title: RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins.
abstract: BACKGROUND
  Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients.
  
  
  OBJECTIVE
  To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype.
  
  
  METHODS
  We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells.
  
  
  RESULTS
  We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1.
  
  
  CONCLUSIONS
  RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Okada
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
authors.forename: T
authors.initials: T
authors.lastname: Nakai
authors.forename: Y
authors.initials: Y
authors.lastname: Kamikawa
authors.forename: M
authors.initials: M
authors.lastname: Shimizu
authors.forename: Y
authors.initials: Y
authors.lastname: Fukuhara
authors.forename: M
authors.initials: M
authors.lastname: Yonekura
authors.forename: E
authors.initials: E
authors.lastname: Matsuura
authors.forename: Y
authors.initials: Y
authors.lastname: Hoshika
authors.forename: R
authors.initials: R
authors.lastname: Nagai
authors.forename: W C
authors.initials: WC
authors.lastname: Aird
authors.forename: T
authors.initials: T
authors.lastname: Doi
mesh_terms: D001791:Blood Platelet Disorders / Q000235:genetics*; D002460:Cell Line; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D009154:Mutation; D010978:Platelet Factor 4 / Q000235:genetics*; D050783:Proto-Oncogene Proteins c-ets / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D050676:Core Binding Factor Alpha 2 Subunit; D050783:Proto-Oncogene Proteins c-ets; C493728:RUNX1 protein, human; D010978:Platelet Factor 4
keywords: ETS1 protein, human; FLI1 protein, human; RUNX1 protein, human; megakaryocytes; platelet disorder, familial, with associated myeloid malignancy; platelet factor 4