title: Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site.
abstract: In previous studies on the mechanism underlying megakaryocyte-specific gene expression, several ETS motifs were found in each megakaryocyte-specific gene promoter. Although these studies suggested that several ETS family proteins regulate megakaryocyte-specific gene expression, only a few ETS family proteins have been identified. Platelet factor 4 (PF4) is a megakaryocyte-specific gene and its promoter includes multiple ETS motifs. We had previously shown that ETS-1 binds to an ETS motif in the PF4 promoter. However, the functions of the other ETS motifs are still unclear. The goal of this study was to investigate a novel functional ETS motif in the PF4 promoter and identify proteins binding to the motif. In electrophoretic mobility shift assays and a chromatin immunoprecipitation assay, FLI-1, ELF-1, and GABP bound to the -51 ETS site. Expression of FLI-1, ELF-1, and GABP activated the PF4 promoter in HepG2 cells. Mutation of a -51 ETS site attenuated FLI-1-, ELF-1-, and GABP-mediated transactivation of the promoter. siRNA analysis demonstrated that FLI-1, ELF-1, and GABP regulate PF4 gene expression in HEL cells. Among these three proteins, only FLI-1 synergistically activated the promoter with GATA-1. In addition, only FLI-1 expression was increased during megakaryocytic differentiation. Finally, the importance of the -51 ETS site for the activation of the PF4 promoter during physiological megakaryocytic differentiation was confirmed by a novel reporter gene assay using in vitro ES cell differentiation system. Together, these data suggest that FLI-1, ELF-1, and GABP regulate PF4 gene expression through the -51 ETS site in megakaryocytes and implicate the differentiation stage-specific regulation of PF4 gene expression by multiple ETS factors.
authors.affiliation: Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. okadabos@phs.osaka-u.ac.jp
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Okada
authors.forename: Haruaki
authors.initials: H
authors.lastname: Nobori
authors.forename: Mikiko
authors.initials: M
authors.lastname: Shimizu
authors.forename: Miho
authors.initials: M
authors.lastname: Watanabe
authors.forename: Masaaki
authors.initials: M
authors.lastname: Yonekura
authors.forename: Tomoko
authors.initials: T
authors.lastname: Nakai
authors.forename: Yuko
authors.initials: Y
authors.lastname: Kamikawa
authors.forename: Atsuko
authors.initials: A
authors.lastname: Wakimura
authors.forename: Nobuaki
authors.initials: N
authors.lastname: Funahashi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Naruse
authors.forename: Ayako
authors.initials: A
authors.lastname: Watanabe
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamasaki
authors.forename: So-ichiro
authors.initials: S
authors.lastname: Fukada
authors.forename: Kazuta
authors.initials: K
authors.lastname: Yasui
authors.forename: Kayoko
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Takahiro
authors.initials: T
authors.lastname: Sato
authors.forename: Kenji
authors.initials: K
authors.lastname: Kitajima
authors.forename: Toru
authors.initials: T
authors.lastname: Nakano
authors.forename: William C
authors.initials: WC
authors.lastname: Aird
authors.forename: Takefumi
authors.initials: T
authors.lastname: Doi
mesh_terms: D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D051268:GA-Binding Protein Transcription Factor / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008533:Megakaryocytes / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D010978:Platelet Factor 4 / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D050783:Proto-Oncogene Proteins c-ets / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D051381:Rats; D060888:Real-Time Polymerase Chain Reaction; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators
chemical_list: C098179:FLII protein, human; D051268:GA-Binding Protein Transcription Factor; D008840:Microfilament Proteins; D050783:Proto-Oncogene Proteins c-ets; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; D010978:Platelet Factor 4