RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins. (12th September 2013)

• Record Type: Journal Article
• Authors: 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.
• Is Part Of: Journal of thrombosis and haemostasis. Volume 11:Number 9(2013:Sep.)
• Page Start: 1742
• Page End: 1750
• Publication Date: 2013-09-12
• Subjects: Thrombosis -- Periodicals; Hemostasis -- Periodicals; Blood coagulation disorders -- Periodicals; 616.1
• DOI: 10.1111/jth.12355
• Languages: English
• ISSNs: 1538-7933

Abstract

<abstract abstract-type="main" id="jth12355-abs-0001"> <title>Summary</title> <sec id="jth12355-sec-0001" sec-type="section"> <title>Background</title> <p>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 (<italic>RUNX1</italic>) of FPD patients.</p> </sec> <sec id="jth12355-sec-0002" sec-type="section"> <title>Objectives</title> <p>To elucidate the role of <italic>RUNX1</italic> in the regulation of expression of platelet factor 4 (<italic>PF4</italic>) and to propose a plausible mechanism underlying <italic>RUNX1</italic>‐mediated induction of the FPD phenotype.</p> </sec> <sec id="jth12355-sec-0003" sec-type="section"> <title>Methods</title> <p>We assessed whether <italic>RUNX1</italic> 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 <italic>PF4</italic> expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous <italic>RUNX1</italic> and <italic>PF4</italic> 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.</p> </sec> <sec id="jth12355-sec-0004" sec-type="section"> <title>Results</title> <p>We demonstrated that <italic>RUNX1</italic> activates endogenous <italic>PF4</italic> expression in megakaryocytic differentiation. <italic>RUNX1</italic>, but not its mutants, in combination with ETS‐1 and CBFβ, or FLI‐1, synergistically activated the <italic>PF4</italic> 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.</p> </sec> <sec id="jth12355-sec-0005" sec-type="section"> <title>Conclusions</title> <p> <italic>RUNX1</italic>, but not its mutants, strongly and synergistically activates <italic>PF4</italic> expression along with ETS family proteins. Furthermore, loss of the <italic>RUNX1</italic> transcriptional activation function is induced by various functional abnormalities.</p> </sec> </abstract>