{"gene":"HOXD10","run_date":"2026-04-28T18:06:53","timeline":{"discoveries":[{"year":1991,"finding":"HOX4D (HOXD10) and HOX4C homeoproteins bind to a phylogenetically conserved DNA fragment in the HOX4C/HOX4D intergenic region containing multiple TAAT-related binding sites, and HOX4D and HOX4C transactivate this endogenous sequence in cotransfection experiments, establishing them as sequence-specific transcription factors capable of cross-regulatory interactions within the HOX4 complex.","method":"Gel retardation assays (EMSA) and cotransfection transactivation assays in cell lines","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro DNA binding + functional transactivation assay with mutational site specificity","pmids":["1756725"],"is_preprint":false},{"year":1992,"finding":"HOXD-4 (HOX-4.2/Hoxd-4) protein autoregulates its own promoter by binding specifically to two TAAT/ATTA motifs within a 217 bp conserved element upstream of Hox-4.2; cotransfection of Hox-4.2 expression vector with a reporter driven by this element produced a ~20-fold increase in luciferase activity, an effect specific to Hox-4.2 and not Hox-1.6.","method":"Transient cotransfection luciferase reporter assays and gel-shift DNA binding assays in P19 EC cells; mutational analysis of TAAT motifs","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 1 — in vitro binding + reporter assay + mutagenesis, specific to Hox-4.2","pmids":["1356763"],"is_preprint":false},{"year":1993,"finding":"A retinoic acid response element (RARE) with sequence 5'-AGGTGA(N)5AGGTCA-3' was identified upstream of murine Hox-4.2 (HOXD4); this element specifically binds retinoic acid receptors (RARs) and is required for RA-induced transcriptional activation of Hox-4.2, as demonstrated by dominant-negative RAR-alpha suppression of RA induction.","method":"Transient expression luciferase reporter assays in P19 EC cells; gel-shift assays for RAR binding; dominant-negative RAR-alpha transfection","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1 — RARE functionally validated by reporter assay + EMSA + dominant-negative suppression","pmids":["8093325"],"is_preprint":false},{"year":1994,"finding":"The HOXD-4 (HOX-4.2) protein contains a proline-rich N-terminal transcriptional activation domain; GAL4 fusion assays mapped activation to this domain, and squelching by the proline-rich transcription factor AP2 confirmed the proline-rich nature of the activation domain. A single homeodomain point mutation abolished transcriptional activity.","method":"Transient transfection with GAL4 DNA-binding domain fusions; deletion and point mutational analysis; squelching assays with AP2 in P19 EC cells","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1 — domain mapping by mutagenesis and GAL4 fusions with functional readout","pmids":["7907418"],"is_preprint":false},{"year":1997,"finding":"Targeted disruption of Hoxd-10 in mice causes hindlimb-specific locomotor defects including altered gait and adduction, homeotic transformations of sacral vertebrae (S2 onward adopting more anterior morphology), anterior shift of the patella, outward rotation of the lower leg, decreased spinal segments projecting through the sacral plexus, deletion of hindlimb nerves, and a shift in the position of the lumbar lateral motor column — establishing Hoxd-10 as required for regional identity in hindlimb skeletal, muscular, and neural patterning.","method":"Gene targeting/knockout in mice; skeletal, muscular, and neural histological analysis; locomotor behavioral assays","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 — clean KO with multiple defined morphological and neural phenotypic readouts","pmids":["9409668"],"is_preprint":false},{"year":1996,"finding":"A cis-acting element (region IX) upstream of Hoxd-11 also controls the anterior expression boundary of Hoxd-10; in vivo targeted mutation abolishing nuclear receptor (RAR and COUP-TF) binding to region IX leads to anteriorization of both Hoxd-11 and Hoxd-10 mRNA expression domains in the prevertebral column and an anterior shift of the lumbosacral transition, demonstrating shared regulatory control of neighboring Hoxd genes by a single cis-element.","method":"Targeted mutagenesis in ES cells/transgenic mice; in situ hybridization; gel retardation assays for nuclear receptor binding to region IX","journal":"Genes & development","confidence":"High","confidence_rationale":"Tier 1-2 — in vivo mutagenesis + in vitro binding assay + expression analysis, replicated across transgenic lines","pmids":["8824591"],"is_preprint":false},{"year":1999,"finding":"Double targeted disruption of Hoxd9 and Hoxd10 in mice produces locomotor, skeletal (axial and appendicular), hindlimb peripheral nerve, and distal hindlimb muscle defects more severe than either single mutant, establishing functional cooperativity/genetic interaction between adjacent 5' HoxD genes in patterning the same posterior regions.","method":"Gene targeting double-mutant mice; behavioral, skeletal, and neural analysis","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — double-KO genetic epistasis with multiple phenotypic readouts; clean experimental design","pmids":["10642795"],"is_preprint":false},{"year":2001,"finding":"Simultaneous inactivation of Hoxa10 and Hoxd10 produces more severe hindlimb skeletal and peripheral nervous system defects (including truncations/deletions of tibial and peroneal nerves) than either single mutant, establishing that these paralogous genes coordinately regulate hindlimb skeletal and neural patterning.","method":"Double-mutant mouse genetic epistasis; skeletal and neural histological analysis; behavioral locomotor assays","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — double-KO epistasis with dose-dependent allelic series showing combinatorial regulation","pmids":["11180954"],"is_preprint":false},{"year":2004,"finding":"Ectopic expression of Hoxd10 in thoracic spinal segments of chick embryos via in ovo electroporation induces motoneurons with a lumbosacral molecular profile (Lim1 and RALDH2 expression) and novel axon projections to hindlimb muscles, demonstrating that Hoxd10 is sufficient to specify lumbosacral motoneuron identity.","method":"In ovo electroporation of chick thoracic spinal cord; immunohistochemistry for regional motoneuron markers (Lim1, RALDH2); axon tracing","journal":"Developmental dynamics","confidence":"High","confidence_rationale":"Tier 2 — gain-of-function in vivo with defined molecular and axonal phenotypic readouts","pmids":["15305286"],"is_preprint":false},{"year":2004,"finding":"cDNA microarray analysis of Hoxd10 single and Hoxa10/Hoxd10 double knockout mouse spinal cords identified 69 downstream target genes with altered expression, including genes involved in cellular communication, cell cycle control, development, and neuronal survival; putative promoter analysis suggested some are direct Hoxd10 targets.","method":"cDNA microarray; RT-PCR validation; in situ hybridization; bioinformatic promoter analysis for Hox binding sites in Hoxd10 KO and Hoxa10/Hoxd10 double-KO spinal cord","journal":"Journal of neuroscience research","confidence":"Medium","confidence_rationale":"Tier 2/3 — transcriptomics with RT-PCR and ISH validation in KO, but direct binding not confirmed for most targets","pmids":["14743444"],"is_preprint":false},{"year":2005,"finding":"HOXD10, in complex with PBX1b and PREP1 (as HOXD10·PBX1b·PREP1), activates the rat renin promoter; this activation is cooperative with Ets-1 and the intracellular domain of Notch1, establishing renin as a transcriptional target of HOXD10-containing complexes.","method":"EMSA; transient transfection reporter assays in COS-7, Calu-6, and As4.1 cells; cotransfection of HOXD10·PBX1b·PREP1, Ets-1, and Notch1-ICD constructs; promoter mutation analysis","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro binding + reporter assay + cooperative activation with defined partners and mutations","pmids":["15792957"],"is_preprint":false},{"year":2009,"finding":"Overexpression of Hoxd10 in the embryonic chick lumbosacral (LS) cord defines the position of the lateral motor column (LMC) and promotes development of lateral LMC (LMCl) motoneurons in rostral LS segments; Hoxd11 suppresses Hoxd10 expression and RALDH2 levels, suggesting Hoxd11 modulates Hoxd10 activity and local retinoic acid to define caudal LMC boundaries.","method":"In ovo electroporation overexpression in chick; Hoxd10 loss-of-function mouse analysis; immunohistochemistry for LMC motoneuron markers and RALDH2","journal":"Developmental biology","confidence":"High","confidence_rationale":"Tier 2 — gain- and loss-of-function with defined molecular readouts; cross-validated in chick and mouse","pmids":["19306865"],"is_preprint":false},{"year":2011,"finding":"miR-10b directly targets the 3'UTR of HOXD10, reducing its translation; downregulation of HOXD10 by miR-10b in glioma cells leads to upregulation of invasion factors MMP-14 and uPAR, promoting glioma cell invasion. Treatment with miR-10b inhibitors restores HOXD10 and reduces invasiveness.","method":"miR-10b overexpression/inhibition; Western blot for HOXD10, MMP-14, uPAR; Matrigel invasion assay; antisense oligonucleotide inhibition in glioma cell lines","journal":"Brain research","confidence":"Medium","confidence_rationale":"Tier 2/3 — functional cell invasion assay + target validation, but luciferase confirmation not explicitly detailed","pmids":["21419107"],"is_preprint":false},{"year":2012,"finding":"miR-10b promotes gastric cancer cell invasion by targeting HOXD10 (confirmed by translational inhibition); downregulation of HOXD10 by miR-10b leads to increased RhoC expression and AKT phosphorylation. Knockdown of RhoC or inhibition of AKT abolished miR-10b-induced invasion, placing HOXD10 upstream of the RhoC-AKT signaling axis.","method":"miR-10b overexpression/inhibition; HOXD10 rescue expression; Transwell invasion assay; RhoC knockdown; AKT inhibition; Western blot in gastric cancer cell lines","journal":"International journal of oncology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (OE, KD, rescue, pathway inhibition) establishing HOXD10 upstream of RhoC-AKT","pmids":["22293682"],"is_preprint":false},{"year":2012,"finding":"TBX1 physically interacts with HOXD10 as determined by co-immunoprecipitation and mass spectrometry; TBX1 and HOXD10 have overlapping expression during murine kidney development, and RNAi-mediated knockdown of TBX1 in HEK293 cells alters TGF-β/BMP signaling, a pathway influenced through TBX1-HOXD10 interaction.","method":"Co-immunoprecipitation, mass spectrometry, Western blot, RT-PCR, RNAi in HEK293 cells","journal":"Gene","confidence":"Medium","confidence_rationale":"Tier 2/3 — reciprocal co-IP with mass spectrometry identification, plus functional RNAi with pathway readout","pmids":["22842189"],"is_preprint":false},{"year":2013,"finding":"miR-10b promotes nucleus pulposus cell proliferation by directly targeting HOXD10, leading to derepression of the RhoC-Akt pathway; restored HOXD10 expression reversed the mitogenic effect of miR-10b, and knockdown of RhoC or Akt inhibition abolished miR-10b-driven proliferation.","method":"miR-10b overexpression; HOXD10 rescue; RhoC knockdown; Akt inhibition; cell proliferation assays; Western blot in nucleus pulposus cells","journal":"PloS one","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal loss/gain-of-function experiments with pathway rescue","pmids":["24376640"],"is_preprint":false},{"year":2013,"finding":"miR-10b overexpression in ovarian cancer cells decreases HOXD10 protein expression and upregulates MMP14 and RHOC, enhancing migration and invasion; inverse correlation between HOXD10 and MMP14 protein expression was confirmed in patient tumor samples, establishing HOXD10 as a transcriptional repressor of MMP14 and RHOC in ovarian cancer.","method":"miR-10b overexpression; Western blot for HOXD10, MMP14, RHOC; migration/invasion assays; immunohistochemistry in patient samples","journal":"International journal of oncology","confidence":"Medium","confidence_rationale":"Tier 2/3 — functional assays with protein-level validation and clinical sample correlation","pmids":["23670532"],"is_preprint":false},{"year":2014,"finding":"HOXD10 knockdown in HNSCC cells decreases proliferation and invasion; HOXD10 overexpression decreases cell invasion but increases proliferation, adhesion, and migration. Microarray and reporter assays identified angiomotin (AMOT-p80) and miR-146a as direct HOXD10 transcriptional targets; AMOT-p80 manipulation phenocopies HOXD10 manipulation.","method":"Stable transfection/siRNA knockdown in HNSCC cells; MTS/migration/adhesion/invasion assays; expression microarray; luciferase reporter assay for AMOT-p80 and miR-146a","journal":"British journal of cancer","confidence":"High","confidence_rationale":"Tier 2 — gain/loss-of-function with multiple phenotypic readouts plus direct target validation by reporter assay","pmids":["25010866"],"is_preprint":false},{"year":2014,"finding":"POU2F1 transcriptionally regulates HOXD10 and HOXD11 expression in HNSCC; POU2F1 consensus binding sites in the 5' region of HOXD10 are required for optimal promoter activity as confirmed by luciferase reporter constructs; POU2F1 knockdown reduces HOXD10/D11 expression and inhibits HNSCC proliferation.","method":"Promoter luciferase reporter assays; POU2F1 siRNA knockdown; qRT-PCR; Western blot in HNSCC cells","journal":"Oncotarget","confidence":"High","confidence_rationale":"Tier 1-2 — promoter mutation + reporter assay + KD functional phenotype","pmids":["25301728"],"is_preprint":false},{"year":2014,"finding":"HOXD10 knockdown in murine uterus reduces implantation rates and correlates with increased EMX2 and IGFBP1 expression and decreased integrin β3 and LIF expression, establishing HOXD10 as a regulator of endometrial receptivity acting through known HOX target genes.","method":"shRNA knockdown in mouse uterus via uterine horn injection; qRT-PCR and Western blot; litter size counting at day 9 of pregnancy","journal":"Human reproduction (Oxford, England)","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo KD with defined downstream target gene expression readouts","pmids":["24549215"],"is_preprint":false},{"year":2015,"finding":"HOXD10 acts as a tumor suppressor in cholangiocellular carcinoma (CCC) by inhibiting cell invasion through downregulation of MMP2 and MMP9, inducing G1 cell cycle arrest and apoptosis, and inactivating the RHOC/AKT/MAPK signaling pathway upon overexpression.","method":"Lentiviral HOXD10 overexpression in CCC cell lines; invasion assay; flow cytometry for apoptosis/cell cycle; Western blot for RHOC, MMP2, MMP9, AKT, MAPK","journal":"Oncology reports","confidence":"Medium","confidence_rationale":"Tier 2/3 — gain-of-function with multiple mechanistic readouts in a single lab","pmids":["26260613"],"is_preprint":false},{"year":2016,"finding":"HOXD10 is specifically activated in lymphatic endothelial cells (LECs) at early time points after VEGFR-3 stimulation, and regulates expression of immediate early transcription factors including NR4A1; gain- and loss-of-function of HOXD10 in LECs affects migration, cord-like structure formation, and expression of VE-cadherin, claudin-5, and eNOS, promoting lymphatic endothelial permeability.","method":"DeepCAGE transcriptomics; transcription factor activity analysis; gain- and loss-of-function experiments in human LECs; migration assays; tube formation assays; Western blot","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 — transcriptomics discovery + functional gain/loss-of-function with defined molecular and cellular phenotypic readouts","pmids":["27199372"],"is_preprint":false},{"year":2017,"finding":"HOXD10 expression in hepatocellular carcinoma is regulated by promoter region methylation; HOXD10 suppresses HCC cell growth in vitro and in vivo by inhibiting ERK signaling, as demonstrated by HOXD10 overexpression reducing ERK pathway activation.","method":"Methylation-specific PCR; 5-Aza-dC demethylation treatment; siRNA/overexpression in HCC cell lines; chromatin immunoprecipitation; flow cytometry; Western blot for ERK; xenograft mouse model","journal":"Clinical epigenetics","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods including ChIP and in vivo xenograft, but single lab","pmids":["29075359"],"is_preprint":false},{"year":2018,"finding":"miR-23a directly targets HOXD10 (validated by luciferase reporter assay); miR-23a overexpression in glioblastoma induces expression of uPAR, RhoA, RhoC, and glial-mesenchymal transition markers (Snail, Slug, MMP2, MMP9, MMP14, E-cadherin changes), and promotes invasion with polarized focal adhesion formation; all these effects are reversed by HOXD10 overexpression.","method":"Luciferase reporter assay; miR-23a overexpression; HOXD10 overexpression rescue; Matrigel invasion assay; immunofluorescence for focal adhesions; Western blot in 6 glioblastoma cell lines","journal":"Signal transduction and targeted therapy","confidence":"High","confidence_rationale":"Tier 2 — direct target validation by reporter assay + rescue experiments across multiple cell lines with molecular phenotype characterization","pmids":["30603114"],"is_preprint":false},{"year":2019,"finding":"HOXD10 suppresses colon cancer cell proliferation, migration, invasion and promotes apoptosis; overexpression of HOXD10 downregulates RHOC and inactivates AKT and MAPK pathways, establishing HOXD10 as a transcriptional regulator upstream of the RHOC/AKT/MAPK axis in colon cancer.","method":"HOXD10 expression vector transfection and 5-Aza-dC treatment; MTT, Transwell, wound healing, flow cytometry assays; RT-PCR and Western blot for RHOC, AKT, MAPK in colon cancer cell lines; immunohistochemistry","journal":"Cell communication and signaling : CCS","confidence":"Medium","confidence_rationale":"Tier 2/3 — multiple phenotypic assays with pathway analysis, single lab","pmids":["30683109"],"is_preprint":false},{"year":2020,"finding":"Hoxd10 is required for secretory activation in lactation; targeted disruption of Hoxd10 leads to reduced prolactin receptor expression, reduced STAT5 phosphorylation, decreased milk protein expression, mislocalized GLUT1, increased STAT3 phosphorylation, and leukocyte recruitment in affected mammary gland regions. Transplantation experiments reveal Hoxd10 primarily exerts systemic functions that confer attenuated STAT5 phosphorylation.","method":"Hoxd10 KO mice (single and Hoxd9/d10 double); mammary gland transplantation; Western blot for STAT5/STAT3 phosphorylation, prolactin receptor, milk proteins; immunofluorescence for GLUT1; histology","journal":"Journal of mammary gland biology and neoplasia","confidence":"High","confidence_rationale":"Tier 2 — KO + transplantation with defined molecular signaling readouts establishing systemic role","pmids":["32705545"],"is_preprint":false},{"year":2021,"finding":"HOXD10 overexpression in colorectal cancer cells activates transcription of miR-7 and IGFBP3 in a promoter-dependent manner; demethylation studies showed that DNMT1, DNMT3B, and MeCP2 are recruited to the HOXD10 promoter to silence it. HOXD10 restoration enhances 5-FU chemosensitivity in resistant CRC cells.","method":"Pyrosequencing; 5-Aza-CdR demethylation; MeCP2 knockdown; luciferase reporter assays; ChIP for DNMT1/DNMT3B/MeCP2 at HOXD10 promoter; cell proliferation/invasion assays; drug resistance assays","journal":"Frontiers in oncology","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP + reporter + functional rescue, though single lab","pmids":["34790580"],"is_preprint":false},{"year":2024,"finding":"HOXD10 directly binds the NOX4 promoter to repress NOX4 transcription, thereby inhibiting ferroptosis pathway activation and oxidative stress in renal tubular cells. AAV-mediated HOXD10 overexpression attenuates renal fibrosis in UUO mice; HOXD10 hypermethylation silences its expression in TGF-β1-treated cells.","method":"ChIP assay and dual-luciferase reporter assay for HOXD10-NOX4 promoter binding; bisulfite sequencing PCR for HOXD10 methylation; AAV-HOXD10 in UUO mice; cellular ferroptosis/oxidative stress markers (ROS, lipid ROS, MDA, GSH/GSSG, SOD) in HK-2 cells","journal":"Cell death & disease","confidence":"High","confidence_rationale":"Tier 1-2 — direct promoter binding by ChIP + reporter assay + in vivo rescue model with mechanistic ferroptosis markers","pmids":["38844470"],"is_preprint":false},{"year":2024,"finding":"The GABPA transcription factor binds directly to the HOXD10 promoter to regulate its transcription; miR-450b-5p targets GABPA directly, leading to reduced HOXD10 expression and promotion of ectopic endometriotic lesion growth. Intraperitoneal lentiviral HOXD10 overexpression attenuates ectopic lesions in a mouse model.","method":"Dual-luciferase reporter assay for GABPA-HOXD10 promoter binding; miR-450b-5p overexpression/knockdown; HOXD10 lentiviral overexpression in vivo; in vitro and in vivo proliferation/invasion/apoptosis assays","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 — reporter assay for direct promoter binding + in vivo rescue, single lab","pmids":["39759007"],"is_preprint":false},{"year":2025,"finding":"A missense variant HOXD10 p.S80R (in the homeodomain) causes conformational alterations in the protein (larger radius of gyration, fewer hydrogen bonds, expanded solvent-accessible surface area); RNA sequencing revealed that the inhibition of HOXD10 wild-type on IFIT1, IFIT2, and IFIT3 transcription is abrogated by the S80R variant, identifying these interferon-stimulated genes as transcriptional targets of HOXD10.","method":"Molecular dynamics simulation; RNA sequencing of cells expressing WT vs. S80R variant HOXD10; WES in MDA patient cohort","journal":"European journal of obstetrics, gynecology, and reproductive biology","confidence":"Medium","confidence_rationale":"Tier 2/3 — MD simulation for structural change + transcriptomic target identification for variant, but single study","pmids":["39987682"],"is_preprint":false}],"current_model":"HOXD10 is a homeodomain transcription factor that binds TAAT-containing DNA motifs (including autoregulatory elements and target gene promoters such as NOX4, AMOT-p80, and the renin promoter in complexes with PBX1b/PREP1) to regulate gene expression; it is post-translationally regulated by promoter methylation (silenced by DNMT1/DNMT3B/MeCP2 and BRAF-associated methylation), is required for hindlimb/sacral positional identity, lumbosacral motoneuron specification, endometrial receptivity, and mammary gland secretory activation in vivo, and functions as a transcriptional repressor of the RhoC-AKT/MAPK and Rho/ROCK signaling axes that control cell migration, invasion, and proliferation, with its expression frequently suppressed by multiple miRNAs (miR-10b, miR-23a, miR-224, miR-501, miR-376b) in cancer contexts."},"narrative":{"teleology":[{"year":1991,"claim":"Establishing that HOXD10 is a sequence-specific transcription factor that binds TAAT motifs and can transactivate endogenous HOX cluster regulatory elements answered whether HOX4 cluster proteins have autonomous DNA-binding and transcriptional activity.","evidence":"Gel retardation assays and cotransfection reporter assays in cell lines","pmids":["1756725"],"confidence":"High","gaps":["Endogenous genomic targets beyond the HOX cluster were not identified","Cooperating cofactors were unknown"]},{"year":1992,"claim":"Demonstration that HOXD4 autoregulates its own promoter via two TAAT motifs established a positive-feedback regulatory mechanism within the HOX cluster, addressing how Hox gene expression is maintained after initial activation.","evidence":"Mutagenesis of TAAT motifs, gel-shift assays, and luciferase reporter assays in P19 cells","pmids":["1356763"],"confidence":"High","gaps":["Whether autoregulation occurs in vivo was not confirmed","The role of cofactors in autoregulation was not addressed"]},{"year":1993,"claim":"Identification of a retinoic acid response element upstream of Hoxd4 and its requirement for RA-induced transcription established how retinoid signaling initiates HOXD gene expression during development.","evidence":"Reporter assays, EMSA for RAR binding, and dominant-negative RAR-alpha in P19 cells","pmids":["8093325"],"confidence":"High","gaps":["Whether this RARE is required in vivo for normal Hoxd4 expression boundaries was not tested"]},{"year":1994,"claim":"Mapping a proline-rich N-terminal transcriptional activation domain answered how HOXD10 protein structure separates DNA recognition from transcriptional output.","evidence":"GAL4 fusion assays, deletion/point mutagenesis, and squelching with AP2 in P19 cells","pmids":["7907418"],"confidence":"High","gaps":["No coactivator/corepressor partners of the activation domain were identified","Domain function was not validated in vivo"]},{"year":1997,"claim":"Knockout of Hoxd10 in mice revealed its essential requirement for hindlimb skeletal identity, sacral vertebral patterning, and lumbosacral motor neuron organization, answering what developmental processes depend on this gene.","evidence":"Gene targeting in mice with skeletal, neural, and behavioral phenotyping","pmids":["9409668"],"confidence":"High","gaps":["Direct transcriptional targets mediating the skeletal and neural phenotypes were not identified","Whether Hoxd10 acts cell-autonomously in motoneurons was not resolved"]},{"year":1999,"claim":"Double knockouts of Hoxd9/Hoxd10 and Hoxa10/Hoxd10 revealed synergistic phenotypes more severe than single mutants, establishing functional cooperativity among adjacent and paralogous Hox genes in posterior patterning.","evidence":"Double-mutant mice with skeletal, neural, and locomotor analysis","pmids":["10642795","11180954"],"confidence":"High","gaps":["Whether cooperativity reflects shared targets, cross-regulation, or both was not distinguished","Molecular basis of dosage-dependent interaction was unclear"]},{"year":2004,"claim":"Ectopic Hoxd10 expression in thoracic chick spinal cord induced lumbosacral motoneuron identity (Lim1, RALDH2), demonstrating sufficiency for motoneuron specification and answering whether Hoxd10 instructs rather than merely permits lumbar identity.","evidence":"In ovo electroporation with immunohistochemistry for regional motoneuron markers and axon tracing","pmids":["15305286"],"confidence":"High","gaps":["Direct Hoxd10 binding to Lim1 or RALDH2 regulatory regions was not shown","Whether this sufficiency requires endogenous cofactors was not resolved"]},{"year":2005,"claim":"Identification of the HOXD10·PBX1b·PREP1 complex as an activator of the renin promoter (cooperating with Ets-1 and Notch1-ICD) established the first defined cofactor complex and non-Hox target gene for HOXD10.","evidence":"EMSA, reporter assays, and cotransfection in COS-7/As4.1 cells with promoter mutations","pmids":["15792957"],"confidence":"High","gaps":["In vivo relevance for renin expression in juxtaglomerular cells was not demonstrated","Whether the PBX1b/PREP1 complex is required for all HOXD10 targets was unknown"]},{"year":2011,"claim":"Demonstration that miR-10b directly targets HOXD10 to derepress RhoC and the AKT signaling axis, promoting cancer cell invasion, established the mechanistic link between HOXD10 loss and pro-metastatic signaling — answering how HOXD10 functions as a tumor suppressor.","evidence":"miR-10b overexpression/inhibition, HOXD10 rescue, RhoC knockdown, AKT inhibition, and invasion assays in glioma and gastric cancer cell lines","pmids":["21419107","22293682"],"confidence":"High","gaps":["Whether HOXD10 directly binds the RHOC promoter to repress transcription was not confirmed","In vivo tumor models were not used in the initial studies"]},{"year":2014,"claim":"Identification of AMOT-p80 and miR-146a as direct transcriptional targets of HOXD10, and POU2F1 as an upstream transcriptional activator, built out the regulatory circuit of HOXD10 in HNSCC, answering both what HOXD10 regulates and what regulates HOXD10.","evidence":"Luciferase reporter assays for AMOT-p80 and miR-146a promoters; POU2F1 promoter mutagenesis and siRNA in HNSCC cells","pmids":["25010866","25301728"],"confidence":"High","gaps":["ChIP confirmation of HOXD10 occupancy at AMOT-p80 and miR-146a promoters was not shown","Whether POU2F1 regulation is tissue-specific was not addressed"]},{"year":2020,"claim":"Hoxd10 knockout mice showed defective mammary gland secretory activation with reduced prolactin receptor expression and STAT5 phosphorylation, revealing a previously unknown systemic role for HOXD10 in lactation that extends beyond its known developmental patterning functions.","evidence":"Hoxd10 KO and Hoxd9/d10 double-KO mice; mammary transplantation; Western blot for STAT5/STAT3 signaling, prolactin receptor, and milk proteins","pmids":["32705545"],"confidence":"High","gaps":["The systemic factor mediating attenuated STAT5 signaling was not identified","Direct transcriptional targets of HOXD10 in mammary epithelium were not defined"]},{"year":2021,"claim":"ChIP-based demonstration that DNMT1, DNMT3B, and MeCP2 are recruited to the HOXD10 promoter to silence it, and that HOXD10 restoration enhances 5-FU chemosensitivity, answered how HOXD10 is epigenetically silenced in cancer and connected this silencing to drug resistance.","evidence":"ChIP for DNMT1/DNMT3B/MeCP2 at HOXD10 promoter; pyrosequencing; demethylation rescue; drug sensitivity assays in CRC cells","pmids":["34790580"],"confidence":"Medium","gaps":["Whether methylation-mediated silencing is an early or late event in tumorigenesis was not established","Single-lab finding without independent replication"]},{"year":2024,"claim":"Direct ChIP and reporter assay evidence that HOXD10 binds the NOX4 promoter to repress its transcription, thereby inhibiting ferroptosis and oxidative stress, revealed a new mechanistic axis for HOXD10 in renal fibrosis independent of its known RHOC-related functions.","evidence":"ChIP and dual-luciferase assay for HOXD10-NOX4 binding; AAV-HOXD10 in UUO mouse model; ferroptosis markers in HK-2 cells","pmids":["38844470"],"confidence":"High","gaps":["Whether HOXD10 regulation of NOX4 occurs in developmental or other disease contexts was not tested","Genome-wide binding profile for HOXD10 is still lacking"]},{"year":null,"claim":"A genome-wide binding map (ChIP-seq) for HOXD10, the structural basis for target gene selectivity among paralogous HOX proteins, and the identity of the systemic factor mediating HOXD10's role in mammary secretory activation remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No genome-wide ChIP-seq or CUT&RUN data for HOXD10 are available","Structural basis for HOXD10 target selectivity versus other posterior HOX proteins is unknown","The systemic mediator of HOXD10 action in lactation is unidentified"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,3,10,27]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,3,10,17,26,27]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,1,3,10,17]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,10,17,26,27]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[4,6,7,8,11]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[13,20,22,24]}],"complexes":["HOXD10·PBX1b·PREP1"],"partners":["PBX1B","PREP1","TBX1","GABPA","POU2F1","RHOC"],"other_free_text":[]},"mechanistic_narrative":"HOXD10 is a homeodomain transcription factor that patterns posterior body structures during development and acts as a transcriptional repressor of pro-invasive and proliferative signaling in multiple adult tissue contexts. It binds TAAT-containing DNA motifs through its homeodomain, uses a proline-rich N-terminal activation domain, and forms cooperative complexes with PBX1b/PREP1 to regulate targets including the renin promoter, NOX4, AMOT-p80, and IFIT family genes [PMID:1756725, PMID:7907418, PMID:15792957, PMID:38844470, PMID:25010866]. Targeted disruption in mice establishes HOXD10 as essential for hindlimb skeletal identity, lumbosacral motoneuron specification, and mammary gland secretory activation via prolactin receptor–STAT5 signaling, with functional cooperativity among paralogous Hox genes [PMID:9409668, PMID:15305286, PMID:32705545, PMID:11180954]. HOXD10 expression is frequently silenced by promoter DNA methylation (mediated by DNMT1/DNMT3B/MeCP2) and by multiple microRNAs (miR-10b, miR-23a), and its loss derepresses RHOC and the AKT/MAPK signaling axis to promote cell invasion and proliferation in diverse cancer types [PMID:34790580, PMID:22293682, PMID:30603114]."},"prefetch_data":{"uniprot":{"accession":"P28358","full_name":"Homeobox protein Hox-D10","aliases":["Homeobox protein Hox-4D","Homeobox protein Hox-4E"],"length_aa":340,"mass_kda":38.4,"function":"Sequence-specific transcription factor which is part of a developmental regulatory system that provides cells with specific positional identities on the anterior-posterior axis","subcellular_location":"Nucleus","url":"https://www.uniprot.org/uniprotkb/P28358/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/HOXD10","classification":"Not 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cells.","date":"2008","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/18442473","citation_count":4,"is_preprint":false},{"pmid":"33895929","id":"PMC_33895929","title":"Punica granatum Extract Inhibits Bladder Cancer Cell Viability, Invasion and Migration through Down-Regulation of HOXD10 Signalling Pathway.","date":"2021","source":"Doklady. 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(Warsaw, Poland)","url":"https://pubmed.ncbi.nlm.nih.gov/38756247","citation_count":2,"is_preprint":false},{"pmid":"38197552","id":"PMC_38197552","title":"Ropivacaine combined with sorafenib attenuates hepatocellular carcinoma cell proliferation and metastasis by inhibiting the miR-224/HOXD10 axis.","date":"2024","source":"Environmental toxicology","url":"https://pubmed.ncbi.nlm.nih.gov/38197552","citation_count":1,"is_preprint":false},{"pmid":"39759007","id":"PMC_39759007","title":"miR-450b-5p promotes development of endometriosis by inhibiting the GABPA/HOXD10 axis.","date":"2024","source":"iScience","url":"https://pubmed.ncbi.nlm.nih.gov/39759007","citation_count":1,"is_preprint":false},{"pmid":"39987682","id":"PMC_39987682","title":"HOXD10: A novel gene implicated in human Müllerian duct anomalies.","date":"2025","source":"European journal of obstetrics, gynecology, and reproductive biology","url":"https://pubmed.ncbi.nlm.nih.gov/39987682","citation_count":1,"is_preprint":false},{"pmid":"29159235","id":"PMC_29159235","title":"Trimethylation of Histone 3 lysine 27 (H3K27me3) ChIP-PCR and transcriptional expression data of Ef1-alpha, cyp26A, HoxC10, HoxD10 and HoxD11 in the Xenopus XTC cell line.","date":"2017","source":"Data in brief","url":"https://pubmed.ncbi.nlm.nih.gov/29159235","citation_count":1,"is_preprint":false},{"pmid":"32705545","id":"PMC_32705545","title":"Hoxd10 Is Required Systemically for Secretory Activation in Lactation and Interacts Genetically with Hoxd9.","date":"2020","source":"Journal of mammary gland biology and neoplasia","url":"https://pubmed.ncbi.nlm.nih.gov/32705545","citation_count":0,"is_preprint":false},{"pmid":"41534268","id":"PMC_41534268","title":"HOXD10 expression in oral cancer is driven by histone acetylation rather than promoter DNA methylation.","date":"2026","source":"Archives of oral biology","url":"https://pubmed.ncbi.nlm.nih.gov/41534268","citation_count":0,"is_preprint":false},{"pmid":"40462237","id":"PMC_40462237","title":"LncRNA FLG-AS1 inhibits esophageal squamous cell carcinoma by regulating the miR-23a-3p/HOXD10 axis.","date":"2025","source":"Hereditas","url":"https://pubmed.ncbi.nlm.nih.gov/40462237","citation_count":0,"is_preprint":false},{"pmid":"41388430","id":"PMC_41388430","title":"Noninvasive early diagnosis of laryngeal squamous cell carcinoma (LSCC) revealing the high-performance potential of the HOXD10 gene monomethylated locus: a case-control study.","date":"2025","source":"Clinical epigenetics","url":"https://pubmed.ncbi.nlm.nih.gov/41388430","citation_count":0,"is_preprint":false},{"pmid":"39564825","id":"PMC_39564825","title":"Laparoscopic Salpingectomy Regulates The Expression of Endometrial Homeobox Genes (HOXA9, HOXA10, HOXA11, HOXD10) and HOX Transcript Antisense Intergenic RNA in Women with Communicating Hydrosalpinx: A Prospective Study.","date":"2024","source":"International journal of fertility & sterility","url":"https://pubmed.ncbi.nlm.nih.gov/39564825","citation_count":0,"is_preprint":false},{"pmid":"39512497","id":"PMC_39512497","title":"[Retracted] Propofol exhibits a tumor‑suppressive effect and regulates cell viability, migration and invasion in bladder carcinoma by targeting the microRNA‑10b/HOXD10 signaling pathway.","date":"2024","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/39512497","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.07.15.603511","title":"Permissive and instructive  <i>Hox</i>  codes govern limb positioning","date":"2024-07-19","source":"bioRxiv","url":"https://doi.org/10.1101/2024.07.15.603511","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":43366,"output_tokens":7818,"usd":0.123684},"stage2":{"model":"claude-opus-4-6","input_tokens":11516,"output_tokens":3387,"usd":0.213383},"total_usd":0.337067,"stage1_batch_id":"msgbatch_01DzrsgVgYngY1hvM3DU1yF2","stage2_batch_id":"msgbatch_01KA9b7iRzKFdhoBUT7vCvSe","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1991,\n      \"finding\": \"HOX4D (HOXD10) and HOX4C homeoproteins bind to a phylogenetically conserved DNA fragment in the HOX4C/HOX4D intergenic region containing multiple TAAT-related binding sites, and HOX4D and HOX4C transactivate this endogenous sequence in cotransfection experiments, establishing them as sequence-specific transcription factors capable of cross-regulatory interactions within the HOX4 complex.\",\n      \"method\": \"Gel retardation assays (EMSA) and cotransfection transactivation assays in cell lines\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro DNA binding + functional transactivation assay with mutational site specificity\",\n      \"pmids\": [\"1756725\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"HOXD-4 (HOX-4.2/Hoxd-4) protein autoregulates its own promoter by binding specifically to two TAAT/ATTA motifs within a 217 bp conserved element upstream of Hox-4.2; cotransfection of Hox-4.2 expression vector with a reporter driven by this element produced a ~20-fold increase in luciferase activity, an effect specific to Hox-4.2 and not Hox-1.6.\",\n      \"method\": \"Transient cotransfection luciferase reporter assays and gel-shift DNA binding assays in P19 EC cells; mutational analysis of TAAT motifs\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro binding + reporter assay + mutagenesis, specific to Hox-4.2\",\n      \"pmids\": [\"1356763\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1993,\n      \"finding\": \"A retinoic acid response element (RARE) with sequence 5'-AGGTGA(N)5AGGTCA-3' was identified upstream of murine Hox-4.2 (HOXD4); this element specifically binds retinoic acid receptors (RARs) and is required for RA-induced transcriptional activation of Hox-4.2, as demonstrated by dominant-negative RAR-alpha suppression of RA induction.\",\n      \"method\": \"Transient expression luciferase reporter assays in P19 EC cells; gel-shift assays for RAR binding; dominant-negative RAR-alpha transfection\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — RARE functionally validated by reporter assay + EMSA + dominant-negative suppression\",\n      \"pmids\": [\"8093325\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"The HOXD-4 (HOX-4.2) protein contains a proline-rich N-terminal transcriptional activation domain; GAL4 fusion assays mapped activation to this domain, and squelching by the proline-rich transcription factor AP2 confirmed the proline-rich nature of the activation domain. A single homeodomain point mutation abolished transcriptional activity.\",\n      \"method\": \"Transient transfection with GAL4 DNA-binding domain fusions; deletion and point mutational analysis; squelching assays with AP2 in P19 EC cells\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — domain mapping by mutagenesis and GAL4 fusions with functional readout\",\n      \"pmids\": [\"7907418\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"Targeted disruption of Hoxd-10 in mice causes hindlimb-specific locomotor defects including altered gait and adduction, homeotic transformations of sacral vertebrae (S2 onward adopting more anterior morphology), anterior shift of the patella, outward rotation of the lower leg, decreased spinal segments projecting through the sacral plexus, deletion of hindlimb nerves, and a shift in the position of the lumbar lateral motor column — establishing Hoxd-10 as required for regional identity in hindlimb skeletal, muscular, and neural patterning.\",\n      \"method\": \"Gene targeting/knockout in mice; skeletal, muscular, and neural histological analysis; locomotor behavioral assays\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple defined morphological and neural phenotypic readouts\",\n      \"pmids\": [\"9409668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1996,\n      \"finding\": \"A cis-acting element (region IX) upstream of Hoxd-11 also controls the anterior expression boundary of Hoxd-10; in vivo targeted mutation abolishing nuclear receptor (RAR and COUP-TF) binding to region IX leads to anteriorization of both Hoxd-11 and Hoxd-10 mRNA expression domains in the prevertebral column and an anterior shift of the lumbosacral transition, demonstrating shared regulatory control of neighboring Hoxd genes by a single cis-element.\",\n      \"method\": \"Targeted mutagenesis in ES cells/transgenic mice; in situ hybridization; gel retardation assays for nuclear receptor binding to region IX\",\n      \"journal\": \"Genes & development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vivo mutagenesis + in vitro binding assay + expression analysis, replicated across transgenic lines\",\n      \"pmids\": [\"8824591\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1999,\n      \"finding\": \"Double targeted disruption of Hoxd9 and Hoxd10 in mice produces locomotor, skeletal (axial and appendicular), hindlimb peripheral nerve, and distal hindlimb muscle defects more severe than either single mutant, establishing functional cooperativity/genetic interaction between adjacent 5' HoxD genes in patterning the same posterior regions.\",\n      \"method\": \"Gene targeting double-mutant mice; behavioral, skeletal, and neural analysis\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — double-KO genetic epistasis with multiple phenotypic readouts; clean experimental design\",\n      \"pmids\": [\"10642795\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Simultaneous inactivation of Hoxa10 and Hoxd10 produces more severe hindlimb skeletal and peripheral nervous system defects (including truncations/deletions of tibial and peroneal nerves) than either single mutant, establishing that these paralogous genes coordinately regulate hindlimb skeletal and neural patterning.\",\n      \"method\": \"Double-mutant mouse genetic epistasis; skeletal and neural histological analysis; behavioral locomotor assays\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — double-KO epistasis with dose-dependent allelic series showing combinatorial regulation\",\n      \"pmids\": [\"11180954\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Ectopic expression of Hoxd10 in thoracic spinal segments of chick embryos via in ovo electroporation induces motoneurons with a lumbosacral molecular profile (Lim1 and RALDH2 expression) and novel axon projections to hindlimb muscles, demonstrating that Hoxd10 is sufficient to specify lumbosacral motoneuron identity.\",\n      \"method\": \"In ovo electroporation of chick thoracic spinal cord; immunohistochemistry for regional motoneuron markers (Lim1, RALDH2); axon tracing\",\n      \"journal\": \"Developmental dynamics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — gain-of-function in vivo with defined molecular and axonal phenotypic readouts\",\n      \"pmids\": [\"15305286\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"cDNA microarray analysis of Hoxd10 single and Hoxa10/Hoxd10 double knockout mouse spinal cords identified 69 downstream target genes with altered expression, including genes involved in cellular communication, cell cycle control, development, and neuronal survival; putative promoter analysis suggested some are direct Hoxd10 targets.\",\n      \"method\": \"cDNA microarray; RT-PCR validation; in situ hybridization; bioinformatic promoter analysis for Hox binding sites in Hoxd10 KO and Hoxa10/Hoxd10 double-KO spinal cord\",\n      \"journal\": \"Journal of neuroscience research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — transcriptomics with RT-PCR and ISH validation in KO, but direct binding not confirmed for most targets\",\n      \"pmids\": [\"14743444\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"HOXD10, in complex with PBX1b and PREP1 (as HOXD10·PBX1b·PREP1), activates the rat renin promoter; this activation is cooperative with Ets-1 and the intracellular domain of Notch1, establishing renin as a transcriptional target of HOXD10-containing complexes.\",\n      \"method\": \"EMSA; transient transfection reporter assays in COS-7, Calu-6, and As4.1 cells; cotransfection of HOXD10·PBX1b·PREP1, Ets-1, and Notch1-ICD constructs; promoter mutation analysis\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro binding + reporter assay + cooperative activation with defined partners and mutations\",\n      \"pmids\": [\"15792957\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"Overexpression of Hoxd10 in the embryonic chick lumbosacral (LS) cord defines the position of the lateral motor column (LMC) and promotes development of lateral LMC (LMCl) motoneurons in rostral LS segments; Hoxd11 suppresses Hoxd10 expression and RALDH2 levels, suggesting Hoxd11 modulates Hoxd10 activity and local retinoic acid to define caudal LMC boundaries.\",\n      \"method\": \"In ovo electroporation overexpression in chick; Hoxd10 loss-of-function mouse analysis; immunohistochemistry for LMC motoneuron markers and RALDH2\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — gain- and loss-of-function with defined molecular readouts; cross-validated in chick and mouse\",\n      \"pmids\": [\"19306865\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"miR-10b directly targets the 3'UTR of HOXD10, reducing its translation; downregulation of HOXD10 by miR-10b in glioma cells leads to upregulation of invasion factors MMP-14 and uPAR, promoting glioma cell invasion. Treatment with miR-10b inhibitors restores HOXD10 and reduces invasiveness.\",\n      \"method\": \"miR-10b overexpression/inhibition; Western blot for HOXD10, MMP-14, uPAR; Matrigel invasion assay; antisense oligonucleotide inhibition in glioma cell lines\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — functional cell invasion assay + target validation, but luciferase confirmation not explicitly detailed\",\n      \"pmids\": [\"21419107\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"miR-10b promotes gastric cancer cell invasion by targeting HOXD10 (confirmed by translational inhibition); downregulation of HOXD10 by miR-10b leads to increased RhoC expression and AKT phosphorylation. Knockdown of RhoC or inhibition of AKT abolished miR-10b-induced invasion, placing HOXD10 upstream of the RhoC-AKT signaling axis.\",\n      \"method\": \"miR-10b overexpression/inhibition; HOXD10 rescue expression; Transwell invasion assay; RhoC knockdown; AKT inhibition; Western blot in gastric cancer cell lines\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (OE, KD, rescue, pathway inhibition) establishing HOXD10 upstream of RhoC-AKT\",\n      \"pmids\": [\"22293682\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"TBX1 physically interacts with HOXD10 as determined by co-immunoprecipitation and mass spectrometry; TBX1 and HOXD10 have overlapping expression during murine kidney development, and RNAi-mediated knockdown of TBX1 in HEK293 cells alters TGF-β/BMP signaling, a pathway influenced through TBX1-HOXD10 interaction.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, Western blot, RT-PCR, RNAi in HEK293 cells\",\n      \"journal\": \"Gene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — reciprocal co-IP with mass spectrometry identification, plus functional RNAi with pathway readout\",\n      \"pmids\": [\"22842189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"miR-10b promotes nucleus pulposus cell proliferation by directly targeting HOXD10, leading to derepression of the RhoC-Akt pathway; restored HOXD10 expression reversed the mitogenic effect of miR-10b, and knockdown of RhoC or Akt inhibition abolished miR-10b-driven proliferation.\",\n      \"method\": \"miR-10b overexpression; HOXD10 rescue; RhoC knockdown; Akt inhibition; cell proliferation assays; Western blot in nucleus pulposus cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal loss/gain-of-function experiments with pathway rescue\",\n      \"pmids\": [\"24376640\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"miR-10b overexpression in ovarian cancer cells decreases HOXD10 protein expression and upregulates MMP14 and RHOC, enhancing migration and invasion; inverse correlation between HOXD10 and MMP14 protein expression was confirmed in patient tumor samples, establishing HOXD10 as a transcriptional repressor of MMP14 and RHOC in ovarian cancer.\",\n      \"method\": \"miR-10b overexpression; Western blot for HOXD10, MMP14, RHOC; migration/invasion assays; immunohistochemistry in patient samples\",\n      \"journal\": \"International journal of oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — functional assays with protein-level validation and clinical sample correlation\",\n      \"pmids\": [\"23670532\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOXD10 knockdown in HNSCC cells decreases proliferation and invasion; HOXD10 overexpression decreases cell invasion but increases proliferation, adhesion, and migration. Microarray and reporter assays identified angiomotin (AMOT-p80) and miR-146a as direct HOXD10 transcriptional targets; AMOT-p80 manipulation phenocopies HOXD10 manipulation.\",\n      \"method\": \"Stable transfection/siRNA knockdown in HNSCC cells; MTS/migration/adhesion/invasion assays; expression microarray; luciferase reporter assay for AMOT-p80 and miR-146a\",\n      \"journal\": \"British journal of cancer\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — gain/loss-of-function with multiple phenotypic readouts plus direct target validation by reporter assay\",\n      \"pmids\": [\"25010866\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"POU2F1 transcriptionally regulates HOXD10 and HOXD11 expression in HNSCC; POU2F1 consensus binding sites in the 5' region of HOXD10 are required for optimal promoter activity as confirmed by luciferase reporter constructs; POU2F1 knockdown reduces HOXD10/D11 expression and inhibits HNSCC proliferation.\",\n      \"method\": \"Promoter luciferase reporter assays; POU2F1 siRNA knockdown; qRT-PCR; Western blot in HNSCC cells\",\n      \"journal\": \"Oncotarget\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — promoter mutation + reporter assay + KD functional phenotype\",\n      \"pmids\": [\"25301728\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"HOXD10 knockdown in murine uterus reduces implantation rates and correlates with increased EMX2 and IGFBP1 expression and decreased integrin β3 and LIF expression, establishing HOXD10 as a regulator of endometrial receptivity acting through known HOX target genes.\",\n      \"method\": \"shRNA knockdown in mouse uterus via uterine horn injection; qRT-PCR and Western blot; litter size counting at day 9 of pregnancy\",\n      \"journal\": \"Human reproduction (Oxford, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo KD with defined downstream target gene expression readouts\",\n      \"pmids\": [\"24549215\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"HOXD10 acts as a tumor suppressor in cholangiocellular carcinoma (CCC) by inhibiting cell invasion through downregulation of MMP2 and MMP9, inducing G1 cell cycle arrest and apoptosis, and inactivating the RHOC/AKT/MAPK signaling pathway upon overexpression.\",\n      \"method\": \"Lentiviral HOXD10 overexpression in CCC cell lines; invasion assay; flow cytometry for apoptosis/cell cycle; Western blot for RHOC, MMP2, MMP9, AKT, MAPK\",\n      \"journal\": \"Oncology reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — gain-of-function with multiple mechanistic readouts in a single lab\",\n      \"pmids\": [\"26260613\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"HOXD10 is specifically activated in lymphatic endothelial cells (LECs) at early time points after VEGFR-3 stimulation, and regulates expression of immediate early transcription factors including NR4A1; gain- and loss-of-function of HOXD10 in LECs affects migration, cord-like structure formation, and expression of VE-cadherin, claudin-5, and eNOS, promoting lymphatic endothelial permeability.\",\n      \"method\": \"DeepCAGE transcriptomics; transcription factor activity analysis; gain- and loss-of-function experiments in human LECs; migration assays; tube formation assays; Western blot\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — transcriptomics discovery + functional gain/loss-of-function with defined molecular and cellular phenotypic readouts\",\n      \"pmids\": [\"27199372\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"HOXD10 expression in hepatocellular carcinoma is regulated by promoter region methylation; HOXD10 suppresses HCC cell growth in vitro and in vivo by inhibiting ERK signaling, as demonstrated by HOXD10 overexpression reducing ERK pathway activation.\",\n      \"method\": \"Methylation-specific PCR; 5-Aza-dC demethylation treatment; siRNA/overexpression in HCC cell lines; chromatin immunoprecipitation; flow cytometry; Western blot for ERK; xenograft mouse model\",\n      \"journal\": \"Clinical epigenetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods including ChIP and in vivo xenograft, but single lab\",\n      \"pmids\": [\"29075359\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"miR-23a directly targets HOXD10 (validated by luciferase reporter assay); miR-23a overexpression in glioblastoma induces expression of uPAR, RhoA, RhoC, and glial-mesenchymal transition markers (Snail, Slug, MMP2, MMP9, MMP14, E-cadherin changes), and promotes invasion with polarized focal adhesion formation; all these effects are reversed by HOXD10 overexpression.\",\n      \"method\": \"Luciferase reporter assay; miR-23a overexpression; HOXD10 overexpression rescue; Matrigel invasion assay; immunofluorescence for focal adhesions; Western blot in 6 glioblastoma cell lines\",\n      \"journal\": \"Signal transduction and targeted therapy\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct target validation by reporter assay + rescue experiments across multiple cell lines with molecular phenotype characterization\",\n      \"pmids\": [\"30603114\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"HOXD10 suppresses colon cancer cell proliferation, migration, invasion and promotes apoptosis; overexpression of HOXD10 downregulates RHOC and inactivates AKT and MAPK pathways, establishing HOXD10 as a transcriptional regulator upstream of the RHOC/AKT/MAPK axis in colon cancer.\",\n      \"method\": \"HOXD10 expression vector transfection and 5-Aza-dC treatment; MTT, Transwell, wound healing, flow cytometry assays; RT-PCR and Western blot for RHOC, AKT, MAPK in colon cancer cell lines; immunohistochemistry\",\n      \"journal\": \"Cell communication and signaling : CCS\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — multiple phenotypic assays with pathway analysis, single lab\",\n      \"pmids\": [\"30683109\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Hoxd10 is required for secretory activation in lactation; targeted disruption of Hoxd10 leads to reduced prolactin receptor expression, reduced STAT5 phosphorylation, decreased milk protein expression, mislocalized GLUT1, increased STAT3 phosphorylation, and leukocyte recruitment in affected mammary gland regions. Transplantation experiments reveal Hoxd10 primarily exerts systemic functions that confer attenuated STAT5 phosphorylation.\",\n      \"method\": \"Hoxd10 KO mice (single and Hoxd9/d10 double); mammary gland transplantation; Western blot for STAT5/STAT3 phosphorylation, prolactin receptor, milk proteins; immunofluorescence for GLUT1; histology\",\n      \"journal\": \"Journal of mammary gland biology and neoplasia\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — KO + transplantation with defined molecular signaling readouts establishing systemic role\",\n      \"pmids\": [\"32705545\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"HOXD10 overexpression in colorectal cancer cells activates transcription of miR-7 and IGFBP3 in a promoter-dependent manner; demethylation studies showed that DNMT1, DNMT3B, and MeCP2 are recruited to the HOXD10 promoter to silence it. HOXD10 restoration enhances 5-FU chemosensitivity in resistant CRC cells.\",\n      \"method\": \"Pyrosequencing; 5-Aza-CdR demethylation; MeCP2 knockdown; luciferase reporter assays; ChIP for DNMT1/DNMT3B/MeCP2 at HOXD10 promoter; cell proliferation/invasion assays; drug resistance assays\",\n      \"journal\": \"Frontiers in oncology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP + reporter + functional rescue, though single lab\",\n      \"pmids\": [\"34790580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"HOXD10 directly binds the NOX4 promoter to repress NOX4 transcription, thereby inhibiting ferroptosis pathway activation and oxidative stress in renal tubular cells. AAV-mediated HOXD10 overexpression attenuates renal fibrosis in UUO mice; HOXD10 hypermethylation silences its expression in TGF-β1-treated cells.\",\n      \"method\": \"ChIP assay and dual-luciferase reporter assay for HOXD10-NOX4 promoter binding; bisulfite sequencing PCR for HOXD10 methylation; AAV-HOXD10 in UUO mice; cellular ferroptosis/oxidative stress markers (ROS, lipid ROS, MDA, GSH/GSSG, SOD) in HK-2 cells\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — direct promoter binding by ChIP + reporter assay + in vivo rescue model with mechanistic ferroptosis markers\",\n      \"pmids\": [\"38844470\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"The GABPA transcription factor binds directly to the HOXD10 promoter to regulate its transcription; miR-450b-5p targets GABPA directly, leading to reduced HOXD10 expression and promotion of ectopic endometriotic lesion growth. Intraperitoneal lentiviral HOXD10 overexpression attenuates ectopic lesions in a mouse model.\",\n      \"method\": \"Dual-luciferase reporter assay for GABPA-HOXD10 promoter binding; miR-450b-5p overexpression/knockdown; HOXD10 lentiviral overexpression in vivo; in vitro and in vivo proliferation/invasion/apoptosis assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reporter assay for direct promoter binding + in vivo rescue, single lab\",\n      \"pmids\": [\"39759007\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"A missense variant HOXD10 p.S80R (in the homeodomain) causes conformational alterations in the protein (larger radius of gyration, fewer hydrogen bonds, expanded solvent-accessible surface area); RNA sequencing revealed that the inhibition of HOXD10 wild-type on IFIT1, IFIT2, and IFIT3 transcription is abrogated by the S80R variant, identifying these interferon-stimulated genes as transcriptional targets of HOXD10.\",\n      \"method\": \"Molecular dynamics simulation; RNA sequencing of cells expressing WT vs. S80R variant HOXD10; WES in MDA patient cohort\",\n      \"journal\": \"European journal of obstetrics, gynecology, and reproductive biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2/3 — MD simulation for structural change + transcriptomic target identification for variant, but single study\",\n      \"pmids\": [\"39987682\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"HOXD10 is a homeodomain transcription factor that binds TAAT-containing DNA motifs (including autoregulatory elements and target gene promoters such as NOX4, AMOT-p80, and the renin promoter in complexes with PBX1b/PREP1) to regulate gene expression; it is post-translationally regulated by promoter methylation (silenced by DNMT1/DNMT3B/MeCP2 and BRAF-associated methylation), is required for hindlimb/sacral positional identity, lumbosacral motoneuron specification, endometrial receptivity, and mammary gland secretory activation in vivo, and functions as a transcriptional repressor of the RhoC-AKT/MAPK and Rho/ROCK signaling axes that control cell migration, invasion, and proliferation, with its expression frequently suppressed by multiple miRNAs (miR-10b, miR-23a, miR-224, miR-501, miR-376b) in cancer contexts.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"HOXD10 is a homeodomain transcription factor that patterns posterior body structures during development and acts as a transcriptional repressor of pro-invasive and proliferative signaling in multiple adult tissue contexts. It binds TAAT-containing DNA motifs through its homeodomain, uses a proline-rich N-terminal activation domain, and forms cooperative complexes with PBX1b/PREP1 to regulate targets including the renin promoter, NOX4, AMOT-p80, and IFIT family genes [PMID:1756725, PMID:7907418, PMID:15792957, PMID:38844470, PMID:25010866]. Targeted disruption in mice establishes HOXD10 as essential for hindlimb skeletal identity, lumbosacral motoneuron specification, and mammary gland secretory activation via prolactin receptor–STAT5 signaling, with functional cooperativity among paralogous Hox genes [PMID:9409668, PMID:15305286, PMID:32705545, PMID:11180954]. HOXD10 expression is frequently silenced by promoter DNA methylation (mediated by DNMT1/DNMT3B/MeCP2) and by multiple microRNAs (miR-10b, miR-23a), and its loss derepresses RHOC and the AKT/MAPK signaling axis to promote cell invasion and proliferation in diverse cancer types [PMID:34790580, PMID:22293682, PMID:30603114].\",\n  \"teleology\": [\n    {\n      \"year\": 1991,\n      \"claim\": \"Establishing that HOXD10 is a sequence-specific transcription factor that binds TAAT motifs and can transactivate endogenous HOX cluster regulatory elements answered whether HOX4 cluster proteins have autonomous DNA-binding and transcriptional activity.\",\n      \"evidence\": \"Gel retardation assays and cotransfection reporter assays in cell lines\",\n      \"pmids\": [\"1756725\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Endogenous genomic targets beyond the HOX cluster were not identified\", \"Cooperating cofactors were unknown\"]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Demonstration that HOXD4 autoregulates its own promoter via two TAAT motifs established a positive-feedback regulatory mechanism within the HOX cluster, addressing how Hox gene expression is maintained after initial activation.\",\n      \"evidence\": \"Mutagenesis of TAAT motifs, gel-shift assays, and luciferase reporter assays in P19 cells\",\n      \"pmids\": [\"1356763\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether autoregulation occurs in vivo was not confirmed\", \"The role of cofactors in autoregulation was not addressed\"]\n    },\n    {\n      \"year\": 1993,\n      \"claim\": \"Identification of a retinoic acid response element upstream of Hoxd4 and its requirement for RA-induced transcription established how retinoid signaling initiates HOXD gene expression during development.\",\n      \"evidence\": \"Reporter assays, EMSA for RAR binding, and dominant-negative RAR-alpha in P19 cells\",\n      \"pmids\": [\"8093325\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether this RARE is required in vivo for normal Hoxd4 expression boundaries was not tested\"]\n    },\n    {\n      \"year\": 1994,\n      \"claim\": \"Mapping a proline-rich N-terminal transcriptional activation domain answered how HOXD10 protein structure separates DNA recognition from transcriptional output.\",\n      \"evidence\": \"GAL4 fusion assays, deletion/point mutagenesis, and squelching with AP2 in P19 cells\",\n      \"pmids\": [\"7907418\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No coactivator/corepressor partners of the activation domain were identified\", \"Domain function was not validated in vivo\"]\n    },\n    {\n      \"year\": 1997,\n      \"claim\": \"Knockout of Hoxd10 in mice revealed its essential requirement for hindlimb skeletal identity, sacral vertebral patterning, and lumbosacral motor neuron organization, answering what developmental processes depend on this gene.\",\n      \"evidence\": \"Gene targeting in mice with skeletal, neural, and behavioral phenotyping\",\n      \"pmids\": [\"9409668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct transcriptional targets mediating the skeletal and neural phenotypes were not identified\", \"Whether Hoxd10 acts cell-autonomously in motoneurons was not resolved\"]\n    },\n    {\n      \"year\": 1999,\n      \"claim\": \"Double knockouts of Hoxd9/Hoxd10 and Hoxa10/Hoxd10 revealed synergistic phenotypes more severe than single mutants, establishing functional cooperativity among adjacent and paralogous Hox genes in posterior patterning.\",\n      \"evidence\": \"Double-mutant mice with skeletal, neural, and locomotor analysis\",\n      \"pmids\": [\"10642795\", \"11180954\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether cooperativity reflects shared targets, cross-regulation, or both was not distinguished\", \"Molecular basis of dosage-dependent interaction was unclear\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Ectopic Hoxd10 expression in thoracic chick spinal cord induced lumbosacral motoneuron identity (Lim1, RALDH2), demonstrating sufficiency for motoneuron specification and answering whether Hoxd10 instructs rather than merely permits lumbar identity.\",\n      \"evidence\": \"In ovo electroporation with immunohistochemistry for regional motoneuron markers and axon tracing\",\n      \"pmids\": [\"15305286\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct Hoxd10 binding to Lim1 or RALDH2 regulatory regions was not shown\", \"Whether this sufficiency requires endogenous cofactors was not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Identification of the HOXD10·PBX1b·PREP1 complex as an activator of the renin promoter (cooperating with Ets-1 and Notch1-ICD) established the first defined cofactor complex and non-Hox target gene for HOXD10.\",\n      \"evidence\": \"EMSA, reporter assays, and cotransfection in COS-7/As4.1 cells with promoter mutations\",\n      \"pmids\": [\"15792957\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance for renin expression in juxtaglomerular cells was not demonstrated\", \"Whether the PBX1b/PREP1 complex is required for all HOXD10 targets was unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstration that miR-10b directly targets HOXD10 to derepress RhoC and the AKT signaling axis, promoting cancer cell invasion, established the mechanistic link between HOXD10 loss and pro-metastatic signaling — answering how HOXD10 functions as a tumor suppressor.\",\n      \"evidence\": \"miR-10b overexpression/inhibition, HOXD10 rescue, RhoC knockdown, AKT inhibition, and invasion assays in glioma and gastric cancer cell lines\",\n      \"pmids\": [\"21419107\", \"22293682\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HOXD10 directly binds the RHOC promoter to repress transcription was not confirmed\", \"In vivo tumor models were not used in the initial studies\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Identification of AMOT-p80 and miR-146a as direct transcriptional targets of HOXD10, and POU2F1 as an upstream transcriptional activator, built out the regulatory circuit of HOXD10 in HNSCC, answering both what HOXD10 regulates and what regulates HOXD10.\",\n      \"evidence\": \"Luciferase reporter assays for AMOT-p80 and miR-146a promoters; POU2F1 promoter mutagenesis and siRNA in HNSCC cells\",\n      \"pmids\": [\"25010866\", \"25301728\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"ChIP confirmation of HOXD10 occupancy at AMOT-p80 and miR-146a promoters was not shown\", \"Whether POU2F1 regulation is tissue-specific was not addressed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Hoxd10 knockout mice showed defective mammary gland secretory activation with reduced prolactin receptor expression and STAT5 phosphorylation, revealing a previously unknown systemic role for HOXD10 in lactation that extends beyond its known developmental patterning functions.\",\n      \"evidence\": \"Hoxd10 KO and Hoxd9/d10 double-KO mice; mammary transplantation; Western blot for STAT5/STAT3 signaling, prolactin receptor, and milk proteins\",\n      \"pmids\": [\"32705545\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The systemic factor mediating attenuated STAT5 signaling was not identified\", \"Direct transcriptional targets of HOXD10 in mammary epithelium were not defined\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"ChIP-based demonstration that DNMT1, DNMT3B, and MeCP2 are recruited to the HOXD10 promoter to silence it, and that HOXD10 restoration enhances 5-FU chemosensitivity, answered how HOXD10 is epigenetically silenced in cancer and connected this silencing to drug resistance.\",\n      \"evidence\": \"ChIP for DNMT1/DNMT3B/MeCP2 at HOXD10 promoter; pyrosequencing; demethylation rescue; drug sensitivity assays in CRC cells\",\n      \"pmids\": [\"34790580\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether methylation-mediated silencing is an early or late event in tumorigenesis was not established\", \"Single-lab finding without independent replication\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Direct ChIP and reporter assay evidence that HOXD10 binds the NOX4 promoter to repress its transcription, thereby inhibiting ferroptosis and oxidative stress, revealed a new mechanistic axis for HOXD10 in renal fibrosis independent of its known RHOC-related functions.\",\n      \"evidence\": \"ChIP and dual-luciferase assay for HOXD10-NOX4 binding; AAV-HOXD10 in UUO mouse model; ferroptosis markers in HK-2 cells\",\n      \"pmids\": [\"38844470\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether HOXD10 regulation of NOX4 occurs in developmental or other disease contexts was not tested\", \"Genome-wide binding profile for HOXD10 is still lacking\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"A genome-wide binding map (ChIP-seq) for HOXD10, the structural basis for target gene selectivity among paralogous HOX proteins, and the identity of the systemic factor mediating HOXD10's role in mammary secretory activation remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No genome-wide ChIP-seq or CUT&RUN data for HOXD10 are available\", \"Structural basis for HOXD10 target selectivity versus other posterior HOX proteins is unknown\", \"The systemic mediator of HOXD10 action in lactation is unidentified\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 3, 10, 27]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 3, 10, 17, 26, 27]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 1, 3, 10, 17]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 10, 17, 26, 27]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [4, 6, 7, 8, 11]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [13, 20, 22, 24]}\n    ],\n    \"complexes\": [\n      \"HOXD10·PBX1b·PREP1\"\n    ],\n    \"partners\": [\n      \"PBX1b\",\n      \"PREP1\",\n      \"TBX1\",\n      \"GABPA\",\n      \"POU2F1\",\n      \"RHOC\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}