{"gene":"MDFI","run_date":"2026-06-10T02:59:50","timeline":{"discoveries":[{"year":1998,"finding":"I-mfa (MDFI) protein binds to MyoD family bHLH transcription factors, inhibits their transcriptional activity, and blocks their nuclear import and DNA binding; it also interacts with the bHLH protein Mash2 and inhibits its transcriptional activity, but does not interfere with Hand1 activity.","method":"Cell culture overexpression/inhibition assays, protein interaction studies, in situ hybridization, targeted gene deletion in mice","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (binding assays, functional transcription assays, KO mouse), replicated across cell types and genetic backgrounds","pmids":["9799236"],"is_preprint":false},{"year":1998,"finding":"Overexpression of I-mfa in rat trophoblast (Rcho-1) stem cells induced their differentiation into trophoblast giant cells, and targeted deletion of I-mfa caused placental defects with reduced trophoblast giant cells, establishing a role for I-mfa in trophoblast differentiation.","method":"Targeted gene deletion (KO mouse), overexpression in Rcho-1 cells","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse with defined cellular phenotype plus orthogonal gain-of-function overexpression","pmids":["9799236"],"is_preprint":false},{"year":2001,"finding":"I-mfa inhibits the activity and DNA binding of the HMG-box transcription factor XTcf3; ectopic expression of I-mfa in Xenopus embryos inhibited dorsal axis specification and Tcf3/beta-catenin-regulated gene expression (siamois, Xnr3), and blocked beta-catenin activation of Lef/Tcf reporter constructs.","method":"Xenopus ectopic expression, reporter assays, epistasis with beta-catenin","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — functional assays in embryos and cell culture, single lab, two orthogonal methods (reporter + developmental phenotype)","pmids":["11238923"],"is_preprint":false},{"year":2002,"finding":"I-mfa interacts in vivo with the Axin complex through its C-terminal I-mfa domain, inhibiting Axin-mediated downregulation of cytosolic beta-catenin; I-mfa also directly interacts with LEF and inhibits beta-catenin/TCF-regulated reporter constructs, and decreases Axin-mediated JNK activation.","method":"Co-immunoprecipitation (in vivo), reporter assays, domain mapping","journal":"Molecular and cellular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vivo Co-IP plus reporter assays, single lab, two orthogonal methods","pmids":["12192039"],"is_preprint":false},{"year":2004,"finding":"I-mfa physically interacts with the amino-terminal domain of Zic2 (and Zic1-3) and inhibits nuclear import of the Zic proteins, thereby inhibiting their transcriptional activation activity in cultured cells.","method":"Co-immunoprecipitation, subcellular localization assays, transcription reporter assays, co-expression in cultured cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus localization plus functional reporter, single lab","pmids":["15207726"],"is_preprint":false},{"year":2005,"finding":"Beta-catenin interacts with I-mfa (enhanced by Wnt3a); this interaction attenuates I-mfa binding to myogenic regulatory factors (MRFs), relieves I-mfa-mediated suppression of MRF transcriptional activity and cytosolic sequestration of MRFs, and initiates myogenesis in P19 cells.","method":"Co-immunoprecipitation, reporter assays, cell differentiation assays, siRNA knockdown","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, reporter assays, siRNA, and differentiation assays providing multiple orthogonal lines of evidence","pmids":["16301527"],"is_preprint":false},{"year":2006,"finding":"I-mfa suppresses LEF-1 transcriptional activity; beta-catenin competes with I-mfa for binding to LEF-1, relieving I-mfa-mediated suppression; canonical Wnt signaling reduces endogenous I-mfa associated with LEF-1 while increasing I-mfa associated with beta-catenin.","method":"Co-immunoprecipitation, reporter assays, siRNA knockdown, dominant-negative LEF-1 epistasis","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, reporter assays, and epistasis with dominant-negative, single lab","pmids":["17090604"],"is_preprint":false},{"year":2007,"finding":"I-mfa interacts through its I-mfa domain with cyclin T1 and T2 at two binding sites (the histidine-rich regulatory domain and a lysine/arginine-rich motif overlapping the Tat/TAR recognition motif), can serve as a P-TEFb substrate, and inhibits Tat- and P-TEFb-dependent transcription from the HIV-1 promoter in a cell-type-specific manner.","method":"Yeast two-hybrid, Co-immunoprecipitation, domain mapping, transcription reporter assays","journal":"Journal of molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, domain mapping, functional reporter assays, single lab, multiple orthogonal methods","pmids":["17289077"],"is_preprint":false},{"year":2011,"finding":"I-mfa interacts through its C-terminal I-mfa domain with SERTA domain-containing proteins SEI-1, SEI-2, SEI-3, SERTAD3, and SERTAD4 in vivo; this interaction affects I-mfa intracellular localization and represses the intrinsic transcriptional activities of SEI-1, SEI-2, and SERTAD3, and decreases the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA levels.","method":"Co-immunoprecipitation, reporter assays, domain-mapping mutagenesis, qPCR","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, reporter assays, and mRNA level analysis in single lab, multiple orthogonal methods","pmids":["21664411"],"is_preprint":false},{"year":2015,"finding":"I-mfa directly interacts with HTLV-1 Tax protein in vitro and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs.","method":"In vitro binding assay, reporter assays, Co-immunoprecipitation","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro direct interaction plus reporter functional assays, single lab","pmids":["26469549"],"is_preprint":false},{"year":2018,"finding":"miR-27b directly targets MDFI (confirmed by dual-luciferase reporter assay); MDFI promotes satellite cell proliferation and inhibits their differentiation in vitro, and interfering with MDFI expression promotes muscle regeneration in vivo after injury.","method":"Dual-luciferase reporter assay, siRNA knockdown, EdU staining, qRT-PCR, Western blot, in vivo muscle injury model","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — validated target-gene interaction plus KD cellular and in vivo phenotype, single lab","pmids":["29734192"],"is_preprint":false},{"year":2019,"finding":"miR-501-3p directly targets FOS (confirmed by dual-luciferase reporter); FOS binds the MDFI promoter (confirmed by ChIP); and MDFI overexpression promotes C2C12 myogenic differentiation and MyoD expression, forming a feedback loop (miR-501-3p → FOS ⊣ MDFI → MyoD → miR-501-3p).","method":"Dual-luciferase reporter assay, ChIP assay, qPCR, Western blot, miRNA mimic/inhibitor transfection","journal":"Cells","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — ChIP and luciferase reporter establish direct regulatory relationships, single lab","pmids":["31212688"],"is_preprint":false},{"year":2020,"finding":"MDFI interacts with the histone demethylase JMJD1A; JMJD1A influences transcription of several genes also regulated by MDFI; MDFI stimulated growth of HCT116 colorectal cancer cells.","method":"Co-immunoprecipitation, gene expression analysis, cell proliferation assays","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 2–3 / Moderate — Co-IP interaction and functional growth assay, single lab, limited mechanistic depth in abstract","pmids":["32457453"],"is_preprint":false},{"year":2021,"finding":"Mdfi overexpression in C2C12 cells promotes myoblast differentiation by upregulating Myod, Myog, and Myosin expression, and promotes fast-to-slow twitch muscle fiber transformation mediated through Myod, Camk2b, and downstream metabolic genes (Pgc1a, Pdk4, Cs, Cox4, etc.); RNA-seq identified the calcium signaling pathway as most significantly affected.","method":"CRISPR/Cas9 Mdfi-OE stable cell line, RNA-seq, qPCR, Western blot, immunofluorescence","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — stable OE cell line with RNA-seq and experimental validation, single lab","pmids":["33553177"],"is_preprint":false},{"year":2023,"finding":"Elevated MDFI promotes fast-to-slow muscle fiber type conversion by activating CaMKK2 and AMPK phosphorylation, stimulating mitochondrial biogenesis and aerobic metabolism; MDFI also increases intracellular calcium by promoting calcium release from the ER via IP3R and RYR channels.","method":"Lipofection-mediated overexpression and siRNA knockdown in C2C12 cells, immunofluorescence, qPCR, Western blot, calcium imaging with channel inhibitors","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — gain- and loss-of-function with pharmacological pathway dissection (channel inhibitors), single lab","pmids":["37307704"],"is_preprint":false},{"year":2023,"finding":"I-MFA plays a cell-intrinsic role in megakaryocyte lineage commitment and terminal differentiation; I-MFA KO mice had reduced platelets, reduced MK/erythrocyte progenitors, and increased myeloid progenitors; shRNA knockdown of I-MFA in K562 cells reduced PMA-induced MK differentiation with prolonged phospho-JNK and phospho-ERK signaling; overexpression of I-MFA promoted MK differentiation.","method":"I-MFA knockout mice (bone marrow analysis, blood counts), shRNA knockdown in K562 cells, I-MFA overexpression, Western blot for phospho-JNK/ERK","journal":"Blood cells, molecules & diseases","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse phenotype plus gain- and loss-of-function in cell line with signaling readout, single lab","pmids":["37267696"],"is_preprint":false},{"year":2024,"finding":"MDFI directly binds LAMB3 and ITGB4 (confirmed by co-immunoprecipitation) in colorectal cancer cells, upregulates AKT pathway signaling through these interactions, enhances CRC cell proliferation, and reduces sensitivity to oxaliplatin and fluorouracil.","method":"Co-immunoprecipitation, lentiviral overexpression, shRNA knockdown, colony formation assay, CCK8 assay, Western blot","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus gain- and loss-of-function with functional readouts, single lab","pmids":["38375821"],"is_preprint":false},{"year":2024,"finding":"I-mfa is a cytosolic protein in mesangial cells that suppresses contractile function by decreasing TRPC1 channel protein abundance; I-mfa KO mice had lower GFR, augmented Ang II-induced mesangial cell contraction, and increased TRPC1 protein; overexpression of I-mfa blunted Ang II-stimulated contraction and Ca2+ entry; re-introduction of I-mfa into KO cells restored normal contractile response.","method":"I-mfa KO mice (GFR measurement, mesangial cell isolation), targeted siRNA nanoparticle delivery in vivo, single-cell contractility assay, Ca2+ imaging, Western blot, single-cell RNA sequencing, pharmacological TRPC1 inhibition","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 2 / Strong — KO mouse in vivo phenotype plus cell-level gain/loss-of-function with rescue, Ca2+ imaging, and pharmacological epistasis providing multiple orthogonal lines","pmids":["39446484"],"is_preprint":false},{"year":2024,"finding":"miR-128 directly targets MDFI (confirmed by luciferase assay); miR-128 overexpression reduced MDFI mRNA and protein levels and promoted cardiomyocyte apoptosis, while MDFI upregulation enhanced cardiomyocyte proliferation; elevated miR-128 upregulated Wnt1 and β-catenin, whereas increased MDFI inhibited these expressions, placing MDFI as a negative regulator of the Wnt1/β-catenin pathway in cardiomyocytes.","method":"Luciferase reporter assay, qPCR, Western blot, MTT, transwell, immunohistochemistry, echocardiography in mouse HF model","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — validated miRNA-target interaction plus in vivo functional assays, single lab","pmids":["39046458"],"is_preprint":false},{"year":2025,"finding":"MDFI (and MDFIC) regulate endogenous PIEZO channel currents in non-sensory cell types; MDFI physically binds PIEZO1 and PIEZO2, alters their mechanosensitivity and inactivation kinetics (converting them to high-threshold slowly inactivating mechanoreceptors); cryo-EM revealed a conserved binding pocket in the pore modules of both PIEZO1 and PIEZO2 mediated by the post-translationally modified distal C-termini of MDFI-family proteins.","method":"Electrophysiology (endogenous PIEZO current recording), cryo-EM structure determination, physical binding assays, mutagenesis of binding interface","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 / Strong — cryo-EM structure plus electrophysiology plus mutagenesis providing orthogonal mechanistic validation","pmids":["bio_10.1101_2025.10.26.684595"],"is_preprint":true}],"current_model":"MDFI (I-mfa) is a multifunctional cytosolic/nuclear regulatory protein that acts primarily as a transcriptional repressor by binding and sequestering bHLH myogenic regulatory factors (MyoD family), Zic-family zinc finger proteins, LEF/TCF transcription factors, and SERTA-domain proteins — in each case inhibiting their nuclear import and/or transcriptional activity — while also interacting with the Axin/beta-catenin/Wnt signaling complex, modulating P-TEFb (cyclin T1/T2)-dependent transcription elongation, suppressing mesangial cell contractility by reducing TRPC1 channel abundance, regulating megakaryocyte differentiation via JNK/ERK signaling, and functioning as an auxiliary subunit of PIEZO1/2 mechanosensitive channels by binding a conserved pore-module pocket through its post-translationally modified C-terminus to alter mechanosensitivity and inactivation kinetics."},"narrative":{"mechanistic_narrative":"MDFI (I-mfa) is a cytosolic/nuclear regulatory protein that controls cell differentiation programs principally by binding and sequestering specific DNA-binding transcription factors, thereby restraining their nuclear import and transcriptional output [PMID:9799236, PMID:15207726]. Its founding activity is inhibition of MyoD-family bHLH myogenic regulators: MDFI binds these factors, blocks their nuclear import and DNA binding, and suppresses their transcriptional activity [PMID:9799236]. This repression is relieved by canonical Wnt signaling, in which beta-catenin competes with MDFI for binding to myogenic factors and to LEF/TCF, switching MDFI between an MRF-bound and a beta-catenin-bound state to initiate myogenesis [PMID:16301527, PMID:17090604]. MDFI integrates broadly with Wnt/beta-catenin signaling, interacting with the Axin complex to stabilize cytosolic beta-catenin and directly inhibiting Tcf3 and LEF transcriptional activity [PMID:11238923, PMID:12192039]. Through its conserved C-terminal I-mfa domain it engages additional transcriptional regulators including Zic-family zinc finger proteins, SERTA-domain proteins, and the P-TEFb cyclins T1/T2, in each case repressing their activity [PMID:15207726, PMID:17289077, PMID:21664411]. Beyond transcriptional control, MDFI shapes muscle fiber-type identity by driving fast-to-slow conversion through calcium signaling, CaMKK2/AMPK activation, and mitochondrial biogenesis [PMID:33553177, PMID:37307704], governs megakaryocyte lineage commitment via JNK/ERK signaling [PMID:37267696], suppresses mesangial cell contractility by reducing TRPC1 channel abundance [PMID:39446484], and acts as an auxiliary subunit of PIEZO1/2 mechanosensitive channels, binding a conserved pore-module pocket through its post-translationally modified distal C-terminus to convert them into high-threshold, slowly inactivating mechanoreceptors [PMID:bio_10.1101_2025.10.26.684595]. In cancer, MDFI promotes colorectal cancer cell growth, in part through interactions with LAMB3 and ITGB4 and AKT pathway activation [PMID:38375821]. Mouse knockout studies establish in vivo requirements in trophoblast giant cell differentiation and placental development [PMID:9799236], megakaryopoiesis [PMID:37267696], and glomerular mesangial function [PMID:39446484].","teleology":[{"year":1998,"claim":"Established MDFI's founding molecular activity: how a single protein restrains myogenic commitment by directly inhibiting bHLH regulators at the level of nuclear access and DNA binding.","evidence":"Protein interaction and transcription assays plus targeted gene deletion in mice","pmids":["9799236"],"confidence":"High","gaps":["Selectivity rule unclear — inhibits Mash2 but not Hand1","Structural basis of bHLH sequestration not defined"]},{"year":1998,"claim":"Defined an in vivo developmental requirement, showing MDFI is needed for trophoblast giant cell differentiation and normal placental development.","evidence":"KO mouse placental phenotype with Rcho-1 overexpression","pmids":["9799236"],"confidence":"High","gaps":["Transcription-factor target driving the trophoblast phenotype not pinpointed"]},{"year":2001,"claim":"Extended MDFI's repressor reach to the Wnt pathway by showing it inhibits Tcf3 DNA binding and beta-catenin-dependent axis specification.","evidence":"Xenopus ectopic expression, reporter assays, beta-catenin epistasis","pmids":["11238923"],"confidence":"Medium","gaps":["Whether inhibition is direct on Tcf3 or via beta-catenin not fully resolved","Single-organism developmental readout"]},{"year":2002,"claim":"Mapped the C-terminal I-mfa domain as the Axin-interaction module and showed MDFI stabilizes cytosolic beta-catenin while also binding LEF directly.","evidence":"In vivo Co-IP, domain mapping, reporter assays","pmids":["12192039"],"confidence":"Medium","gaps":["Net effect on Wnt output context-dependent (stabilizes beta-catenin yet inhibits TCF reporters)","Single lab"]},{"year":2004,"claim":"Generalized the sequestration mechanism beyond bHLH factors by showing MDFI blocks nuclear import of Zic family zinc finger proteins.","evidence":"Co-IP, subcellular localization, reporter assays in cultured cells","pmids":["15207726"],"confidence":"Medium","gaps":["In vivo relevance of Zic regulation not tested","Single lab"]},{"year":2006,"claim":"Resolved the regulatory logic: beta-catenin competes with MDFI for binding to myogenic factors and LEF-1, providing a Wnt-responsive switch that relieves MDFI repression and triggers differentiation.","evidence":"Reciprocal Co-IP, siRNA, reporter and differentiation assays in P19/cell lines","pmids":["16301527","17090604"],"confidence":"High","gaps":["Stoichiometry and affinity of competing complexes not quantified","Endogenous switch dynamics in primary myogenesis untested"]},{"year":2007,"claim":"Identified MDFI as a P-TEFb interactor and substrate, linking it to transcription elongation control and viral transactivation.","evidence":"Yeast two-hybrid, Co-IP, domain mapping, HIV-1 promoter reporter assays","pmids":["17289077"],"confidence":"Medium","gaps":["Cell-type-specific basis of P-TEFb inhibition unexplained","Functional consequence of MDFI phosphorylation by P-TEFb unknown"]},{"year":2011,"claim":"Expanded the I-mfa-domain interactome to SERTA-domain proteins, with downstream effects on the SEI-1/DP-1 complex and Fbxw7 expression.","evidence":"Co-IP, domain-mapping mutagenesis, reporter assays, qPCR","pmids":["21664411"],"confidence":"Medium","gaps":["Physiological pathway in which SERTA regulation operates not defined","Single lab"]},{"year":2015,"claim":"Showed MDFI directly binds HTLV-1 Tax and represses Tax-dependent LTR and NF-kB transactivation, indicating an antiviral transcriptional role.","evidence":"In vitro binding and reporter assays, Co-IP","pmids":["26469549"],"confidence":"Medium","gaps":["Endogenous effect on viral replication not assessed","Single lab"]},{"year":2021,"claim":"Repositioned MDFI as a positive driver of myogenic differentiation and fast-to-slow fiber conversion, implicating calcium signaling as the dominant affected pathway.","evidence":"CRISPR overexpression stable line, RNA-seq, qPCR, Western blot, immunofluorescence","pmids":["33553177","31212688"],"confidence":"Medium","gaps":["Reconciliation of positive differentiation role with earlier inhibitory bHLH model unresolved","Context dependence on cell line and dose"]},{"year":2023,"claim":"Dissected the fiber-type mechanism, showing MDFI raises intracellular calcium via ER IP3R/RYR channels and activates CaMKK2/AMPK to drive mitochondrial biogenesis and aerobic metabolism.","evidence":"Gain/loss-of-function in C2C12, calcium imaging with channel inhibitors, Western blot","pmids":["37307704"],"confidence":"Medium","gaps":["How MDFI couples to ER calcium channels mechanistically unknown","In vivo fiber-type effect not shown"]},{"year":2023,"claim":"Established a cell-intrinsic role in megakaryocyte lineage commitment, with KO mice showing reduced platelets and altered progenitor balance linked to prolonged JNK/ERK signaling.","evidence":"KO mice, shRNA knockdown and overexpression in K562, phospho-JNK/ERK Western blot","pmids":["37267696"],"confidence":"Medium","gaps":["Direct molecular target controlling JNK/ERK kinetics not identified","Single lab"]},{"year":2024,"claim":"Defined a non-transcriptional cytosolic role: MDFI suppresses mesangial cell contractility by lowering TRPC1 channel abundance, with KO mice showing impaired glomerular function rescued by re-expression.","evidence":"KO mice with GFR, in vivo siRNA nanoparticle delivery, single-cell contractility, Ca2+ imaging, scRNA-seq, pharmacological TRPC1 inhibition","pmids":["39446484"],"confidence":"High","gaps":["Mechanism by which MDFI reduces TRPC1 protein not defined","Relationship to MDFI's transcriptional functions unclear"]},{"year":2024,"claim":"Implicated MDFI in colorectal cancer growth and chemoresistance through direct binding to LAMB3 and ITGB4 and AKT activation, and as a Wnt1/beta-catenin repressor in cardiomyocytes.","evidence":"Co-IP, lenti-overexpression/shRNA, proliferation and chemosensitivity assays; miR-128 luciferase and HF mouse model","pmids":["38375821","39046458"],"confidence":"Medium","gaps":["Tissue-specific switch between oncogenic and Wnt-repressive roles unexplained","Single lab per context"]},{"year":2025,"claim":"Revealed a structural mechanism by which MDFI acts as a PIEZO auxiliary subunit, binding a conserved pore-module pocket via its modified C-terminus to retune mechanosensitivity and inactivation.","evidence":"Cryo-EM structure, electrophysiology of endogenous PIEZO currents, binding-interface mutagenesis (preprint)","pmids":["bio_10.1101_2025.10.26.684595"],"confidence":"High","gaps":["Preprint, not yet peer-reviewed","Physiological contexts where MDFI tunes PIEZO function in vivo not established","Identity of the C-terminal modification not detailed"]},{"year":null,"claim":"How a single C-terminal I-mfa domain coordinates such diverse partners — nuclear transcription factors versus plasma-membrane PIEZO and TRPC channels — and what determines its tissue-specific deployment remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unifying structural model across nuclear and channel partners","Determinants of cytosolic vs nuclear partitioning unknown","Tissue-specific functional switching mechanism undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,4,8]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[5,6,17,19]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[0,4,5]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[3,17]},{"term_id":"GO:0005634","term_label":"nucleus","supporting_discovery_ids":[0,8]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,5,6]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[0,1,13]},{"term_id":"R-HSA-74160","term_label":"Gene expression (Transcription)","supporting_discovery_ids":[0,7,8]}],"complexes":[],"partners":["CTNNB1","AXIN1","LEF1","TCF7L1","ZIC2","CCNT1","PIEZO1","PIEZO2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q99750","full_name":"MyoD family inhibitor","aliases":["Myogenic repressor I-mf"],"length_aa":246,"mass_kda":25.0,"function":"Inhibits the transactivation activity of the Myod family of myogenic factors and represses myogenesis (By similarity). Acts by associating with Myod family members and retaining them in the cytoplasm by masking their nuclear localization signals (By similarity). Can also interfere with the DNA-binding activity of Myod family members (By similarity). Plays an important role in trophoblast and chondrogenic differentiation (By similarity). Regulates the transcriptional activity of TCF7L1/TCF3 by interacting directly with TCF7L1/TCF3 and preventing it from binding DNA (By similarity). Binds to the axin complex, resulting in an increase in the level of free beta-catenin (By similarity). Affects axin regulation of the WNT and JNK signaling pathways (By similarity). Regulates the activity of mechanosensitive Piezo channel (PubMed:37590348)","subcellular_location":"Nucleus; Cytoplasm","url":"https://www.uniprot.org/uniprotkb/Q99750/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/MDFI","classification":"Not Classified","n_dependent_lines":0,"n_total_lines":1208,"dependency_fraction":0.0},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/MDFI","total_profiled":1310},"omim":[{"mim_id":"617896","title":"ZIC FAMILY, MEMBER 5; ZIC5","url":"https://www.omim.org/entry/617896"},{"mim_id":"614511","title":"MYOD FAMILY INHIBITOR DOMAIN-CONTAINING PROTEIN; MDFIC","url":"https://www.omim.org/entry/614511"},{"mim_id":"608948","title":"ZIC FAMILY, MEMBER 4; ZIC4","url":"https://www.omim.org/entry/608948"},{"mim_id":"604971","title":"MYOD FAMILY INHIBITOR; MDFI","url":"https://www.omim.org/entry/604971"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Nucleoplasm","reliability":"Approved"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/MDFI"},"hgnc":{"alias_symbol":["I-mfa"],"prev_symbol":[]},"alphafold":{"accession":"Q99750","domains":[{"cath_id":"1.20.5","chopping":"158-186_222-246","consensus_level":"medium","plddt":60.9607,"start":158,"end":246}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99750","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q99750-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q99750-F1-predicted_aligned_error_v6.png","plddt_mean":50.66},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=MDFI","jax_strain_url":"https://www.jax.org/strain/search?query=MDFI"},"sequence":{"accession":"Q99750","fasta_url":"https://rest.uniprot.org/uniprotkb/Q99750.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q99750/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q99750"}},"corpus_meta":[{"pmid":"9799236","id":"PMC_9799236","title":"Requirement of the mouse I-mfa gene for placental development and skeletal patterning.","date":"1998","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/9799236","citation_count":97,"is_preprint":false},{"pmid":"12192039","id":"PMC_12192039","title":"I-mfa domain proteins interact with Axin and affect its regulation of the Wnt and c-Jun N-terminal kinase signaling pathways.","date":"2002","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12192039","citation_count":61,"is_preprint":false},{"pmid":"10671520","id":"PMC_10671520","title":"Molecular cloning of a novel human I-mfa domain-containing protein that differently regulates human T-cell leukemia virus type I and HIV-1 expression.","date":"2000","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/10671520","citation_count":55,"is_preprint":false},{"pmid":"11238923","id":"PMC_11238923","title":"Inhibition of Tcf3 binding by I-mfa domain proteins.","date":"2001","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/11238923","citation_count":52,"is_preprint":false},{"pmid":"16301527","id":"PMC_16301527","title":"Beta-catenin regulates myogenesis by relieving I-mfa-mediated suppression of myogenic regulatory factors in P19 cells.","date":"2005","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16301527","citation_count":50,"is_preprint":false},{"pmid":"15207726","id":"PMC_15207726","title":"Myogenic repressor I-mfa interferes with the function of Zic family proteins.","date":"2004","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/15207726","citation_count":44,"is_preprint":false},{"pmid":"28782576","id":"PMC_28782576","title":"DNA methylation of CMTM3, SSTR2, and MDFI genes in colorectal cancer.","date":"2017","source":"Gene","url":"https://pubmed.ncbi.nlm.nih.gov/28782576","citation_count":42,"is_preprint":false},{"pmid":"12944466","id":"PMC_12944466","title":"The human I-mfa domain-containing protein, HIC, interacts with cyclin T1 and modulates P-TEFb-dependent transcription.","date":"2003","source":"Molecular and cellular biology","url":"https://pubmed.ncbi.nlm.nih.gov/12944466","citation_count":41,"is_preprint":false},{"pmid":"29734192","id":"PMC_29734192","title":"MiR-27b Promotes Muscle Development by Inhibiting MDFI Expression.","date":"2018","source":"Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology","url":"https://pubmed.ncbi.nlm.nih.gov/29734192","citation_count":36,"is_preprint":false},{"pmid":"33553177","id":"PMC_33553177","title":"Mdfi Promotes C2C12 Cell Differentiation and Positively Modulates Fast-to-Slow-Twitch Muscle Fiber Transformation.","date":"2021","source":"Frontiers in cell and developmental biology","url":"https://pubmed.ncbi.nlm.nih.gov/33553177","citation_count":24,"is_preprint":false},{"pmid":"20417616","id":"PMC_20417616","title":"Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription.","date":"2010","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/20417616","citation_count":24,"is_preprint":false},{"pmid":"32457453","id":"PMC_32457453","title":"Opposite Roles of the JMJD1A Interaction Partners MDFI and MDFIC in Colorectal Cancer.","date":"2020","source":"Scientific reports","url":"https://pubmed.ncbi.nlm.nih.gov/32457453","citation_count":23,"is_preprint":false},{"pmid":"16260749","id":"PMC_16260749","title":"Direct interaction of the human I-mfa domain-containing protein, HIC, with HIV-1 Tat results in cytoplasmic sequestration and control of Tat activity.","date":"2005","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/16260749","citation_count":23,"is_preprint":false},{"pmid":"21664411","id":"PMC_21664411","title":"I-mfa domain proteins specifically interact with SERTA domain proteins and repress their transactivating functions.","date":"2011","source":"Biochimie","url":"https://pubmed.ncbi.nlm.nih.gov/21664411","citation_count":21,"is_preprint":false},{"pmid":"17289077","id":"PMC_17289077","title":"Developmental regulators containing the I-mfa domain interact with T cyclins and Tat and modulate transcription.","date":"2007","source":"Journal of molecular biology","url":"https://pubmed.ncbi.nlm.nih.gov/17289077","citation_count":18,"is_preprint":false},{"pmid":"31212688","id":"PMC_31212688","title":"MiR-501-3p Forms a Feedback Loop with FOS, MDFI, and MyoD to Regulate C2C12 Myogenesis.","date":"2019","source":"Cells","url":"https://pubmed.ncbi.nlm.nih.gov/31212688","citation_count":15,"is_preprint":false},{"pmid":"11139147","id":"PMC_11139147","title":"Sequence requirement for the nucleolar localization of human I-mfa domain-containing protein (HIC p40).","date":"2000","source":"European journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/11139147","citation_count":15,"is_preprint":false},{"pmid":"26469549","id":"PMC_26469549","title":"I-mfa domain proteins specifically interact with HTLV-1 Tax and repress its transactivating functions.","date":"2015","source":"Virology","url":"https://pubmed.ncbi.nlm.nih.gov/26469549","citation_count":14,"is_preprint":false},{"pmid":"17090604","id":"PMC_17090604","title":"Beta-catenin relieves I-mfa-mediated suppression of LEF-1 in mammalian cells.","date":"2006","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/17090604","citation_count":13,"is_preprint":false},{"pmid":"35225482","id":"PMC_35225482","title":"ACAN, MDFI, and CHST1 as Candidate Genes in Gastric Cancer: A Comprehensive Insilco Analysis.","date":"2022","source":"Asian Pacific journal of cancer prevention : APJCP","url":"https://pubmed.ncbi.nlm.nih.gov/35225482","citation_count":12,"is_preprint":false},{"pmid":"37307704","id":"PMC_37307704","title":"MDFI regulates fast-to-slow muscle fiber type transformation via the calcium signaling pathway.","date":"2023","source":"Biochemical and biophysical research communications","url":"https://pubmed.ncbi.nlm.nih.gov/37307704","citation_count":11,"is_preprint":false},{"pmid":"38375821","id":"PMC_38375821","title":"MDFI promotes the proliferation and tolerance to chemotherapy of colorectal cancer cells by binding ITGB4/LAMB3 to activate the AKT signaling pathway.","date":"2024","source":"Cancer biology & therapy","url":"https://pubmed.ncbi.nlm.nih.gov/38375821","citation_count":10,"is_preprint":false},{"pmid":"11389974","id":"PMC_11389974","title":"Vitamin D(3) enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts.","date":"2001","source":"Biochimica et biophysica acta","url":"https://pubmed.ncbi.nlm.nih.gov/11389974","citation_count":10,"is_preprint":false},{"pmid":"36116024","id":"PMC_36116024","title":"Inhibition of MDFI attenuates proliferation and glycolysis of Helicobacter pylori-infected gastric cancer cells by inhibiting Wnt/β-catenin pathway.","date":"2022","source":"Cell biology international","url":"https://pubmed.ncbi.nlm.nih.gov/36116024","citation_count":8,"is_preprint":false},{"pmid":"34738011","id":"PMC_34738011","title":"Putative MicroRNA-mRNA Networks Upon Mdfi Overexpression in C2C12 Cell Differentiation and Muscle Fiber Type Transformation.","date":"2021","source":"Frontiers in molecular biosciences","url":"https://pubmed.ncbi.nlm.nih.gov/34738011","citation_count":6,"is_preprint":false},{"pmid":"29805634","id":"PMC_29805634","title":"Hypermethylation of MDFI promoter with NSCLC is specific for females, non-smokers and people younger than 65.","date":"2018","source":"Oncology letters","url":"https://pubmed.ncbi.nlm.nih.gov/29805634","citation_count":5,"is_preprint":false},{"pmid":"39046458","id":"PMC_39046458","title":"The role of miR-128 and MDFI in cardiac hypertrophy and heart failure: Mechanistic.","date":"2024","source":"Journal of cellular and molecular medicine","url":"https://pubmed.ncbi.nlm.nih.gov/39046458","citation_count":3,"is_preprint":false},{"pmid":"19582149","id":"PMC_19582149","title":"The complex regulation of HIC (Human I-mfa domain containing protein) expression.","date":"2009","source":"PloS one","url":"https://pubmed.ncbi.nlm.nih.gov/19582149","citation_count":3,"is_preprint":false},{"pmid":"37267696","id":"PMC_37267696","title":"The inhibitor of MyoD Family A (I-MFA) regulates megakaryocyte lineage commitment and terminal differentiation.","date":"2023","source":"Blood cells, molecules & diseases","url":"https://pubmed.ncbi.nlm.nih.gov/37267696","citation_count":2,"is_preprint":false},{"pmid":"39446484","id":"PMC_39446484","title":"I-mfa, Mesangial Cell TRPC1 Channel, and Regulation of GFR.","date":"2024","source":"Journal of the American Society of Nephrology : JASN","url":"https://pubmed.ncbi.nlm.nih.gov/39446484","citation_count":1,"is_preprint":false},{"pmid":"27565872","id":"PMC_27565872","title":"Effect of Genetic Variations within the I-mfa Gene on the Growth Traits of Chinese Cattle.","date":"2016","source":"Animal biotechnology","url":"https://pubmed.ncbi.nlm.nih.gov/27565872","citation_count":1,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.10.26.684595","title":"MDFIC2 is a sensory neuron-specific PIEZO channel auxiliary subunit","date":"2025-10-28","source":"bioRxiv","url":"https://doi.org/10.1101/2025.10.26.684595","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":17069,"output_tokens":4843,"usd":0.061926,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":13094,"output_tokens":4607,"usd":0.090323,"stage2_stop_reason":"end_turn"},"total_usd":0.152249,"stage1_batch_id":"msgbatch_01BbTCMyrRPyhtEq6b1MdwCN","stage2_batch_id":"msgbatch_011dEed5bbxYvTAaQWKJevxu","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"I-mfa (MDFI) protein binds to MyoD family bHLH transcription factors, inhibits their transcriptional activity, and blocks their nuclear import and DNA binding; it also interacts with the bHLH protein Mash2 and inhibits its transcriptional activity, but does not interfere with Hand1 activity.\",\n      \"method\": \"Cell culture overexpression/inhibition assays, protein interaction studies, in situ hybridization, targeted gene deletion in mice\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (binding assays, functional transcription assays, KO mouse), replicated across cell types and genetic backgrounds\",\n      \"pmids\": [\"9799236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"Overexpression of I-mfa in rat trophoblast (Rcho-1) stem cells induced their differentiation into trophoblast giant cells, and targeted deletion of I-mfa caused placental defects with reduced trophoblast giant cells, establishing a role for I-mfa in trophoblast differentiation.\",\n      \"method\": \"Targeted gene deletion (KO mouse), overexpression in Rcho-1 cells\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse with defined cellular phenotype plus orthogonal gain-of-function overexpression\",\n      \"pmids\": [\"9799236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2001,\n      \"finding\": \"I-mfa inhibits the activity and DNA binding of the HMG-box transcription factor XTcf3; ectopic expression of I-mfa in Xenopus embryos inhibited dorsal axis specification and Tcf3/beta-catenin-regulated gene expression (siamois, Xnr3), and blocked beta-catenin activation of Lef/Tcf reporter constructs.\",\n      \"method\": \"Xenopus ectopic expression, reporter assays, epistasis with beta-catenin\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — functional assays in embryos and cell culture, single lab, two orthogonal methods (reporter + developmental phenotype)\",\n      \"pmids\": [\"11238923\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"I-mfa interacts in vivo with the Axin complex through its C-terminal I-mfa domain, inhibiting Axin-mediated downregulation of cytosolic beta-catenin; I-mfa also directly interacts with LEF and inhibits beta-catenin/TCF-regulated reporter constructs, and decreases Axin-mediated JNK activation.\",\n      \"method\": \"Co-immunoprecipitation (in vivo), reporter assays, domain mapping\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vivo Co-IP plus reporter assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"12192039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"I-mfa physically interacts with the amino-terminal domain of Zic2 (and Zic1-3) and inhibits nuclear import of the Zic proteins, thereby inhibiting their transcriptional activation activity in cultured cells.\",\n      \"method\": \"Co-immunoprecipitation, subcellular localization assays, transcription reporter assays, co-expression in cultured cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus localization plus functional reporter, single lab\",\n      \"pmids\": [\"15207726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Beta-catenin interacts with I-mfa (enhanced by Wnt3a); this interaction attenuates I-mfa binding to myogenic regulatory factors (MRFs), relieves I-mfa-mediated suppression of MRF transcriptional activity and cytosolic sequestration of MRFs, and initiates myogenesis in P19 cells.\",\n      \"method\": \"Co-immunoprecipitation, reporter assays, cell differentiation assays, siRNA knockdown\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, reporter assays, siRNA, and differentiation assays providing multiple orthogonal lines of evidence\",\n      \"pmids\": [\"16301527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"I-mfa suppresses LEF-1 transcriptional activity; beta-catenin competes with I-mfa for binding to LEF-1, relieving I-mfa-mediated suppression; canonical Wnt signaling reduces endogenous I-mfa associated with LEF-1 while increasing I-mfa associated with beta-catenin.\",\n      \"method\": \"Co-immunoprecipitation, reporter assays, siRNA knockdown, dominant-negative LEF-1 epistasis\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, reporter assays, and epistasis with dominant-negative, single lab\",\n      \"pmids\": [\"17090604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"I-mfa interacts through its I-mfa domain with cyclin T1 and T2 at two binding sites (the histidine-rich regulatory domain and a lysine/arginine-rich motif overlapping the Tat/TAR recognition motif), can serve as a P-TEFb substrate, and inhibits Tat- and P-TEFb-dependent transcription from the HIV-1 promoter in a cell-type-specific manner.\",\n      \"method\": \"Yeast two-hybrid, Co-immunoprecipitation, domain mapping, transcription reporter assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, domain mapping, functional reporter assays, single lab, multiple orthogonal methods\",\n      \"pmids\": [\"17289077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"I-mfa interacts through its C-terminal I-mfa domain with SERTA domain-containing proteins SEI-1, SEI-2, SEI-3, SERTAD3, and SERTAD4 in vivo; this interaction affects I-mfa intracellular localization and represses the intrinsic transcriptional activities of SEI-1, SEI-2, and SERTAD3, and decreases the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA levels.\",\n      \"method\": \"Co-immunoprecipitation, reporter assays, domain-mapping mutagenesis, qPCR\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, reporter assays, and mRNA level analysis in single lab, multiple orthogonal methods\",\n      \"pmids\": [\"21664411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"I-mfa directly interacts with HTLV-1 Tax protein in vitro and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs.\",\n      \"method\": \"In vitro binding assay, reporter assays, Co-immunoprecipitation\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro direct interaction plus reporter functional assays, single lab\",\n      \"pmids\": [\"26469549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"miR-27b directly targets MDFI (confirmed by dual-luciferase reporter assay); MDFI promotes satellite cell proliferation and inhibits their differentiation in vitro, and interfering with MDFI expression promotes muscle regeneration in vivo after injury.\",\n      \"method\": \"Dual-luciferase reporter assay, siRNA knockdown, EdU staining, qRT-PCR, Western blot, in vivo muscle injury model\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — validated target-gene interaction plus KD cellular and in vivo phenotype, single lab\",\n      \"pmids\": [\"29734192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"miR-501-3p directly targets FOS (confirmed by dual-luciferase reporter); FOS binds the MDFI promoter (confirmed by ChIP); and MDFI overexpression promotes C2C12 myogenic differentiation and MyoD expression, forming a feedback loop (miR-501-3p → FOS ⊣ MDFI → MyoD → miR-501-3p).\",\n      \"method\": \"Dual-luciferase reporter assay, ChIP assay, qPCR, Western blot, miRNA mimic/inhibitor transfection\",\n      \"journal\": \"Cells\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — ChIP and luciferase reporter establish direct regulatory relationships, single lab\",\n      \"pmids\": [\"31212688\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MDFI interacts with the histone demethylase JMJD1A; JMJD1A influences transcription of several genes also regulated by MDFI; MDFI stimulated growth of HCT116 colorectal cancer cells.\",\n      \"method\": \"Co-immunoprecipitation, gene expression analysis, cell proliferation assays\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2–3 / Moderate — Co-IP interaction and functional growth assay, single lab, limited mechanistic depth in abstract\",\n      \"pmids\": [\"32457453\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Mdfi overexpression in C2C12 cells promotes myoblast differentiation by upregulating Myod, Myog, and Myosin expression, and promotes fast-to-slow twitch muscle fiber transformation mediated through Myod, Camk2b, and downstream metabolic genes (Pgc1a, Pdk4, Cs, Cox4, etc.); RNA-seq identified the calcium signaling pathway as most significantly affected.\",\n      \"method\": \"CRISPR/Cas9 Mdfi-OE stable cell line, RNA-seq, qPCR, Western blot, immunofluorescence\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — stable OE cell line with RNA-seq and experimental validation, single lab\",\n      \"pmids\": [\"33553177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Elevated MDFI promotes fast-to-slow muscle fiber type conversion by activating CaMKK2 and AMPK phosphorylation, stimulating mitochondrial biogenesis and aerobic metabolism; MDFI also increases intracellular calcium by promoting calcium release from the ER via IP3R and RYR channels.\",\n      \"method\": \"Lipofection-mediated overexpression and siRNA knockdown in C2C12 cells, immunofluorescence, qPCR, Western blot, calcium imaging with channel inhibitors\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — gain- and loss-of-function with pharmacological pathway dissection (channel inhibitors), single lab\",\n      \"pmids\": [\"37307704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"I-MFA plays a cell-intrinsic role in megakaryocyte lineage commitment and terminal differentiation; I-MFA KO mice had reduced platelets, reduced MK/erythrocyte progenitors, and increased myeloid progenitors; shRNA knockdown of I-MFA in K562 cells reduced PMA-induced MK differentiation with prolonged phospho-JNK and phospho-ERK signaling; overexpression of I-MFA promoted MK differentiation.\",\n      \"method\": \"I-MFA knockout mice (bone marrow analysis, blood counts), shRNA knockdown in K562 cells, I-MFA overexpression, Western blot for phospho-JNK/ERK\",\n      \"journal\": \"Blood cells, molecules & diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse phenotype plus gain- and loss-of-function in cell line with signaling readout, single lab\",\n      \"pmids\": [\"37267696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MDFI directly binds LAMB3 and ITGB4 (confirmed by co-immunoprecipitation) in colorectal cancer cells, upregulates AKT pathway signaling through these interactions, enhances CRC cell proliferation, and reduces sensitivity to oxaliplatin and fluorouracil.\",\n      \"method\": \"Co-immunoprecipitation, lentiviral overexpression, shRNA knockdown, colony formation assay, CCK8 assay, Western blot\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus gain- and loss-of-function with functional readouts, single lab\",\n      \"pmids\": [\"38375821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"I-mfa is a cytosolic protein in mesangial cells that suppresses contractile function by decreasing TRPC1 channel protein abundance; I-mfa KO mice had lower GFR, augmented Ang II-induced mesangial cell contraction, and increased TRPC1 protein; overexpression of I-mfa blunted Ang II-stimulated contraction and Ca2+ entry; re-introduction of I-mfa into KO cells restored normal contractile response.\",\n      \"method\": \"I-mfa KO mice (GFR measurement, mesangial cell isolation), targeted siRNA nanoparticle delivery in vivo, single-cell contractility assay, Ca2+ imaging, Western blot, single-cell RNA sequencing, pharmacological TRPC1 inhibition\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — KO mouse in vivo phenotype plus cell-level gain/loss-of-function with rescue, Ca2+ imaging, and pharmacological epistasis providing multiple orthogonal lines\",\n      \"pmids\": [\"39446484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-128 directly targets MDFI (confirmed by luciferase assay); miR-128 overexpression reduced MDFI mRNA and protein levels and promoted cardiomyocyte apoptosis, while MDFI upregulation enhanced cardiomyocyte proliferation; elevated miR-128 upregulated Wnt1 and β-catenin, whereas increased MDFI inhibited these expressions, placing MDFI as a negative regulator of the Wnt1/β-catenin pathway in cardiomyocytes.\",\n      \"method\": \"Luciferase reporter assay, qPCR, Western blot, MTT, transwell, immunohistochemistry, echocardiography in mouse HF model\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — validated miRNA-target interaction plus in vivo functional assays, single lab\",\n      \"pmids\": [\"39046458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MDFI (and MDFIC) regulate endogenous PIEZO channel currents in non-sensory cell types; MDFI physically binds PIEZO1 and PIEZO2, alters their mechanosensitivity and inactivation kinetics (converting them to high-threshold slowly inactivating mechanoreceptors); cryo-EM revealed a conserved binding pocket in the pore modules of both PIEZO1 and PIEZO2 mediated by the post-translationally modified distal C-termini of MDFI-family proteins.\",\n      \"method\": \"Electrophysiology (endogenous PIEZO current recording), cryo-EM structure determination, physical binding assays, mutagenesis of binding interface\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — cryo-EM structure plus electrophysiology plus mutagenesis providing orthogonal mechanistic validation\",\n      \"pmids\": [\"bio_10.1101_2025.10.26.684595\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MDFI (I-mfa) is a multifunctional cytosolic/nuclear regulatory protein that acts primarily as a transcriptional repressor by binding and sequestering bHLH myogenic regulatory factors (MyoD family), Zic-family zinc finger proteins, LEF/TCF transcription factors, and SERTA-domain proteins — in each case inhibiting their nuclear import and/or transcriptional activity — while also interacting with the Axin/beta-catenin/Wnt signaling complex, modulating P-TEFb (cyclin T1/T2)-dependent transcription elongation, suppressing mesangial cell contractility by reducing TRPC1 channel abundance, regulating megakaryocyte differentiation via JNK/ERK signaling, and functioning as an auxiliary subunit of PIEZO1/2 mechanosensitive channels by binding a conserved pore-module pocket through its post-translationally modified C-terminus to alter mechanosensitivity and inactivation kinetics.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"MDFI (I-mfa) is a cytosolic/nuclear regulatory protein that controls cell differentiation programs principally by binding and sequestering specific DNA-binding transcription factors, thereby restraining their nuclear import and transcriptional output [#0, #4]. Its founding activity is inhibition of MyoD-family bHLH myogenic regulators: MDFI binds these factors, blocks their nuclear import and DNA binding, and suppresses their transcriptional activity [#0]. This repression is relieved by canonical Wnt signaling, in which beta-catenin competes with MDFI for binding to myogenic factors and to LEF/TCF, switching MDFI between an MRF-bound and a beta-catenin-bound state to initiate myogenesis [#5, #6]. MDFI integrates broadly with Wnt/beta-catenin signaling, interacting with the Axin complex to stabilize cytosolic beta-catenin and directly inhibiting Tcf3 and LEF transcriptional activity [#2, #3]. Through its conserved C-terminal I-mfa domain it engages additional transcriptional regulators including Zic-family zinc finger proteins, SERTA-domain proteins, and the P-TEFb cyclins T1/T2, in each case repressing their activity [#4, #7, #8]. Beyond transcriptional control, MDFI shapes muscle fiber-type identity by driving fast-to-slow conversion through calcium signaling, CaMKK2/AMPK activation, and mitochondrial biogenesis [#13, #14], governs megakaryocyte lineage commitment via JNK/ERK signaling [#15], suppresses mesangial cell contractility by reducing TRPC1 channel abundance [#17], and acts as an auxiliary subunit of PIEZO1/2 mechanosensitive channels, binding a conserved pore-module pocket through its post-translationally modified distal C-terminus to convert them into high-threshold, slowly inactivating mechanoreceptors [#19]. In cancer, MDFI promotes colorectal cancer cell growth, in part through interactions with LAMB3 and ITGB4 and AKT pathway activation [#16]. Mouse knockout studies establish in vivo requirements in trophoblast giant cell differentiation and placental development [#1], megakaryopoiesis [#15], and glomerular mesangial function [#17].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Established MDFI's founding molecular activity: how a single protein restrains myogenic commitment by directly inhibiting bHLH regulators at the level of nuclear access and DNA binding.\",\n      \"evidence\": \"Protein interaction and transcription assays plus targeted gene deletion in mice\",\n      \"pmids\": [\"9799236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Selectivity rule unclear \\u2014 inhibits Mash2 but not Hand1\", \"Structural basis of bHLH sequestration not defined\"]\n    },\n    {\n      \"year\": 1998,\n      \"claim\": \"Defined an in vivo developmental requirement, showing MDFI is needed for trophoblast giant cell differentiation and normal placental development.\",\n      \"evidence\": \"KO mouse placental phenotype with Rcho-1 overexpression\",\n      \"pmids\": [\"9799236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Transcription-factor target driving the trophoblast phenotype not pinpointed\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Extended MDFI's repressor reach to the Wnt pathway by showing it inhibits Tcf3 DNA binding and beta-catenin-dependent axis specification.\",\n      \"evidence\": \"Xenopus ectopic expression, reporter assays, beta-catenin epistasis\",\n      \"pmids\": [\"11238923\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether inhibition is direct on Tcf3 or via beta-catenin not fully resolved\", \"Single-organism developmental readout\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Mapped the C-terminal I-mfa domain as the Axin-interaction module and showed MDFI stabilizes cytosolic beta-catenin while also binding LEF directly.\",\n      \"evidence\": \"In vivo Co-IP, domain mapping, reporter assays\",\n      \"pmids\": [\"12192039\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Net effect on Wnt output context-dependent (stabilizes beta-catenin yet inhibits TCF reporters)\", \"Single lab\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Generalized the sequestration mechanism beyond bHLH factors by showing MDFI blocks nuclear import of Zic family zinc finger proteins.\",\n      \"evidence\": \"Co-IP, subcellular localization, reporter assays in cultured cells\",\n      \"pmids\": [\"15207726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo relevance of Zic regulation not tested\", \"Single lab\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Resolved the regulatory logic: beta-catenin competes with MDFI for binding to myogenic factors and LEF-1, providing a Wnt-responsive switch that relieves MDFI repression and triggers differentiation.\",\n      \"evidence\": \"Reciprocal Co-IP, siRNA, reporter and differentiation assays in P19/cell lines\",\n      \"pmids\": [\"16301527\", \"17090604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Stoichiometry and affinity of competing complexes not quantified\", \"Endogenous switch dynamics in primary myogenesis untested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identified MDFI as a P-TEFb interactor and substrate, linking it to transcription elongation control and viral transactivation.\",\n      \"evidence\": \"Yeast two-hybrid, Co-IP, domain mapping, HIV-1 promoter reporter assays\",\n      \"pmids\": [\"17289077\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Cell-type-specific basis of P-TEFb inhibition unexplained\", \"Functional consequence of MDFI phosphorylation by P-TEFb unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Expanded the I-mfa-domain interactome to SERTA-domain proteins, with downstream effects on the SEI-1/DP-1 complex and Fbxw7 expression.\",\n      \"evidence\": \"Co-IP, domain-mapping mutagenesis, reporter assays, qPCR\",\n      \"pmids\": [\"21664411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Physiological pathway in which SERTA regulation operates not defined\", \"Single lab\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed MDFI directly binds HTLV-1 Tax and represses Tax-dependent LTR and NF-kB transactivation, indicating an antiviral transcriptional role.\",\n      \"evidence\": \"In vitro binding and reporter assays, Co-IP\",\n      \"pmids\": [\"26469549\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Endogenous effect on viral replication not assessed\", \"Single lab\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Repositioned MDFI as a positive driver of myogenic differentiation and fast-to-slow fiber conversion, implicating calcium signaling as the dominant affected pathway.\",\n      \"evidence\": \"CRISPR overexpression stable line, RNA-seq, qPCR, Western blot, immunofluorescence\",\n      \"pmids\": [\"33553177\", \"31212688\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Reconciliation of positive differentiation role with earlier inhibitory bHLH model unresolved\", \"Context dependence on cell line and dose\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Dissected the fiber-type mechanism, showing MDFI raises intracellular calcium via ER IP3R/RYR channels and activates CaMKK2/AMPK to drive mitochondrial biogenesis and aerobic metabolism.\",\n      \"evidence\": \"Gain/loss-of-function in C2C12, calcium imaging with channel inhibitors, Western blot\",\n      \"pmids\": [\"37307704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How MDFI couples to ER calcium channels mechanistically unknown\", \"In vivo fiber-type effect not shown\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Established a cell-intrinsic role in megakaryocyte lineage commitment, with KO mice showing reduced platelets and altered progenitor balance linked to prolonged JNK/ERK signaling.\",\n      \"evidence\": \"KO mice, shRNA knockdown and overexpression in K562, phospho-JNK/ERK Western blot\",\n      \"pmids\": [\"37267696\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Direct molecular target controlling JNK/ERK kinetics not identified\", \"Single lab\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Defined a non-transcriptional cytosolic role: MDFI suppresses mesangial cell contractility by lowering TRPC1 channel abundance, with KO mice showing impaired glomerular function rescued by re-expression.\",\n      \"evidence\": \"KO mice with GFR, in vivo siRNA nanoparticle delivery, single-cell contractility, Ca2+ imaging, scRNA-seq, pharmacological TRPC1 inhibition\",\n      \"pmids\": [\"39446484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MDFI reduces TRPC1 protein not defined\", \"Relationship to MDFI's transcriptional functions unclear\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Implicated MDFI in colorectal cancer growth and chemoresistance through direct binding to LAMB3 and ITGB4 and AKT activation, and as a Wnt1/beta-catenin repressor in cardiomyocytes.\",\n      \"evidence\": \"Co-IP, lenti-overexpression/shRNA, proliferation and chemosensitivity assays; miR-128 luciferase and HF mouse model\",\n      \"pmids\": [\"38375821\", \"39046458\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Tissue-specific switch between oncogenic and Wnt-repressive roles unexplained\", \"Single lab per context\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Revealed a structural mechanism by which MDFI acts as a PIEZO auxiliary subunit, binding a conserved pore-module pocket via its modified C-terminus to retune mechanosensitivity and inactivation.\",\n      \"evidence\": \"Cryo-EM structure, electrophysiology of endogenous PIEZO currents, binding-interface mutagenesis (preprint)\",\n      \"pmids\": [\"bio_10.1101_2025.10.26.684595\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Preprint, not yet peer-reviewed\", \"Physiological contexts where MDFI tunes PIEZO function in vivo not established\", \"Identity of the C-terminal modification not detailed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How a single C-terminal I-mfa domain coordinates such diverse partners \\u2014 nuclear transcription factors versus plasma-membrane PIEZO and TRPC channels \\u2014 and what determines its tissue-specific deployment remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unifying structural model across nuclear and channel partners\", \"Determinants of cytosolic vs nuclear partitioning unknown\", \"Tissue-specific functional switching mechanism undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 4, 8]},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [5, 6, 17, 19]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [0, 4, 5]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [3, 17]},\n      {\"term_id\": \"GO:0005634\", \"supporting_discovery_ids\": [0, 8]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 5, 6]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [0, 1, 13]},\n      {\"term_id\": \"R-HSA-74160\", \"supporting_discovery_ids\": [0, 7, 8]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\"CTNNB1\", \"AXIN1\", \"LEF1\", \"TCF7L1\", \"ZIC2\", \"CCNT1\", \"PIEZO1\", \"PIEZO2\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":8,"faith_total":8,"faith_pct":100.0}}