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Multiple ETS Family Proteins Regulate PF4 Gene Expression by Binding to the Same ETS Binding Site

2011; Public Library of Science; Volume: 6; Issue: 9 Linguagem: Inglês

10.1371/journal.pone.0024837

1932-6203

Yoshiaki Okada, Haruaki Nobori, Mikiko Shimizu, Miho Watanabe, M. Yonekura, Tomoko Nakai, Yuko Kamikawa, Atsuko Wakimura, Nobuaki Funahashi, Hiroki Naruse, Ayako Watanabe, Daisuke Yamasaki, So‐ichiro Fukada, Kazuta Yasui, Kayoko Matsumoto, Takahiro Sato, Kenji Kitajima, Toru Nakano, William C. Aird, Takefumi Doi,

Antiplatelet Therapy and Cardiovascular Diseases

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.