditis by Regulating Macrophage Polarization.","date":"2022","source":"Frontiers in genetics","url":"https://pubmed.ncbi.nlm.nih.gov/35812737","citation_count":12,"is_preprint":false},{"pmid":"29453404","id":"PMC_29453404","title":"PREP1 tumor suppressor protects the late-replicating DNA by controlling its replication timing and symmetry.","date":"2018","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29453404","citation_count":11,"is_preprint":false},{"pmid":"21900201","id":"PMC_21900201","title":"C-terminal domain of MEIS1 converts PKNOX1 (PREP1) into a HOXA9-collaborating oncoprotein.","date":"2011","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/21900201","citation_count":11,"is_preprint":false},{"pmid":"27123834","id":"PMC_27123834","title":"The miR-17∼92 cluster contributes to MLL leukemia through the repression of MEIS1 competitor PKNOX1.","date":"2016","source":"Leukemia research","url":"https://pubmed.ncbi.nlm.nih.gov/27123834","citation_count":11,"is_preprint":false},{"pmid":"26979610","id":"PMC_26979610","title":"The Interacting Domains of PREP1 and p160 are Endowed with a Remarkable Structural Stability.","date":"2016","source":"Molecular biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/26979610","citation_count":10,"is_preprint":false},{"pmid":"25856340","id":"PMC_25856340","title":"Purification and characterization of a DNA-binding recombinant PREP1:PBX1 complex.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25856340","citation_count":10,"is_preprint":false},{"pmid":"17623278","id":"PMC_17623278","title":"Purification of the Prep1 interactome identifies novel pathways regulated by Prep1.","date":"2007","source":"Proteomics","url":"https://pubmed.ncbi.nlm.nih.gov/17623278","citation_count":10,"is_preprint":false},{"pmid":"34679197","id":"PMC_34679197","title":"Interleukin 6 reduces vascular smooth muscle cell apoptosis via Prep1 and is associated with aging.","date":"2021","source":"FASEB journal : official publication of the Federation of American Societies for Experimental Biology","url":"https://pubmed.ncbi.nlm.nih.gov/34679197","citation_count":9,"is_preprint":false},{"pmid":"25233378","id":"PMC_25233378","title":"Prep1 (pKnox1) regulates mouse embryonic HSC cycling and self-renewal affecting the Stat1-Sca1 IFN-dependent pathway.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/25233378","citation_count":9,"is_preprint":false},{"pmid":"36356379","id":"PMC_36356379","title":"Polyunsaturated fatty acids ameliorate renal stone-induced renal tubular damage via miR-93-5p/Pknox1 axis.","date":"2022","source":"Nutrition (Burbank, Los Angeles County, Calif.)","url":"https://pubmed.ncbi.nlm.nih.gov/36356379","citation_count":9,"is_preprint":false},{"pmid":"20423710","id":"PMC_20423710","title":"prep1.2 and aldh1a2 participate to a positive loop required for branchial arches development in zebrafish.","date":"2010","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/20423710","citation_count":9,"is_preprint":false},{"pmid":"18560600","id":"PMC_18560600","title":"Cytosolic sequestration of Prep1 influences early stages of T cell development.","date":"2008","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/18560600","citation_count":9,"is_preprint":false},{"pmid":"30337366","id":"PMC_30337366","title":"Transcription factor Sp2 potentiates binding of the TALE homeoproteins Pbx1:Prep1 and the histone-fold domain protein Nf-y to composite genomic sites.","date":"2018","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/30337366","citation_count":9,"is_preprint":false},{"pmid":"29474930","id":"PMC_29474930","title":"Prep1 deficiency improves metabolic response in white adipose tissue.","date":"2018","source":"Biochimica et biophysica acta. Molecular and cell biology of lipids","url":"https://pubmed.ncbi.nlm.nih.gov/29474930","citation_count":8,"is_preprint":false},{"pmid":"37194045","id":"PMC_37194045","title":"Bortezomib induced peripheral neuropathy and single nucleotide polymorphisms in PKNOX1.","date":"2023","source":"Biomarker research","url":"https://pubmed.ncbi.nlm.nih.gov/37194045","citation_count":8,"is_preprint":false},{"pmid":"29138456","id":"PMC_29138456","title":"Prep1 prevents premature adipogenesis of mesenchymal progenitors.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/29138456","citation_count":8,"is_preprint":false},{"pmid":"24809472","id":"PMC_24809472","title":"The deficiency of tumor suppressor prep1 accelerates the onset of meis1- hoxa9 leukemogenesis.","date":"2014","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/24809472","citation_count":8,"is_preprint":false},{"pmid":"23133585","id":"PMC_23133585","title":"Reduction of Prep1 levels affects differentiation of normal and malignant B cells and accelerates Myc driven lymphomagenesis.","date":"2012","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23133585","citation_count":8,"is_preprint":false},{"pmid":"37843471","id":"PMC_37843471","title":"Lnc-PKNOX1-1 inhibits tumor progression in cutaneous malignant melanoma by regulating NF-κB/IL-8 axis.","date":"2023","source":"Carcinogenesis","url":"https://pubmed.ncbi.nlm.nih.gov/37843471","citation_count":7,"is_preprint":false},{"pmid":"35550602","id":"PMC_35550602","title":"The transcription factor PREP1(PKNOX1) regulates nuclear stiffness, the expression of LINC complex proteins and mechanotransduction.","date":"2022","source":"Communications biology","url":"https://pubmed.ncbi.nlm.nih.gov/35550602","citation_count":7,"is_preprint":false},{"pmid":"34327205","id":"PMC_34327205","title":"Leptin and TGF-β1 Downregulate PREP1 Expression in Human Adipose-Derived Mesenchymal Stem Cells and Mature Adipocytes.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/34327205","citation_count":7,"is_preprint":false},{"pmid":"28094776","id":"PMC_28094776","title":"New Insights into Cooperative Binding of Homeodomain Transcription Factors PREP1 and PBX1 to DNA.","date":"2017","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/28094776","citation_count":7,"is_preprint":false},{"pmid":"30604851","id":"PMC_30604851","title":"The genetics and the molecular functions of the PREP1 homeodomain transcription factor.","date":"2018","source":"The International journal of developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/30604851","citation_count":6,"is_preprint":false},{"pmid":"29293683","id":"PMC_29293683","title":"Germ cell-intrinsic requirement for the homeodomain transcription factor PKnox1/Prep1 in adult spermatogenesis.","date":"2018","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/29293683","citation_count":6,"is_preprint":false},{"pmid":"36237510","id":"PMC_36237510","title":"Stable and reproducible expression of bacterial ipt gene under the control of SAM-specific promoter (pKNOX1) with interference of developmental patterns in transgenic Peperomia pellucida plants.","date":"2022","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/36237510","citation_count":5,"is_preprint":false},{"pmid":"30604852","id":"PMC_30604852","title":"Prep1 (pKnox1) transcription factor contributes to pubertal mammary gland branching morphogenesis.","date":"2018","source":"The International journal of developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/30604852","citation_count":5,"is_preprint":false},{"pmid":"26285139","id":"PMC_26285139","title":"Loss of the Homeodomain Transcription Factor Prep1 Perturbs Adult Hematopoiesis in the Bone Marrow.","date":"2015","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/26285139","citation_count":5,"is_preprint":false},{"pmid":"31349607","id":"PMC_31349607","title":"The Role of Prep1 in the Regulation of Mesenchymal Stromal Cells.","date":"2019","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/31349607","citation_count":4,"is_preprint":false},{"pmid":"38640751","id":"PMC_38640751","title":"MicroRNA-1258 suppresses oxidative stress and inflammation in septic acute lung injury through the Pknox1-regulated TGF-β1/SMAD3 cascade.","date":"2024","source":"Clinics (Sao Paulo, Brazil)","url":"https://pubmed.ncbi.nlm.nih.gov/38640751","citation_count":3,"is_preprint":false},{"pmid":"21203543","id":"PMC_21203543","title":"Characterization of the regulatory region of the zebrafish Prep1.1 gene: analogies to the promoter of the human PREP1.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/21203543","citation_count":3,"is_preprint":false},{"pmid":"37569266","id":"PMC_37569266","title":"Resveratrol Improves Endothelial Function by A PREP1-Mediated Pathway in Mouse Aortic Endothelial Cells.","date":"2023","source":"International journal of molecular sciences","url":"https://pubmed.ncbi.nlm.nih.gov/37569266","citation_count":2,"is_preprint":false},{"pmid":"37864517","id":"PMC_37864517","title":"PKNOX1 acts as a transcription factor of DHH and promotes the progression of stomach adenocarcinoma by regulating the Hedgehog signalling pathway.","date":"2023","source":"International journal of immunopathology and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/37864517","citation_count":2,"is_preprint":false},{"pmid":"15468914","id":"PMC_15468914","title":"[Molecular cloning for an alternatively splicing cDNA of human PKNOX1 gene and it's expression analysis].","date":"2004","source":"Yi chuan xue bao = Acta genetica Sinica","url":"https://pubmed.ncbi.nlm.nih.gov/15468914","citation_count":0,"is_preprint":false},{"pmid":"38682403","id":"PMC_38682403","title":"The Characterization of Multifaceted PREP1 Peptides Provides Insights into Correlations between Spectroscopic and Structural Properties of Amyloid-like Assemblies.","date":"2024","source":"Chemistry (Weinheim an der Bergstrasse, Germany)","url":"https://pubmed.ncbi.nlm.nih.gov/38682403","citation_count":0,"is_preprint":false},{"pmid":"41821002","id":"PMC_41821002","title":"ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis.","date":"2026","source":"Cancer cell international","url":"https://pubmed.ncbi.nlm.nih.gov/41821002","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":45738,"output_tokens":12815,"usd":0.164719,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":24339,"output_tokens":6036,"usd":0.136297,"stage2_stop_reason":"end_turn"},"total_usd":0.301016,"stage1_batch_id":"msgbatch_01CNoDbkChHEkgrJnL7BwtGJ","stage2_batch_id":"msgbatch_016Y4x9E9Wg5VQDANj9quSQD","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"PREP1 forms a stable heterodimer with PBX proteins independent of DNA binding, requiring sequences in the N-terminal portions of both proteins (HR1/HR2 domains). The PREP1-PBX interaction is essential for DNA-binding activity, and PREP1 enhances the ability of the HOXB1-PBX1 complex to activate transcription cooperatively, including formation of a ternary PREP1-PBX1-HOXB1 complex on a HOXB1-responsive target in vitro.\",\n      \"method\": \"Co-immunoprecipitation, in vitro protein-protein interaction assays, EMSA, transient transfection transcriptional assays, mutagenesis of homeodomain and N-terminal interaction surfaces\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (co-IP, EMSA, mutagenesis, transcriptional assays) in founding paper, replicated across multiple subsequent studies\",\n      \"pmids\": [\"9482740\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"PREP1 was cloned as a subunit of the UEF3 complex (urokinase plasminogen activator enhancer factor 3). PREP1 is a TALE-class homeodomain protein that forms a stable complex with PBX proteins in solution independent of DNA, and the heterodimer binds the TGACAG motif in the uPA promoter with high affinity. The other UEF3 subunit was identified as a PBX family member.\",\n      \"method\": \"cDNA cloning, EMSA, co-immunoprecipitation, in vitro binding assays\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — founding biochemical characterization with multiple orthogonal methods; replicated by companion paper\",\n      \"pmids\": [\"9482739\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"PKNOX1 (PREP1) dimerizes with PBX1 on the TGATTGAC motif and binds DNA cooperatively. The PKNOX1/MEIS1-interaction domain in PBX1 resides in a conserved N-terminal PBX domain deleted in the leukemic oncoprotein E2a-PBX1; consequently, PKNOX1 and MEIS1 fail to dimerize significantly with E2a-PBX1.\",\n      \"method\": \"EMSA, GST pull-down, nuclear extract binding assays\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reconstitution-level biochemical assays with domain mapping; replicated by multiple subsequent studies\",\n      \"pmids\": [\"9405651\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PREP1 is cytoplasmic and requires association with PBX/EXD for nuclear localization. Coexpression of EXD/PBX with PREP1/HTH causes nuclear localization of their heterodimers in both mammalian fibroblasts and Drosophila Schneider cells. Heterodimerization with PBX induces nuclear translocation of PREP1 by blocking nuclear export of PBX.\",\n      \"method\": \"Immunofluorescence microscopy, subcellular fractionation, co-expression experiments in multiple cell contexts\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct localization experiments with functional consequence, tested in multiple cell contexts, replicated independently\",\n      \"pmids\": [\"10215622\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PREP1-PBX heterodimers bind the UE-A element of the somatostatin promoter cooperatively (neither protein alone can bind), and both PBX1 and PREP1 are required for transcriptional activation from this element. In the context of the intact somatostatin mini-enhancer, PREP1-PBX1 cooperates with the pancreatic homeodomain factor PDX1 to produce drastic transcriptional activation.\",\n      \"method\": \"EMSA with recombinant proteins, transient transfection reporter assays, co-expression experiments\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution with recombinant proteins plus cellular reporter assays, multiple orthogonal methods\",\n      \"pmids\": [\"9933599\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PREP1-PBX complexes bind Pbx-Meinox (PM) sites in the Hoxb2 r4 enhancer, and the PM site is essential in vivo for r4-restricted expression. Both PM and PH (Pbx-Hox) sites are required for formation and binding of a ternary HOXB1-PBX1a-PREP1 complex in vitro. A similar ternary complex forms in nuclear extracts from retinoic acid-induced P19 cells, containing PREP1 (or MEIS1), PBX, and HOXB1.\",\n      \"method\": \"EMSA, transgenic mouse enhancer assays, nuclear extract binding assays, site-directed mutagenesis of enhancer elements\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro biochemical assays combined with in vivo transgenic validation, multiple orthogonal methods\",\n      \"pmids\": [\"10654609\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PREP1 is present in mouse embryos from at least E9.5, is ubiquitously nuclear in embryonic tissues (E7.5–17.5), and forms tissue-specific DNA-binding complexes with different PBX family members depending on cell type. PREP1 super-activates the PBX-HOXB1 autoregulated Hoxb-1 promoter, and all three proteins (PREP1, PBX, HOXB1) are co-localized in rhombomere 4 in vivo.\",\n      \"method\": \"Co-immunoprecipitation, EMSA, immunohistochemistry, Northern blot, in situ hybridization\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods, in vivo validation, replicated across tissues\",\n      \"pmids\": [\"10381567\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"PBX-MEIS1/PREP1 heterodimers cooperate with myogenic bHLH proteins (MyoD, myogenin, Mrf-4, Myf-5) for DNA binding. A conserved tryptophan motif N-terminal to the DNA-binding domain of each myogenic bHLH protein is required for cooperative DNA binding with PBX-MEIS1/PREP1. MyoD requires this tryptophan motif to evoke chromatin remodeling at the Myogenin promoter.\",\n      \"method\": \"EMSA, mutagenesis, chromatin remodeling assays, transcriptional reporter assays\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods including mutagenesis and chromatin assays; mechanistically defined interaction surface\",\n      \"pmids\": [\"10471746\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"PREP1-PBX heterodimers bind the glucagon gene promoter (domain B of the G3 enhancer and the cAMP response element and G5 element) and restrict transcription in non-glucagon-producing cells. Coexpression of Pbx1a/1b-Prep1 inhibited the glucagon promoter activated by Pax6 in non-glucagon-producing cells, but had no effect in glucagon-producing islet cells.\",\n      \"method\": \"EMSA with specific antiserum and in vitro translated proteins, transient transfection reporter assays\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA with antibody supershift and functional reporter assays, single lab\",\n      \"pmids\": [\"10869353\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PREP1 overexpression increases PBX-2 protein levels by preventing its proteasomal degradation. Dimerization with PREP1 stabilizes PBX-2 protein against cycloheximide-induced decay; the proteasome inhibitor MG132 prevents PBX-2 decay in control cells, indicating PBX-2 is normally degraded by the proteasome and PREP1 binding protects it.\",\n      \"method\": \"Cycloheximide chase, proteasome inhibitor (MG132) treatment, Western blot, EMSA, stable transfection\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — biochemical mechanism established with multiple orthogonal methods (cycloheximide chase, proteasome inhibition, Western blot); replicated in subsequent studies\",\n      \"pmids\": [\"12871956\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"PREP1 binds the FSHβ promoter in a complex with SMAD4 and PBX1 at the -134 activin response element; PBX1 and PREP1 interact physically with SMAD2 and SMAD3 as shown by GST pull-down. Pbx1 and Prep1 bind the mouse FSHβ gene in vivo, and mutation of the -134 site abrogates activin responsiveness.\",\n      \"method\": \"EMSA, GST interaction assays, transient transfection, chromatin immunoprecipitation (in vivo binding)\",\n      \"journal\": \"Molecular endocrinology (Baltimore, Md.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (GST pulldown, EMSA with antibody supershift, ChIP, reporter assays) establishing physical and functional interaction with SMADs\",\n      \"pmids\": [\"14764653\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"PREP1-PBX1 (PM) binding sites in the Hoxb1 autoregulatory enhancer include three sites (PM1, PM2, and R2/PM3). PM1 and PM2 cooperate to modulate in vivo regulatory activity. R2/PM3 has high binding affinity for PREP1-PBX1 dimers and, in combination with PM1 and R3, inhibits ternary PREP1-PBX1-HOXB1 complex formation in vitro and restricts reporter expression in transgenic embryos.\",\n      \"method\": \"EMSA, site-directed mutagenesis, transgenic chicken and mouse embryo reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro biochemical assays combined with in vivo transgenic validation in two species\",\n      \"pmids\": [\"16166636\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"PREP1 deficiency in hypomorphic Prep1(i/i) mice causes an overall decrease in protein levels of MEIS1, PBX1, and PBX2, strongly reducing total PREP1/MEIS-PBX DNA-binding activity. PREP1 acts upstream of a PBX-MEIS network. The levels of cMyb and Pax6 are significantly decreased in fetal liver and retina of Prep1(i/i) embryos, correlating with hematopoietic and eye phenotypes.\",\n      \"method\": \"Western blot, EMSA, immunohistochemistry, hypomorphic mouse model\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo genetic model with multiple biochemical readouts, replicated across tissues\",\n      \"pmids\": [\"16847320\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"The fetal adrenal enhancer (FAdE) in intron 4 of Ad4BP/SF-1 contains binding sites for PBX-PREP1, PBX-HOX, and Ad4BP/SF-1. PBX/PREP1 and PBX/HOX sites initiate transcription prior to the establishment of the Ad4BP/SF-1 autoregulatory loop during fetal adrenal development.\",\n      \"method\": \"Transgenic mouse assays, site-directed mutagenesis of enhancer elements, EMSA\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — transgenic in vivo validation with mutagenesis, single lab\",\n      \"pmids\": [\"16705164\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"p160 Myb-binding protein (p160) is a novel PREP1-interacting protein that competes with PBX1 for PREP1 binding in vitro. The N-terminal truncated form p67 binds the sequence 63-LFPLL-67 in the HR1 domain of PREP1; mutation of L63 and L66 impairs binding to both p160/p67 and PBX1. p160 inhibits PREP1-dependent HoxB2 expression.\",\n      \"method\": \"Co-immunoprecipitation, in vitro competition assay, site-directed mutagenesis, immunofluorescence colocalization, RT-PCR\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — mutagenesis identifying specific binding residues, competition assay, co-IP, and functional gene expression assay; replicated in multiple subsequent studies\",\n      \"pmids\": [\"17875935\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Tandem affinity purification of PREP1 from nuclear and cytoplasmic fractions identified its interactome, including PBX1b, PBX2, p160 Myb-binding protein (p160), beta-actin, and NMMHCIIA as co-purifying partners.\",\n      \"method\": \"Tandem affinity purification (TAP), mass spectrometry\",\n      \"journal\": \"Proteomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — TAP/MS interactome from functional tagged protein, single lab\",\n      \"pmids\": [\"17623278\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"IL-10 production by macrophages in response to apoptotic cells is regulated at transcription via the apoptotic-cell-response element (ACRE) in the human IL10 promoter; PREP1 (Pbx-regulating protein 1) and PBX1b bind ACRE and mediate transcriptional activation of IL-10 in response to apoptotic cells in a p38 MAPK-dependent manner.\",\n      \"method\": \"Reporter assay (ACRE mapping), EMSA, ChIP, gene knockdown, p38 inhibitor treatment\",\n      \"journal\": \"Immunity\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — multiple orthogonal methods (reporter assay, EMSA, ChIP, functional knockdown) in peer-reviewed high-impact journal\",\n      \"pmids\": [\"18093541\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PREP1 directly controls the intrinsic apoptotic pathway by regulating Bcl-x gene expression. Prep1(i/i) MEFs have decreased Bcl-X(L) mRNA and protein. Chromatin immunoprecipitation and transient transfection identified the Bcl-x promoter as a direct transcriptional target of PREP1. Overexpression of Bcl-x rescues the apoptotic defect of Prep1(i/i) MEFs.\",\n      \"method\": \"ChIP, transient transfection reporter assay, Western blot, annexin V assay, caspase activity assay, Bcl-x overexpression rescue\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP plus reporter assay plus genetic rescue, multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"19103748\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PREP1 deficiency in skeletal muscle increases insulin sensitivity through a p160-GLUT4 pathway. Reduced Prep1 decreases p160 levels, leading to increased PGC-1α and GLUT4 expression. PREP1 controls the stability of the p160 protein. Overexpression of Prep1 or p160 (but not PBX1) reduces GLUT4 and insulin sensitivity; in vivo delivery of p160 cDNA reverses the molecular phenotype of Prep1(i/i) muscle.\",\n      \"method\": \"Hypomorphic mouse model, L6 cell overexpression/knockdown, Western blot, glucose uptake assay, in vivo muscle cDNA delivery\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro and in vivo experiments, genetic rescue, pathway placement established\",\n      \"pmids\": [\"18644868\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Cytoplasmic PREP1 in mouse oocytes interacts with 4EHP (eukaryotic translation initiation factor 4E homolog protein) to repress translation of Hoxb4 mRNA. PREP1 binds the Hoxb4 3'UTR (RNA EMSA), inhibits Hoxb4 mRNA translation >95% in vitro in the presence of 4EHP, and a functional 4EHP-binding motif in PREP1 was identified by mutagenesis. Prep1(i/i) oocytes show oocyte growth failure and increased Hoxb4 protein.\",\n      \"method\": \"Confocal microscopy, co-immunoprecipitation, GST pull-down, mutagenesis of 4EHP-binding motif, RNA EMSA, in vitro translation assay, immunohistochemistry\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — in vitro reconstitution of translational repression, mutagenesis of functional motif, multiple orthogonal methods, in vivo genetic validation\",\n      \"pmids\": [\"19365557\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PREP1 transcriptionally activates the SHP1 tyrosine phosphatase gene via a promoter element between -2113 and -1778. Both PREP1 and PBX1 bind the SHP1 promoter in vivo and cooperatively induce luciferase activity 30-fold. This leads to attenuation of insulin receptor/IRS1/2 phosphorylation and reduced glycogen storage in liver. Antisense silencing of SHP1 (but not SYP) rescues insulin-dependent IR phosphorylation in PREP1-overexpressing cells.\",\n      \"method\": \"ChIP, luciferase reporter assay, Western blot, euglycemic hyperinsulinemic clamp, antisense knockdown, HepG2 overexpression\",\n      \"journal\": \"Diabetes\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP plus reporter assay plus functional rescue, in vivo and in vitro orthogonal methods\",\n      \"pmids\": [\"20864515\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Prep1 is required for genomic stability. Prep1(i/i) fetal liver cells and MEFs exhibit increased basal DNA damage and chromosomal aberrations/aneuploidy. Acute PREP1 knockdown by siRNA in human fibroblasts induces DNA damage response and increases heterochromatin-associated histone methylation with decreased satellite DNA transcription. Ectopic PREP1 expression rescues DNA damage and heterochromatin methylation. PREP1 deficiency facilitates immortalization and H-Ras(V12)-dependent transformation, partially rescued by Prep1 restoration.\",\n      \"method\": \"γH2AX immunofluorescence, cytogenetic analysis, siRNA knockdown, ChIP, satellite DNA transcription assay, transformation assay, rescue experiment\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vivo and in vitro methods with genetic rescue; establishes genomic stability function\",\n      \"pmids\": [\"21715654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Absence of Prep1 in null embryos causes p53-dependent apoptosis of epiblast cells. The embryonic lethality of Prep1(-/-) embryos is partially rescued in a p53(-/-) background but not in a p16(-/-) background, placing PREP1 upstream of p53-dependent apoptosis. Atm downregulation exacerbates the Prep1(-/-) phenotype, suggesting DNA damage as the cause of apoptosis.\",\n      \"method\": \"Genetic epistasis (Prep1(-/-)/p53(-/-) double mutants), TUNEL staining, cleaved caspase 3 immunostaining, immunohistochemistry\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with double mutant rescue, multiple mutant backgrounds tested\",\n      \"pmids\": [\"20826531\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Excess PREP1 increases sensitivity of cells to genotoxic stress via p53. PREP1 overexpression induces p53, and p53 depletion reverts the apoptotic phenotype of PREP1-overexpressing cells. p53 is identified as a direct transcriptional target of PREP1. PREP1 overexpression also increases anti-apoptotic Bcl-X(L) levels, but pro-apoptotic p53 induction dominates.\",\n      \"method\": \"Apoptosis assays (genotoxic stress), p53 siRNA rescue, Western blot, transient transfection reporter assay (implied by 'direct target')\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic rescue experiments and functional assays; abstract implies direct target relationship but does not explicitly describe ChIP for p53\",\n      \"pmids\": [\"20110257\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PREP1 deficiency results in premature cycling and exhaustion of fetal hematopoietic stem cells (HSCs) through induction of interferon signaling (Stat1-Sca1 IFN-dependent pathway). Prep1 controls the self-renewal ability of FL HSCs, as shown by serial transplantation experiments.\",\n      \"method\": \"Hypomorphic mouse model, serial transplantation, flow cytometry, gene expression analysis\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo serial transplantation with pathway identification, single lab\",\n      \"pmids\": [\"25233378\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PREP1 regulates mitochondrial oxidative phosphorylation components in skeletal muscle through direct and indirect mechanisms. Muscle-specific ablation of Prep1 increases expression of respiratory chain subunits and mitochondrial enzyme activity. PREP1 stabilizes p160 Mybbp1a, a known inhibitor of PGC-1α activity; Prep1 ablation reduces p160, elevating PGC-1α. ChIP-seq identifies direct PREP1 binding sites in genes encoding mitochondrial components (e.g., Ndufs2).\",\n      \"method\": \"Muscle-specific conditional knockout, electron microscopy, treadmill exercise testing, Western blot, ChIP-seq\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — tissue-specific KO, genome-wide ChIP-seq, multiple phenotypic and biochemical readouts; establishes both direct and indirect mechanisms\",\n      \"pmids\": [\"24216763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PREP1/PBX1 complex transcriptionally activates the SHIP2 (Inppl1) promoter, and the PREP1-HR mutant (unable to bind PBX1) has no effect on Ship2, demonstrating that PREP1-PBX1 dimerization is required for this transcriptional activation. PREP1 deficiency reduces SHIP2, leading to increased PKCζ/LKB1/AMPK/ACC phosphorylation and reduced hepatic lipogenesis.\",\n      \"method\": \"ChIP, luciferase reporter assay, PBX1-binding mutant (HR mutant), Western blot, hypomorphic mouse model\",\n      \"journal\": \"Diabetologia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP plus reporter assay plus dimerization-deficient mutant establishing PBX1-dependence; multiple orthogonal methods\",\n      \"pmids\": [\"24052111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PREP1 and MEIS1 compete for PBX1 binding in mouse embryonic fibroblasts. PREP1 posttranslationally controls MEIS1 levels by decreasing its stability through sequestering PBX1. MEIS1 alone transforms Prep1-deficient fibroblasts; PREP1 overexpression inhibits MEIS1 tumorigenicity. PREP1 also modifies the DNA binding landscape of MEIS1 and prevents MEIS1 interaction with DDX3X and DDX5.\",\n      \"method\": \"Co-immunoprecipitation, protein stability assays, transformation assay, transcriptional profiling, MEF genetic model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods establishing competition mechanism, post-translational stability control, and functional consequences; peer-reviewed PNAS\",\n      \"pmids\": [\"24578510\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"PREP1 is a novel EMT inducer and prometastatic transcription factor in non-small cell lung cancer. PREP1 overexpression triggers EMT, and PREP1 down-regulation inhibits TGF-β-induced EMT. Mechanistically, PREP1 induces SMAD3 nuclear translocation through transactivation of a regulatory element in the SMAD3 first intron. PREP1 also stabilizes and accumulates PBX1 and induces FRA-1 expression; both FRA-1 and PBX1 are required for PREP1-induced mesenchymal changes.\",\n      \"method\": \"Overexpression/knockdown, reporter assay (SMAD3 intron element), Western blot, immunofluorescence (SMAD3 localization), lung colonization assay\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — reporter assay establishing direct transcriptional target, subcellular localization experiments, functional rescue with FRA-1/PBX1 knockdown, in vivo colonization assay\",\n      \"pmids\": [\"25157139\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In embryonic stem cells, PREP1 binds ~2,000 novel genomic sites compared to embryo trunk, including a higher proportion of enhancers. RNA-seq identifies ~1800 genes down-regulated in Prep1(-/-) ES cells, including essential components of Wnt and Fgf pathways. Prep1(-/-) ES cells show deficient embryoid body formation and differentiation, rescued by re-expression of PREP1.\",\n      \"method\": \"ChIP-seq, RNA-seq, embryoid body differentiation assay, Prep1 knockout ES cells, rescue experiment\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genome-wide ChIP-seq and RNA-seq with genetic rescue; identifies Wnt/Fgf as PREP1 target pathways in ES cells\",\n      \"pmids\": [\"25875616\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"PREP1 and PBX1 homeodomains (HDs) bind DNA cooperatively. The kinetics of individual PREP1 and PBX1 HDs and their combination show cooperative DNA binding. A novel PREP1 motif flanking the HD at the C-terminus is required for cooperativity.\",\n      \"method\": \"NMR spectroscopy, biophysical characterization of recombinant PREP1 HD, DNA binding kinetics\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — structural (NMR) plus biochemical characterization, identification of novel cooperativity motif; single lab but rigorous biophysical approach\",\n      \"pmids\": [\"25856340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"High glucose exposure increases NF-κB p65 binding and recruitment of SET7 histone methyltransferase and p300 acetyltransferase to the 5' region of Prep1, leading to enhanced H3K4 methylation and H3K9/14 acetylation and increased Prep1 transcription. Prep1-induced recruitment of MEF2/HDAC5 repressor complex at the Glut4 promoter represses Glut4 expression.\",\n      \"method\": \"Chromatin immunoprecipitation (ChIP), Western blot, L6 cell model, streptozotocin diabetic mouse model\",\n      \"journal\": \"Diabetologia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP establishing histone modifications and transcription factor binding at endogenous loci, validated in vivo in diabetic mice\",\n      \"pmids\": [\"26453063\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NMR spectroscopy of the PREP1 homeodomain shows that residues flanking the HD participate in DNA binding. Isolated PREP1 and PBX1 HDs bind DNA cooperatively, and a novel PREP1 motif C-terminal to the HD is required for cooperativity with PBX1.\",\n      \"method\": \"NMR spectroscopy, surface plasmon resonance/kinetic binding assays, mutagenesis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — NMR structural characterization with functional validation by binding kinetics; first biophysical characterization of PREP1 HD\",\n      \"pmids\": [\"28094776\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The synthetic PREP1(54-72) peptide mimicking the PREP1 region involved in interaction with p160 disrupts the PREP1-p160 interaction in vitro (IC50 = 0.20 μM) and restores insulin signaling impaired by ceramide in L6 skeletal muscle cells. The PREP1-p160 interaction is induced by ceramide treatment.\",\n      \"method\": \"In vitro binding competition assay (IC50 determination), co-immunoprecipitation, insulin signaling Western blot, glucose uptake assay, glycogen assay\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — IC50 of peptide inhibitor determined in vitro, mechanistic rescue in cellular model, multiple orthogonal readouts\",\n      \"pmids\": [\"29069751\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"PREP1 down-regulation in mesenchymal stromal cells and 3T3-L1 preadipocytes increases adipogenic differentiation. PREP1 restricts DNA binding of C/EBPβ to adipogenic enhancers without affecting C/EBPβ expression or activation, thereby suppressing adipogenesis.\",\n      \"method\": \"Prep1 siRNA knockdown, adipogenic differentiation assay, ChIP (C/EBPβ binding), ex vivo bone marrow-derived MSC\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP establishing mechanism of C/EBPβ binding restriction, single lab with ex vivo and in vitro models\",\n      \"pmids\": [\"29138456\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PREP1 depletion affects DNA replication timing of middle-to-late-replicating DNA. PREP1 down-regulation increases replication rate, number of simultaneously firing origins, and asymmetry of DNA replication, leading to DNA damage. Genome-wide Repli-seq shows 25% of genome (LADs, LRRs, TTRs) replicates earlier upon PREP1 depletion. PREP1 down-regulation also decreases Lamin B1 levels, suggesting DNA release from the nuclear lamina.\",\n      \"method\": \"DNA combing, Repli-seq (genome-wide replication timing), Western blot (Lamin B1), DNA damage assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genome-wide replication timing assay plus DNA combing with quantitative analysis; establishes novel function in DNA replication regulation\",\n      \"pmids\": [\"29453404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PKnox1/PREP1 is required in a germ cell-intrinsic manner for adult spermatogenesis. Tamoxifen-induced or germ cell-specific PKnox1 loss causes arrest at the c-Kit+ spermatogonia stage with loss of meiotic spermatocytes and compromised c-Kit+ spermatogonia differentiation.\",\n      \"method\": \"Conditional/inducible knockout (tamoxifen-Cre, germ cell-specific Cre), immunohistochemistry, flow cytometry\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific conditional KO with defined stage of arrest; germ cell-intrinsic requirement established\",\n      \"pmids\": [\"29293683\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PREP1 regulates angiogenesis through the p160/PGC-1α pathway. PREP1 overexpression in mouse aortic endothelial cells increases p160 binding and levels (~4-fold) and reduces PGC-1α (~70%), stimulating angiogenic migration, proliferation, and tube formation. The PREP1(54-72) peptide blocking PREP1-p160 interaction reverts the proangiogenic effects. PREP1 levels increase during bFGF-mediated endothelial colony activation.\",\n      \"method\": \"Endothelial cell overexpression, PREP1(54-72) peptide inhibition, migration/proliferation/tube formation assays, Western blot, co-immunoprecipitation, hypomorphic mouse Matrigel plug assay\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal in vitro and in vivo methods, pharmacological disruption of PREP1-p160 with functional readout\",\n      \"pmids\": [\"31618597\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PREP1 deficiency reduces olfactory bulb development and impairs BDNF-TrkB signaling: Prep1(i/+) mice show reduced TrkB receptor levels and decreased BDNF-induced ERK1/2 activation. Overexpression of Prep1 in N2A neuronal cells increases TrkB expression levels, BDNF-induced ERK phosphorylation, and cell viability.\",\n      \"method\": \"Immunohistochemistry, cytochrome C oxidase activity, Western blot, overexpression in N2A cells, olfactory perception behavioral test\",\n      \"journal\": \"Molecular neurobiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro overexpression and in vivo hypomorphic model, single lab, links PREP1 to TrkB pathway\",\n      \"pmids\": [\"29349576\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"PREP1 and p160 domains interact with a KD of approximately 0.3–1 μM. Both PREP1 (residues 45–155) and p160 (residues 20–160) form folded, monomeric, predominantly alpha-helical domains with unusually high thermal stability.\",\n      \"method\": \"Recombinant protein expression, CD spectroscopy, thermal stability assay, binding affinity measurement (KD determination)\",\n      \"journal\": \"Molecular biotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct biophysical characterization of recombinant domains; single lab, first quantification of binding affinity\",\n      \"pmids\": [\"26979610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"SP2 transcription factor forms a complex with PREP1-PBX1 and NF-Y at composite genomic motifs where TALE and NF-Y recognition sequences are separated by 11 bp. SP2 potentiates binding of PBX1:PREP1 and NF-Y to co-occupied regulatory elements; reciprocally, PBX1:PREP1 together with NF-Y recruits SP2 to these sites.\",\n      \"method\": \"ChIP-exo sequencing, co-immunoprecipitation, ChIP, motif analysis in MEFs\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — ChIP-exo at nucleotide resolution, co-IP, multiple binding/functional assays establishing novel complex and mutual potentiation\",\n      \"pmids\": [\"30337366\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"PREP1 (PKNOX1) regulates nuclear stiffness, expression of LINC complex proteins, and YAP-TAZ mechanotransduction. PREP1 depletion upsets nuclear membrane protein stoichiometry, renders nuclei soft, leads to fortified actomyosin networks with larger focal adhesions and greater traction forces, but impairs YAP-TAZ nuclear translocation.\",\n      \"method\": \"PREP1 knockdown, atomic force microscopy (nuclear stiffness), traction force microscopy, YAP-TAZ immunofluorescence, Western blot of LINC complex proteins\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biophysical and cell biological methods in single lab; establishes novel PREP1 function in mechanosignaling\",\n      \"pmids\": [\"35550602\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"PKNOX1 acts as a transcription factor for the DHH (desert hedgehog) gene, directly binding the DHH promoter and promoting its expression, thereby activating Hedgehog signaling in stomach adenocarcinoma. ANKRD49 physically interacts with PKNOX1 (co-IP) and uses PKNOX1-mediated transcription of TGF-β1 to activate SMAD signaling and promote EMT.\",\n      \"method\": \"Dual-luciferase reporter assay (DHH promoter), ChIP (PKNOX1 binding to TGF-β1 promoter), co-immunoprecipitation (ANKRD49-PKNOX1), Western blot, knockdown experiments\",\n      \"journal\": \"Cancer cell international\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase establishing direct transcriptional target relationship, co-IP for protein interaction; single lab, recent paper\",\n      \"pmids\": [\"41821002\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PBX1 and PREP1 bind the PBX consensus element (PCE, TGATTGAC) in the long terminal repeat of Moloney murine leukemia virus (MLV), confirmed by gel shift assay. Overexpression of PBX1 and PREP1 together enhances MLV transcription, and mutations in the PCE completely inhibit viral transcription.\",\n      \"method\": \"EMSA (gel shift), overexpression, site-directed mutagenesis of PCE, transcriptional reporter assay\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA and functional overexpression assay, mutagenesis of binding site; single lab\",\n      \"pmids\": [\"12529389\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"PBX2-PREP1 heterodimer binds to the Pbx site in the UGT2B17 promoter and interferes with HNF1α binding to the adjacent HNF1 site, resulting in down-regulation of HNF1α-mediated activation of the UGT2B17 promoter.\",\n      \"method\": \"EMSA, GST interaction assays, transient transfection reporter assays\",\n      \"journal\": \"Molecular pharmacology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gel shift, GST pulldown, functional reporter assay; single lab\",\n      \"pmids\": [\"12065766\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"PREP1/PBX2 complex preferentially binds the -2578 G allele in the CCL2 promoter (created by the -2578 G polymorphism) and suppresses basal CCL2 promoter activity compared to the ancestral A allele in astrocytes.\",\n      \"method\": \"EMSA, transient transfection reporter assay, supershift with specific antibodies\",\n      \"journal\": \"Genes and immunity\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA with allele-specific binding and functional reporter assay; single lab\",\n      \"pmids\": [\"18480829\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2003,\n      \"finding\": \"PKNOX1 (PREP1) specifically binds the Pbx/POU binding site of the FABP7 promoter in vitro, and overexpression of PKNOX1 in neuroblastoma cells trans-activates the FABP7 promoter.\",\n      \"method\": \"EMSA (in vitro binding), transient transfection reporter assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, two methods (EMSA and reporter assay) but limited mechanistic depth\",\n      \"pmids\": [\"12771203\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The MEIS1 C-terminal transactivating domain, when fused to full-length PKNOX1, confers oncogenic activity: the chimeric PKNOX1-MC protein accelerates onset of HOXA9-induced leukemia in a mouse bone marrow transduction/transplantation model. The absence of a transactivating domain in native PKNOX1 accounts for its lack of pro-leukemic activity despite similar DNA-binding properties.\",\n      \"method\": \"Bone marrow transduction/transplantation leukemia model, gene expression profiling, chimeric protein construction\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — in vivo leukemia model with chimeric protein establishing structure-function relationship; clean genetic experiment\",\n      \"pmids\": [\"21900201\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"miR-17-5p and miR-19a-3p of the miR-17~92 cluster target PKNOX1, validated as a direct target. PKNOX1 competitively inhibits MEIS1-PBX complex formation; PKNOX1 acts antagonistically to leukemia in an MLL-AF9 murine model.\",\n      \"method\": \"miRNA target validation (luciferase reporter), co-immunoprecipitation (competitive PBX binding), murine MLL-AF9 leukemia model, knockdown studies\",\n      \"journal\": \"Leukemia research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reporter assay for miRNA targeting, co-IP for competitive binding, in vivo leukemia model; single lab\",\n      \"pmids\": [\"27123834\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PREP1 forms complexes with SNAIL2 in mammary stem/progenitor cells and protects SNAIL2 from proteasome-mediated degradation. Prep1-deficient mammary progenitor cells show reduced Snail2, Snail1, and vimentin expression, reduced mammosphere formation and branching in 3D assay, and increased TP53-dependent apoptosis.\",\n      \"method\": \"Co-immunoprecipitation (PREP1-SNAIL2 complex), proteasome inhibitor rescue, mammosphere assay, 3D branching assay, Western blot, conditional KO mouse model\",\n      \"journal\": \"The International journal of developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — co-IP establishing complex, proteasomal stabilization mechanism; single lab with in vitro and in vivo data\",\n      \"pmids\": [\"30604852\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"PKNOX1/PREP1 is a ubiquitous TALE-class homeodomain transcription factor that functions primarily as a stable heterodimer with PBX proteins (requiring N-terminal interaction domains in both proteins), which are mutually stabilized against proteasomal degradation; the PREP1-PBX heterodimer binds specific DNA motifs (TGACAG/TGATTGAC) cooperatively and can form ternary complexes with HOX proteins to regulate developmental gene expression (Hox, SF-1, FSHβ, Bcl-x, SHP1, SHIP2, SMAD3, DHH promoters), while competing with MEIS1 for PBX1 binding to control MEIS1 stability and suppress tumorigenesis; additionally, cytoplasmic PREP1 represses translation of Hoxb4 mRNA via 4EHP interaction in oocytes, PREP1 regulates genomic stability and DNA replication timing of lamin-associated domains (via Lamin B1), controls nuclear stiffness and YAP-TAZ mechanotransduction through LINC complex protein expression, and modulates insulin sensitivity through the p160-PGC-1α-GLUT4 axis in skeletal muscle and SHP1/SHIP2 in liver.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PKNOX1/PREP1 is a ubiquitous TALE-class homeodomain transcription factor that controls developmental gene expression as an obligate, stabilizing heterodimer with PBX proteins and governs cellular fate, genomic stability, and metabolic homeostasis [#0, #1, #21]. PREP1 binds PBX through N-terminal HR1/HR2 interaction surfaces independent of DNA, an association that is required for high-affinity, cooperative DNA binding at TGACAG/TGATTGAC-class motifs and that mutually stabilizes both partners against proteasomal degradation [#0, #9, #30]; because PREP1 is intrinsically cytoplasmic, dimerization with PBX also drives its nuclear translocation by blocking PBX export [#3]. The PREP1-PBX dimer nucleates higher-order complexes — ternary assemblies with HOX proteins on developmental enhancers (Hoxb1, Hoxb2, SF-1/FAdE) and composite complexes with SMADs, NF-Y/SP2, and myogenic bHLH factors — to activate or restrict transcription in a context-dependent manner [#5, #7, #10, #13, #40]. Through this network PREP1 acts upstream of a PBX-MEIS module, and by sequestering PBX1 it competes with MEIS1, destabilizing MEIS1 and suppressing its oncogenic and pro-leukemic activity [#12, #27, #47]. PREP1 directly governs cell survival and genome maintenance, transactivating Bcl-x and p53 and being required to prevent DNA damage, chromosomal instability, and aberrant replication timing of lamin-associated domains via Lamin B1 [#17, #21, #22, #35]. In metabolic and signaling tissues, PREP1 controls insulin sensitivity and mitochondrial biogenesis by stabilizing the p160/Mybbp1a inhibitor of PGC-1\\u03b1 and transactivating the phosphatases SHP1 and SHIP2 [#18, #20, #25, #26], and it additionally functions in the cytoplasm of oocytes by binding 4EHP to repress Hoxb4 mRNA translation [#19]. PREP1 is required in vivo for hematopoietic stem cell self-renewal, spermatogenesis, embryonic stem cell differentiation, and epiblast survival [#22, #24, #29, #36].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established the founding biochemical identity of PREP1: how does this TALE homeodomain protein engage DNA and partner proteins to regulate transcription?\",\n      \"evidence\": \"cDNA cloning, co-IP, EMSA, and transcriptional assays identifying PREP1 as a UEF3 subunit forming a DNA-independent heterodimer with PBX that binds the TGACAG motif\",\n      \"pmids\": [\"9482740\", \"9482739\", \"9405651\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of cooperative DNA binding\", \"Physiological target genes beyond uPA/Hox reporters not yet defined\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Explained the subcellular logic of the dimer: why does PREP1 require PBX for function?\",\n      \"evidence\": \"Immunofluorescence and fractionation in mammalian and Drosophila cells showing PBX/EXD blocks PREP1 nuclear export, driving heterodimer nuclear translocation\",\n      \"pmids\": [\"10215622\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"NLS/NES sequences mediating the switch not mapped\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Defined PREP1-PBX as a developmental enhancer organizer that nucleates ternary HOX complexes and tissue-restricted expression in vivo.\",\n      \"evidence\": \"EMSA, transgenic mouse enhancer assays, and IHC at Hoxb1/Hoxb2 r4 enhancers and the somatostatin and glucagon promoters showing PM-site-dependent ternary PREP1-PBX-HOX complexes\",\n      \"pmids\": [\"10654609\", \"10381567\", \"9933599\", \"10869353\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PREP1 vs MEIS1 occupancy at PM sites is regulated was not resolved\", \"Co-activator recruitment mechanism not defined\"]\n    },\n    {\n      \"year\": 2003,\n      \"claim\": \"Revealed mutual protein stabilization as a core mechanism: PREP1 dimerization protects PBX from proteasomal degradation.\",\n      \"evidence\": \"Cycloheximide chase plus MG132 treatment and Western blot showing PREP1 prevents PBX-2 proteasomal decay\",\n      \"pmids\": [\"12871956\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"E3 ligase targeting free PBX not identified\", \"Reciprocal stabilization of PREP1 by PBX not quantified here\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Placed PREP1 atop a PBX-MEIS regulatory network in vivo via a hypomorphic mouse, linking its dosage to network-wide protein levels and tissue phenotypes.\",\n      \"evidence\": \"Prep1(i/i) hypomorphic mice with Western blot, EMSA, and IHC showing reduced MEIS1/PBX1/PBX2 and downstream cMyb/Pax6, plus FAdE/SF-1 enhancer transgenic assays\",\n      \"pmids\": [\"16847320\", \"16705164\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect contributions to each downstream factor not fully separated\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Expanded the PREP1 interactome beyond PBX, identifying p160/Mybbp1a as a competitive partner and immune/cytoskeletal associates.\",\n      \"evidence\": \"Co-IP, in vitro competition with mapped HR1 residues, TAP-MS interactome, and ACRE reporter/ChIP in macrophages\",\n      \"pmids\": [\"17875935\", \"17623278\", \"18093541\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional significance of beta-actin/NMMHCIIA association not pursued\", \"Stoichiometry of p160 vs PBX competition in vivo unknown\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Connected PREP1 to cell survival and metabolism: direct transcriptional control of Bcl-x and a p160-GLUT4 axis governing insulin sensitivity.\",\n      \"evidence\": \"ChIP, reporter, and Bcl-x rescue in Prep1(i/i) MEFs; hypomorphic muscle plus L6 cells, glucose uptake, and in vivo p160 cDNA rescue\",\n      \"pmids\": [\"19103748\", \"18644868\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How PREP1 dosage toggles between pro- and anti-apoptotic outputs not resolved at this stage\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Uncovered a non-transcriptional cytoplasmic function: PREP1 represses specific mRNA translation.\",\n      \"evidence\": \"Co-IP, GST pull-down, 4EHP-motif mutagenesis, RNA EMSA, and in vitro translation showing PREP1 binds Hoxb4 3'UTR and represses translation via 4EHP in oocytes\",\n      \"pmids\": [\"19365557\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this translational repression occurs in somatic tissues unknown\", \"Breadth of PREP1-bound mRNAs not defined\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Established PREP1 as a guardian of genome integrity acting through p53 and heterochromatin, and a hepatic insulin-signaling regulator via SHP1.\",\n      \"evidence\": \"\\u03b3H2AX/cytogenetics, siRNA, ChIP and transformation rescue in MEFs/fibroblasts; Prep1(-/-)/p53(-/-) epistasis; p53 induction assays; SHP1 ChIP/reporter with hyperinsulinemic clamp\",\n      \"pmids\": [\"21715654\", \"20826531\", \"20110257\", \"20864515\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism linking PREP1 loss to heterochromatin remodeling not molecularly defined\", \"Whether p53 is a direct ChIP-confirmed target (Medium-confidence #23) not settled\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defined genome-wide direct targeting and the p160-PGC-1\\u03b1 mechanism for mitochondrial and lipid metabolism.\",\n      \"evidence\": \"Muscle-specific KO with ChIP-seq identifying direct OXPHOS gene binding; SHIP2 ChIP/reporter with PBX1-binding-deficient HR mutant in liver\",\n      \"pmids\": [\"24216763\", \"24052111\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs p160-mediated indirect effects on PGC-1\\u03b1 targets not fully partitioned\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Resolved the PREP1-MEIS1-PBX1 competition as a tumor-suppressive mechanism and identified PREP1 as an EMT/metastasis driver via SMAD3.\",\n      \"evidence\": \"Co-IP, stability assays, and transformation in MEFs showing PREP1 sequesters PBX1 to destabilize MEIS1; reporter/IF/colonization assays for SMAD3 intron transactivation in NSCLC\",\n      \"pmids\": [\"24578510\", \"25157139\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Contradiction between tumor-suppressive (MEIS1 sequestration) and pro-metastatic (EMT) roles not mechanistically reconciled\", \"Tissue determinants of opposing outputs unknown\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Provided structural and genome-scale views: a C-terminal homeodomain motif enabling cooperativity, ES-cell-specific enhancer binding, and Wnt/Fgf target pathways.\",\n      \"evidence\": \"NMR/biophysics of PREP1 HD; ChIP-seq/RNA-seq with rescue in Prep1(-/-) ES cells; ChIP at Glut4 with MEF2/HDAC5 recruitment in diabetic models\",\n      \"pmids\": [\"25856340\", \"25875616\", \"26453063\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full structure of the PREP1-PBX-DNA ternary complex still unresolved\", \"How high-glucose epigenetic induction of Prep1 integrates with target repression not fully mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Extended PREP1 function to nuclear mechanics and DNA replication control, plus tissue-specific roles in spermatogenesis, neurotrophin signaling, and angiogenesis.\",\n      \"evidence\": \"Repli-seq/DNA combing with Lamin B1 Western; AFM/traction-force microscopy and LINC/YAP-TAZ readouts; conditional germ cell KO; N2A overexpression with BDNF-TrkB assays; endothelial p160/PGC-1\\u03b1 assays with PREP1(54-72) peptide; biophysical PREP1-p160 KD\",\n      \"pmids\": [\"29453404\", \"35550602\", \"29293683\", \"29349576\", \"31618597\", \"29069751\", \"26979610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanistic link between PREP1, Lamin B1, and replication timing not established at molecular level\", \"Whether nuclear-stiffness role is transcriptional or structural unresolved\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identified a composite SP2-PBX1:PREP1-NF-Y complex defining a higher-order combinatorial code for genomic occupancy.\",\n      \"evidence\": \"ChIP-exo, co-IP, and motif analysis in MEFs showing mutual potentiation of binding at spaced TALE/NF-Y motifs\",\n      \"pmids\": [\"30337366\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional transcriptional output of the SP2-containing complex not defined\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Generalized the protein-stabilization mechanism to additional partners controlling stemness and EMT.\",\n      \"evidence\": \"Co-IP and proteasome-inhibitor rescue showing PREP1 stabilizes SNAIL2 in mammary progenitors, with mammosphere/branching and TP53-apoptosis readouts in conditional KO\",\n      \"pmids\": [\"30604852\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab co-IP without structural mapping of the PREP1-SNAIL2 interface\", \"Whether stabilization is direct or via an intermediary not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Linked PKNOX1 to Hedgehog and TGF-\\u03b2/SMAD signaling in gastric cancer via direct promoter targeting and a new partner.\",\n      \"evidence\": \"Luciferase, ChIP, and co-IP in stomach adenocarcinoma showing PKNOX1 transactivates DHH and TGF-\\u03b21 and interacts with ANKRD49\",\n      \"pmids\": [\"41821002\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study; ANKRD49-PKNOX1 interaction not reciprocally validated\", \"Direct vs indirect TGF-\\u03b21 regulation needs orthogonal confirmation\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How PREP1 dosage and partner choice (PBX vs MEIS vs p160 vs SNAIL2) are switched to produce its opposing tumor-suppressive versus pro-metastatic and pro-survival versus pro-apoptotic outputs in a given tissue remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying regulatory model for context-dependent partner selection\", \"Structure of the PREP1-PBX-HOX-DNA ternary complex still lacking\", \"Molecular link between PREP1, Lamin B1, and replication timing undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 4, 5, 10, 17, 20, 25, 26, 28, 31, 40, 42]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 2, 30, 32]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [0, 27, 40]},\n      {\"term_id\": \"GO:0045182\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0003723\", \"supporting_discovery_ids\": [19]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [9, 27, 49]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [3, 6]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 19]},\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [40]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 5, 10, 17, 20, 40]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [5, 6, 11, 13, 29, 36]},\n      {\"term_id\": \"R-HSA-5357801\", \"supporting_discovery_ids\": [17, 22, 23]},\n      {\"term_id\": \"R-HSA-73894\", \"supporting_discovery_ids\": [21, 22]},\n      {\"term_id\": \"R-HSA-69306\", \"supporting_discovery_ids\": [35]},\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [18, 20, 25, 26, 31]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [9, 27, 49]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [28, 38, 42]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [27, 28, 47, 48]}\n    ],\n    \"complexes\": [\n      \"PREP1-PBX heterodimer\",\n      \"PREP1-PBX-HOX ternary complex\",\n      \"SP2-PBX1:PREP1-NF-Y complex\",\n      \"UEF3 complex\"\n    ],\n    \"partners\": [\n      \"PBX1\",\n      \"PBX2\",\n      \"MEIS1\",\n      \"MYBBP1A\",\n      \"SMAD3\",\n      \"EIF4E2\",\n      \"SP2\",\n      \"SNAI2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"faith_supported":7,"faith_total":7,"faith_pct":100.0}}