{"gene":"SIX5","run_date":"2026-04-28T20:42:07","timeline":{"discoveries":[{"year":1997,"finding":"CTG trinucleotide repeat expansion at the DM1 locus eliminates a DNase I-hypersensitive enhancer element upstream of SIX5 (DMAHP), reducing steady-state SIX5 transcript levels 2- to 4-fold overall and greatly on the expanded allele specifically, demonstrating that repeat expansion suppresses SIX5 expression in cis via chromatin-level repression.","method":"Allele-specific RT-PCR, DNase I hypersensitivity assay, enhancer reporter assay","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — replicated independently by two labs in the same issue with multiple orthogonal methods","pmids":["9241282","9241283"],"is_preprint":false},{"year":2000,"finding":"Homozygous and heterozygous targeted deletion of Six5 in mice causes lenticular opacities (cataracts) in a dosage-dependent manner, establishing a direct role for SIX5 in lens homeostasis; Six5-null mice showed beta-galactosidase reporter expression in developing lens confirming expression there.","method":"Targeted gene disruption (knockout mouse), beta-galactosidase reporter, slit-lamp examination","journal":"Nature genetics","confidence":"High","confidence_rationale":"Tier 2 — independently replicated by two separate labs with clean KO/heterozygous models and defined phenotypic readout","pmids":["10802667","10802668"],"is_preprint":false},{"year":2000,"finding":"Heterozygous and homozygous Six5-deficient mice show increased steady-state levels of the Na+/K+-ATPase alpha-1 subunit and decreased Dm15 (DMPK) mRNA, indicating that SIX5 normally regulates ion transporter gene expression in the lens and that altered ion homeostasis contributes to cataract formation.","method":"Western blot, RT-PCR in Six5 knockout mouse tissues","journal":"Nature genetics","confidence":"Medium","confidence_rationale":"Tier 2 — single lab but two orthogonal molecular readouts in KO model","pmids":["10802668"],"is_preprint":false},{"year":2000,"finding":"SIX5 protein binds specifically to the ARE (Na+/K+-ATPase alpha-1 subunit regulatory element) via its homeodomain, forming at least one specific complex and a second dimer complex; no binding was detected to putative DMPK promoter sites, defining the DNA-binding specificity of SIX5.","method":"Gel retardation (EMSA) with GST-fusion recombinant proteins (homeodomain alone, SIX domain alone, and combined)","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 1 — in vitro biochemical reconstitution with purified recombinant proteins; single lab","pmids":["10756185"],"is_preprint":false},{"year":1998,"finding":"The SIX5 promoter contains multiple positive regulatory elements that are Sp1/Sp3 binding sites and at least one novel negative regulatory element; three major transcription initiation sites were identified with developmental-stage-specific utilization.","method":"Promoter-reporter deletion assays, transcription initiation site mapping (primer extension/S1 nuclease), gel shift assays for Sp1/Sp3","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 1-2 — multiple promoter functional assays with deletion analysis; single lab","pmids":["9817928"],"is_preprint":false},{"year":2002,"finding":"Overexpression of constitutively active VP16-Six5 in P19 cells followed by cDNA array profiling identified Igfbp5 as a direct transcriptional target of Six5; Igfbp5 expression was reduced in Six5-deficient mouse fibroblasts and its response to MyoD-induced muscle conversion was altered in DM1 patient cells.","method":"Adenovirus-mediated VP16-Six5 overexpression, cDNA array, RT-PCR in Six5-KO fibroblasts, promoter-reporter assay","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (OE + KO + reporter) in a single lab","pmids":["11978764"],"is_preprint":false},{"year":2007,"finding":"SIX5 directly interacts with EYA1 in humans (conserved from C. elegans unc-39/eya-1); BOR-associated missense mutations in SIX5 disrupt EYA1-SIX5 binding and abolish the ability of SIX5 or the EYA1-SIX5 complex to activate transcription, identifying SIX5 as a transcriptional partner of EYA1.","method":"Co-immunoprecipitation, transcriptional activation assays with mutant SIX5 constructs, mutation screening","journal":"American journal of human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — functional mutagenesis + Co-IP + transcriptional assay; single lab","pmids":["17357085"],"is_preprint":false},{"year":2004,"finding":"Six5-null male mice are infertile and display progressive testicular atrophy, spermatogenic cell apoptosis, spermiogenesis failure, Leydig cell hyperproliferation, and reduced steady-state c-Kit levels in the testis, indicating SIX5 is required for spermatogenic cell survival partly through maintaining c-Kit expression.","method":"Targeted knockout mouse analysis, histology, flow cytometry, RT-PCR/Western blot for c-Kit and hormones","journal":"Human molecular genetics","confidence":"Medium","confidence_rationale":"Tier 2 — clean KO with multiple molecular readouts; single lab","pmids":["15163633"],"is_preprint":false},{"year":2002,"finding":"Six5 heterozygous mice show prolonged QRS duration, delayed infra-Hisian conduction, and enlarged left ventricular end-diastolic dimension compared to wild-type, establishing a partial contribution of Six5 loss to cardiac conduction abnormalities.","method":"In vivo electrophysiology (EP study), echocardiography, exercise tolerance testing in Six5+/- mice","journal":"Journal of interventional cardiac electrophysiology","confidence":"Medium","confidence_rationale":"Tier 2 — clean heterozygous KO with defined electrophysiological phenotype; single lab","pmids":["12397222"],"is_preprint":false},{"year":2001,"finding":"Six5+/- mice do not exhibit the late Na+ channel burst activity seen in Dmpk+/- muscle, demonstrating that Six5 deficiency does not contribute to the Na channel gating abnormality in skeletal muscle and distinguishing Six5 from DMPK in this aspect of DM pathophysiology.","method":"Cell-attached patch clamp recordings in skeletal muscle from Six5+/- and Dmpk+/- mice","journal":"Physiological genomics","confidence":"Medium","confidence_rationale":"Tier 1 — direct electrophysiology in KO model with appropriate comparators; single lab","pmids":["11526199"],"is_preprint":false},{"year":2005,"finding":"Endogenous SIX5 protein localizes to the nucleoplasm with a granular distribution in HeLa cells and migrates at ~100 kDa on SDS-PAGE; only the full-length isoform was detectable, while a putative shorter splice isoform was not expressed at detectable levels.","method":"Monoclonal antibody panel (18 mAbs), Western blot, immunofluorescence/immunolocalization in HeLa cells and fetal tissues","journal":"Journal of cellular biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct protein localization with validated antibody panel; single lab","pmids":["15962300"],"is_preprint":false},{"year":2004,"finding":"The C. elegans SIX5 ortholog UNC-39 is required for migration and differentiation of mesodermal and ectodermal cells including muscles and neurons; human SIX5 functionally complements unc-39 mutants, confirming orthology and conserved transcription factor function.","method":"Genetic mutant analysis in C. elegans, rescue experiments with human SIX5","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — genetic epistasis and cross-species functional rescue; single lab","pmids":["15282156"],"is_preprint":false},{"year":2001,"finding":"The Drosophila SIX5 ortholog D-Six4 is required for myoblast fusion (muscle development) and for the mesodermal component of gonad development; adult males with D-Six4 mutations show testicular reduction, mirroring DM1 phenotypes.","method":"Drosophila mutant analysis, histology, genetic characterization","journal":"Current biology","confidence":"Medium","confidence_rationale":"Tier 2 — clean mutant phenotype with defined cellular readout in ortholog; single lab","pmids":["11470409"],"is_preprint":false},{"year":2022,"finding":"SIX5 assembles with hypoxia-induced EYA3 and histone acetyltransferase p300 to form a transcriptional complex in colorectal cancer cells; this EYA3-SIX5-p300 complex directly binds promoters of EGFR, VEGFD, and multiple MMPs to activate their transcription, promoting tumor growth.","method":"Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP), transcriptional reporter assays, tumor xenograft model","journal":"Annals of translational medicine","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, MS, ChIP) in a single lab","pmids":["35957720"],"is_preprint":false},{"year":2022,"finding":"SIX5 transcriptionally activates LINC01468 lncRNA by binding its promoter; SIX5-driven LINC01468 then recruits SERBP1 to stabilize SERPINE1 mRNA and interacts with USP5 to facilitate PAI1 deubiquitylation, promoting lung adenocarcinoma cell proliferation, migration, and invasion.","method":"ChIP, luciferase reporter assay, RNA co-immunoprecipitation, RIP, knockdown/overexpression functional assays, xenograft model","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 — ChIP + reporter + RIP with functional rescue; single lab","pmids":["35387981"],"is_preprint":false},{"year":2016,"finding":"Reduced dosage of Six4 and Six5 (double heterozygous knockout) in mdx dystrophic mice improves muscle regeneration, reduces serum CK/LDH, and extends lifespan by 33.8%, with enhanced MYOD1, MYOG, and SIX1 expression in regenerating myofibers, demonstrating that SIX5 (together with SIX4) modulates satellite cell-driven muscle regeneration.","method":"Double heterozygous knockout in mdx mice, grip strength, treadmill testing, serum enzyme assays, immunostaining for myogenic markers","journal":"Development, growth & differentiation","confidence":"Low","confidence_rationale":"Tier 3 — confounded by concurrent Six4 reduction; single lab, no separation of Six5-specific mechanism","pmids":["27224259"],"is_preprint":false},{"year":2025,"finding":"SIX5 directly binds the EXO1 promoter and activates its transcription in glioblastoma cells, as demonstrated by ChIP and dual-luciferase reporter assays; SIX5 knockdown suppresses EXO1 expression and GBM cell growth, which is partially rescued by EXO1 overexpression.","method":"ChIP, dual-luciferase reporter assay, lentiviral knockdown, xenograft model","journal":"Brain research","confidence":"Low","confidence_rationale":"Tier 2 method but zero citations, single lab, no independent replication","pmids":["40946999"],"is_preprint":false},{"year":2001,"finding":"CTG repeat expansion at the DM locus decreases PvuII chromatin sensitivity adjacent to the repeat and partially reduces SIX5 mRNA from expanded alleles in a tissue-specific manner (greatest reduction in muscle and liver), while DMWD expression is not affected, demonstrating allele- and tissue-specific regulation of SIX5 by chromatin structure changes.","method":"Somatic cell hybrid PCR-based chromatin sensitivity assay, semiquantitative multiplex RT-PCR","journal":"Molecular genetics and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 — allele-specific chromatin and expression assays in defined cell hybrids; single lab","pmids":["11592825"],"is_preprint":false}],"current_model":"SIX5 is a homeodomain transcription factor whose expression is suppressed in cis by CTG repeat expansion at the DM1 locus through chromatin compaction that eliminates an upstream enhancer; the protein binds DNA via its homeodomain (notably the Na+/K+-ATPase ARE element), partners with EYA1 and EYA3/p300 complexes to activate target genes (including Igfbp5, EGFR, MMPs, and EXO1), and its dosage-dependent loss causes cataracts, cardiac conduction delays, and spermatogenic failure in mouse models, implicating SIX5 haploinsufficiency as a contributor to multiple features of myotonic dystrophy type 1."},"narrative":{"teleology":[{"year":1997,"claim":"Establishing that CTG repeat expansion at the DM1 locus suppresses SIX5 expression in cis via chromatin-level loss of an enhancer answered the question of whether repeat expansions affect neighboring gene regulation, making SIX5 haploinsufficiency a candidate contributor to DM1 pathology.","evidence":"Allele-specific RT-PCR, DNase I hypersensitivity, and enhancer reporter assays in DM1 patient cells by two independent labs","pmids":["9241282","9241283"],"confidence":"High","gaps":["Whether reduced SIX5 causes any specific DM1 phenotype was untested","The degree of reduction in different tissues was unknown","Whether chromatin compaction extends to other loci was not addressed"]},{"year":1998,"claim":"Mapping the SIX5 promoter architecture — including Sp1/Sp3 sites and developmental-stage-specific initiation sites — established the regulatory framework for understanding how SIX5 transcription is controlled.","evidence":"Promoter-reporter deletion assays, primer extension, and gel shift assays","pmids":["9817928"],"confidence":"Medium","gaps":["Tissue-specific regulatory elements were not fully delineated","In vivo relevance of individual promoter elements untested"]},{"year":2000,"claim":"Demonstrating that Six5 knockout mice develop cataracts in a dosage-dependent manner directly linked SIX5 deficiency to lens pathology and provided the first phenotypic evidence that SIX5 haploinsufficiency contributes to DM1-associated cataracts.","evidence":"Targeted Six5 disruption in two independent mouse lines with slit-lamp examination and beta-galactosidase reporter","pmids":["10802667","10802668"],"confidence":"High","gaps":["Molecular targets of SIX5 in the lens beyond Na+/K+-ATPase were undefined","Whether cataracts arise from ion transport dysregulation or other mechanisms was uncertain"]},{"year":2000,"claim":"Identifying that SIX5 binds the Na+/K+-ATPase alpha-1 ARE element via its homeodomain and that Six5-deficient lenses have elevated Na+/K+-ATPase alpha-1 protein established a direct transcriptional target and a mechanism (ion homeostasis disruption) for the cataract phenotype.","evidence":"EMSA with recombinant GST-fusion proteins and Western blot/RT-PCR in Six5 knockout lens tissue","pmids":["10756185","10802668"],"confidence":"Medium","gaps":["Whether SIX5 directly represses or indirectly modulates Na+/K+-ATPase transcription was not resolved","No ChIP confirmation of in vivo promoter occupancy was provided"]},{"year":2001,"claim":"Showing that CTG expansion reduces SIX5 mRNA in a tissue-specific manner (greatest in muscle and liver) via chromatin compaction, while distinguishing SIX5 from DMPK by demonstrating that Six5 deficiency does not cause Na+ channel gating abnormalities, refined which DM1 features are attributable to SIX5 loss versus DMPK loss.","evidence":"Somatic cell hybrid chromatin sensitivity assay, multiplex RT-PCR, cell-attached patch clamp in Six5+/- and Dmpk+/- mice","pmids":["11592825","11526199"],"confidence":"Medium","gaps":["Tissue-specific transcriptional targets of SIX5 in muscle were not identified","Why reduction varies across tissues remained unexplained"]},{"year":2002,"claim":"Identifying Igfbp5 as a direct SIX5 transcriptional target and demonstrating cardiac conduction defects in Six5 heterozygotes expanded the known downstream targets and phenotypic consequences of SIX5 haploinsufficiency beyond the lens.","evidence":"VP16-Six5 overexpression with cDNA array profiling, RT-PCR in Six5-KO fibroblasts, in vivo cardiac electrophysiology in Six5+/- mice","pmids":["11978764","12397222"],"confidence":"Medium","gaps":["Whether Igfbp5 mediates any DM1 phenotype was untested","Whether cardiac conduction delay reflects direct transcriptional targets or secondary effects was unknown"]},{"year":2004,"claim":"Demonstrating that Six5-null males are infertile with spermatogenic apoptosis and reduced c-Kit, together with conservation of gonadal and mesodermal functions in the Drosophila ortholog D-Six4 and rescue of C. elegans unc-39 by human SIX5, established SIX5 as an evolutionarily conserved regulator of mesodermal/gonadal development.","evidence":"Six5 KO mouse testicular histology and molecular analysis, Drosophila D-Six4 mutant characterization, C. elegans unc-39 rescue with human SIX5","pmids":["15163633","11470409","15282156"],"confidence":"Medium","gaps":["Direct transcriptional targets of SIX5 in spermatogenesis beyond c-Kit were not identified","Whether SIX5 testicular function requires EYA cofactors was unknown"]},{"year":2007,"claim":"Demonstrating that SIX5 physically interacts with EYA1 and that BOR-associated SIX5 mutations disrupt this interaction and abolish transcriptional activation identified the EYA1-SIX5 partnership as a functional transcriptional unit and linked SIX5 to branchio-oto-renal syndrome pathophysiology.","evidence":"Co-immunoprecipitation, transcriptional activation assays with mutant SIX5 constructs, mutation screening in BOR patients","pmids":["17357085"],"confidence":"Medium","gaps":["Genome-wide targets of the EYA1-SIX5 complex were not mapped","Whether other EYA family members are redundant cofactors for SIX5 in specific tissues was not addressed"]},{"year":2022,"claim":"Identifying that SIX5 assembles with EYA3 and p300 to activate EGFR/MMP/VEGFD transcription in colorectal cancer, and separately activates LINC01468 in lung adenocarcinoma, revealed SIX5 as a transcriptional activator co-opted in cancer and expanded the repertoire of SIX5 cofactor complexes.","evidence":"Co-IP, mass spectrometry, ChIP, luciferase reporter, xenograft models in colorectal and lung cancer cells","pmids":["35957720","35387981"],"confidence":"Medium","gaps":["Whether SIX5 functions as an oncogene or context-dependent factor in normal versus cancerous tissue is unresolved","Structural basis for SIX5-EYA3-p300 complex assembly is lacking"]},{"year":null,"claim":"Key open questions include the genome-wide binding landscape of SIX5 in primary human tissues, the structural basis of SIX5-EYA complex selectivity, and whether therapeutic restoration of SIX5 levels can ameliorate DM1 features.","evidence":"","pmids":[],"confidence":"Low","gaps":["No genome-wide ChIP-seq or CUT&RUN map for SIX5 in primary tissues exists","No structural model of SIX5 alone or in complex with EYA cofactors has been determined","No therapeutic strategy to restore SIX5 dosage has been tested in DM1 models"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[3,13,14,16]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[5,6,13,14,16]}],"localization":[{"term_id":"GO:0005654","term_label":"nucleoplasm","supporting_discovery_ids":[10]}],"pathway":[{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[5,6,13,14,16]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,7,11,12]}],"complexes":["EYA1-SIX5","EYA3-SIX5-p300"],"partners":["EYA1","EYA3","EP300"],"other_free_text":[]},"mechanistic_narrative":"SIX5 is a homeodomain transcription factor that functions as a transcriptional activator in developmental and homeostatic programs of the lens, heart, testis, and skeletal muscle. SIX5 binds DNA through its homeodomain — notably the Na+/K+-ATPase ARE element — and partners with the EYA1 and EYA3/p300 coactivator complexes to drive expression of target genes including Igfbp5, EGFR, MMPs, EXO1, and c-Kit [PMID:10756185, PMID:17357085, PMID:35957720, PMID:11978764, PMID:40946999]. CTG trinucleotide repeat expansion at the DM1 locus eliminates an upstream enhancer and reduces SIX5 expression in cis, and dosage-dependent loss of SIX5 in mouse models causes cataracts, cardiac conduction defects, and male infertility, implicating SIX5 haploinsufficiency as a contributor to multiple features of myotonic dystrophy type 1 [PMID:9241282, PMID:10802667, PMID:12397222, PMID:15163633]. The developmental requirement for SIX5 in mesodermal and ectodermal cell differentiation is evolutionarily conserved, as human SIX5 rescues the C. elegans ortholog unc-39 [PMID:15282156]."},"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":"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":"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":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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mutation","url":"https://pubmed.ncbi.nlm.nih.gov/21280147","citation_count":78,"is_preprint":false},{"pmid":"29481865","id":"PMC_29481865","title":"The Fusarium oxysporum Avr2-Six5 Effector Pair Alters Plasmodesmatal Exclusion Selectivity to Facilitate Cell-to-Cell Movement of Avr2.","date":"2018","source":"Molecular plant","url":"https://pubmed.ncbi.nlm.nih.gov/29481865","citation_count":77,"is_preprint":false},{"pmid":"9949207","id":"PMC_9949207","title":"Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy.","date":"1999","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/9949207","citation_count":63,"is_preprint":false},{"pmid":"11470409","id":"PMC_11470409","title":"Drosophila homolog of the myotonic dystrophy-associated gene, SIX5, is required for muscle and gonad development.","date":"2001","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/11470409","citation_count":45,"is_preprint":false},{"pmid":"15163633","id":"PMC_15163633","title":"Six5 is required for spermatogenic cell survival and spermiogenesis.","date":"2004","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/15163633","citation_count":40,"is_preprint":false},{"pmid":"23840632","id":"PMC_23840632","title":"Mutational analysis of EYA1, SIX1 and SIX5 genes and strategies for management of hearing loss in patients with BOR/BO syndrome.","date":"2013","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/23840632","citation_count":34,"is_preprint":false},{"pmid":"12397222","id":"PMC_12397222","title":"Characterization of cardiac conduction system abnormalities in mice with targeted disruption of Six5 gene.","date":"2002","source":"Journal of interventional cardiac electrophysiology : an international journal of arrhythmias and pacing","url":"https://pubmed.ncbi.nlm.nih.gov/12397222","citation_count":33,"is_preprint":false},{"pmid":"11592825","id":"PMC_11592825","title":"Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy.","date":"2001","source":"Molecular genetics and metabolism","url":"https://pubmed.ncbi.nlm.nih.gov/11592825","citation_count":33,"is_preprint":false},{"pmid":"10332033","id":"PMC_10332033","title":"Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression.","date":"1999","source":"Human molecular genetics","url":"https://pubmed.ncbi.nlm.nih.gov/10332033","citation_count":30,"is_preprint":false},{"pmid":"22447252","id":"PMC_22447252","title":"Mutation screening of the EYA1, SIX1, and SIX5 genes in an East Asian cohort with branchio-oto-renal syndrome.","date":"2012","source":"The 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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":15,"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":"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":"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":2,"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":17870,"output_tokens":3966,"usd":0.05655},"stage2":{"model":"claude-opus-4-6","input_tokens":7417,"output_tokens":2736,"usd":0.158227},"total_usd":0.214777,"stage1_batch_id":"msgbatch_011rkcQdVV1RaFq9XhcG6df6","stage2_batch_id":"msgbatch_01RzgrJafdF4EWHhj5mNi6dh","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 enhancer element upstream of SIX5 (DMAHP), reducing steady-state SIX5 transcript levels 2- to 4-fold overall and greatly on the expanded allele specifically, demonstrating that repeat expansion suppresses SIX5 expression in cis via chromatin-level repression.\",\n      \"method\": \"Allele-specific RT-PCR, DNase I hypersensitivity assay, enhancer reporter assay\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — replicated independently by two labs in the same issue with multiple orthogonal methods\",\n      \"pmids\": [\"9241282\", \"9241283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Homozygous and heterozygous targeted deletion of Six5 in mice causes lenticular opacities (cataracts) in a dosage-dependent manner, establishing a direct role for SIX5 in lens homeostasis; Six5-null mice showed beta-galactosidase reporter expression in developing lens confirming expression there.\",\n      \"method\": \"Targeted gene disruption (knockout mouse), beta-galactosidase reporter, slit-lamp examination\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — independently replicated by two separate labs with clean KO/heterozygous models and defined phenotypic readout\",\n      \"pmids\": [\"10802667\", \"10802668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"Heterozygous and homozygous Six5-deficient mice show increased steady-state levels of the Na+/K+-ATPase alpha-1 subunit and decreased Dm15 (DMPK) mRNA, indicating that SIX5 normally regulates ion transporter gene expression in the lens and that altered ion homeostasis contributes to cataract formation.\",\n      \"method\": \"Western blot, RT-PCR in Six5 knockout mouse tissues\",\n      \"journal\": \"Nature genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — single lab but two orthogonal molecular readouts in KO model\",\n      \"pmids\": [\"10802668\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2000,\n      \"finding\": \"SIX5 protein binds specifically to the ARE (Na+/K+-ATPase alpha-1 subunit regulatory element) via its homeodomain, forming at least one specific complex and a second dimer complex; no binding was detected to putative DMPK promoter sites, defining the DNA-binding specificity of SIX5.\",\n      \"method\": \"Gel retardation (EMSA) with GST-fusion recombinant proteins (homeodomain alone, SIX domain alone, and combined)\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — in vitro biochemical reconstitution with purified recombinant proteins; single lab\",\n      \"pmids\": [\"10756185\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"The SIX5 promoter contains multiple positive regulatory elements that are Sp1/Sp3 binding sites and at least one novel negative regulatory element; three major transcription initiation sites were identified with developmental-stage-specific utilization.\",\n      \"method\": \"Promoter-reporter deletion assays, transcription initiation site mapping (primer extension/S1 nuclease), gel shift assays for Sp1/Sp3\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1-2 — multiple promoter functional assays with deletion analysis; single lab\",\n      \"pmids\": [\"9817928\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Overexpression of constitutively active VP16-Six5 in P19 cells followed by cDNA array profiling identified Igfbp5 as a direct transcriptional target of Six5; Igfbp5 expression was reduced in Six5-deficient mouse fibroblasts and its response to MyoD-induced muscle conversion was altered in DM1 patient cells.\",\n      \"method\": \"Adenovirus-mediated VP16-Six5 overexpression, cDNA array, RT-PCR in Six5-KO fibroblasts, promoter-reporter assay\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (OE + KO + reporter) in a single lab\",\n      \"pmids\": [\"11978764\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SIX5 directly interacts with EYA1 in humans (conserved from C. elegans unc-39/eya-1); BOR-associated missense mutations in SIX5 disrupt EYA1-SIX5 binding and abolish the ability of SIX5 or the EYA1-SIX5 complex to activate transcription, identifying SIX5 as a transcriptional partner of EYA1.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional activation assays with mutant SIX5 constructs, mutation screening\",\n      \"journal\": \"American journal of human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — functional mutagenesis + Co-IP + transcriptional assay; single lab\",\n      \"pmids\": [\"17357085\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"Six5-null male mice are infertile and display progressive testicular atrophy, spermatogenic cell apoptosis, spermiogenesis failure, Leydig cell hyperproliferation, and reduced steady-state c-Kit levels in the testis, indicating SIX5 is required for spermatogenic cell survival partly through maintaining c-Kit expression.\",\n      \"method\": \"Targeted knockout mouse analysis, histology, flow cytometry, RT-PCR/Western blot for c-Kit and hormones\",\n      \"journal\": \"Human molecular genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with multiple molecular readouts; single lab\",\n      \"pmids\": [\"15163633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Six5 heterozygous mice show prolonged QRS duration, delayed infra-Hisian conduction, and enlarged left ventricular end-diastolic dimension compared to wild-type, establishing a partial contribution of Six5 loss to cardiac conduction abnormalities.\",\n      \"method\": \"In vivo electrophysiology (EP study), echocardiography, exercise tolerance testing in Six5+/- mice\",\n      \"journal\": \"Journal of interventional cardiac electrophysiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean heterozygous KO with defined electrophysiological phenotype; single lab\",\n      \"pmids\": [\"12397222\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"Six5+/- mice do not exhibit the late Na+ channel burst activity seen in Dmpk+/- muscle, demonstrating that Six5 deficiency does not contribute to the Na channel gating abnormality in skeletal muscle and distinguishing Six5 from DMPK in this aspect of DM pathophysiology.\",\n      \"method\": \"Cell-attached patch clamp recordings in skeletal muscle from Six5+/- and Dmpk+/- mice\",\n      \"journal\": \"Physiological genomics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 — direct electrophysiology in KO model with appropriate comparators; single lab\",\n      \"pmids\": [\"11526199\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Endogenous SIX5 protein localizes to the nucleoplasm with a granular distribution in HeLa cells and migrates at ~100 kDa on SDS-PAGE; only the full-length isoform was detectable, while a putative shorter splice isoform was not expressed at detectable levels.\",\n      \"method\": \"Monoclonal antibody panel (18 mAbs), Western blot, immunofluorescence/immunolocalization in HeLa cells and fetal tissues\",\n      \"journal\": \"Journal of cellular biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct protein localization with validated antibody panel; single lab\",\n      \"pmids\": [\"15962300\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"The C. elegans SIX5 ortholog UNC-39 is required for migration and differentiation of mesodermal and ectodermal cells including muscles and neurons; human SIX5 functionally complements unc-39 mutants, confirming orthology and conserved transcription factor function.\",\n      \"method\": \"Genetic mutant analysis in C. elegans, rescue experiments with human SIX5\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — genetic epistasis and cross-species functional rescue; single lab\",\n      \"pmids\": [\"15282156\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"The Drosophila SIX5 ortholog D-Six4 is required for myoblast fusion (muscle development) and for the mesodermal component of gonad development; adult males with D-Six4 mutations show testicular reduction, mirroring DM1 phenotypes.\",\n      \"method\": \"Drosophila mutant analysis, histology, genetic characterization\",\n      \"journal\": \"Current biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean mutant phenotype with defined cellular readout in ortholog; single lab\",\n      \"pmids\": [\"11470409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIX5 assembles with hypoxia-induced EYA3 and histone acetyltransferase p300 to form a transcriptional complex in colorectal cancer cells; this EYA3-SIX5-p300 complex directly binds promoters of EGFR, VEGFD, and multiple MMPs to activate their transcription, promoting tumor growth.\",\n      \"method\": \"Co-immunoprecipitation, mass spectrometry, chromatin immunoprecipitation (ChIP), transcriptional reporter assays, tumor xenograft model\",\n      \"journal\": \"Annals of translational medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, MS, ChIP) in a single lab\",\n      \"pmids\": [\"35957720\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"SIX5 transcriptionally activates LINC01468 lncRNA by binding its promoter; SIX5-driven LINC01468 then recruits SERBP1 to stabilize SERPINE1 mRNA and interacts with USP5 to facilitate PAI1 deubiquitylation, promoting lung adenocarcinoma cell proliferation, migration, and invasion.\",\n      \"method\": \"ChIP, luciferase reporter assay, RNA co-immunoprecipitation, RIP, knockdown/overexpression functional assays, xenograft model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — ChIP + reporter + RIP with functional rescue; single lab\",\n      \"pmids\": [\"35387981\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Reduced dosage of Six4 and Six5 (double heterozygous knockout) in mdx dystrophic mice improves muscle regeneration, reduces serum CK/LDH, and extends lifespan by 33.8%, with enhanced MYOD1, MYOG, and SIX1 expression in regenerating myofibers, demonstrating that SIX5 (together with SIX4) modulates satellite cell-driven muscle regeneration.\",\n      \"method\": \"Double heterozygous knockout in mdx mice, grip strength, treadmill testing, serum enzyme assays, immunostaining for myogenic markers\",\n      \"journal\": \"Development, growth & differentiation\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — confounded by concurrent Six4 reduction; single lab, no separation of Six5-specific mechanism\",\n      \"pmids\": [\"27224259\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SIX5 directly binds the EXO1 promoter and activates its transcription in glioblastoma cells, as demonstrated by ChIP and dual-luciferase reporter assays; SIX5 knockdown suppresses EXO1 expression and GBM cell growth, which is partially rescued by EXO1 overexpression.\",\n      \"method\": \"ChIP, dual-luciferase reporter assay, lentiviral knockdown, xenograft model\",\n      \"journal\": \"Brain research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 method but zero citations, single lab, no independent replication\",\n      \"pmids\": [\"40946999\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"CTG repeat expansion at the DM locus decreases PvuII chromatin sensitivity adjacent to the repeat and partially reduces SIX5 mRNA from expanded alleles in a tissue-specific manner (greatest reduction in muscle and liver), while DMWD expression is not affected, demonstrating allele- and tissue-specific regulation of SIX5 by chromatin structure changes.\",\n      \"method\": \"Somatic cell hybrid PCR-based chromatin sensitivity assay, semiquantitative multiplex RT-PCR\",\n      \"journal\": \"Molecular genetics and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — allele-specific chromatin and expression assays in defined cell hybrids; single lab\",\n      \"pmids\": [\"11592825\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SIX5 is a homeodomain transcription factor whose expression is suppressed in cis by CTG repeat expansion at the DM1 locus through chromatin compaction that eliminates an upstream enhancer; the protein binds DNA via its homeodomain (notably the Na+/K+-ATPase ARE element), partners with EYA1 and EYA3/p300 complexes to activate target genes (including Igfbp5, EGFR, MMPs, and EXO1), and its dosage-dependent loss causes cataracts, cardiac conduction delays, and spermatogenic failure in mouse models, implicating SIX5 haploinsufficiency as a contributor to multiple features of myotonic dystrophy type 1.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SIX5 is a homeodomain transcription factor that functions as a transcriptional activator in developmental and homeostatic programs of the lens, heart, testis, and skeletal muscle. SIX5 binds DNA through its homeodomain — notably the Na+/K+-ATPase ARE element — and partners with the EYA1 and EYA3/p300 coactivator complexes to drive expression of target genes including Igfbp5, EGFR, MMPs, EXO1, and c-Kit [PMID:10756185, PMID:17357085, PMID:35957720, PMID:11978764, PMID:40946999]. CTG trinucleotide repeat expansion at the DM1 locus eliminates an upstream enhancer and reduces SIX5 expression in cis, and dosage-dependent loss of SIX5 in mouse models causes cataracts, cardiac conduction defects, and male infertility, implicating SIX5 haploinsufficiency as a contributor to multiple features of myotonic dystrophy type 1 [PMID:9241282, PMID:10802667, PMID:12397222, PMID:15163633]. The developmental requirement for SIX5 in mesodermal and ectodermal cell differentiation is evolutionarily conserved, as human SIX5 rescues the C. elegans ortholog unc-39 [PMID:15282156].\",\n  \"teleology\": [\n    {\n      \"year\": 1997,\n      \"claim\": \"Establishing that CTG repeat expansion at the DM1 locus suppresses SIX5 expression in cis via chromatin-level loss of an enhancer answered the question of whether repeat expansions affect neighboring gene regulation, making SIX5 haploinsufficiency a candidate contributor to DM1 pathology.\",\n      \"evidence\": \"Allele-specific RT-PCR, DNase I hypersensitivity, and enhancer reporter assays in DM1 patient cells by two independent labs\",\n      \"pmids\": [\"9241282\", \"9241283\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether reduced SIX5 causes any specific DM1 phenotype was untested\", \"The degree of reduction in different tissues was unknown\", \"Whether chromatin compaction extends to other loci was not addressed\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Mapping the SIX5 promoter architecture — including Sp1/Sp3 sites and developmental-stage-specific initiation sites — established the regulatory framework for understanding how SIX5 transcription is controlled.\",\n      \"evidence\": \"Promoter-reporter deletion assays, primer extension, and gel shift assays\",\n      \"pmids\": [\"9817928\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific regulatory elements were not fully delineated\", \"In vivo relevance of individual promoter elements untested\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Demonstrating that Six5 knockout mice develop cataracts in a dosage-dependent manner directly linked SIX5 deficiency to lens pathology and provided the first phenotypic evidence that SIX5 haploinsufficiency contributes to DM1-associated cataracts.\",\n      \"evidence\": \"Targeted Six5 disruption in two independent mouse lines with slit-lamp examination and beta-galactosidase reporter\",\n      \"pmids\": [\"10802667\", \"10802668\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular targets of SIX5 in the lens beyond Na+/K+-ATPase were undefined\", \"Whether cataracts arise from ion transport dysregulation or other mechanisms was uncertain\"]\n    },\n    {\n      \"year\": 2000,\n      \"claim\": \"Identifying that SIX5 binds the Na+/K+-ATPase alpha-1 ARE element via its homeodomain and that Six5-deficient lenses have elevated Na+/K+-ATPase alpha-1 protein established a direct transcriptional target and a mechanism (ion homeostasis disruption) for the cataract phenotype.\",\n      \"evidence\": \"EMSA with recombinant GST-fusion proteins and Western blot/RT-PCR in Six5 knockout lens tissue\",\n      \"pmids\": [\"10756185\", \"10802668\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SIX5 directly represses or indirectly modulates Na+/K+-ATPase transcription was not resolved\", \"No ChIP confirmation of in vivo promoter occupancy was provided\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Showing that CTG expansion reduces SIX5 mRNA in a tissue-specific manner (greatest in muscle and liver) via chromatin compaction, while distinguishing SIX5 from DMPK by demonstrating that Six5 deficiency does not cause Na+ channel gating abnormalities, refined which DM1 features are attributable to SIX5 loss versus DMPK loss.\",\n      \"evidence\": \"Somatic cell hybrid chromatin sensitivity assay, multiplex RT-PCR, cell-attached patch clamp in Six5+/- and Dmpk+/- mice\",\n      \"pmids\": [\"11592825\", \"11526199\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific transcriptional targets of SIX5 in muscle were not identified\", \"Why reduction varies across tissues remained unexplained\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identifying Igfbp5 as a direct SIX5 transcriptional target and demonstrating cardiac conduction defects in Six5 heterozygotes expanded the known downstream targets and phenotypic consequences of SIX5 haploinsufficiency beyond the lens.\",\n      \"evidence\": \"VP16-Six5 overexpression with cDNA array profiling, RT-PCR in Six5-KO fibroblasts, in vivo cardiac electrophysiology in Six5+/- mice\",\n      \"pmids\": [\"11978764\", \"12397222\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Igfbp5 mediates any DM1 phenotype was untested\", \"Whether cardiac conduction delay reflects direct transcriptional targets or secondary effects was unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Demonstrating that Six5-null males are infertile with spermatogenic apoptosis and reduced c-Kit, together with conservation of gonadal and mesodermal functions in the Drosophila ortholog D-Six4 and rescue of C. elegans unc-39 by human SIX5, established SIX5 as an evolutionarily conserved regulator of mesodermal/gonadal development.\",\n      \"evidence\": \"Six5 KO mouse testicular histology and molecular analysis, Drosophila D-Six4 mutant characterization, C. elegans unc-39 rescue with human SIX5\",\n      \"pmids\": [\"15163633\", \"11470409\", \"15282156\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets of SIX5 in spermatogenesis beyond c-Kit were not identified\", \"Whether SIX5 testicular function requires EYA cofactors was unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Demonstrating that SIX5 physically interacts with EYA1 and that BOR-associated SIX5 mutations disrupt this interaction and abolish transcriptional activation identified the EYA1-SIX5 partnership as a functional transcriptional unit and linked SIX5 to branchio-oto-renal syndrome pathophysiology.\",\n      \"evidence\": \"Co-immunoprecipitation, transcriptional activation assays with mutant SIX5 constructs, mutation screening in BOR patients\",\n      \"pmids\": [\"17357085\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Genome-wide targets of the EYA1-SIX5 complex were not mapped\", \"Whether other EYA family members are redundant cofactors for SIX5 in specific tissues was not addressed\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identifying that SIX5 assembles with EYA3 and p300 to activate EGFR/MMP/VEGFD transcription in colorectal cancer, and separately activates LINC01468 in lung adenocarcinoma, revealed SIX5 as a transcriptional activator co-opted in cancer and expanded the repertoire of SIX5 cofactor complexes.\",\n      \"evidence\": \"Co-IP, mass spectrometry, ChIP, luciferase reporter, xenograft models in colorectal and lung cancer cells\",\n      \"pmids\": [\"35957720\", \"35387981\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether SIX5 functions as an oncogene or context-dependent factor in normal versus cancerous tissue is unresolved\", \"Structural basis for SIX5-EYA3-p300 complex assembly is lacking\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include the genome-wide binding landscape of SIX5 in primary human tissues, the structural basis of SIX5-EYA complex selectivity, and whether therapeutic restoration of SIX5 levels can ameliorate DM1 features.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No genome-wide ChIP-seq or CUT&RUN map for SIX5 in primary tissues exists\", \"No structural model of SIX5 alone or in complex with EYA cofactors has been determined\", \"No therapeutic strategy to restore SIX5 dosage has been tested in DM1 models\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [3, 13, 14, 16]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [5, 6, 13, 14, 16]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005654\", \"supporting_discovery_ids\": [10]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [5, 6, 13, 14, 16]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 7, 11, 12]}\n    ],\n    \"complexes\": [\n      \"EYA1-SIX5\",\n      \"EYA3-SIX5-p300\"\n    ],\n    \"partners\": [\n      \"EYA1\",\n      \"EYA3\",\n      \"EP300\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}