{"gene":"SIX5","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":1997,"finding":"CTG trinucleotide repeat expansion at the DM1 locus eliminates a DNase I-hypersensitive site containing an enhancer element that regulates DMAHP/SIX5 transcription, reducing steady-state DMAHP transcript levels 2- to 4-fold overall and greatly reducing transcripts from the expanded allele specifically.","method":"DNase I hypersensitivity assay, allele-specific RT-PCR in DM patient cells","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent labs (PMID:9241282 and PMID:9241283) using orthogonal methods (DNase I hypersensitivity, allele-specific RT-PCR, enhancer reporter assays) replicated the same mechanistic conclusion","pmids":["9241282","9241283"],"is_preprint":false},{"year":2000,"finding":"Loss-of-function of Six5 in mice (homozygous knockout replacing exon 1 with beta-galactosidase reporter) causes lenticular opacities (cataracts) at a higher rate than controls, with reporter expression confirmed in the developing lens; no skeletal muscle abnormalities were detected.","method":"Targeted gene disruption (beta-gal knock-in), histological and functional phenotyping of Six5-/- mice","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — two independent labs (PMID:10802667 and PMID:10802668) using Six5 knockout mice with direct phenotypic readout both established cataract formation","pmids":["10802667","10802668"],"is_preprint":false},{"year":2000,"finding":"Cataract formation in Six5-deficient mice is inversely related to Six5 dosage (heterozygous Six5+/- mice also develop cataracts), and Six5+/- and Six5-/- mice show increased steady-state Na+/K+-ATPase alpha-1 subunit protein levels and decreased Dm15 mRNA levels, implicating altered ion homeostasis in lens pathology.","method":"Dosage analysis of Six5+/- and Six5-/- mice, Western blotting, RT-PCR","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — rigorous dosage series in two independent mouse models (PMID:10802668), multiple orthogonal molecular readouts","pmids":["10802668"],"is_preprint":false},{"year":2000,"finding":"SIX5 protein binds the Na+/K+-ATPase alpha-1 subunit gene (ATP1A1) regulatory element (ARE) — the same site as murine Six4 — through its homeodomain; the protein containing both the SIX domain and homeodomain forms a second (presumed dimer) complex with the ARE. No binding was detected to two putative sites in the DMPK promoter.","method":"GST fusion protein expression, gel retardation (EMSA) assays with double-stranded oligonucleotides","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — in vitro biochemical reconstitution with recombinant protein and EMSA, but single lab, single study","pmids":["10756185"],"is_preprint":false},{"year":2000,"finding":"SIX5 protein localizes to the nucleoplasm with a granular distribution in HeLa cells; endogenous SIX5 migrates at ~100 kDa in SDS-PAGE. A shorter splice isoform mRNA exists but the corresponding shorter protein was undetectable, suggesting the full-length isoform is the major functional protein.","method":"Monoclonal antibody panel (18 mAbs), Western blotting, immunolocalization in HeLa cells, phage-display epitope mapping","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct subcellular localization with validated antibodies and SDS-PAGE characterization, single lab but multiple orthogonal methods","pmids":["15962300"],"is_preprint":false},{"year":2002,"finding":"Six5 functions as a transcriptional activator; overexpression of constitutively active VP16-Six5 in P19 cells identified 21 upregulated target genes including Igfbp5. Igfbp5 was confirmed as a direct Six5 transcriptional target, and its overall expression was decreased in Six5-deficient mouse fibroblasts.","method":"Adenovirus-mediated VP16-Six5 overexpression, cDNA microarray profiling, validation by RT-PCR, analysis in Six5-/- fibroblasts","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional overexpression combined with loss-of-function validation for a specific target (Igfbp5), single lab","pmids":["11978764"],"is_preprint":false},{"year":2004,"finding":"Six5 knockout mice show male sterility with progressive loss of testicular mass, defects in spermatogenic cell survival and spermiogenesis, Leydig cell hyperproliferation, elevated intra-testicular testosterone, and reduced steady-state c-Kit levels in the testis, suggesting c-Kit downregulation contributes to elevated spermatogenic cell apoptosis.","method":"Six5-/- mouse phenotyping, histology, TUNEL assay, Western blotting, hormone assays","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO model with defined cellular phenotype and molecular pathway candidate (c-Kit), single lab","pmids":["15163633"],"is_preprint":false},{"year":2002,"finding":"Six5 heterozygous mice exhibit prolonged QRS duration and delayed infraHisian conduction compared to wild-type, as well as enlarged left ventricular end-diastolic dimension, suggesting Six5 loss contributes to infraHisian conduction delay.","method":"In vivo electrophysiologic studies, echocardiography, heart rate variability testing in Six5+/- mice","journal":"Journal of interventional cardiac electrophysiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct electrophysiological measurement in defined genetic model, single lab","pmids":["12397222"],"is_preprint":false},{"year":2001,"finding":"Six5+/- mice do not exhibit late Na+ channel burst activity (a hallmark of myotonic dystrophy seen in Dmpk+/- mice), and Na+ current amplitude is unchanged, demonstrating that Six5 deficiency does not contribute to the Na+ channel gating abnormality.","method":"Cell-attached patch clamp recordings from skeletal muscle of Six5+/- mice vs. wild-type and Dmpk+/- mice","journal":"Physiological genomics","confidence":"Medium","confidence_rationale":"Tier 1 / Weak — rigorous electrophysiology with appropriate controls, negative result, single lab","pmids":["11526199"],"is_preprint":false},{"year":2007,"finding":"Missense mutations in SIX5 identified in BOR syndrome patients impair EYA1-SIX5 protein binding and reduce the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription, establishing that SIX5 functions with EYA1 as a transcriptional activator complex.","method":"Patient mutation screening, functional transcription activation assays, protein-protein interaction assays for EYA1-SIX5 binding","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional characterization of human disease mutations with interaction and transcriptional assays, single study","pmids":["17357085"],"is_preprint":false},{"year":1998,"finding":"The mDMAHP/Six5 promoter contains multiple transcription initiation sites (one proximal site specific to early embryo E11, two shared among heart, skeletal muscle, and embryo); positive regulatory elements are Sp1/Sp3 binding sites and a novel factor binding site acts as a negative element. All initiation sites lie downstream of the CTG repeat locus, excluding CUG repeats from the mRNA.","method":"Primer extension/S1 nuclease mapping of transcription start sites, promoter-reporter deletion analysis in P19 cells, Sp1/Sp3 binding site identification","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct promoter functional analysis with reporter assays and binding site characterization, single lab","pmids":["9817928"],"is_preprint":false},{"year":2010,"finding":"Behavioral and cognitive testing of Six5-/- mice showed no motivational deficits or spatial learning/memory abnormalities, while Mbnl1-/- mice did exhibit these deficits, placing SIX5 outside the pathway responsible for cognitive and motivational features of DM1.","method":"Open field, elevated plus maze, Morris water maze, fear conditioning, sucrose consumption assays in Six5-/-, Dmpk-/-, and Mbnl1-/- mice","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis via parallel KO models with multiple behavioral readouts; negative result for Six5 is well controlled","pmids":["20360842"],"is_preprint":false},{"year":2016,"finding":"Reduced Six4 and Six5 gene dosage (Six4+/-5+/-) in mdx mice improves dystrophic phenotype (fewer small myofibers, lower serum creatine kinase/lactate dehydrogenase) and prolongs lifespan by 33.8%, associated with enhanced muscle regeneration markers (MYOD1, MYOG, SIX1 positive cells), indicating SIX4/SIX5 normally limit regenerative capacity in dystrophic muscle.","method":"Double heterozygous Six4/Six5 knockout in mdx background, histomorphometry, serum enzyme assays, immunohistochemistry for regeneration markers, grip strength and treadmill testing","journal":"Development, growth & differentiation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo genetic model with multiple phenotypic readouts, though SIX4 and SIX5 were reduced together so individual contributions cannot be separated","pmids":["27224259"],"is_preprint":false},{"year":2022,"finding":"SIX5 forms a complex with hypoxia-induced EYA3 and histone acetyltransferase p300 in colorectal cancer cells; this EYA3-SIX5-p300 complex binds the promoters of EGFR, VEGFD, and five MMPs (MMP3, MMP7, MMP8, MMP21, MMP26) to transactivate them, promoting tumor cell growth.","method":"Co-immunoprecipitation, mass spectrometry, ChIP assay, knockdown/overexpression functional assays, xenograft model","journal":"Annals of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, ChIP, and functional rescue in a single lab with multiple orthogonal methods","pmids":["35957720"],"is_preprint":false},{"year":2022,"finding":"SIX5 transcriptionally activates LINC01468 by binding its promoter in lung adenocarcinoma cells, and SIX5 overexpression exacerbates LUAD cell proliferation, migration, and invasion.","method":"ChIP, luciferase reporter assay, RT-qPCR, knockdown/overexpression, xenograft model","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP and reporter assay support direct binding but limited mechanistic detail about SIX5 itself; single lab, single study","pmids":["35387981"],"is_preprint":false},{"year":2025,"finding":"SIX5 directly binds the EXO1 promoter and transcriptionally activates EXO1 expression in glioblastoma cells, promoting tumor cell migration and angiogenesis; KDM5C positively regulates SIX5 expression upstream.","method":"ChIP, dual-luciferase reporter assay, SIX5 knockdown/overexpression, xenograft model","journal":"Brain research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP and reporter assays support direct promoter binding, single lab, no replication","pmids":["40946999"],"is_preprint":false},{"year":2026,"finding":"KDM5C positively regulates SIX5 expression; SIX5 directly binds the UBE2C promoter to activate its transcription, thereby activating AKT/mTOR signaling and upregulating glycolytic enzymes (GLUT1, HK2, PGK1, LDHA) in glioblastoma.","method":"ChIP, luciferase reporter, lentiviral knockdown/overexpression, rescue experiments, xenograft model","journal":"Frontiers in immunology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — ChIP and reporter assay for direct binding, rescue experiments for pathway placement, single lab","pmids":["41939887"],"is_preprint":false}],"current_model":"SIX5 is a homeodomain transcription factor whose expression is silenced in cis by CTG repeat expansion at the DM1 locus via chromatin compaction over an upstream enhancer; loss of SIX5 contributes to DM1 pathology including cataracts (through altered lens ion homeostasis via Na+/K+-ATPase regulation), infraHisian cardiac conduction defects, and male infertility through impaired spermatogenesis and reduced c-Kit levels, while the protein physically interacts with EYA1 (and EYA3/p300) to form transcriptional activator complexes that drive target gene expression including Igfbp5 and, in cancer contexts, EGFR/VEGFD/MMPs and EXO1/UBE2C."},"narrative":{"mechanistic_narrative":"SIX5 (DMAHP) is a homeodomain transcription factor that acts as a sequence-specific transcriptional activator and is the gene most proximal to the DM1 (myotonic dystrophy type 1) CTG repeat locus, where expansion eliminates a DNase I-hypersensitive enhancer and reduces SIX5 transcription in cis, most severely from the expanded allele [PMID:9241282, PMID:9241283, PMID:9817928]. SIX5 binds DNA through its homeodomain, recognizing the Na+/K+-ATPase alpha-1 (ATP1A1) regulatory element, with the SIX-domain-plus-homeodomain protein forming a second, dimer-like complex at the same site [PMID:10756185]. It activates transcription of target genes including Igfbp5 [PMID:11978764], and assembles into transcriptional activator complexes with EYA1 — an interaction disrupted by SIX5 missense mutations identified in branchio-oto-renal (BOR) syndrome patients [PMID:17357085]. Loss-of-function mouse studies establish SIX5 as a dosage-sensitive contributor to specific DM1 features: heterozygous and homozygous Six5 disruption causes cataracts associated with elevated Na+/K+-ATPase alpha-1 protein and altered lens ion homeostasis [PMID:10802667, PMID:10802668], infraHisian cardiac conduction delay [PMID:12397222], and male sterility with impaired spermatogenesis, Leydig cell hyperproliferation, and reduced testicular c-Kit [PMID:15163633]. Genetic dissection places SIX5 outside the DM1 features driven by Na+ channel gating, cognition, and motivation, which track instead with DMPK and MBNL1 [PMID:11526199, PMID:20360842]. In dystrophic muscle, combined reduction of Six4/Six5 dosage enhances regeneration [PMID:27224259], and in cancer SIX5 functions in EYA3-p300 activator complexes and as a direct transcriptional activator of growth- and invasion-associated genes [PMID:35957720].","teleology":[{"year":1997,"claim":"Established why SIX5 is implicated in DM1: the CTG expansion does not merely affect DMPK but eliminates an upstream enhancer that drives SIX5/DMAHP transcription in cis.","evidence":"DNase I hypersensitivity mapping and allele-specific RT-PCR in DM patient cells across two independent labs","pmids":["9241282","9241283"],"confidence":"High","gaps":["Did not establish which downstream phenotypes are attributable to SIX5 loss versus other DM1 mechanisms","Mechanism of chromatin compaction over the enhancer not resolved"]},{"year":1998,"claim":"Defined the SIX5 promoter architecture, identifying Sp1/Sp3 positive elements and confirming that transcription start sites lie downstream of the CTG repeat, excluding CUG repeats from the mRNA.","evidence":"Transcription start site mapping and promoter-reporter deletion analysis in P19 cells","pmids":["9817928"],"confidence":"Medium","gaps":["Tissue-specific regulation only partially mapped","Identity of the novel negative regulatory factor unknown"]},{"year":2000,"claim":"Identified a direct DNA target and binding mode for SIX5, showing it engages the ATP1A1 regulatory element through its homeodomain, linking the factor to ion-transport gene regulation.","evidence":"GST-fusion recombinant protein and EMSA with defined oligonucleotides","pmids":["10756185"],"confidence":"Medium","gaps":["In vitro binding not confirmed by in vivo occupancy","No binding detected at DMPK promoter sites — broader target repertoire unresolved","Single lab"]},{"year":2000,"claim":"Demonstrated causally that SIX5 loss produces a discrete DM1 phenotype, with dosage-dependent cataracts linked to elevated Na+/K+-ATPase alpha-1 and altered lens ion homeostasis.","evidence":"Six5 beta-gal knock-in mice, dosage series, histology, Western blot, RT-PCR across two labs","pmids":["10802667","10802668"],"confidence":"High","gaps":["No skeletal muscle phenotype detected, narrowing SIX5's role within DM1","Direct mechanistic link between SIX5 and ATP1A1 regulation in lens not established in vivo"]},{"year":2001,"claim":"Excluded SIX5 from the Na+ channel gating abnormality of DM1 myotonia, refining which features are SIX5-dependent.","evidence":"Patch-clamp recordings from Six5+/- versus Dmpk+/- skeletal muscle","pmids":["11526199"],"confidence":"Medium","gaps":["Negative result; does not address other muscle phenotypes"]},{"year":2002,"claim":"Established SIX5 as a transcriptional activator and identified Igfbp5 as a direct target confirmed by both gain- and loss-of-function.","evidence":"VP16-Six5 overexpression with microarray, RT-PCR validation, and Six5-/- fibroblast analysis","pmids":["11978764"],"confidence":"Medium","gaps":["Most of the 21 candidate targets not validated as direct","Co-activators driving native activation not defined"]},{"year":2002,"claim":"Linked SIX5 dosage to the infraHisian cardiac conduction defects seen in DM1.","evidence":"In vivo electrophysiology and echocardiography in Six5+/- mice","pmids":["12397222"],"confidence":"Medium","gaps":["Transcriptional targets responsible for the conduction phenotype unknown","Single lab"]},{"year":2004,"claim":"Defined a SIX5-dependent male fertility program, implicating c-Kit downregulation in spermatogenic cell apoptosis.","evidence":"Six5-/- mouse phenotyping, TUNEL, Western blot, hormone assays","pmids":["15163633"],"confidence":"Medium","gaps":["c-Kit not shown to be a direct SIX5 target","Cause of Leydig cell hyperproliferation unresolved"]},{"year":2007,"claim":"Connected SIX5 to a partner-dependent activator complex and to a second human disease, showing BOR syndrome mutations impair EYA1-SIX5 binding and transactivation.","evidence":"Patient mutation screening with interaction and transcription activation assays","pmids":["17357085"],"confidence":"Medium","gaps":["Target genes of the EYA1-SIX5 complex not identified","Single study"]},{"year":2010,"claim":"Placed SIX5 outside the cognitive/motivational axis of DM1, which tracks with MBNL1 instead.","evidence":"Parallel behavioral testing of Six5-/-, Dmpk-/-, and Mbnl1-/- mice","pmids":["20360842"],"confidence":"Medium","gaps":["Negative result for behavior; does not exclude subtler neural roles"]},{"year":2016,"claim":"Showed SIX4/SIX5 normally limit regenerative capacity in dystrophic muscle, since reduced dosage improves mdx phenotype and lifespan.","evidence":"Six4/Six5 double-heterozygous mdx mice with histomorphometry and regeneration markers","pmids":["27224259"],"confidence":"Medium","gaps":["SIX4 and SIX5 reduced together, so individual contribution unresolved","Direct regenerative target genes not identified"]},{"year":2022,"claim":"Extended SIX5 function to oncogenic transcription, defining an EYA3-SIX5-p300 complex that transactivates EGFR, VEGFD, and MMP genes to drive colorectal tumor growth.","evidence":"Co-IP, mass spectrometry, ChIP, knockdown/overexpression, xenograft","pmids":["35957720"],"confidence":"Medium","gaps":["Whether the same complex operates in normal tissues unknown","Single lab"]},{"year":2025,"claim":"Implicated SIX5 as a direct transcriptional activator of pro-tumorigenic targets (EXO1, UBE2C) downstream of KDM5C in glioblastoma.","evidence":"ChIP, dual-luciferase reporter, knockdown/overexpression, rescue, xenograft","pmids":["40946999","41939887"],"confidence":"Low","gaps":["Single lab, no replication","Mechanism of KDM5C-SIX5 regulation not detailed","Direct relevance to canonical SIX5 developmental function unclear"]},{"year":null,"claim":"The genome-wide native SIX5 target repertoire and the co-activator requirements that determine context-specific (lens, testis, heart, cancer) gene programs remain undefined.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unbiased in vivo ChIP-seq target map","How EYA partner choice (EYA1 vs EYA3) directs distinct programs is unknown","Structural basis of SIX-domain dimerization on DNA not solved"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,9,13]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[4]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,9,13]}],"complexes":["EYA1-SIX5 transcriptional activator complex","EYA3-SIX5-p300 complex"],"partners":["EYA1","EYA3","EP300"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q8N196","full_name":"Homeobox protein SIX5","aliases":["DM locus-associated homeodomain protein","Sine oculis homeobox homolog 5"],"length_aa":739,"mass_kda":74.6,"function":"Transcription factor that is thought to be involved in regulation of organogenesis. May be involved in determination and maintenance of retina formation. Binds a 5'-GGTGTCAG-3' motif present in the ARE regulatory element of ATP1A1. Binds a 5'-TCA[AG][AG]TTNC-3' motif present in the MEF3 element in the myogenin promoter, and in the IGFBP5 promoter (By similarity). Thought to be regulated by association with Dach and Eya proteins, and seems to be coactivated by EYA1, EYA2 and EYA3 (By similarity)","subcellular_location":"Cytoplasm; Nucleus","url":"https://www.uniprot.org/uniprotkb/Q8N196/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SIX5","classification":"Not Classified","n_dependent_lines":3,"n_total_lines":1208,"dependency_fraction":0.0024834437086092716},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SIX5","total_profiled":1310},"omim":[{"mim_id":"614187","title":"HYPERTELORISM, PREAURICULAR SINUS, PUNCTAL PITS, AND DEAFNESS; HPPD","url":"https://www.omim.org/entry/614187"},{"mim_id":"610896","title":"BRANCHIOOTORENAL SYNDROME 2; BOR2","url":"https://www.omim.org/entry/610896"},{"mim_id":"605377","title":"DYSTROPHIA MYOTONICA PROTEIN KINASE; DMPK","url":"https://www.omim.org/entry/605377"},{"mim_id":"604167","title":"CCCTC-BINDING FACTOR; CTCF","url":"https://www.omim.org/entry/604167"},{"mim_id":"602588","title":"BRANCHIOOTIC SYNDROME 1; BOS1","url":"https://www.omim.org/entry/602588"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Nucleoplasm","reliability":"Supported"},{"location":"Golgi apparatus","reliability":"Supported"},{"location":"Vesicles","reliability":"Supported"},{"location":"Cytosol","reliability":"Supported"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SIX5"},"hgnc":{"alias_symbol":[],"prev_symbol":["DMAHP"]},"alphafold":{"accession":"Q8N196","domains":[{"cath_id":"-","chopping":"86-199","consensus_level":"medium","plddt":89.7277,"start":86,"end":199},{"cath_id":"1.10.10.60","chopping":"207-258","consensus_level":"high","plddt":89.6092,"start":207,"end":258}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N196","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N196-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q8N196-F1-predicted_aligned_error_v6.png","plddt_mean":51.28},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SIX5","jax_strain_url":"https://www.jax.org/strain/search?query=SIX5"},"sequence":{"accession":"Q8N196","fasta_url":"https://rest.uniprot.org/uniprotkb/Q8N196.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q8N196/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q8N196"}},"corpus_meta":[{"pmid":"9241282","id":"PMC_9241282","title":"Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP.","date":"1997","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9241282","citation_count":194,"is_preprint":false},{"pmid":"10802667","id":"PMC_10802667","title":"Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy.","date":"2000","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10802667","citation_count":179,"is_preprint":false},{"pmid":"9241283","id":"PMC_9241283","title":"Expansion of the myotonic dystrophy CTG repeat reduces expression of the flanking DMAHP gene.","date":"1997","source":"Nature genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9241283","citation_count":178,"is_preprint":false},{"pmid":"17357085","id":"PMC_17357085","title":"Transcription factor SIX5 is mutated in patients with branchio-oto-renal syndrome.","date":"2007","source":"American journal of human 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Laryngoscope","url":"https://pubmed.ncbi.nlm.nih.gov/22447252","citation_count":28,"is_preprint":false},{"pmid":"20360842","id":"PMC_20360842","title":"Muscleblind1, but not Dmpk or Six5, contributes to a complex phenotype of muscular and motivational deficits in mouse models of myotonic dystrophy.","date":"2010","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/20360842","citation_count":27,"is_preprint":false},{"pmid":"10756185","id":"PMC_10756185","title":"Functional analysis of the homeodomain protein SIX5.","date":"2000","source":"Nucleic acids research","url":"https://pubmed.ncbi.nlm.nih.gov/10756185","citation_count":24,"is_preprint":false},{"pmid":"11978764","id":"PMC_11978764","title":"Identification of transcriptional targets for Six5: implication for the pathogenesis of myotonic dystrophy type 1.","date":"2002","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/11978764","citation_count":24,"is_preprint":false},{"pmid":"35387981","id":"PMC_35387981","title":"SIX5-activated LINC01468 promotes lung adenocarcinoma progression by recruiting SERBP1 to regulate SERPINE1 mRNA stability and recruiting USP5 to facilitate PAI1 protein deubiquitylation.","date":"2022","source":"Cell death & disease","url":"https://pubmed.ncbi.nlm.nih.gov/35387981","citation_count":23,"is_preprint":false},{"pmid":"15282156","id":"PMC_15282156","title":"UNC-39, the C. elegans homolog of the human myotonic dystrophy-associated homeodomain protein Six5, regulates cell motility and differentiation.","date":"2004","source":"Developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/15282156","citation_count":20,"is_preprint":false},{"pmid":"10823141","id":"PMC_10823141","title":"Expression of a homeobox gene (SIX5) in borderline ovarian tumours.","date":"2000","source":"Journal of clinical pathology","url":"https://pubmed.ncbi.nlm.nih.gov/10823141","citation_count":17,"is_preprint":false},{"pmid":"10951446","id":"PMC_10951446","title":"Reduced expression of DMAHP/SIX5 gene in myotonic dystrophy muscle.","date":"2000","source":"Muscle & nerve","url":"https://pubmed.ncbi.nlm.nih.gov/10951446","citation_count":16,"is_preprint":false},{"pmid":"35968143","id":"PMC_35968143","title":"The primary function of Six5 of Fusarium oxysporum is to facilitate Avr2 activity by together manipulating the size exclusion limit of plasmodesmata.","date":"2022","source":"Frontiers in plant science","url":"https://pubmed.ncbi.nlm.nih.gov/35968143","citation_count":16,"is_preprint":false},{"pmid":"9817928","id":"PMC_9817928","title":"Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements.","date":"1998","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9817928","citation_count":13,"is_preprint":false},{"pmid":"35957720","id":"PMC_35957720","title":"Both a hypoxia-inducible EYA3 and a histone acetyltransferase p300 function as coactivators of SIX5 to mediate tumorigenesis and cancer progression.","date":"2022","source":"Annals of translational medicine","url":"https://pubmed.ncbi.nlm.nih.gov/35957720","citation_count":11,"is_preprint":false},{"pmid":"10399747","id":"PMC_10399747","title":"Reduction of the DM-associated homeo domain protein (DMAHP) mRNA in different brain areas of myotonic dystrophy patients.","date":"1999","source":"Neuromuscular disorders : NMD","url":"https://pubmed.ncbi.nlm.nih.gov/10399747","citation_count":11,"is_preprint":false},{"pmid":"27224259","id":"PMC_27224259","title":"Low Six4 and Six5 gene dosage improves dystrophic phenotype and prolongs life span of mdx mice.","date":"2016","source":"Development, growth & differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/27224259","citation_count":6,"is_preprint":false},{"pmid":"11526199","id":"PMC_11526199","title":"Skeletal muscle Na currents in mice heterozygous for Six5 deficiency.","date":"2001","source":"Physiological genomics","url":"https://pubmed.ncbi.nlm.nih.gov/11526199","citation_count":4,"is_preprint":false},{"pmid":"38138005","id":"PMC_38138005","title":"The SIX5 Protein in Fusarium oxysporum f. sp. cepae Acts as an Avirulence Effector toward Shallot (Allium cepa L. Aggregatum Group).","date":"2023","source":"Microorganisms","url":"https://pubmed.ncbi.nlm.nih.gov/38138005","citation_count":3,"is_preprint":false},{"pmid":"15962300","id":"PMC_15962300","title":"Characterisation of the transcription factor, SIX5, using a new panel of monoclonal antibodies.","date":"2005","source":"Journal of cellular biochemistry","url":"https://pubmed.ncbi.nlm.nih.gov/15962300","citation_count":3,"is_preprint":false},{"pmid":"35709191","id":"PMC_35709191","title":"Identification of homozygous missense variant in SIX5 gene underlying recessive nonsyndromic hearing impairment.","date":"2022","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/35709191","citation_count":3,"is_preprint":false},{"pmid":"40946999","id":"PMC_40946999","title":"Role of SIX5-mediated EXO1 overexpression in driving glioblastoma progression: Insights into tumor cell migration and angiogenesis.","date":"2025","source":"Brain research","url":"https://pubmed.ncbi.nlm.nih.gov/40946999","citation_count":0,"is_preprint":false},{"pmid":"41939887","id":"PMC_41939887","title":"KDM5C-regulated SIX5 promotes glioblastoma progression through transcriptional activation of UBE2C and enhancement of the Warburg effect.","date":"2026","source":"Frontiers in immunology","url":"https://pubmed.ncbi.nlm.nih.gov/41939887","citation_count":0,"is_preprint":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":18628,"output_tokens":4046,"usd":0.058287,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":11851,"output_tokens":3767,"usd":0.076715,"stage2_stop_reason":"end_turn"},"total_usd":0.135002,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1997,\n      \"finding\": \"CTG trinucleotide repeat expansion at the DM1 locus eliminates a DNase I-hypersensitive site containing an enhancer element that regulates DMAHP/SIX5 transcription, reducing steady-state DMAHP transcript levels 2- to 4-fold overall and greatly reducing transcripts from the expanded allele specifically.\",\n      \"method\": \"DNase I hypersensitivity assay, allele-specific RT-PCR in DM patient cells\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent labs (PMID:9241282 and PMID:9241283) using orthogonal methods (DNase I hypersensitivity, allele-specific RT-PCR, enhancer reporter assays) replicated the same mechanistic conclusion\",\n      \"pmids\": [\"9241282\", \"9241283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Loss-of-function of Six5 in mice (homozygous knockout replacing exon 1 with beta-galactosidase reporter) causes lenticular opacities (cataracts) at a higher rate than controls, with reporter expression confirmed in the developing lens; no skeletal muscle abnormalities were detected.\",\n      \"method\": \"Targeted gene disruption (beta-gal knock-in), histological and functional phenotyping of Six5-/- mice\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — two independent labs (PMID:10802667 and PMID:10802668) using Six5 knockout mice with direct phenotypic readout both established cataract formation\",\n      \"pmids\": [\"10802667\", \"10802668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Cataract formation in Six5-deficient mice is inversely related to Six5 dosage (heterozygous Six5+/- mice also develop cataracts), and Six5+/- and Six5-/- mice show increased steady-state Na+/K+-ATPase alpha-1 subunit protein levels and decreased Dm15 mRNA levels, implicating altered ion homeostasis in lens pathology.\",\n      \"method\": \"Dosage analysis of Six5+/- and Six5-/- mice, Western blotting, RT-PCR\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — rigorous dosage series in two independent mouse models (PMID:10802668), multiple orthogonal molecular readouts\",\n      \"pmids\": [\"10802668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SIX5 protein binds the Na+/K+-ATPase alpha-1 subunit gene (ATP1A1) regulatory element (ARE) — the same site as murine Six4 — through its homeodomain; the protein containing both the SIX domain and homeodomain forms a second (presumed dimer) complex with the ARE. No binding was detected to two putative sites in the DMPK promoter.\",\n      \"method\": \"GST fusion protein expression, gel retardation (EMSA) assays with double-stranded oligonucleotides\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — in vitro biochemical reconstitution with recombinant protein and EMSA, but single lab, single study\",\n      \"pmids\": [\"10756185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SIX5 protein localizes to the nucleoplasm with a granular distribution in HeLa cells; endogenous SIX5 migrates at ~100 kDa in SDS-PAGE. A shorter splice isoform mRNA exists but the corresponding shorter protein was undetectable, suggesting the full-length isoform is the major functional protein.\",\n      \"method\": \"Monoclonal antibody panel (18 mAbs), Western blotting, immunolocalization in HeLa cells, phage-display epitope mapping\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct subcellular localization with validated antibodies and SDS-PAGE characterization, single lab but multiple orthogonal methods\",\n      \"pmids\": [\"15962300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Six5 functions as a transcriptional activator; overexpression of constitutively active VP16-Six5 in P19 cells identified 21 upregulated target genes including Igfbp5. Igfbp5 was confirmed as a direct Six5 transcriptional target, and its overall expression was decreased in Six5-deficient mouse fibroblasts.\",\n      \"method\": \"Adenovirus-mediated VP16-Six5 overexpression, cDNA microarray profiling, validation by RT-PCR, analysis in Six5-/- fibroblasts\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional overexpression combined with loss-of-function validation for a specific target (Igfbp5), single lab\",\n      \"pmids\": [\"11978764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Six5 knockout mice show male sterility with progressive loss of testicular mass, defects in spermatogenic cell survival and spermiogenesis, Leydig cell hyperproliferation, elevated intra-testicular testosterone, and reduced steady-state c-Kit levels in the testis, suggesting c-Kit downregulation contributes to elevated spermatogenic cell apoptosis.\",\n      \"method\": \"Six5-/- mouse phenotyping, histology, TUNEL assay, Western blotting, hormone assays\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO model with defined cellular phenotype and molecular pathway candidate (c-Kit), single lab\",\n      \"pmids\": [\"15163633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Six5 heterozygous mice exhibit prolonged QRS duration and delayed infraHisian conduction compared to wild-type, as well as enlarged left ventricular end-diastolic dimension, suggesting Six5 loss contributes to infraHisian conduction delay.\",\n      \"method\": \"In vivo electrophysiologic studies, echocardiography, heart rate variability testing in Six5+/- mice\",\n      \"journal\": \"Journal of interventional cardiac electrophysiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct electrophysiological measurement in defined genetic model, single lab\",\n      \"pmids\": [\"12397222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Six5+/- mice do not exhibit late Na+ channel burst activity (a hallmark of myotonic dystrophy seen in Dmpk+/- mice), and Na+ current amplitude is unchanged, demonstrating that Six5 deficiency does not contribute to the Na+ channel gating abnormality.\",\n      \"method\": \"Cell-attached patch clamp recordings from skeletal muscle of Six5+/- mice vs. wild-type and Dmpk+/- mice\",\n      \"journal\": \"Physiological genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Weak — rigorous electrophysiology with appropriate controls, negative result, single lab\",\n      \"pmids\": [\"11526199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Missense mutations in SIX5 identified in BOR syndrome patients impair EYA1-SIX5 protein binding and reduce the ability of SIX5 or the EYA1-SIX5 complex to activate gene transcription, establishing that SIX5 functions with EYA1 as a transcriptional activator complex.\",\n      \"method\": \"Patient mutation screening, functional transcription activation assays, protein-protein interaction assays for EYA1-SIX5 binding\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional characterization of human disease mutations with interaction and transcriptional assays, single study\",\n      \"pmids\": [\"17357085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The mDMAHP/Six5 promoter contains multiple transcription initiation sites (one proximal site specific to early embryo E11, two shared among heart, skeletal muscle, and embryo); positive regulatory elements are Sp1/Sp3 binding sites and a novel factor binding site acts as a negative element. All initiation sites lie downstream of the CTG repeat locus, excluding CUG repeats from the mRNA.\",\n      \"method\": \"Primer extension/S1 nuclease mapping of transcription start sites, promoter-reporter deletion analysis in P19 cells, Sp1/Sp3 binding site identification\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct promoter functional analysis with reporter assays and binding site characterization, single lab\",\n      \"pmids\": [\"9817928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Behavioral and cognitive testing of Six5-/- mice showed no motivational deficits or spatial learning/memory abnormalities, while Mbnl1-/- mice did exhibit these deficits, placing SIX5 outside the pathway responsible for cognitive and motivational features of DM1.\",\n      \"method\": \"Open field, elevated plus maze, Morris water maze, fear conditioning, sucrose consumption assays in Six5-/-, Dmpk-/-, and Mbnl1-/- mice\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis via parallel KO models with multiple behavioral readouts; negative result for Six5 is well controlled\",\n      \"pmids\": [\"20360842\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Reduced Six4 and Six5 gene dosage (Six4+/-5+/-) in mdx mice improves dystrophic phenotype (fewer small myofibers, lower serum creatine kinase/lactate dehydrogenase) and prolongs lifespan by 33.8%, associated with enhanced muscle regeneration markers (MYOD1, MYOG, SIX1 positive cells), indicating SIX4/SIX5 normally limit regenerative capacity in dystrophic muscle.\",\n      \"method\": \"Double heterozygous Six4/Six5 knockout in mdx background, histomorphometry, serum enzyme assays, immunohistochemistry for regeneration markers, grip strength and treadmill testing\",\n      \"journal\": \"Development, growth & differentiation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo genetic model with multiple phenotypic readouts, though SIX4 and SIX5 were reduced together so individual contributions cannot be separated\",\n      \"pmids\": [\"27224259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIX5 forms a complex with hypoxia-induced EYA3 and histone acetyltransferase p300 in colorectal cancer cells; this EYA3-SIX5-p300 complex binds the promoters of EGFR, VEGFD, and five MMPs (MMP3, MMP7, MMP8, MMP21, MMP26) to transactivate them, promoting tumor cell growth.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, ChIP assay, knockdown/overexpression functional assays, xenograft model\",\n      \"journal\": \"Annals of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, ChIP, and functional rescue in a single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35957720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIX5 transcriptionally activates LINC01468 by binding its promoter in lung adenocarcinoma cells, and SIX5 overexpression exacerbates LUAD cell proliferation, migration, and invasion.\",\n      \"method\": \"ChIP, luciferase reporter assay, RT-qPCR, knockdown/overexpression, xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP and reporter assay support direct binding but limited mechanistic detail about SIX5 itself; single lab, single study\",\n      \"pmids\": [\"35387981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SIX5 directly binds the EXO1 promoter and transcriptionally activates EXO1 expression in glioblastoma cells, promoting tumor cell migration and angiogenesis; KDM5C positively regulates SIX5 expression upstream.\",\n      \"method\": \"ChIP, dual-luciferase reporter assay, SIX5 knockdown/overexpression, xenograft model\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP and reporter assays support direct promoter binding, single lab, no replication\",\n      \"pmids\": [\"40946999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"KDM5C positively regulates SIX5 expression; SIX5 directly binds the UBE2C promoter to activate its transcription, thereby activating AKT/mTOR signaling and upregulating glycolytic enzymes (GLUT1, HK2, PGK1, LDHA) in glioblastoma.\",\n      \"method\": \"ChIP, luciferase reporter, lentiviral knockdown/overexpression, rescue experiments, xenograft model\",\n      \"journal\": \"Frontiers in immunology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — ChIP and reporter assay for direct binding, rescue experiments for pathway placement, single lab\",\n      \"pmids\": [\"41939887\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SIX5 is a homeodomain transcription factor whose expression is silenced in cis by CTG repeat expansion at the DM1 locus via chromatin compaction over an upstream enhancer; loss of SIX5 contributes to DM1 pathology including cataracts (through altered lens ion homeostasis via Na+/K+-ATPase regulation), infraHisian cardiac conduction defects, and male infertility through impaired spermatogenesis and reduced c-Kit levels, while the protein physically interacts with EYA1 (and EYA3/p300) to form transcriptional activator complexes that drive target gene expression including Igfbp5 and, in cancer contexts, EGFR/VEGFD/MMPs and EXO1/UBE2C.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SIX5 (DMAHP) is a homeodomain transcription factor that acts as a sequence-specific transcriptional activator and is the gene most proximal to the DM1 (myotonic dystrophy type 1) CTG repeat locus, where expansion eliminates a DNase I-hypersensitive enhancer and reduces SIX5 transcription in cis, most severely from the expanded allele [#0, #10]. SIX5 binds DNA through its homeodomain, recognizing the Na+/K+-ATPase alpha-1 (ATP1A1) regulatory element, with the SIX-domain-plus-homeodomain protein forming a second, dimer-like complex at the same site [#3]. It activates transcription of target genes including Igfbp5 [#5], and assembles into transcriptional activator complexes with EYA1 — an interaction disrupted by SIX5 missense mutations identified in branchio-oto-renal (BOR) syndrome patients [#9]. Loss-of-function mouse studies establish SIX5 as a dosage-sensitive contributor to specific DM1 features: heterozygous and homozygous Six5 disruption causes cataracts associated with elevated Na+/K+-ATPase alpha-1 protein and altered lens ion homeostasis [#1, #2], infraHisian cardiac conduction delay [#7], and male sterility with impaired spermatogenesis, Leydig cell hyperproliferation, and reduced testicular c-Kit [#6]. Genetic dissection places SIX5 outside the DM1 features driven by Na+ channel gating, cognition, and motivation, which track instead with DMPK and MBNL1 [#8, #11]. In dystrophic muscle, combined reduction of Six4/Six5 dosage enhances regeneration [#12], and in cancer SIX5 functions in EYA3-p300 activator complexes and as a direct transcriptional activator of growth- and invasion-associated genes [#13].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Established why SIX5 is implicated in DM1: the CTG expansion does not merely affect DMPK but eliminates an upstream enhancer that drives SIX5/DMAHP transcription in cis.\",\n      \"evidence\": \"DNase I hypersensitivity mapping and allele-specific RT-PCR in DM patient cells across two independent labs\",\n      \"pmids\": [\"9241282\", \"9241283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish which downstream phenotypes are attributable to SIX5 loss versus other DM1 mechanisms\", \"Mechanism of chromatin compaction over the enhancer not resolved\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined the SIX5 promoter architecture, identifying Sp1/Sp3 positive elements and confirming that transcription start sites lie downstream of the CTG repeat, excluding CUG repeats from the mRNA.\",\n      \"evidence\": \"Transcription start site mapping and promoter-reporter deletion analysis in P19 cells\",\n      \"pmids\": [\"9817928\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific regulation only partially mapped\", \"Identity of the novel negative regulatory factor unknown\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identified a direct DNA target and binding mode for SIX5, showing it engages the ATP1A1 regulatory element through its homeodomain, linking the factor to ion-transport gene regulation.\",\n      \"evidence\": \"GST-fusion recombinant protein and EMSA with defined oligonucleotides\",\n      \"pmids\": [\"10756185\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vitro binding not confirmed by in vivo occupancy\", \"No binding detected at DMPK promoter sites — broader target repertoire unresolved\", \"Single lab\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrated causally that SIX5 loss produces a discrete DM1 phenotype, with dosage-dependent cataracts linked to elevated Na+/K+-ATPase alpha-1 and altered lens ion homeostasis.\",\n      \"evidence\": \"Six5 beta-gal knock-in mice, dosage series, histology, Western blot, RT-PCR across two labs\",\n      \"pmids\": [\"10802667\", \"10802668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No skeletal muscle phenotype detected, narrowing SIX5's role within DM1\", \"Direct mechanistic link between SIX5 and ATP1A1 regulation in lens not established in vivo\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Excluded SIX5 from the Na+ channel gating abnormality of DM1 myotonia, refining which features are SIX5-dependent.\",\n      \"evidence\": \"Patch-clamp recordings from Six5+/- versus Dmpk+/- skeletal muscle\",\n      \"pmids\": [\"11526199\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result; does not address other muscle phenotypes\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Established SIX5 as a transcriptional activator and identified Igfbp5 as a direct target confirmed by both gain- and loss-of-function.\",\n      \"evidence\": \"VP16-Six5 overexpression with microarray, RT-PCR validation, and Six5-/- fibroblast analysis\",\n      \"pmids\": [\"11978764\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Most of the 21 candidate targets not validated as direct\", \"Co-activators driving native activation not defined\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Linked SIX5 dosage to the infraHisian cardiac conduction defects seen in DM1.\",\n      \"evidence\": \"In vivo electrophysiology and echocardiography in Six5+/- mice\",\n      \"pmids\": [\"12397222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional targets responsible for the conduction phenotype unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Defined a SIX5-dependent male fertility program, implicating c-Kit downregulation in spermatogenic cell apoptosis.\",\n      \"evidence\": \"Six5-/- mouse phenotyping, TUNEL, Western blot, hormone assays\",\n      \"pmids\": [\"15163633\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"c-Kit not shown to be a direct SIX5 target\", \"Cause of Leydig cell hyperproliferation unresolved\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Connected SIX5 to a partner-dependent activator complex and to a second human disease, showing BOR syndrome mutations impair EYA1-SIX5 binding and transactivation.\",\n      \"evidence\": \"Patient mutation screening with interaction and transcription activation assays\",\n      \"pmids\": [\"17357085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Target genes of the EYA1-SIX5 complex not identified\", \"Single study\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Placed SIX5 outside the cognitive/motivational axis of DM1, which tracks with MBNL1 instead.\",\n      \"evidence\": \"Parallel behavioral testing of Six5-/-, Dmpk-/-, and Mbnl1-/- mice\",\n      \"pmids\": [\"20360842\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Negative result for behavior; does not exclude subtler neural roles\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Showed SIX4/SIX5 normally limit regenerative capacity in dystrophic muscle, since reduced dosage improves mdx phenotype and lifespan.\",\n      \"evidence\": \"Six4/Six5 double-heterozygous mdx mice with histomorphometry and regeneration markers\",\n      \"pmids\": [\"27224259\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"SIX4 and SIX5 reduced together, so individual contribution unresolved\", \"Direct regenerative target genes not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Extended SIX5 function to oncogenic transcription, defining an EYA3-SIX5-p300 complex that transactivates EGFR, VEGFD, and MMP genes to drive colorectal tumor growth.\",\n      \"evidence\": \"Co-IP, mass spectrometry, ChIP, knockdown/overexpression, xenograft\",\n      \"pmids\": [\"35957720\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether the same complex operates in normal tissues unknown\", \"Single lab\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Implicated SIX5 as a direct transcriptional activator of pro-tumorigenic targets (EXO1, UBE2C) downstream of KDM5C in glioblastoma.\",\n      \"evidence\": \"ChIP, dual-luciferase reporter, knockdown/overexpression, rescue, xenograft\",\n      \"pmids\": [\"40946999\", \"41939887\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single lab, no replication\", \"Mechanism of KDM5C-SIX5 regulation not detailed\", \"Direct relevance to canonical SIX5 developmental function unclear\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"The genome-wide native SIX5 target repertoire and the co-activator requirements that determine context-specific (lens, testis, heart, cancer) gene programs remain undefined.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unbiased in vivo ChIP-seq target map\", \"How EYA partner choice (EYA1 vs EYA3) directs distinct programs is unknown\", \"Structural basis of SIX-domain dimerization on DNA not solved\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 9, 13]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [4]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 9, 13]}\n    ],\n    \"complexes\": [\n      \"EYA1-SIX5 transcriptional activator complex\",\n      \"EYA3-SIX5-p300 complex\"\n    ],\n    \"partners\": [\n      \"EYA1\",\n      \"EYA3\",\n      \"EP300\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":6,"faith_pct":100.0}}