{"gene":"MDFI","run_date":"2026-04-28T18:30:28","timeline":{"discoveries":[{"year":1998,"finding":"I-mfa (MDFI) binds to MyoD family bHLH transcription factors, inhibits their transcriptional activity, and blocks their nuclear import and DNA binding. I-mfa also interacts with the bHLH protein Mash2 and inhibits its transcriptional activity, but does not interfere with Hand1 activity, demonstrating selectivity among bHLH proteins.","method":"Cell culture overexpression, nuclear import assays, transcriptional reporter assays, targeted gene deletion in mice with placental and skeletal phenotypes","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (KO mice with defined phenotype, transcriptional assays, nuclear localization studies), replicated across labs","pmids":["9799236"],"is_preprint":false},{"year":1998,"finding":"I-mfa is required for placental trophoblast giant cell differentiation; targeted deletion causes embryonic lethality with placental defects, and overexpression in Rcho-1 trophoblast stem cells induces differentiation into giant cells.","method":"Targeted gene deletion in mice, overexpression in rat trophoblast stem cells, in situ hybridization","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 — clean KO with defined cellular phenotype plus gain-of-function validation","pmids":["9799236"],"is_preprint":false},{"year":2001,"finding":"I-mfa inhibits the activity and DNA binding of the HMG box transcription factor Tcf3 (XTcf3), and ectopic expression of I-mfa in Xenopus embryos inhibited dorsal axis specification and Tcf3/beta-catenin-regulated gene expression (siamois, Xnr3), placing I-mfa as a regulator of both Wnt signaling and bHLH proteins.","method":"Ectopic expression in Xenopus embryos, transcriptional reporter assays, DNA-binding assays","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — epistasis in Xenopus embryo model with multiple downstream readouts","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-reporter constructs. Both I-mfa and HIC decrease Axin-mediated JNK activation.","method":"Co-immunoprecipitation, transcriptional reporter assays, dominant-negative domain experiments","journal":"Molecular and cellular biology","confidence":"High","confidence_rationale":"Tier 2 — reciprocal Co-IP with functional reporter validation, multiple pathway readouts","pmids":["12192039"],"is_preprint":false},{"year":2004,"finding":"I-mfa physically interacts with the amino-terminal domain of Zic family proteins (Zic1-3) and inhibits their nuclear import in cultured cells, thereby inhibiting Zic-mediated transcriptional activation.","method":"Yeast two-hybrid screen, co-immunoprecipitation, nuclear localization assays, transcriptional reporter assays in cultured cells","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — Co-IP plus functional nuclear localization and transcription assays, single lab","pmids":["15207726"],"is_preprint":false},{"year":2005,"finding":"Beta-catenin directly interacts with I-mfa (MDFI), and this interaction (enhanced by Wnt3a) attenuates I-mfa binding to myogenic regulatory factors (MRFs), relieving I-mfa-mediated transcriptional suppression and cytosolic sequestration of MRFs to initiate myogenesis in P19 cells.","method":"Co-immunoprecipitation, siRNA knockdown, transcriptional reporter assays, P19 cell myogenic differentiation model","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (Co-IP, siRNA, reporters, differentiation assay) in a single study","pmids":["16301527"],"is_preprint":false},{"year":2006,"finding":"I-mfa suppresses myogenesis by inhibiting TCF/LEF-1, and canonical Wnt signaling relieves I-mfa-mediated suppression of LEF-1 by elevating beta-catenin levels which compete with LEF-1 for I-mfa binding; knockdown of endogenous I-mfa mimics canonical Wnt treatment in P19 cells.","method":"siRNA knockdown, co-immunoprecipitation competition assays, transcriptional reporter assays, dominant-negative LEF-1 rescue","journal":"Journal of cell science","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods with epistasis validation (dominant-negative rescue), moderate-strong evidence","pmids":["17090604"],"is_preprint":false},{"year":2007,"finding":"I-mfa interacts with cyclin T1 and cyclin T2 (P-TEFb subunits) through its I-mfa domain at two binding sites (the histidine-rich regulatory domain and a lysine/arginine-rich motif); I-mfa 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, in vitro binding assays, transcriptional reporter assays, phosphorylation assays","journal":"Journal of molecular biology","confidence":"High","confidence_rationale":"Tier 1-2 — in vitro and in vivo binding with domain mapping and functional transcription assays","pmids":["17289077"],"is_preprint":false},{"year":2011,"finding":"I-mfa interacts with SERTA domain proteins (SEI-1, SEI-2, SEI-3, SERTAD3, SERTAD4) through its I-mfa domain in vivo, affects intracellular localization of I-mfa, and represses the intrinsic transcriptional activities of SEI-1, SEI-2, and SERTAD3; I-mfa also decreases the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA levels.","method":"Co-immunoprecipitation, transcriptional reporter assays, Western blot, qRT-PCR","journal":"Biochimie","confidence":"Medium","confidence_rationale":"Tier 2-3 — multiple Co-IPs with functional assays, single lab","pmids":["21664411"],"is_preprint":false},{"year":2015,"finding":"I-mfa (MDFI) directly interacts in vitro and in vivo with HTLV-1 Tax protein through its I-mfa domain, and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs.","method":"In vitro binding assays, co-immunoprecipitation, transcriptional reporter assays in COS-1, Jurkat, and HTLV-1-infected T cells","journal":"Virology","confidence":"Medium","confidence_rationale":"Tier 2 — in vitro plus in vivo binding with functional reporters, single lab","pmids":["26469549"],"is_preprint":false},{"year":2018,"finding":"MDFI promotes pig muscle satellite cell (PSC) proliferation and inhibits PSC differentiation in vitro; miR-27b targets the MDFI 3'UTR directly (validated by dual-luciferase reporter assay) and promotes PSC myogenesis by suppressing MDFI expression. In vivo, interfering with MDFI expression promotes mouse muscle regeneration after injury.","method":"Dual-luciferase reporter assay, siRNA/miRNA transfection, EdU staining, qRT-PCR, Western blot, H&E staining in vivo","journal":"Cellular physiology and biochemistry","confidence":"Medium","confidence_rationale":"Tier 2 — direct target validation by luciferase + in vitro and in vivo functional assays","pmids":["29734192"],"is_preprint":false},{"year":2020,"finding":"MDFI interacts with the histone demethylase JMJD1A in colorectal cancer cells; MDFI stimulates and MDFIC inhibits growth of HCT116 cells. JMJD1A influences transcription of several genes also regulated by MDFI or MDFIC.","method":"Co-immunoprecipitation, cell growth assays, gene expression analysis","journal":"Scientific reports","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with functional cell growth readout, single lab","pmids":["32457453"],"is_preprint":false},{"year":2021,"finding":"MDFI overexpression in C2C12 cells promotes myoblast differentiation by upregulating MyoD, Myogenin, and Myosin expression, and promotes fast-to-slow-twitch muscle fiber transformation via a pathway involving MyoD, CaMK2b, and downstream metabolic genes (Pgc1a, Pdk4, Cs, Cox4, etc.).","method":"CRISPR/Cas9-mediated MDFI overexpression, RNA-seq, qRT-PCR, Western blot, immunofluorescence","journal":"Frontiers in cell and developmental biology","confidence":"Medium","confidence_rationale":"Tier 2 — clean OE model with RNA-seq plus experimental validation of pathway components","pmids":["33553177"],"is_preprint":false},{"year":2023,"finding":"MDFI promotes fast-to-slow muscle fiber type transformation by activating the calcium signaling pathway: elevated MDFI stimulates CaMKK2 and AMPK phosphorylation, promotes mitochondrial biogenesis and aerobic metabolism, increases intracellular calcium via IP3R and RYR channels from the ER, driving conversion of C2C12 cells from fast glycolytic to slow oxidative type.","method":"Lipofection-based OE/knockdown in C2C12, immunofluorescence, qPCR, Western blot, pharmacological channel inhibition (IP3R and RYR inhibitors)","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 — multiple methods with pharmacological rescue, single lab","pmids":["37307704"],"is_preprint":false},{"year":2023,"finding":"I-MFA (MDFI) plays a cell-intrinsic role in megakaryocyte lineage commitment and terminal differentiation: I-MFA knockout mice show reduced platelets, reduced MK/erythrocyte progenitors, and increased myeloid progenitors; shRNA knockdown of I-MFA in K562 cells reduces PMA-induced MK differentiation with prolonged phospho-JNK and phospho-ERK signaling, while I-MFA overexpression promotes MK differentiation.","method":"Knockout mouse analysis (bone marrow, blood counts), shRNA knockdown in K562 cells, overexpression, flow cytometry, phospho-kinase Western blot","journal":"Blood cells, molecules & diseases","confidence":"Medium","confidence_rationale":"Tier 2 — KO mouse with defined phenotype plus gain/loss of function in cell line with signaling readout","pmids":["37267696"],"is_preprint":false},{"year":2024,"finding":"I-mfa (MDFI) promotes glomerular filtration rate by suppressing contractile function of mesangial cells through decreasing TRPC1 channel protein abundance, thereby reducing angiotensin II-stimulated calcium entry and cell contraction. Knockdown of I-mfa in mesangial cells decreases GFR, and I-mfa KO mice show significantly lower GFR with increased TRPC1 protein.","method":"I-mfa KO mice with transdermal GFR measurement, mesangial cell-targeted siRNA nanoparticle delivery, single-cell RNA sequencing, Western blot, Ca2+ imaging, contractility assay, TRPC1 inhibitor (Pico145) pharmacology","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (KO, targeted KD, OE, Ca2+ imaging, pharmacology) with clear mechanism","pmids":["39446484"],"is_preprint":false},{"year":2024,"finding":"MDFI directly interacts with LAMB3 and ITGB4 in colorectal cancer cells (validated by co-immunoprecipitation), upregulates the AKT signaling pathway through this interaction, enhances CRC cell proliferation, and reduces sensitivity to oxaliplatin and fluorouracil.","method":"Co-immunoprecipitation, lentiviral overexpression, shRNA knockdown, colony formation assay, CCK8 assay, single-cell RNA sequencing data analysis","journal":"Cancer biology & therapy","confidence":"Medium","confidence_rationale":"Tier 3 — Co-IP with functional assays, single lab","pmids":["38375821"],"is_preprint":false},{"year":2024,"finding":"miR-128 directly targets MDFI (validated by luciferase assay), reduces MDFI expression, promotes cardiomyocyte apoptosis and attenuates proliferation; MDFI upregulation inhibits Wnt1 and beta-catenin expression, while miR-128 elevation upregulates these, placing MDFI as a negative regulator of the Wnt1/beta-catenin pathway in cardiomyocytes.","method":"Luciferase reporter assay, qPCR, Western blot, MTT assay, transwell assay, echocardiography, histology","journal":"Journal of cellular and molecular medicine","confidence":"Medium","confidence_rationale":"Tier 3 — direct target validation plus functional and signaling assays, single lab","pmids":["39046458"],"is_preprint":false},{"year":2025,"finding":"MDFI (and MDFIC) physically bind to PIEZO1 and PIEZO2 channels through a conserved binding pocket in the pore modules of PIEZO1/2, mediated by the post-translationally modified distal C-termini of MyoD-family inhibitor proteins. MDFI regulates endogenous PIEZO channel currents in non-sensory cell types, altering mechanosensitivity and inactivation kinetics, converting PIEZO channels into high-threshold slowly inactivating mechanoreceptors.","method":"Cryo-EM structural determination, electrophysiology (patch clamp), co-immunoprecipitation/pulldown, overexpression in multiple cell types","journal":"bioRxiv","confidence":"High","confidence_rationale":"Tier 1 — cryo-EM structure with functional validation by electrophysiology, multiple cell types","pmids":["bio_10.1101_2025.10.26.684595"],"is_preprint":true}],"current_model":"MDFI (I-mfa) is a multifunctional cysteine-rich domain protein that acts primarily as a transcriptional repressor by binding and sequestering bHLH myogenic regulatory factors (blocking their nuclear import), TCF/LEF transcription factors, Zic proteins, and SERTA domain proteins in the cytoplasm; it modulates Wnt/beta-catenin signaling through interaction with the Axin complex, LEF-1, and beta-catenin; it regulates P-TEFb (cyclin T1/T2) and viral transactivators (Tat, Tax); it promotes fast-to-slow muscle fiber transformation via calcium/CaMKK2/AMPK signaling; it suppresses mesangial cell contractility by reducing TRPC1 channel abundance to promote glomerular filtration; and its I-mfa domain structurally docks into a conserved binding pocket in the pore modules of PIEZO1/2 mechanosensitive channels to alter their inactivation kinetics and mechanosensitivity."},"narrative":{"teleology":[{"year":1998,"claim":"Establishing MDFI as a selective inhibitor of bHLH myogenic factors and a required developmental gene resolved how myogenic transcription factors are held inactive in the cytoplasm and revealed an essential role in trophoblast differentiation.","evidence":"Targeted gene deletion in mice (embryonic lethality with placental defects), nuclear import assays, transcriptional reporters, and gain-of-function in trophoblast stem cells","pmids":["9799236"],"confidence":"High","gaps":["Mechanism by which MDFI blocks nuclear import not structurally defined","Selectivity determinants among bHLH proteins not mapped at residue level","Whether MDFI loss-of-function placental phenotype is solely due to Mash2 inhibition unclear"]},{"year":2001,"claim":"Demonstrating that MDFI inhibits TCF3/β-catenin-dependent transcription and Wnt target genes in Xenopus expanded its role from a myogenic repressor to a Wnt signaling modulator.","evidence":"Ectopic expression in Xenopus embryos with dorsal axis and Wnt target gene readouts, DNA-binding assays","pmids":["11238923"],"confidence":"High","gaps":["Whether MDFI acts on TCF/LEF in mammalian Wnt contexts not yet tested","Relative contribution of bHLH vs. TCF inhibition to embryonic phenotype unknown"]},{"year":2002,"claim":"Identifying MDFI as an interactor of the Axin destruction complex and a direct LEF-1 binding partner established it as a multi-node regulator within canonical Wnt signaling and JNK pathways.","evidence":"Reciprocal co-immunoprecipitation, TCF/LEF reporter assays, Axin-mediated β-catenin and JNK readouts","pmids":["12192039"],"confidence":"High","gaps":["Whether MDFI modulates Axin complex stability or only signaling output not resolved","Stoichiometry and competition between MDFI–Axin and MDFI–LEF interactions unknown"]},{"year":2004,"claim":"Finding that MDFI binds Zic family zinc-finger proteins and blocks their nuclear import generalized its mechanism of cytoplasmic sequestration beyond bHLH and HMG-box factors.","evidence":"Yeast two-hybrid, co-immunoprecipitation, nuclear localization and reporter assays","pmids":["15207726"],"confidence":"Medium","gaps":["Single-lab observation not independently replicated","In vivo relevance for Zic-dependent processes (e.g., neural crest) not tested"]},{"year":2005,"claim":"Showing that β-catenin directly binds MDFI and competes with MRFs for MDFI binding revealed how Wnt signaling derepresses myogenic transcription factors to initiate myogenesis.","evidence":"Co-immunoprecipitation, siRNA knockdown, and myogenic differentiation in P19 cells","pmids":["16301527"],"confidence":"High","gaps":["Binding site on β-catenin for MDFI not structurally mapped","Whether this competition operates in adult muscle stem cells in vivo not established"]},{"year":2006,"claim":"Epistasis experiments demonstrated that canonical Wnt signaling relieves MDFI-mediated suppression of LEF-1 by elevating β-catenin, integrating MDFI into a coherent Wnt-to-myogenesis relay.","evidence":"siRNA knockdown mimicking Wnt treatment, dominant-negative LEF-1 rescue, competition co-immunoprecipitation in P19 cells","pmids":["17090604"],"confidence":"High","gaps":["Whether MDFI–LEF-1 interaction is regulated by post-translational modifications unknown"]},{"year":2007,"claim":"Identifying P-TEFb (cyclin T1/T2) as MDFI partners and showing MDFI inhibits HIV-1 Tat/P-TEFb-dependent transcription extended MDFI function to transcription elongation control and viral gene regulation.","evidence":"Yeast two-hybrid, in vitro binding, co-immunoprecipitation, phosphorylation assays, HIV-1 promoter reporters","pmids":["17289077"],"confidence":"High","gaps":["Physiological significance of MDFI–P-TEFb interaction in non-viral contexts unknown","Whether MDFI regulates global Pol II elongation not tested"]},{"year":2011,"claim":"Demonstrating that MDFI binds and represses SERTA domain proteins and affects Fbxw7 mRNA levels expanded the repertoire of MDFI targets to cell cycle regulators.","evidence":"Co-immunoprecipitation, transcriptional reporters, qRT-PCR in cell lines","pmids":["21664411"],"confidence":"Medium","gaps":["Single-lab study","Mechanism linking MDFI–SEI interaction to Fbxw7 mRNA regulation not defined","In vivo cell cycle consequences not tested"]},{"year":2015,"claim":"Showing MDFI directly binds HTLV-1 Tax and represses Tax-dependent HTLV-1 LTR and NF-κB transcription broadened its antiviral repressive capacity beyond HIV-1.","evidence":"In vitro binding, co-immunoprecipitation, reporters in COS-1, Jurkat, and HTLV-1-infected T cells","pmids":["26469549"],"confidence":"Medium","gaps":["Single-lab finding","Whether MDFI affects HTLV-1 viral replication or latency in vivo unknown"]},{"year":2021,"claim":"Demonstrating that MDFI overexpression drives fast-to-slow muscle fiber transformation via MyoD/CaMK2b and downstream metabolic gene induction established MDFI as a fiber-type specification factor.","evidence":"CRISPR/Cas9-mediated overexpression in C2C12, RNA-seq, qRT-PCR, Western blot","pmids":["33553177"],"confidence":"Medium","gaps":["In vivo fiber-type switching not demonstrated","Mechanism connecting MDFI to CaMK2b activation not resolved"]},{"year":2023,"claim":"Identifying that MDFI elevates intracellular calcium via IP3R/RYR channels and activates CaMKK2/AMPK signaling provided a mechanistic pathway for MDFI-driven slow-fiber transformation and mitochondrial biogenesis.","evidence":"Overexpression/knockdown in C2C12 with pharmacological IP3R and RYR inhibitors, Western blot, immunofluorescence","pmids":["37307704"],"confidence":"Medium","gaps":["How MDFI increases ER calcium release mechanistically unresolved","Not replicated in primary muscle fibers or in vivo"]},{"year":2023,"claim":"Showing that MDFI knockout mice have reduced platelets and impaired megakaryocyte progenitors, with altered JNK/ERK signaling, revealed a non-muscle developmental role for MDFI in hematopoietic lineage commitment.","evidence":"Knockout mouse bone marrow analysis, shRNA knockdown and overexpression in K562 cells, flow cytometry, phospho-kinase Western blot","pmids":["37267696"],"confidence":"Medium","gaps":["Whether MDFI acts via bHLH sequestration or an independent pathway in megakaryopoiesis not determined","Conditional KO not performed to exclude non-cell-autonomous effects"]},{"year":2024,"claim":"Establishing that MDFI suppresses mesangial cell contractility by reducing TRPC1 channel abundance and calcium entry defined a kidney-specific function regulating glomerular filtration rate.","evidence":"I-mfa KO mice with transdermal GFR measurement, siRNA nanoparticle delivery to mesangial cells, scRNA-seq, calcium imaging, TRPC1 inhibitor rescue","pmids":["39446484"],"confidence":"High","gaps":["Mechanism by which MDFI reduces TRPC1 protein levels (transcriptional vs. post-translational) not resolved","Whether MDFI regulates other TRP channels in kidney not explored"]},{"year":null,"claim":"Key open questions include: (1) how MDFI's I-mfa domain structurally engages its diverse transcription factor and channel targets, (2) whether the cryo-EM-resolved PIEZO channel interaction operates in vivo to tune mechanosensation, (3) what post-translational modifications regulate MDFI partner selectivity, and (4) whether MDFI's numerous binding activities are coordinated or context-exclusive in specific tissues.","evidence":"","pmids":[],"confidence":"Low","gaps":["No high-resolution structure of MDFI alone or in complex with transcription factors","In vivo validation of PIEZO channel modulation pending peer review","Tissue-specific regulation of MDFI activity largely uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,2,3,4,5,6,7,8,9]},{"term_id":"GO:0140110","term_label":"transcription regulator activity","supporting_discovery_ids":[0,2,6,7,9]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[0,4,5]}],"pathway":[{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[2,3,5,6,17]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[1,14]},{"term_id":"R-HSA-397014","term_label":"Muscle contraction","supporting_discovery_ids":[12,13]}],"complexes":[],"partners":["MYOD1","LEF1","CTNNB1","AXIN1","CCNT1","ZIC1","TRPC1","JMJD1A"],"other_free_text":[]},"mechanistic_narrative":"MDFI (I-mfa) is a cysteine-rich transcriptional repressor that functions broadly as a cytoplasmic sequestration factor for multiple classes of transcription factors and as a modulator of calcium-dependent signaling in muscle and kidney. MDFI binds and inhibits bHLH myogenic regulatory factors (MyoD family), TCF/LEF transcription factors, and Zic proteins by blocking their nuclear import and DNA binding, thereby regulating myogenesis and Wnt/β-catenin signaling; β-catenin competes with MRFs for MDFI binding, and canonical Wnt signaling relieves MDFI-mediated repression to initiate myogenic differentiation [PMID:9799236, PMID:11238923, PMID:16301527, PMID:17090604]. Beyond transcription factor sequestration, MDFI interacts with cyclin T1/T2 (P-TEFb subunits) and viral transactivators to repress HIV-1 and HTLV-1 promoter activity [PMID:17289077, PMID:26469549], promotes fast-to-slow muscle fiber transformation through CaMKK2/AMPK-dependent calcium signaling [PMID:37307704], and suppresses mesangial cell contractility by reducing TRPC1 channel abundance to regulate glomerular filtration rate [PMID:39446484]. MDFI is also required for placental trophoblast giant cell differentiation and megakaryocyte lineage commitment, with knockout mice exhibiting embryonic lethality (placental) and thrombocytopenia, respectively [PMID:9799236, PMID:37267696]."},"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":60,"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":"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":"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":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":"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":"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":23,"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":"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":"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":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":"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":"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":2,"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":"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":"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":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},{"pmid":null,"id":"bio_10.1101_2024.05.06.592657","title":"Screening and identification of key biomarkers associated with endometriosis using bioinformatics and next generation sequencing data analysis","date":"2024-05-08","source":"bioRxiv","url":"https://doi.org/10.1101/2024.05.06.592657","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":16935,"output_tokens":4618,"usd":0.060038},"stage2":{"model":"claude-opus-4-6","input_tokens":8093,"output_tokens":6555,"usd":0.30651},"total_usd":0.366548,"stage1_batch_id":"msgbatch_01Mgbec4vM6c9vXht7JVJzss","stage2_batch_id":"msgbatch_01JkuuH5RB2BdJNsgnhjbC6s","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 1998,\n      \"finding\": \"I-mfa (MDFI) binds to MyoD family bHLH transcription factors, inhibits their transcriptional activity, and blocks their nuclear import and DNA binding. I-mfa also interacts with the bHLH protein Mash2 and inhibits its transcriptional activity, but does not interfere with Hand1 activity, demonstrating selectivity among bHLH proteins.\",\n      \"method\": \"Cell culture overexpression, nuclear import assays, transcriptional reporter assays, targeted gene deletion in mice with placental and skeletal phenotypes\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KO mice with defined phenotype, transcriptional assays, nuclear localization studies), replicated across labs\",\n      \"pmids\": [\"9799236\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1998,\n      \"finding\": \"I-mfa is required for placental trophoblast giant cell differentiation; targeted deletion causes embryonic lethality with placental defects, and overexpression in Rcho-1 trophoblast stem cells induces differentiation into giant cells.\",\n      \"method\": \"Targeted gene deletion in mice, overexpression in rat trophoblast stem cells, in situ hybridization\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean KO with defined cellular phenotype plus gain-of-function validation\",\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 Tcf3 (XTcf3), and ectopic expression of I-mfa in Xenopus embryos inhibited dorsal axis specification and Tcf3/beta-catenin-regulated gene expression (siamois, Xnr3), placing I-mfa as a regulator of both Wnt signaling and bHLH proteins.\",\n      \"method\": \"Ectopic expression in Xenopus embryos, transcriptional reporter assays, DNA-binding assays\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — epistasis in Xenopus embryo model with multiple downstream readouts\",\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-reporter constructs. Both I-mfa and HIC decrease Axin-mediated JNK activation.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, dominant-negative domain experiments\",\n      \"journal\": \"Molecular and cellular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal Co-IP with functional reporter validation, multiple pathway readouts\",\n      \"pmids\": [\"12192039\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"I-mfa physically interacts with the amino-terminal domain of Zic family proteins (Zic1-3) and inhibits their nuclear import in cultured cells, thereby inhibiting Zic-mediated transcriptional activation.\",\n      \"method\": \"Yeast two-hybrid screen, co-immunoprecipitation, nuclear localization assays, transcriptional reporter assays in cultured cells\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — Co-IP plus functional nuclear localization and transcription assays, single lab\",\n      \"pmids\": [\"15207726\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Beta-catenin directly interacts with I-mfa (MDFI), and this interaction (enhanced by Wnt3a) attenuates I-mfa binding to myogenic regulatory factors (MRFs), relieving I-mfa-mediated transcriptional suppression and cytosolic sequestration of MRFs to initiate myogenesis in P19 cells.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, transcriptional reporter assays, P19 cell myogenic differentiation model\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (Co-IP, siRNA, reporters, differentiation assay) in a single study\",\n      \"pmids\": [\"16301527\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"I-mfa suppresses myogenesis by inhibiting TCF/LEF-1, and canonical Wnt signaling relieves I-mfa-mediated suppression of LEF-1 by elevating beta-catenin levels which compete with LEF-1 for I-mfa binding; knockdown of endogenous I-mfa mimics canonical Wnt treatment in P19 cells.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation competition assays, transcriptional reporter assays, dominant-negative LEF-1 rescue\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods with epistasis validation (dominant-negative rescue), moderate-strong evidence\",\n      \"pmids\": [\"17090604\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"I-mfa interacts with cyclin T1 and cyclin T2 (P-TEFb subunits) through its I-mfa domain at two binding sites (the histidine-rich regulatory domain and a lysine/arginine-rich motif); I-mfa 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, in vitro binding assays, transcriptional reporter assays, phosphorylation assays\",\n      \"journal\": \"Journal of molecular biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 — in vitro and in vivo binding with domain mapping and functional transcription assays\",\n      \"pmids\": [\"17289077\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"I-mfa interacts with SERTA domain proteins (SEI-1, SEI-2, SEI-3, SERTAD3, SERTAD4) through its I-mfa domain in vivo, affects intracellular localization of I-mfa, and represses the intrinsic transcriptional activities of SEI-1, SEI-2, and SERTAD3; I-mfa also decreases the SEI-1·DP-1 complex and endogenous Fbxw7 mRNA levels.\",\n      \"method\": \"Co-immunoprecipitation, transcriptional reporter assays, Western blot, qRT-PCR\",\n      \"journal\": \"Biochimie\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2-3 — multiple Co-IPs with functional assays, single lab\",\n      \"pmids\": [\"21664411\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"I-mfa (MDFI) directly interacts in vitro and in vivo with HTLV-1 Tax protein through its I-mfa domain, and represses Tax-dependent transactivation of HTLV-1 LTR and NF-κB reporter constructs.\",\n      \"method\": \"In vitro binding assays, co-immunoprecipitation, transcriptional reporter assays in COS-1, Jurkat, and HTLV-1-infected T cells\",\n      \"journal\": \"Virology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vitro plus in vivo binding with functional reporters, single lab\",\n      \"pmids\": [\"26469549\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"MDFI promotes pig muscle satellite cell (PSC) proliferation and inhibits PSC differentiation in vitro; miR-27b targets the MDFI 3'UTR directly (validated by dual-luciferase reporter assay) and promotes PSC myogenesis by suppressing MDFI expression. In vivo, interfering with MDFI expression promotes mouse muscle regeneration after injury.\",\n      \"method\": \"Dual-luciferase reporter assay, siRNA/miRNA transfection, EdU staining, qRT-PCR, Western blot, H&E staining in vivo\",\n      \"journal\": \"Cellular physiology and biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct target validation by luciferase + in vitro and in vivo functional assays\",\n      \"pmids\": [\"29734192\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"MDFI interacts with the histone demethylase JMJD1A in colorectal cancer cells; MDFI stimulates and MDFIC inhibits growth of HCT116 cells. JMJD1A influences transcription of several genes also regulated by MDFI or MDFIC.\",\n      \"method\": \"Co-immunoprecipitation, cell growth assays, gene expression analysis\",\n      \"journal\": \"Scientific reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional cell growth readout, single lab\",\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, Myogenin, and Myosin expression, and promotes fast-to-slow-twitch muscle fiber transformation via a pathway involving MyoD, CaMK2b, and downstream metabolic genes (Pgc1a, Pdk4, Cs, Cox4, etc.).\",\n      \"method\": \"CRISPR/Cas9-mediated MDFI overexpression, RNA-seq, qRT-PCR, Western blot, immunofluorescence\",\n      \"journal\": \"Frontiers in cell and developmental biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean OE model with RNA-seq plus experimental validation of pathway components\",\n      \"pmids\": [\"33553177\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"MDFI promotes fast-to-slow muscle fiber type transformation by activating the calcium signaling pathway: elevated MDFI stimulates CaMKK2 and AMPK phosphorylation, promotes mitochondrial biogenesis and aerobic metabolism, increases intracellular calcium via IP3R and RYR channels from the ER, driving conversion of C2C12 cells from fast glycolytic to slow oxidative type.\",\n      \"method\": \"Lipofection-based OE/knockdown in C2C12, immunofluorescence, qPCR, Western blot, pharmacological channel inhibition (IP3R and RYR inhibitors)\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple methods with pharmacological rescue, single lab\",\n      \"pmids\": [\"37307704\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"I-MFA (MDFI) plays a cell-intrinsic role in megakaryocyte lineage commitment and terminal differentiation: I-MFA knockout mice show reduced platelets, reduced MK/erythrocyte progenitors, and increased myeloid progenitors; shRNA knockdown of I-MFA in K562 cells reduces PMA-induced MK differentiation with prolonged phospho-JNK and phospho-ERK signaling, while I-MFA overexpression promotes MK differentiation.\",\n      \"method\": \"Knockout mouse analysis (bone marrow, blood counts), shRNA knockdown in K562 cells, overexpression, flow cytometry, phospho-kinase Western blot\",\n      \"journal\": \"Blood cells, molecules & diseases\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — KO mouse with defined phenotype plus gain/loss of function in cell line with signaling readout\",\n      \"pmids\": [\"37267696\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"I-mfa (MDFI) promotes glomerular filtration rate by suppressing contractile function of mesangial cells through decreasing TRPC1 channel protein abundance, thereby reducing angiotensin II-stimulated calcium entry and cell contraction. Knockdown of I-mfa in mesangial cells decreases GFR, and I-mfa KO mice show significantly lower GFR with increased TRPC1 protein.\",\n      \"method\": \"I-mfa KO mice with transdermal GFR measurement, mesangial cell-targeted siRNA nanoparticle delivery, single-cell RNA sequencing, Western blot, Ca2+ imaging, contractility assay, TRPC1 inhibitor (Pico145) pharmacology\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (KO, targeted KD, OE, Ca2+ imaging, pharmacology) with clear mechanism\",\n      \"pmids\": [\"39446484\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"MDFI directly interacts with LAMB3 and ITGB4 in colorectal cancer cells (validated by co-immunoprecipitation), upregulates the AKT signaling pathway through this interaction, enhances CRC cell proliferation, and reduces sensitivity to oxaliplatin and fluorouracil.\",\n      \"method\": \"Co-immunoprecipitation, lentiviral overexpression, shRNA knockdown, colony formation assay, CCK8 assay, single-cell RNA sequencing data analysis\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — Co-IP with functional assays, single lab\",\n      \"pmids\": [\"38375821\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"miR-128 directly targets MDFI (validated by luciferase assay), reduces MDFI expression, promotes cardiomyocyte apoptosis and attenuates proliferation; MDFI upregulation inhibits Wnt1 and beta-catenin expression, while miR-128 elevation upregulates these, placing MDFI as a negative regulator of the Wnt1/beta-catenin pathway in cardiomyocytes.\",\n      \"method\": \"Luciferase reporter assay, qPCR, Western blot, MTT assay, transwell assay, echocardiography, histology\",\n      \"journal\": \"Journal of cellular and molecular medicine\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 3 — direct target validation plus functional and signaling assays, single lab\",\n      \"pmids\": [\"39046458\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"MDFI (and MDFIC) physically bind to PIEZO1 and PIEZO2 channels through a conserved binding pocket in the pore modules of PIEZO1/2, mediated by the post-translationally modified distal C-termini of MyoD-family inhibitor proteins. MDFI regulates endogenous PIEZO channel currents in non-sensory cell types, altering mechanosensitivity and inactivation kinetics, converting PIEZO channels into high-threshold slowly inactivating mechanoreceptors.\",\n      \"method\": \"Cryo-EM structural determination, electrophysiology (patch clamp), co-immunoprecipitation/pulldown, overexpression in multiple cell types\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — cryo-EM structure with functional validation by electrophysiology, multiple cell types\",\n      \"pmids\": [\"bio_10.1101_2025.10.26.684595\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"MDFI (I-mfa) is a multifunctional cysteine-rich domain protein that acts primarily as a transcriptional repressor by binding and sequestering bHLH myogenic regulatory factors (blocking their nuclear import), TCF/LEF transcription factors, Zic proteins, and SERTA domain proteins in the cytoplasm; it modulates Wnt/beta-catenin signaling through interaction with the Axin complex, LEF-1, and beta-catenin; it regulates P-TEFb (cyclin T1/T2) and viral transactivators (Tat, Tax); it promotes fast-to-slow muscle fiber transformation via calcium/CaMKK2/AMPK signaling; it suppresses mesangial cell contractility by reducing TRPC1 channel abundance to promote glomerular filtration; and its I-mfa domain structurally docks into a conserved binding pocket in the pore modules of PIEZO1/2 mechanosensitive channels to alter their inactivation kinetics and mechanosensitivity.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"MDFI (I-mfa) is a cysteine-rich transcriptional repressor that functions broadly as a cytoplasmic sequestration factor for multiple classes of transcription factors and as a modulator of calcium-dependent signaling in muscle and kidney. MDFI binds and inhibits bHLH myogenic regulatory factors (MyoD family), TCF/LEF transcription factors, and Zic proteins by blocking their nuclear import and DNA binding, thereby regulating myogenesis and Wnt/β-catenin signaling; β-catenin competes with MRFs for MDFI binding, and canonical Wnt signaling relieves MDFI-mediated repression to initiate myogenic differentiation [PMID:9799236, PMID:11238923, PMID:16301527, PMID:17090604]. Beyond transcription factor sequestration, MDFI interacts with cyclin T1/T2 (P-TEFb subunits) and viral transactivators to repress HIV-1 and HTLV-1 promoter activity [PMID:17289077, PMID:26469549], promotes fast-to-slow muscle fiber transformation through CaMKK2/AMPK-dependent calcium signaling [PMID:37307704], and suppresses mesangial cell contractility by reducing TRPC1 channel abundance to regulate glomerular filtration rate [PMID:39446484]. MDFI is also required for placental trophoblast giant cell differentiation and megakaryocyte lineage commitment, with knockout mice exhibiting embryonic lethality (placental) and thrombocytopenia, respectively [PMID:9799236, PMID:37267696].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing MDFI as a selective inhibitor of bHLH myogenic factors and a required developmental gene resolved how myogenic transcription factors are held inactive in the cytoplasm and revealed an essential role in trophoblast differentiation.\",\n      \"evidence\": \"Targeted gene deletion in mice (embryonic lethality with placental defects), nuclear import assays, transcriptional reporters, and gain-of-function in trophoblast stem cells\",\n      \"pmids\": [\"9799236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MDFI blocks nuclear import not structurally defined\", \"Selectivity determinants among bHLH proteins not mapped at residue level\", \"Whether MDFI loss-of-function placental phenotype is solely due to Mash2 inhibition unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that MDFI inhibits TCF3/β-catenin-dependent transcription and Wnt target genes in Xenopus expanded its role from a myogenic repressor to a Wnt signaling modulator.\",\n      \"evidence\": \"Ectopic expression in Xenopus embryos with dorsal axis and Wnt target gene readouts, DNA-binding assays\",\n      \"pmids\": [\"11238923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MDFI acts on TCF/LEF in mammalian Wnt contexts not yet tested\", \"Relative contribution of bHLH vs. TCF inhibition to embryonic phenotype unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identifying MDFI as an interactor of the Axin destruction complex and a direct LEF-1 binding partner established it as a multi-node regulator within canonical Wnt signaling and JNK pathways.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, TCF/LEF reporter assays, Axin-mediated β-catenin and JNK readouts\",\n      \"pmids\": [\"12192039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MDFI modulates Axin complex stability or only signaling output not resolved\", \"Stoichiometry and competition between MDFI–Axin and MDFI–LEF interactions unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Finding that MDFI binds Zic family zinc-finger proteins and blocks their nuclear import generalized its mechanism of cytoplasmic sequestration beyond bHLH and HMG-box factors.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, nuclear localization and reporter assays\",\n      \"pmids\": [\"15207726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab observation not independently replicated\", \"In vivo relevance for Zic-dependent processes (e.g., neural crest) not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showing that β-catenin directly binds MDFI and competes with MRFs for MDFI binding revealed how Wnt signaling derepresses myogenic transcription factors to initiate myogenesis.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, and myogenic differentiation in P19 cells\",\n      \"pmids\": [\"16301527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding site on β-catenin for MDFI not structurally mapped\", \"Whether this competition operates in adult muscle stem cells in vivo not established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Epistasis experiments demonstrated that canonical Wnt signaling relieves MDFI-mediated suppression of LEF-1 by elevating β-catenin, integrating MDFI into a coherent Wnt-to-myogenesis relay.\",\n      \"evidence\": \"siRNA knockdown mimicking Wnt treatment, dominant-negative LEF-1 rescue, competition co-immunoprecipitation in P19 cells\",\n      \"pmids\": [\"17090604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MDFI–LEF-1 interaction is regulated by post-translational modifications unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identifying P-TEFb (cyclin T1/T2) as MDFI partners and showing MDFI inhibits HIV-1 Tat/P-TEFb-dependent transcription extended MDFI function to transcription elongation control and viral gene regulation.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, co-immunoprecipitation, phosphorylation assays, HIV-1 promoter reporters\",\n      \"pmids\": [\"17289077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological significance of MDFI–P-TEFb interaction in non-viral contexts unknown\", \"Whether MDFI regulates global Pol II elongation not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that MDFI binds and represses SERTA domain proteins and affects Fbxw7 mRNA levels expanded the repertoire of MDFI targets to cell cycle regulators.\",\n      \"evidence\": \"Co-immunoprecipitation, transcriptional reporters, qRT-PCR in cell lines\",\n      \"pmids\": [\"21664411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Mechanism linking MDFI–SEI interaction to Fbxw7 mRNA regulation not defined\", \"In vivo cell cycle consequences not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showing MDFI directly binds HTLV-1 Tax and represses Tax-dependent HTLV-1 LTR and NF-κB transcription broadened its antiviral repressive capacity beyond HIV-1.\",\n      \"evidence\": \"In vitro binding, co-immunoprecipitation, reporters in COS-1, Jurkat, and HTLV-1-infected T cells\",\n      \"pmids\": [\"26469549\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding\", \"Whether MDFI affects HTLV-1 viral replication or latency in vivo unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that MDFI overexpression drives fast-to-slow muscle fiber transformation via MyoD/CaMK2b and downstream metabolic gene induction established MDFI as a fiber-type specification factor.\",\n      \"evidence\": \"CRISPR/Cas9-mediated overexpression in C2C12, RNA-seq, qRT-PCR, Western blot\",\n      \"pmids\": [\"33553177\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo fiber-type switching not demonstrated\", \"Mechanism connecting MDFI to CaMK2b activation not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying that MDFI elevates intracellular calcium via IP3R/RYR channels and activates CaMKK2/AMPK signaling provided a mechanistic pathway for MDFI-driven slow-fiber transformation and mitochondrial biogenesis.\",\n      \"evidence\": \"Overexpression/knockdown in C2C12 with pharmacological IP3R and RYR inhibitors, Western blot, immunofluorescence\",\n      \"pmids\": [\"37307704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How MDFI increases ER calcium release mechanistically unresolved\", \"Not replicated in primary muscle fibers or in vivo\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showing that MDFI knockout mice have reduced platelets and impaired megakaryocyte progenitors, with altered JNK/ERK signaling, revealed a non-muscle developmental role for MDFI in hematopoietic lineage commitment.\",\n      \"evidence\": \"Knockout mouse bone marrow analysis, shRNA knockdown and overexpression in K562 cells, flow cytometry, phospho-kinase Western blot\",\n      \"pmids\": [\"37267696\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MDFI acts via bHLH sequestration or an independent pathway in megakaryopoiesis not determined\", \"Conditional KO not performed to exclude non-cell-autonomous effects\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Establishing that MDFI suppresses mesangial cell contractility by reducing TRPC1 channel abundance and calcium entry defined a kidney-specific function regulating glomerular filtration rate.\",\n      \"evidence\": \"I-mfa KO mice with transdermal GFR measurement, siRNA nanoparticle delivery to mesangial cells, scRNA-seq, calcium imaging, TRPC1 inhibitor rescue\",\n      \"pmids\": [\"39446484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MDFI reduces TRPC1 protein levels (transcriptional vs. post-translational) not resolved\", \"Whether MDFI regulates other TRP channels in kidney not explored\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: (1) how MDFI's I-mfa domain structurally engages its diverse transcription factor and channel targets, (2) whether the cryo-EM-resolved PIEZO channel interaction operates in vivo to tune mechanosensation, (3) what post-translational modifications regulate MDFI partner selectivity, and (4) whether MDFI's numerous binding activities are coordinated or context-exclusive in specific tissues.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of MDFI alone or in complex with transcription factors\", \"In vivo validation of PIEZO channel modulation pending peer review\", \"Tissue-specific regulation of MDFI activity largely uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 3, 4, 5, 6, 7, 8, 9]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 6, 7, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0162582\", \"supporting_discovery_ids\": [2, 3, 5, 6, 17]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 5, 6, 17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [12, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MYOD1\",\n      \"LEF1\",\n      \"CTNNB1\",\n      \"AXIN1\",\n      \"CCNT1\",\n      \"ZIC1\",\n      \"TRPC1\",\n      \"JMJD1A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```\n\nWait, I need to fix the pathway term_ids. Let me correct them - \"GO:0162582\" is not valid. Let me use only the allowed Reactome IDs.\n\n```json\n{\n  \"mechanistic_narrative\": \"MDFI (I-mfa) is a cysteine-rich transcriptional repressor that functions broadly as a cytoplasmic sequestration factor for multiple classes of transcription factors and as a modulator of calcium-dependent signaling in muscle and kidney. MDFI binds and inhibits bHLH myogenic regulatory factors (MyoD family), TCF/LEF transcription factors, and Zic proteins by blocking their nuclear import and DNA binding, thereby regulating myogenesis and Wnt/β-catenin signaling; β-catenin competes with MRFs for MDFI binding, and canonical Wnt signaling relieves MDFI-mediated repression to initiate myogenic differentiation [PMID:9799236, PMID:11238923, PMID:16301527, PMID:17090604]. Beyond transcription factor sequestration, MDFI interacts with cyclin T1/T2 (P-TEFb subunits) and viral transactivators to repress HIV-1 and HTLV-1 promoter activity [PMID:17289077, PMID:26469549], promotes fast-to-slow muscle fiber transformation through CaMKK2/AMPK-dependent calcium signaling [PMID:37307704], and suppresses mesangial cell contractility by reducing TRPC1 channel abundance to regulate glomerular filtration rate [PMID:39446484]. MDFI is also required for placental trophoblast giant cell differentiation and megakaryocyte lineage commitment, with knockout mice exhibiting embryonic lethality (placental) and thrombocytopenia, respectively [PMID:9799236, PMID:37267696].\",\n  \"teleology\": [\n    {\n      \"year\": 1998,\n      \"claim\": \"Establishing MDFI as a selective inhibitor of bHLH myogenic factors and a required developmental gene resolved how myogenic transcription factors are held inactive in the cytoplasm and revealed an essential role in trophoblast differentiation.\",\n      \"evidence\": \"Targeted gene deletion in mice (embryonic lethality with placental defects), nuclear import assays, transcriptional reporters, and gain-of-function in trophoblast stem cells\",\n      \"pmids\": [\"9799236\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MDFI blocks nuclear import not structurally defined\", \"Selectivity determinants among bHLH proteins not mapped at residue level\", \"Whether MDFI loss-of-function placental phenotype is solely due to Mash2 inhibition unclear\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"Demonstrating that MDFI inhibits TCF3/β-catenin-dependent transcription and Wnt target genes in Xenopus expanded its role from a myogenic repressor to a Wnt signaling modulator.\",\n      \"evidence\": \"Ectopic expression in Xenopus embryos with dorsal axis and Wnt target gene readouts, DNA-binding assays\",\n      \"pmids\": [\"11238923\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MDFI acts on TCF/LEF in mammalian Wnt contexts not yet tested at this point\", \"Relative contribution of bHLH vs. TCF inhibition to embryonic phenotype unknown\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Identifying MDFI as an interactor of the Axin destruction complex and a direct LEF-1 binding partner established it as a multi-node regulator within canonical Wnt signaling and JNK pathways.\",\n      \"evidence\": \"Reciprocal co-immunoprecipitation, TCF/LEF reporter assays, Axin-mediated β-catenin and JNK readouts\",\n      \"pmids\": [\"12192039\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MDFI modulates Axin complex stability or only signaling output not resolved\", \"Stoichiometry and competition between MDFI–Axin and MDFI–LEF interactions unknown\"]\n    },\n    {\n      \"year\": 2004,\n      \"claim\": \"Finding that MDFI binds Zic family zinc-finger proteins and blocks their nuclear import generalized its mechanism of cytoplasmic sequestration beyond bHLH and HMG-box factors.\",\n      \"evidence\": \"Yeast two-hybrid, co-immunoprecipitation, nuclear localization and reporter assays\",\n      \"pmids\": [\"15207726\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab observation not independently replicated\", \"In vivo relevance for Zic-dependent processes (e.g., neural crest) not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Showing that β-catenin directly binds MDFI and competes with MRFs for MDFI binding revealed how Wnt signaling derepresses myogenic transcription factors to initiate myogenesis.\",\n      \"evidence\": \"Co-immunoprecipitation, siRNA knockdown, and myogenic differentiation in P19 cells\",\n      \"pmids\": [\"16301527\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Binding site on β-catenin for MDFI not structurally mapped\", \"Whether this competition operates in adult muscle stem cells in vivo not established\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Epistasis experiments demonstrated that canonical Wnt signaling relieves MDFI-mediated suppression of LEF-1 by elevating β-catenin, integrating MDFI into a coherent Wnt-to-myogenesis relay.\",\n      \"evidence\": \"siRNA knockdown mimicking Wnt treatment, dominant-negative LEF-1 rescue, competition co-immunoprecipitation in P19 cells\",\n      \"pmids\": [\"17090604\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether MDFI–LEF-1 interaction is regulated by post-translational modifications unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Identifying P-TEFb (cyclin T1/T2) as MDFI partners and showing MDFI inhibits HIV-1 Tat/P-TEFb-dependent transcription extended MDFI function to transcription elongation control and viral gene regulation.\",\n      \"evidence\": \"Yeast two-hybrid, in vitro binding, co-immunoprecipitation, phosphorylation assays, HIV-1 promoter reporters\",\n      \"pmids\": [\"17289077\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Physiological significance of MDFI–P-TEFb interaction in non-viral contexts unknown\", \"Whether MDFI regulates global Pol II elongation not tested\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Demonstrating that MDFI binds and represses SERTA domain proteins and affects Fbxw7 mRNA levels expanded the repertoire of MDFI targets to cell cycle regulators.\",\n      \"evidence\": \"Co-immunoprecipitation, transcriptional reporters, qRT-PCR in cell lines\",\n      \"pmids\": [\"21664411\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study\", \"Mechanism linking MDFI–SEI interaction to Fbxw7 mRNA regulation not defined\", \"In vivo cell cycle consequences not tested\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showing MDFI directly binds HTLV-1 Tax and represses Tax-dependent HTLV-1 LTR and NF-κB transcription broadened its antiviral repressive capacity beyond HIV-1.\",\n      \"evidence\": \"In vitro binding, co-immunoprecipitation, reporters in COS-1, Jurkat, and HTLV-1-infected T cells\",\n      \"pmids\": [\"26469549\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab finding\", \"Whether MDFI affects HTLV-1 viral replication or latency in vivo unknown\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Demonstrating that MDFI overexpression drives fast-to-slow muscle fiber transformation via MyoD/CaMK2b and downstream metabolic gene induction established MDFI as a fiber-type specification factor.\",\n      \"evidence\": \"CRISPR/Cas9-mediated overexpression in C2C12, RNA-seq, qRT-PCR, Western blot\",\n      \"pmids\": [\"33553177\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"In vivo fiber-type switching not demonstrated\", \"Mechanism connecting MDFI to CaMK2b activation not resolved\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Identifying that MDFI elevates intracellular calcium via IP3R/RYR channels and activates CaMKK2/AMPK signaling provided a mechanistic pathway for MDFI-driven slow-fiber transformation and mitochondrial biogenesis.\",\n      \"evidence\": \"Overexpression/knockdown in C2C12 with pharmacological IP3R and RYR inhibitors, Western blot, immunofluorescence\",\n      \"pmids\": [\"37307704\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"How MDFI increases ER calcium release mechanistically unresolved\", \"Not replicated in primary muscle fibers or in vivo\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Showing that MDFI knockout mice have reduced platelets and impaired megakaryocyte progenitors, with altered JNK/ERK signaling, revealed a non-muscle developmental role for MDFI in hematopoietic lineage commitment.\",\n      \"evidence\": \"Knockout mouse bone marrow analysis, shRNA knockdown and overexpression in K562 cells, flow cytometry, phospho-kinase Western blot\",\n      \"pmids\": [\"37267696\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether MDFI acts via bHLH sequestration or an independent pathway in megakaryopoiesis not determined\", \"Conditional KO not performed to exclude non-cell-autonomous effects\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Establishing that MDFI suppresses mesangial cell contractility by reducing TRPC1 channel abundance and calcium entry defined a kidney-specific function regulating glomerular filtration rate.\",\n      \"evidence\": \"I-mfa KO mice with transdermal GFR measurement, siRNA nanoparticle delivery to mesangial cells, scRNA-seq, calcium imaging, TRPC1 inhibitor rescue\",\n      \"pmids\": [\"39446484\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism by which MDFI reduces TRPC1 protein levels (transcriptional vs. post-translational) not resolved\", \"Whether MDFI regulates other TRP channels in kidney not explored\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key open questions include: (1) how MDFI's I-mfa domain structurally engages its diverse transcription factor and channel targets, (2) what post-translational modifications regulate MDFI partner selectivity, and (3) whether MDFI's numerous binding activities are coordinated or context-exclusive in specific tissues.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No high-resolution structure of MDFI alone or in complex with transcription factors from peer-reviewed literature\", \"Tissue-specific regulation of MDFI activity largely uncharacterized\", \"Relative physiological importance of its many binding partners not ranked by in vivo studies\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 2, 3, 4, 5, 6, 7, 8, 9]},\n      {\"term_id\": \"GO:0140110\", \"supporting_discovery_ids\": [0, 2, 6, 7, 9]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0, 4, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [2, 3, 5, 6, 17]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [1, 14]},\n      {\"term_id\": \"R-HSA-397014\", \"supporting_discovery_ids\": [12, 13]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"MYOD1\",\n      \"LEF1\",\n      \"CTNNB1\",\n      \"AXIN1\",\n      \"CCNT1\",\n      \"ZIC1\",\n      \"TRPC1\",\n      \"KDM3A\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}