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RUNX 1 , but not its familial platelet disorder mutants, synergistically activates PF 4 gene expression in combination with ETS family proteins
Author(s) -
Okada Y.,
Watanabe M.,
Nakai T.,
Kamikawa Y.,
Shimizu M.,
Fukuhara Y.,
Yonekura M.,
Matsuura E.,
Hoshika Y.,
Nagai R.,
Aird W. C.,
Doi T.
Publication year - 2013
Publication title -
journal of thrombosis and haemostasis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.947
H-Index - 178
eISSN - 1538-7836
pISSN - 1538-7933
DOI - 10.1111/jth.12355
Subject(s) - runx1 , transcription factor , microbiology and biotechnology , biology , mutant , electrophoretic mobility shift assay , platelet disorder , core binding factor , reporter gene , gene expression , gene , platelet , genetics , immunology
Summary 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 ( RUNX 1 ) of FPD patients. Objectives To elucidate the role of RUNX 1 in the regulation of expression of platelet factor 4 ( PF 4 ) and to propose a plausible mechanism underlying RUNX 1 ‐mediated induction of the FPD phenotype. Methods We assessed whether RUNX 1 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 PF 4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX 1 and PF 4 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 RUNX 1 activates endogenous PF 4 expression in megakaryocytic differentiation. RUNX 1 , but not its mutants, in combination with ETS ‐1 and CBF β, or FLI ‐1, synergistically activated the PF 4 promoter. Each RUNX 1 mutant harbors various functional abnormalities, including loss of DNA ‐binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS ‐1, CBF β, and FLI ‐1. Conclusions RUNX 1 , but not its mutants, strongly and synergistically activates PF 4 expression along with ETS family proteins. Furthermore, loss of the RUNX 1 transcriptional activation function is induced by various functional abnormalities.