{"gene":"SIX4","run_date":"2026-06-10T07:46:32","timeline":{"discoveries":[{"year":1996,"finding":"SIX4 (AREC3) protein contains a homeodomain required for specific DNA binding to the ARE (Na,K-ATPase alpha1 subunit gene regulatory element), and a transactivation domain in the C-terminal region identified by GAL4-fusion reporter assays. The protein localizes to both nucleus and cytoplasm of myoblast C2C12 cells, with the 115 kDa form increased in cytoplasmic extract and the 67 kDa form increased in both nuclear and cytoplasmic extracts during muscle differentiation.","method":"Reporter gene assays with GAL4-AREC3 fusion constructs, immunohistochemistry, Western blot analysis of nuclear and cytoplasmic fractions during differentiation","journal":"Nucleic acids research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct DNA-binding assay, transactivation assay, and subcellular fractionation in single lab with multiple orthogonal methods","pmids":["8628654"],"is_preprint":false},{"year":2004,"finding":"SIX4 is the transcriptional regulatory element X (Trex)-binding factor (TrexBF) in the Muscle Creatine Kinase (MCK) enhancer in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle. SIX4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. In adult mouse heart, Six5 (not Six4) is the major TrexBF.","method":"Quantitative proteomics of oligonucleotide-affinity-purified proteins, gel shift assays, Six-specific antisera, cotransfection reporter assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — quantitative proteomics identification combined with gel-shift confirmation with specific antisera and functional reporter assays; multiple orthogonal methods in one rigorous study","pmids":["14966291"],"is_preprint":false},{"year":2005,"finding":"In Six1/Six4 double knockout mice, Pax3 expression is impaired in limb bud somitic cells, blocking myogenic cell delamination and migration; within the myotome, myogenin, Myod1 and Mrf4 expression are abolished, and Myf5 becomes restricted to the caudal somite region, placing Six1 and Six4 upstream of these myogenic regulatory factors.","method":"Double knockout mouse genetics, in situ hybridization, immunohistochemistry for myogenic markers","journal":"Development (Cambridge, England)","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in double-KO mice with multiple molecular marker readouts, replicated across developmental contexts","pmids":["15788460"],"is_preprint":false},{"year":2005,"finding":"Six1 and Six4 differentially regulate a set of target genes. The promoter of Slc12a2 (NKCC1) contains multiple Six1-binding sites and one common binding site for both Six1 and Six4 by gel-retardation assay, indicating distinct DNA-binding specificities. In vivo, Slc12a2 expression is reduced in the dorsal root ganglia of Six1/Six4 double-null mice.","method":"Gel-retardation assays (EMSA), target gene screening, in situ hybridization in double-KO mice","journal":"The FEBS journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — EMSA for direct DNA binding plus in vivo validation in KO mice, single lab, two orthogonal methods","pmids":["15955062"],"is_preprint":false},{"year":2006,"finding":"In Drosophila, D-Six4 is required for development of non-dorsal mesodermal cell types (fat body, somatic gonadal cells, specific somatic muscles). Misexpression analysis shows D-Six4 and its cofactor Eyes absent are sufficient to impose these fates on other mesodermal cells. Tinman (tin) function is required for full D-six4 expression at stage 9.","method":"Loss-of-function genetics, misexpression analysis, epistasis between tin and D-six4","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function and gain-of-function with cellular phenotype readouts and epistasis, single lab","pmids":["16595131"],"is_preprint":false},{"year":2007,"finding":"Six1 and Six4 cooperate in the metanephric mesenchyme to regulate Gdnf expression; Six1/Six4 double-null mice lack ureteric bud formation and show complete absence of Pax2, Pax8, and Gdnf expression in metanephric mesenchyme, whereas Six1 deficiency alone only partially reduces these markers.","method":"Double knockout mouse genetics, in situ hybridization, molecular marker analysis","journal":"Mechanisms of development","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis in double-KO with specific molecular readouts; replicated in multiple studies","pmids":["17300925"],"is_preprint":false},{"year":2007,"finding":"In Drosophila, Six4 is required in somatic gonadal precursors (SGPs) for expression of Hmgcr (HMG-CoA reductase), which is necessary for attraction of primordial germ cells (PGCs) to SGPs. Six4 affects male-specific SGP migration by a different (Hmgcr-independent) pathway. SGPs also fail to coalesce into unified gonads without functional Six4.","method":"Live time-lapse fluorescence imaging of wild-type and mutant embryos, in vivo genetic analysis, epistasis with Hmgcr","journal":"BMC developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — live imaging combined with genetic epistasis in single lab","pmids":["17517128"],"is_preprint":false},{"year":2008,"finding":"Six1 and Six4 are required synergistically for olfactory placode formation; embryos lacking both Six1 and Six4 fail to form the olfactory placode despite normal specification of the preplacodal region (marked by Eya2). Six1 and Six4 act at the top of events controlling olfactory placode specification and patterning through Fgf and Bmp signaling pathways.","method":"Double knockout mouse genetics, in situ hybridization, molecular marker analysis (Eya2, Mash1, Sox2)","journal":"Developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in double-KO mice with multiple molecular marker readouts, single lab","pmids":["19027001"],"is_preprint":false},{"year":2013,"finding":"Six1 and Six4 homeoproteins are required together for male sex determination: double loss leads to male-to-female sex reversal in XY mice due to failure of Sry activation. Forced Sry transgene expression rescues testicular development but not precursor cell growth. Two downstream pathways are identified: Six1/Six4 regulate Fog2 (Zfpm2) to induce Sry expression, and regulate Nr5a1 (Ad4BP/Sf1) to control gonadal precursor formation.","method":"Double knockout mouse genetics, transgenic rescue with Sry, in situ hybridization and gene expression analysis of Fog2 and Nr5a1","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic epistasis with transgenic rescue plus identification of two distinct downstream targets, multiple orthogonal methods","pmids":["23987514"],"is_preprint":false},{"year":2015,"finding":"During adult skeletal muscle regeneration, Six4 co-occupies a core set of muscle gene loci genome-wide together with MyoD and the histone H3K27me3 demethylase Utx. Six4 and MyoD cooperation is associated with removal of the H3K27me3 repressive chromatin mark. In vivo RNAi of Six4 reveals an uncompensated function in muscle regeneration.","method":"ChIP-seq (genome-wide occupancy of Six4, MyoD, Utx), in vivo RNAi knockdown with muscle regeneration phenotype","journal":"FASEB journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — genome-wide ChIP-seq with multiple factors plus in vivo functional validation, multiple orthogonal methods","pmids":["26229056"],"is_preprint":false},{"year":2016,"finding":"Six1 or Six4 are required for MyoD-mediated reprogramming of mouse embryonic fibroblasts toward myogenesis. Genome-wide analysis identified >700 genes co-regulated by Six and MyoD, with MyoD ChIP-seq data showing co-localization of MyoD and MEF3 (Six-binding) sites at >1000 genomic regions. The Six/MyoD synergistic activation involves a feedforward mechanism recruiting Mef2, Pbx-Meis, and EBF co-factors.","method":"Microarray expression profiling, MyoD ChIP-seq, genome-wide MEF3 site search, luciferase reporter assays for individual CRMs, MEF reprogramming assay","journal":"Nucleic acids research","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — genome-wide ChIP-seq, microarray, and functional reporter assays with multiple orthogonal methods","pmids":["27302134"],"is_preprint":false},{"year":2019,"finding":"SIX4 directly binds to the promoters of YAP1 and MET (c-MET) to transactivate their expression in hepatocellular carcinoma cells. HGF upregulates SIX4 expression through the ERK/NF-κB pathway, forming a positive feedback loop (HGF→SIX4→c-MET). Knockdown of both YAP1 and c-MET inhibits SIX4-mediated HCC metastasis.","method":"ChIP assay (SIX4 binding to YAP1 and MET promoters), luciferase reporter assays, rescue experiments with YAP1/c-MET knockdown and overexpression, ERK/NF-κB pathway inhibition","journal":"Oncogene","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and reporter assays for direct target binding, plus genetic rescue; single lab","pmids":["33046796"],"is_preprint":false},{"year":2019,"finding":"SIX4 increases expression of VEGF-A by coordinating with HIF-1α, and upregulates HIF-1α expression in an Akt-dependent manner, thereby promoting tumor angiogenesis in colorectal cancer cells.","method":"Overexpression and knockdown of SIX4, in vitro angiogenesis assay, Western blot for HIF-1α/Akt/VEGF-A, in vivo tumor growth assay","journal":"Experimental cell research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single lab, Western blot and overexpression/knockdown without direct binding evidence for SIX4-HIF-1α interaction","pmids":["31301290"],"is_preprint":false},{"year":2019,"finding":"SIX4 directly interacts with STAT3 protein and promotes phosphorylated STAT3 nuclear translocation, thereby inducing EMT program activation (via Snai1 induction) and breast cancer metastasis.","method":"Co-immunoprecipitation (SIX4-STAT3 interaction), knockdown/overexpression with migration/invasion assays, in vivo lung metastasis assay","journal":"American journal of cancer research","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP demonstrating direct interaction plus functional in vivo validation, single lab","pmids":["32064163"],"is_preprint":false},{"year":2019,"finding":"SIX4 chromatin co-occupancy with Pax3 is demonstrated genome-wide in mesodermal cells. Pax3 cooperates with Six4 (and Tead2) factors and involves chromatin remodeling (increased chromatin accessibility at bound elements) to activate the skeletal myogenic lineage.","method":"ATAC-seq (chromatin accessibility), ChIP-seq (Pax3 binding), transcriptome profiling in Pax3-induced ESCs and Pax3-null embryos","journal":"PLoS biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-wide chromatin and binding data with transcriptome, single lab with multiple orthogonal methods","pmids":["30807574"],"is_preprint":false},{"year":2020,"finding":"In Six1/Six4 double knockout mice, fewer PAX7+ satellite cells occupy their normal position at E18; the remaining mutant PAX7+ cells can divide and contribute to muscle growth but form hypotrophic, non-innervated myofibers after transplantation and retain self-renewal capacity.","method":"Double knockout mouse genetics, immunofluorescence for PAX7+ cell counting, transcriptome analysis, transplantation assay in adult regenerating muscle","journal":"Development (Cambridge, England)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO genetics plus transplantation assay and transcriptome, single lab","pmids":["32591430"],"is_preprint":false},{"year":2021,"finding":"In Drosophila type II neuroblast lineages, Six4 prevents supernumerary type II neuroblasts and premature INP differentiation. Six4 inhibits the expression and activity of PntP1 in immature INPs (imINPs) in part by forming a trimeric complex with Earmuff and PntP1. Six4 also prevents premature differentiation by suppressing ectopic Prospero expression in imINPs.","method":"Loss-of-function genetics (six4 mutants), genetic epistasis with pntP1 and earmuff, complex formation demonstrated","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis with trimeric complex identification, single lab with multiple genetic combinations","pmids":["33556050"],"is_preprint":false},{"year":2023,"finding":"SIX4 directly transcribes STING (cGAS/STING pathway) in colon cancer cells: SIX4 knockout decreases STING mRNA and protein, ectopic SIX4 increases STING expression, and reexpression of SIX4 or STING in SIX4 KO cells reverses the effect. SIX4 depletion attenuates STING activation and downstream signaling, and reduces CD8+ T cell tumor infiltration and anti-PD-1 efficacy in vivo.","method":"CRISPR knockout and ectopic expression of SIX4, qPCR/Western blot for STING levels, STING activation assays (DMXAA/cGAMP), in vivo tumor immunology experiments","journal":"Cancer research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic KO and rescue with functional STING activation readouts and in vivo immune infiltration, single lab","pmids":["37888903"],"is_preprint":false},{"year":2023,"finding":"EYA3 isoforms (regulated by RBFOX2-controlled alternative splicing) interact with SIX4 as a major transcription factor partner during myogenesis. Mass spectrometry-based proteomics and genome-wide transcriptomics identified SIX4 as a primary EYA3-interacting protein that dictates gene expression during muscle cell differentiation.","method":"Mass spectrometry proteomics (EYA3 interactome), genome-wide transcriptomic analysis, myoblast proliferation/differentiation assays","journal":"iScience","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — mass spectrometry identification of SIX4-EYA3 interaction plus transcriptomic functional validation, single lab","pmids":["38026174"],"is_preprint":false},{"year":2023,"finding":"MKRN2 interacts with STAT1 (shown by Co-IP), and MKRN2 regulates SIX4 expression via the EBF2 transcription factor in mouse testis and MEF cells; loss of MKRN2 significantly decreases SIX4 expression.","method":"MKRN2 knockout mouse model, Co-IP (MKRN2-STAT1 interaction), expression analysis of SIX4 in KO testis","journal":"Frontiers in endocrinology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — SIX4 regulation by MKRN2/EBF2 is inferred from expression changes in KO tissue; the Co-IP is for MKRN2-STAT1, not SIX4 directly; single lab, single method for SIX4","pmids":["36967804"],"is_preprint":false},{"year":2024,"finding":"SIX4 is transcriptionally activated by the IL-6/STAT3 signaling pathway in colorectal epithelium, and activated SIX4 binds to c-Jun to transcribe IL-6, forming a positive IL-6/STAT3/SIX4/c-Jun feedback loop that drives intestinal inflammation. SIX4 also binds the DeltaNp63 promoter (but not wild-type p63) to induce tumor stemness signaling.","method":"ChIP assay (SIX4 binding to c-Jun and DeltaNp63 promoters), reporter assays, in vivo DSS/AOM-DSS mouse models, siRNA knockdown","journal":"International journal of biological sciences","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP for direct promoter binding plus in vivo validation in inflammation models, single lab","pmids":["39309424"],"is_preprint":false},{"year":2025,"finding":"In alveolar rhabdomyosarcoma, SIX4 is identified as a key transcription factor that mediates CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes and tumor cell proliferation. The maximal anti-tumor activity of CDK8 inhibitors requires the Mediator kinase module and transcriptional cooperation with the SAGA complex.","method":"Genome-scale CRISPR-Cas9 drug modifier screen, CDK8 knockout and pharmacologic inhibition, transcriptional profiling, in vitro and in vivo tumor models","journal":"bioRxiv","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genome-scale CRISPR screen plus functional validation; preprint, single lab","pmids":["bio_10.1101_2025.07.14.663986"],"is_preprint":true},{"year":2018,"finding":"MyoD binds directly at MyoD and E-box recognition sites in the core promoter region (-522/-193) of bovine SIX4, as demonstrated by EMSA and ChIP; site-directed mutagenesis and siRNA interference confirm that MyoD regulates SIX4 transcription through both direct and indirect mechanisms.","method":"EMSA, chromatin immunoprecipitation (ChIP), luciferase reporter assays with 5'-deletion constructs, site-directed mutation, siRNA knockdown of MyoD","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — multiple direct binding methods (EMSA, ChIP, mutagenesis) in single lab; bovine system","pmids":["29307818"],"is_preprint":false},{"year":2022,"finding":"FOXA1 directly regulates SIX4 transcription in cervical cancer; FOXA1 binds the SIX4 promoter as demonstrated by ChIP and dual-luciferase assay. SIX4 overexpression promotes phosphorylation of PI3K and AKT, activating the PI3K/AKT signaling pathway, and reverses FOXA1 knockdown effects on cell growth and chemoresistance.","method":"ChIP assay, dual-luciferase reporter assay (FOXA1 binding SIX4 promoter), siRNA knockdown, PI3K/AKT Western blot","journal":"Analytical cellular pathology (Amsterdam)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase confirm direct FOXA1-SIX4 promoter interaction; downstream PI3K/AKT by Western blot; single lab","pmids":["35498155"],"is_preprint":false}],"current_model":"SIX4 is a homeodomain transcription factor that binds MEF3/Trex DNA elements (with its Six domain and homeodomain) to directly activate muscle-specific genes (MCK enhancer, Aldolase A, Cardiac troponin C, Slc12a2, YAP1, c-MET, STING, DeltaNp63, IL-6), cooperates genome-wide with MyoD and the H3K27me3 demethylase Utx to activate the skeletal myogenic program, physically interacts with STAT3 and EYA3 as a transcriptional co-regulator, and functions epistatically upstream of Pax3, Myogenin, MyoD, Mrf4, Gdnf, Fog2, and Nr5a1 in skeletal muscle, kidney, and gonadal development, with its transcription in muscle being directly driven by MyoD binding to E-box/MEF3 sites in its own promoter."},"narrative":{"mechanistic_narrative":"SIX4 is a homeodomain transcription factor that binds MEF3/Trex DNA elements through its homeodomain and transactivates muscle-specific target genes via a C-terminal transactivation domain [PMID:8628654, PMID:14966291]. Acting redundantly with its paralog Six1, SIX4 sits at the top of the developmental hierarchy that establishes skeletal muscle, kidney, gonad, and olfactory placode lineages: in Six1/Six4 double-null mice, Pax3 and the myogenic regulatory factors Myogenin, Myod1, and Mrf4 fail to be expressed and myogenic cells fail to delaminate and migrate [PMID:15788460], Gdnf and ureteric bud formation are lost in the metanephric mesenchyme [PMID:17300925], the olfactory placode fails to form [PMID:19027001], and male sex determination fails through loss of Sry activation downstream of Fog2 and Nr5a1 [PMID:23987514]. Mechanistically, SIX4 cooperates genome-wide with MyoD and Pax3, co-occupying muscle gene loci and increasing chromatin accessibility, and its synergy with MyoD is coupled to removal of the repressive H3K27me3 mark by the demethylase Utx [PMID:26229056, PMID:27302134, PMID:30807574]; SIX4 transcription is itself directly driven by MyoD binding E-box/MEF3 sites in the SIX4 promoter, creating a feedforward loop [PMID:29307818]. SIX4 functions with cofactors of the Eyes absent family, interacting directly with EYA3 during myogenesis [PMID:38026174]. Beyond development, SIX4 acts as a pro-tumorigenic and pro-inflammatory transcription factor: it binds the promoters of YAP1 and MET to drive hepatocellular carcinoma metastasis within an HGF feedback loop [PMID:33046796], physically interacts with STAT3 to promote its nuclear translocation and EMT [PMID:32064163], directly transcribes STING to shape anti-tumor immunity [PMID:37888903], and participates in an IL-6/STAT3/SIX4/c-Jun feedback loop while inducing DeltaNp63 in colorectal contexts [PMID:39309424].","teleology":[{"year":1996,"claim":"Establishing that SIX4 is a sequence-specific DNA-binding transactivator answered whether it acts directly on gene regulatory elements rather than as an accessory factor.","evidence":"GAL4-fusion reporter assays, DNA-binding to the Na,K-ATPase ARE element, and subcellular fractionation in C2C12 myoblasts","pmids":["8628654"],"confidence":"Medium","gaps":["Physiological target genes not yet defined","Dual nuclear/cytoplasmic localization and processed forms not mechanistically explained"]},{"year":2004,"claim":"Identifying SIX4 as the Trex/MEF3-binding factor in the MCK enhancer connected its DNA-binding activity to a defined muscle-specific regulatory grammar.","evidence":"Oligonucleotide-affinity proteomics, gel-shift with Six-specific antisera, and reporter assays on MCK, Aldolase A, and Cardiac troponin C elements","pmids":["14966291"],"confidence":"High","gaps":["Distinction of Six4 vs Six5 roles across tissues incomplete","Cofactor requirements for transactivation not resolved"]},{"year":2005,"claim":"Double-knockout genetics placed Six1/Six4 upstream of the core myogenic transcription factors and distinguished their target specificity from Six1.","evidence":"Six1/Six4 double-KO mouse genetics with in situ markers (Pax3, Myogenin, Myod1, Mrf4) and EMSA on the Slc12a2 promoter","pmids":["15788460","15955062"],"confidence":"High","gaps":["Direct vs indirect regulation of each myogenic factor not separated","Redundancy boundaries between Six1 and Six4 unquantified"]},{"year":2006,"claim":"Drosophila genetics established a deeply conserved role for Six4/Eya in mesodermal and gonadal cell-fate specification.","evidence":"Loss- and gain-of-function genetics, misexpression with Eyes absent cofactor, and tin epistasis in fly embryos","pmids":["16595131","17517128"],"confidence":"Medium","gaps":["Direct transcriptional targets in fly not mapped","Relationship to vertebrate target sets unclear"]},{"year":2007,"claim":"Extending the Six1/Six4 requirement to kidney and olfactory placode showed SIX4 operates as a master upstream regulator across multiple organ primordia.","evidence":"Six1/Six4 double-KO mice with marker analysis of Gdnf, Pax2/Pax8 (kidney) and Eya2/Sox2 (olfactory placode)","pmids":["17300925","19027001"],"confidence":"High","gaps":["Whether SIX4 binds these target promoters directly not shown","Signaling integration (Fgf/Bmp) downstream vs upstream unresolved"]},{"year":2013,"claim":"Defining the Fog2 and Nr5a1 branches downstream of Six1/Six4 explained how these factors control both Sry activation and gonadal precursor growth in sex determination.","evidence":"Double-KO mouse genetics with Sry transgenic rescue and expression analysis of Fog2 and Nr5a1","pmids":["23987514"],"confidence":"High","gaps":["Direct SIX4 occupancy at Fog2/Nr5a1 loci not demonstrated","Mechanism limiting rescue of precursor cell growth unknown"]},{"year":2016,"claim":"Genome-wide co-occupancy with MyoD, Pax3, and Utx, plus a self-promoter loop, revealed how SIX4 mechanistically drives the myogenic program through chromatin remodeling and feedforward circuitry.","evidence":"ChIP-seq of Six4/MyoD/Utx and Pax3, ATAC-seq, MEF reprogramming assays, EMSA/ChIP on the SIX4 promoter, and in vivo regeneration RNAi","pmids":["26229056","27302134","30807574","29307818"],"confidence":"High","gaps":["Direct recruitment mechanism of Utx by SIX4 not established","Quantitative contribution of each cofactor (Mef2, Pbx-Meis, EBF) unresolved"]},{"year":2020,"claim":"Satellite cell analysis clarified that SIX4 is required for proper positioning and functional competence of muscle stem cells, beyond initial lineage specification.","evidence":"Six1/Six4 double-KO PAX7+ cell quantification, transcriptome, and transplantation into regenerating muscle","pmids":["32591430"],"confidence":"Medium","gaps":["Transcriptional targets governing satellite cell positioning unmapped","Cell-autonomous vs niche contributions not fully separated"]},{"year":2023,"claim":"Identifying EYA3 as a primary SIX4 partner and a Drosophila trimeric complex defined the cofactor architecture through which SIX4 regulates differentiation.","evidence":"Mass spectrometry of the EYA3 interactome with transcriptomics in myogenesis, and genetic/complex analysis of Six4-Earmuff-PntP1 in fly neuroblasts","pmids":["38026174","33556050"],"confidence":"Medium","gaps":["Structural basis of SIX4-EYA3 interaction unknown","Whether neuroblast and myogenic cofactor logic is shared unclear"]},{"year":2024,"claim":"Multiple cancer studies established SIX4 as a pro-tumorigenic transcription factor acting through STAT3, YAP1/MET, STING, and IL-6/c-Jun circuits.","evidence":"ChIP/reporter assays on YAP1, MET, STING, c-Jun, and DeltaNp63 promoters; Co-IP with STAT3; CRISPR KO/rescue and in vivo tumor and inflammation models","pmids":["33046796","32064163","37888903","39309424"],"confidence":"Medium","gaps":["Whether oncogenic targets share the MEF3 binding grammar of developmental targets unclear","Most cancer findings rest on single-lab studies without reciprocal validation"]},{"year":2025,"claim":"Drug-modifier screening linked SIX4 to CDK8/Mediator-kinase-dependent transcriptional rewiring in rhabdomyosarcoma, connecting its myogenic activity to therapeutic vulnerability.","evidence":"Genome-scale CRISPR screen with CDK8 inhibition and transcriptional profiling (preprint)","pmids":["bio_10.1101_2025.07.14.663986"],"confidence":"Medium","gaps":["Preprint not peer-reviewed","Direct SIX4-Mediator interaction not demonstrated"]},{"year":null,"claim":"How a single MEF3-binding homeodomain factor selects developmental versus oncogenic target gene sets, and which cofactors and chromatin states toggle that selection, remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No structural model of SIX4 on DNA with EYA cofactors","Rules governing context-specific target choice unknown","Direct in vivo binding at most cancer targets not validated"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,1,2,9,10,11,17,20]},{"term_id":"GO:0003677","term_label":"DNA binding","supporting_discovery_ids":[0,1,3,22]}],"localization":[{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[2,5,7,8,9,10,14]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,1,11,17,20]},{"term_id":"R-HSA-1643685","term_label":"Disease","supporting_discovery_ids":[11,13,17,20]}],"complexes":[],"partners":["MYOD","EYA3","STAT3","UTX","PAX3","C-JUN"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q9UIU6","full_name":"Homeobox protein SIX4","aliases":["Sine oculis homeobox homolog 4"],"length_aa":781,"mass_kda":82.9,"function":"Transcriptional regulator which can act as both a transcriptional repressor and activator by binding a DNA sequence on these target genes and is involved in processes like cell differentiation, cell migration and cell survival. Transactivates gene expression by binding a 5'-[CAT]A[CT][CT][CTG]GA[GAT]-3' motif present in the Trex site and a 5'-TCA[AG][AG]TTNC-3' motif present in the MEF3 site of the muscle-specific genes enhancer. Acts cooperatively with EYA proteins to transactivate their target genes through interaction and nuclear translocation of EYA protein. Acts synergistically with SIX1 to regulate target genes involved in formation of various organs, including muscle, kidney, gonad, ganglia, olfactory epithelium and cranial skeleton. Plays a role in several important steps of muscle development. Controls the genesis of hypaxial myogenic progenitors in the dermomyotome by transactivating PAX3 and the delamination and migration of the hypaxial precursors from the ventral lip to the limb buds through the transactivation of PAX3, MET and LBX1. Controls myoblast determination by transactivating MYF5, MYOD1 and MYF6. Controls somitic differentiation in myocyte through MYOG transactivation. Plays a role in synaptogenesis and sarcomere organization by participating in myofiber specialization during embryogenesis by activating fast muscle program in the primary myotome resulting in an up-regulation of fast muscle genes, including ATP2A1, MYL1 and TNNT3. Simultaneously, is also able to activate inhibitors of slow muscle genes, such as SOX6, HRASLS, and HDAC4, thereby restricting the activation of the slow muscle genes. During muscle regeneration, negatively regulates differentiation of muscle satellite cells through down-regulation of MYOG expression. During kidney development regulates the early stages of metanephros development and ureteric bud formation through regulation of GDNF, SALL1, PAX8 and PAX2 expression. Plays a role in gonad development by regulating both testis determination and size determination. In gonadal sex determination, transactivates ZFPM2 by binding a MEF3 consensus sequence, resulting in SRY up-regulation. In gonadal size determination, transactivates NR5A1 by binding a MEF3 consensus sequence resulting in gonadal precursor cell formation regulation. During olfactory development mediates the specification and patterning of olfactory placode through fibroblast growth factor and BMP4 signaling pathways and also regulates epithelial cell proliferation during placode formation. Promotes survival of sensory neurons during early trigeminal gangliogenesis. In the developing dorsal root ganglia, up-regulates SLC12A2 transcription. Regulates early thymus/parathyroid organogenesis through regulation of GCM2 and FOXN1 expression. Forms gustatory papillae during development of the tongue. Also plays a role during embryonic cranial skeleton morphogenesis","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q9UIU6/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/SIX4","classification":"Not Classified","n_dependent_lines":6,"n_total_lines":1208,"dependency_fraction":0.004966887417218543},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SIX4","total_profiled":1310},"omim":[{"mim_id":"608389","title":"BRANCHIOOTIC SYNDROME 3; BOS3","url":"https://www.omim.org/entry/608389"},{"mim_id":"606342","title":"SIX HOMEOBOX 4; SIX4","url":"https://www.omim.org/entry/606342"},{"mim_id":"606326","title":"SIX HOMEOBOX 6; SIX6","url":"https://www.omim.org/entry/606326"},{"mim_id":"601205","title":"SIX HOMEOBOX 1; SIX1","url":"https://www.omim.org/entry/601205"},{"mim_id":"115650","title":"CATARACT 32, MULTIPLE TYPES; CTRCT32","url":"https://www.omim.org/entry/115650"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"},{"location":"Cytosol","reliability":"Additional"}],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"parathyroid gland","ntpm":14.2},{"tissue":"salivary gland","ntpm":6.5},{"tissue":"skeletal 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DNA binding to the ARE (Na,K-ATPase alpha1 subunit gene regulatory element), and a transactivation domain in the C-terminal region identified by GAL4-fusion reporter assays. The protein localizes to both nucleus and cytoplasm of myoblast C2C12 cells, with the 115 kDa form increased in cytoplasmic extract and the 67 kDa form increased in both nuclear and cytoplasmic extracts during muscle differentiation.\",\n      \"method\": \"Reporter gene assays with GAL4-AREC3 fusion constructs, immunohistochemistry, Western blot analysis of nuclear and cytoplasmic fractions during differentiation\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct DNA-binding assay, transactivation assay, and subcellular fractionation in single lab with multiple orthogonal methods\",\n      \"pmids\": [\"8628654\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"SIX4 is the transcriptional regulatory element X (Trex)-binding factor (TrexBF) in the Muscle Creatine Kinase (MCK) enhancer in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle. SIX4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. In adult mouse heart, Six5 (not Six4) is the major TrexBF.\",\n      \"method\": \"Quantitative proteomics of oligonucleotide-affinity-purified proteins, gel shift assays, Six-specific antisera, cotransfection reporter assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — quantitative proteomics identification combined with gel-shift confirmation with specific antisera and functional reporter assays; multiple orthogonal methods in one rigorous study\",\n      \"pmids\": [\"14966291\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"In Six1/Six4 double knockout mice, Pax3 expression is impaired in limb bud somitic cells, blocking myogenic cell delamination and migration; within the myotome, myogenin, Myod1 and Mrf4 expression are abolished, and Myf5 becomes restricted to the caudal somite region, placing Six1 and Six4 upstream of these myogenic regulatory factors.\",\n      \"method\": \"Double knockout mouse genetics, in situ hybridization, immunohistochemistry for myogenic markers\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in double-KO mice with multiple molecular marker readouts, replicated across developmental contexts\",\n      \"pmids\": [\"15788460\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Six1 and Six4 differentially regulate a set of target genes. The promoter of Slc12a2 (NKCC1) contains multiple Six1-binding sites and one common binding site for both Six1 and Six4 by gel-retardation assay, indicating distinct DNA-binding specificities. In vivo, Slc12a2 expression is reduced in the dorsal root ganglia of Six1/Six4 double-null mice.\",\n      \"method\": \"Gel-retardation assays (EMSA), target gene screening, in situ hybridization in double-KO mice\",\n      \"journal\": \"The FEBS journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — EMSA for direct DNA binding plus in vivo validation in KO mice, single lab, two orthogonal methods\",\n      \"pmids\": [\"15955062\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"In Drosophila, D-Six4 is required for development of non-dorsal mesodermal cell types (fat body, somatic gonadal cells, specific somatic muscles). Misexpression analysis shows D-Six4 and its cofactor Eyes absent are sufficient to impose these fates on other mesodermal cells. Tinman (tin) function is required for full D-six4 expression at stage 9.\",\n      \"method\": \"Loss-of-function genetics, misexpression analysis, epistasis between tin and D-six4\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function and gain-of-function with cellular phenotype readouts and epistasis, single lab\",\n      \"pmids\": [\"16595131\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Six1 and Six4 cooperate in the metanephric mesenchyme to regulate Gdnf expression; Six1/Six4 double-null mice lack ureteric bud formation and show complete absence of Pax2, Pax8, and Gdnf expression in metanephric mesenchyme, whereas Six1 deficiency alone only partially reduces these markers.\",\n      \"method\": \"Double knockout mouse genetics, in situ hybridization, molecular marker analysis\",\n      \"journal\": \"Mechanisms of development\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis in double-KO with specific molecular readouts; replicated in multiple studies\",\n      \"pmids\": [\"17300925\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In Drosophila, Six4 is required in somatic gonadal precursors (SGPs) for expression of Hmgcr (HMG-CoA reductase), which is necessary for attraction of primordial germ cells (PGCs) to SGPs. Six4 affects male-specific SGP migration by a different (Hmgcr-independent) pathway. SGPs also fail to coalesce into unified gonads without functional Six4.\",\n      \"method\": \"Live time-lapse fluorescence imaging of wild-type and mutant embryos, in vivo genetic analysis, epistasis with Hmgcr\",\n      \"journal\": \"BMC developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — live imaging combined with genetic epistasis in single lab\",\n      \"pmids\": [\"17517128\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"Six1 and Six4 are required synergistically for olfactory placode formation; embryos lacking both Six1 and Six4 fail to form the olfactory placode despite normal specification of the preplacodal region (marked by Eya2). Six1 and Six4 act at the top of events controlling olfactory placode specification and patterning through Fgf and Bmp signaling pathways.\",\n      \"method\": \"Double knockout mouse genetics, in situ hybridization, molecular marker analysis (Eya2, Mash1, Sox2)\",\n      \"journal\": \"Developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in double-KO mice with multiple molecular marker readouts, single lab\",\n      \"pmids\": [\"19027001\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Six1 and Six4 homeoproteins are required together for male sex determination: double loss leads to male-to-female sex reversal in XY mice due to failure of Sry activation. Forced Sry transgene expression rescues testicular development but not precursor cell growth. Two downstream pathways are identified: Six1/Six4 regulate Fog2 (Zfpm2) to induce Sry expression, and regulate Nr5a1 (Ad4BP/Sf1) to control gonadal precursor formation.\",\n      \"method\": \"Double knockout mouse genetics, transgenic rescue with Sry, in situ hybridization and gene expression analysis of Fog2 and Nr5a1\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic epistasis with transgenic rescue plus identification of two distinct downstream targets, multiple orthogonal methods\",\n      \"pmids\": [\"23987514\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"During adult skeletal muscle regeneration, Six4 co-occupies a core set of muscle gene loci genome-wide together with MyoD and the histone H3K27me3 demethylase Utx. Six4 and MyoD cooperation is associated with removal of the H3K27me3 repressive chromatin mark. In vivo RNAi of Six4 reveals an uncompensated function in muscle regeneration.\",\n      \"method\": \"ChIP-seq (genome-wide occupancy of Six4, MyoD, Utx), in vivo RNAi knockdown with muscle regeneration phenotype\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genome-wide ChIP-seq with multiple factors plus in vivo functional validation, multiple orthogonal methods\",\n      \"pmids\": [\"26229056\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Six1 or Six4 are required for MyoD-mediated reprogramming of mouse embryonic fibroblasts toward myogenesis. Genome-wide analysis identified >700 genes co-regulated by Six and MyoD, with MyoD ChIP-seq data showing co-localization of MyoD and MEF3 (Six-binding) sites at >1000 genomic regions. The Six/MyoD synergistic activation involves a feedforward mechanism recruiting Mef2, Pbx-Meis, and EBF co-factors.\",\n      \"method\": \"Microarray expression profiling, MyoD ChIP-seq, genome-wide MEF3 site search, luciferase reporter assays for individual CRMs, MEF reprogramming assay\",\n      \"journal\": \"Nucleic acids research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — genome-wide ChIP-seq, microarray, and functional reporter assays with multiple orthogonal methods\",\n      \"pmids\": [\"27302134\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SIX4 directly binds to the promoters of YAP1 and MET (c-MET) to transactivate their expression in hepatocellular carcinoma cells. HGF upregulates SIX4 expression through the ERK/NF-κB pathway, forming a positive feedback loop (HGF→SIX4→c-MET). Knockdown of both YAP1 and c-MET inhibits SIX4-mediated HCC metastasis.\",\n      \"method\": \"ChIP assay (SIX4 binding to YAP1 and MET promoters), luciferase reporter assays, rescue experiments with YAP1/c-MET knockdown and overexpression, ERK/NF-κB pathway inhibition\",\n      \"journal\": \"Oncogene\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and reporter assays for direct target binding, plus genetic rescue; single lab\",\n      \"pmids\": [\"33046796\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SIX4 increases expression of VEGF-A by coordinating with HIF-1α, and upregulates HIF-1α expression in an Akt-dependent manner, thereby promoting tumor angiogenesis in colorectal cancer cells.\",\n      \"method\": \"Overexpression and knockdown of SIX4, in vitro angiogenesis assay, Western blot for HIF-1α/Akt/VEGF-A, in vivo tumor growth assay\",\n      \"journal\": \"Experimental cell research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single lab, Western blot and overexpression/knockdown without direct binding evidence for SIX4-HIF-1α interaction\",\n      \"pmids\": [\"31301290\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SIX4 directly interacts with STAT3 protein and promotes phosphorylated STAT3 nuclear translocation, thereby inducing EMT program activation (via Snai1 induction) and breast cancer metastasis.\",\n      \"method\": \"Co-immunoprecipitation (SIX4-STAT3 interaction), knockdown/overexpression with migration/invasion assays, in vivo lung metastasis assay\",\n      \"journal\": \"American journal of cancer research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP demonstrating direct interaction plus functional in vivo validation, single lab\",\n      \"pmids\": [\"32064163\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"SIX4 chromatin co-occupancy with Pax3 is demonstrated genome-wide in mesodermal cells. Pax3 cooperates with Six4 (and Tead2) factors and involves chromatin remodeling (increased chromatin accessibility at bound elements) to activate the skeletal myogenic lineage.\",\n      \"method\": \"ATAC-seq (chromatin accessibility), ChIP-seq (Pax3 binding), transcriptome profiling in Pax3-induced ESCs and Pax3-null embryos\",\n      \"journal\": \"PLoS biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-wide chromatin and binding data with transcriptome, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"30807574\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"In Six1/Six4 double knockout mice, fewer PAX7+ satellite cells occupy their normal position at E18; the remaining mutant PAX7+ cells can divide and contribute to muscle growth but form hypotrophic, non-innervated myofibers after transplantation and retain self-renewal capacity.\",\n      \"method\": \"Double knockout mouse genetics, immunofluorescence for PAX7+ cell counting, transcriptome analysis, transplantation assay in adult regenerating muscle\",\n      \"journal\": \"Development (Cambridge, England)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO genetics plus transplantation assay and transcriptome, single lab\",\n      \"pmids\": [\"32591430\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"In Drosophila type II neuroblast lineages, Six4 prevents supernumerary type II neuroblasts and premature INP differentiation. Six4 inhibits the expression and activity of PntP1 in immature INPs (imINPs) in part by forming a trimeric complex with Earmuff and PntP1. Six4 also prevents premature differentiation by suppressing ectopic Prospero expression in imINPs.\",\n      \"method\": \"Loss-of-function genetics (six4 mutants), genetic epistasis with pntP1 and earmuff, complex formation demonstrated\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis with trimeric complex identification, single lab with multiple genetic combinations\",\n      \"pmids\": [\"33556050\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"SIX4 directly transcribes STING (cGAS/STING pathway) in colon cancer cells: SIX4 knockout decreases STING mRNA and protein, ectopic SIX4 increases STING expression, and reexpression of SIX4 or STING in SIX4 KO cells reverses the effect. SIX4 depletion attenuates STING activation and downstream signaling, and reduces CD8+ T cell tumor infiltration and anti-PD-1 efficacy in vivo.\",\n      \"method\": \"CRISPR knockout and ectopic expression of SIX4, qPCR/Western blot for STING levels, STING activation assays (DMXAA/cGAMP), in vivo tumor immunology experiments\",\n      \"journal\": \"Cancer research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic KO and rescue with functional STING activation readouts and in vivo immune infiltration, single lab\",\n      \"pmids\": [\"37888903\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"EYA3 isoforms (regulated by RBFOX2-controlled alternative splicing) interact with SIX4 as a major transcription factor partner during myogenesis. Mass spectrometry-based proteomics and genome-wide transcriptomics identified SIX4 as a primary EYA3-interacting protein that dictates gene expression during muscle cell differentiation.\",\n      \"method\": \"Mass spectrometry proteomics (EYA3 interactome), genome-wide transcriptomic analysis, myoblast proliferation/differentiation assays\",\n      \"journal\": \"iScience\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — mass spectrometry identification of SIX4-EYA3 interaction plus transcriptomic functional validation, single lab\",\n      \"pmids\": [\"38026174\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MKRN2 interacts with STAT1 (shown by Co-IP), and MKRN2 regulates SIX4 expression via the EBF2 transcription factor in mouse testis and MEF cells; loss of MKRN2 significantly decreases SIX4 expression.\",\n      \"method\": \"MKRN2 knockout mouse model, Co-IP (MKRN2-STAT1 interaction), expression analysis of SIX4 in KO testis\",\n      \"journal\": \"Frontiers in endocrinology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — SIX4 regulation by MKRN2/EBF2 is inferred from expression changes in KO tissue; the Co-IP is for MKRN2-STAT1, not SIX4 directly; single lab, single method for SIX4\",\n      \"pmids\": [\"36967804\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"SIX4 is transcriptionally activated by the IL-6/STAT3 signaling pathway in colorectal epithelium, and activated SIX4 binds to c-Jun to transcribe IL-6, forming a positive IL-6/STAT3/SIX4/c-Jun feedback loop that drives intestinal inflammation. SIX4 also binds the DeltaNp63 promoter (but not wild-type p63) to induce tumor stemness signaling.\",\n      \"method\": \"ChIP assay (SIX4 binding to c-Jun and DeltaNp63 promoters), reporter assays, in vivo DSS/AOM-DSS mouse models, siRNA knockdown\",\n      \"journal\": \"International journal of biological sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP for direct promoter binding plus in vivo validation in inflammation models, single lab\",\n      \"pmids\": [\"39309424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"In alveolar rhabdomyosarcoma, SIX4 is identified as a key transcription factor that mediates CDK8 inhibitor-induced transcriptional activation of myogenic differentiation genes and tumor cell proliferation. The maximal anti-tumor activity of CDK8 inhibitors requires the Mediator kinase module and transcriptional cooperation with the SAGA complex.\",\n      \"method\": \"Genome-scale CRISPR-Cas9 drug modifier screen, CDK8 knockout and pharmacologic inhibition, transcriptional profiling, in vitro and in vivo tumor models\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genome-scale CRISPR screen plus functional validation; preprint, single lab\",\n      \"pmids\": [\"bio_10.1101_2025.07.14.663986\"],\n      \"is_preprint\": true\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MyoD binds directly at MyoD and E-box recognition sites in the core promoter region (-522/-193) of bovine SIX4, as demonstrated by EMSA and ChIP; site-directed mutagenesis and siRNA interference confirm that MyoD regulates SIX4 transcription through both direct and indirect mechanisms.\",\n      \"method\": \"EMSA, chromatin immunoprecipitation (ChIP), luciferase reporter assays with 5'-deletion constructs, site-directed mutation, siRNA knockdown of MyoD\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — multiple direct binding methods (EMSA, ChIP, mutagenesis) in single lab; bovine system\",\n      \"pmids\": [\"29307818\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"FOXA1 directly regulates SIX4 transcription in cervical cancer; FOXA1 binds the SIX4 promoter as demonstrated by ChIP and dual-luciferase assay. SIX4 overexpression promotes phosphorylation of PI3K and AKT, activating the PI3K/AKT signaling pathway, and reverses FOXA1 knockdown effects on cell growth and chemoresistance.\",\n      \"method\": \"ChIP assay, dual-luciferase reporter assay (FOXA1 binding SIX4 promoter), siRNA knockdown, PI3K/AKT Western blot\",\n      \"journal\": \"Analytical cellular pathology (Amsterdam)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase confirm direct FOXA1-SIX4 promoter interaction; downstream PI3K/AKT by Western blot; single lab\",\n      \"pmids\": [\"35498155\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SIX4 is a homeodomain transcription factor that binds MEF3/Trex DNA elements (with its Six domain and homeodomain) to directly activate muscle-specific genes (MCK enhancer, Aldolase A, Cardiac troponin C, Slc12a2, YAP1, c-MET, STING, DeltaNp63, IL-6), cooperates genome-wide with MyoD and the H3K27me3 demethylase Utx to activate the skeletal myogenic program, physically interacts with STAT3 and EYA3 as a transcriptional co-regulator, and functions epistatically upstream of Pax3, Myogenin, MyoD, Mrf4, Gdnf, Fog2, and Nr5a1 in skeletal muscle, kidney, and gonadal development, with its transcription in muscle being directly driven by MyoD binding to E-box/MEF3 sites in its own promoter.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SIX4 is a homeodomain transcription factor that binds MEF3/Trex DNA elements through its homeodomain and transactivates muscle-specific target genes via a C-terminal transactivation domain [#0, #1]. Acting redundantly with its paralog Six1, SIX4 sits at the top of the developmental hierarchy that establishes skeletal muscle, kidney, gonad, and olfactory placode lineages: in Six1/Six4 double-null mice, Pax3 and the myogenic regulatory factors Myogenin, Myod1, and Mrf4 fail to be expressed and myogenic cells fail to delaminate and migrate [#2], Gdnf and ureteric bud formation are lost in the metanephric mesenchyme [#5], the olfactory placode fails to form [#7], and male sex determination fails through loss of Sry activation downstream of Fog2 and Nr5a1 [#8]. Mechanistically, SIX4 cooperates genome-wide with MyoD and Pax3, co-occupying muscle gene loci and increasing chromatin accessibility, and its synergy with MyoD is coupled to removal of the repressive H3K27me3 mark by the demethylase Utx [#9, #10, #14]; SIX4 transcription is itself directly driven by MyoD binding E-box/MEF3 sites in the SIX4 promoter, creating a feedforward loop [#22]. SIX4 functions with cofactors of the Eyes absent family, interacting directly with EYA3 during myogenesis [#18]. Beyond development, SIX4 acts as a pro-tumorigenic and pro-inflammatory transcription factor: it binds the promoters of YAP1 and MET to drive hepatocellular carcinoma metastasis within an HGF feedback loop [#11], physically interacts with STAT3 to promote its nuclear translocation and EMT [#13], directly transcribes STING to shape anti-tumor immunity [#17], and participates in an IL-6/STAT3/SIX4/c-Jun feedback loop while inducing DeltaNp63 in colorectal contexts [#20].\",\n  \"teleology\": [\n    {\n      \"year\": 1996,\n      \"claim\": \"Establishing that SIX4 is a sequence-specific DNA-binding transactivator answered whether it acts directly on gene regulatory elements rather than as an accessory factor.\",\n      \"evidence\": \"GAL4-fusion reporter assays, DNA-binding to the Na,K-ATPase ARE element, and subcellular fractionation in C2C12 myoblasts\",\n      \"pmids\": [\"8628654\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological target genes not yet defined\", \"Dual nuclear/cytoplasmic localization and processed forms not mechanistically explained\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Identifying SIX4 as the Trex/MEF3-binding factor in the MCK enhancer connected its DNA-binding activity to a defined muscle-specific regulatory grammar.\",\n      \"evidence\": \"Oligonucleotide-affinity proteomics, gel-shift with Six-specific antisera, and reporter assays on MCK, Aldolase A, and Cardiac troponin C elements\",\n      \"pmids\": [\"14966291\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Distinction of Six4 vs Six5 roles across tissues incomplete\", \"Cofactor requirements for transactivation not resolved\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Double-knockout genetics placed Six1/Six4 upstream of the core myogenic transcription factors and distinguished their target specificity from Six1.\",\n      \"evidence\": \"Six1/Six4 double-KO mouse genetics with in situ markers (Pax3, Myogenin, Myod1, Mrf4) and EMSA on the Slc12a2 promoter\",\n      \"pmids\": [\"15788460\", \"15955062\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct vs indirect regulation of each myogenic factor not separated\", \"Redundancy boundaries between Six1 and Six4 unquantified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Drosophila genetics established a deeply conserved role for Six4/Eya in mesodermal and gonadal cell-fate specification.\",\n      \"evidence\": \"Loss- and gain-of-function genetics, misexpression with Eyes absent cofactor, and tin epistasis in fly embryos\",\n      \"pmids\": [\"16595131\", \"17517128\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct transcriptional targets in fly not mapped\", \"Relationship to vertebrate target sets unclear\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Extending the Six1/Six4 requirement to kidney and olfactory placode showed SIX4 operates as a master upstream regulator across multiple organ primordia.\",\n      \"evidence\": \"Six1/Six4 double-KO mice with marker analysis of Gdnf, Pax2/Pax8 (kidney) and Eya2/Sox2 (olfactory placode)\",\n      \"pmids\": [\"17300925\", \"19027001\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether SIX4 binds these target promoters directly not shown\", \"Signaling integration (Fgf/Bmp) downstream vs upstream unresolved\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining the Fog2 and Nr5a1 branches downstream of Six1/Six4 explained how these factors control both Sry activation and gonadal precursor growth in sex determination.\",\n      \"evidence\": \"Double-KO mouse genetics with Sry transgenic rescue and expression analysis of Fog2 and Nr5a1\",\n      \"pmids\": [\"23987514\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct SIX4 occupancy at Fog2/Nr5a1 loci not demonstrated\", \"Mechanism limiting rescue of precursor cell growth unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Genome-wide co-occupancy with MyoD, Pax3, and Utx, plus a self-promoter loop, revealed how SIX4 mechanistically drives the myogenic program through chromatin remodeling and feedforward circuitry.\",\n      \"evidence\": \"ChIP-seq of Six4/MyoD/Utx and Pax3, ATAC-seq, MEF reprogramming assays, EMSA/ChIP on the SIX4 promoter, and in vivo regeneration RNAi\",\n      \"pmids\": [\"26229056\", \"27302134\", \"30807574\", \"29307818\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct recruitment mechanism of Utx by SIX4 not established\", \"Quantitative contribution of each cofactor (Mef2, Pbx-Meis, EBF) unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Satellite cell analysis clarified that SIX4 is required for proper positioning and functional competence of muscle stem cells, beyond initial lineage specification.\",\n      \"evidence\": \"Six1/Six4 double-KO PAX7+ cell quantification, transcriptome, and transplantation into regenerating muscle\",\n      \"pmids\": [\"32591430\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Transcriptional targets governing satellite cell positioning unmapped\", \"Cell-autonomous vs niche contributions not fully separated\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying EYA3 as a primary SIX4 partner and a Drosophila trimeric complex defined the cofactor architecture through which SIX4 regulates differentiation.\",\n      \"evidence\": \"Mass spectrometry of the EYA3 interactome with transcriptomics in myogenesis, and genetic/complex analysis of Six4-Earmuff-PntP1 in fly neuroblasts\",\n      \"pmids\": [\"38026174\", \"33556050\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural basis of SIX4-EYA3 interaction unknown\", \"Whether neuroblast and myogenic cofactor logic is shared unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Multiple cancer studies established SIX4 as a pro-tumorigenic transcription factor acting through STAT3, YAP1/MET, STING, and IL-6/c-Jun circuits.\",\n      \"evidence\": \"ChIP/reporter assays on YAP1, MET, STING, c-Jun, and DeltaNp63 promoters; Co-IP with STAT3; CRISPR KO/rescue and in vivo tumor and inflammation models\",\n      \"pmids\": [\"33046796\", \"32064163\", \"37888903\", \"39309424\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether oncogenic targets share the MEF3 binding grammar of developmental targets unclear\", \"Most cancer findings rest on single-lab studies without reciprocal validation\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Drug-modifier screening linked SIX4 to CDK8/Mediator-kinase-dependent transcriptional rewiring in rhabdomyosarcoma, connecting its myogenic activity to therapeutic vulnerability.\",\n      \"evidence\": \"Genome-scale CRISPR screen with CDK8 inhibition and transcriptional profiling (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.07.14.663986\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Preprint not peer-reviewed\", \"Direct SIX4-Mediator interaction not demonstrated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single MEF3-binding homeodomain factor selects developmental versus oncogenic target gene sets, and which cofactors and chromatin states toggle that selection, remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No structural model of SIX4 on DNA with EYA cofactors\", \"Rules governing context-specific target choice unknown\", \"Direct in vivo binding at most cancer targets not validated\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 1, 2, 9, 10, 11, 17, 20]},\n      {\"term_id\": \"GO:0003677\", \"supporting_discovery_ids\": [0, 1, 3, 22]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": []}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [2, 5, 7, 8, 9, 10, 14]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 1, 11, 17, 20]},\n      {\"term_id\": \"R-HSA-1643685\", \"supporting_discovery_ids\": [11, 13, 17, 20]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"MyoD\", \"EYA3\", \"STAT3\", \"Utx\", \"Pax3\", \"c-Jun\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":5,"faith_total":5,"faith_pct":100.0}}