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.
Highlights
Megakaryocytic differentiation is accompanied by drastic morphological changes that are induced by endomitosis and proplatelet formation
Detectable expression of the megakaryocytic transcription factor, GATA-1, coincided with that of platelet factor 4 (PF4). These results are consistent with a role for FLI-1 and PU.1 in regulating PF4 gene expression
Consistent with the Electrophoretic mobility shift assay (EMSA), a chromatin immunoprecipitation (ChIP) assay using HEL cells demonstrated the bindings of FLI-1, ELF-1, and GABP to the 251 ETS site (Figure 4G). These findings suggest that FLI-1, ELF-1, and GABP bind to the 251 ETS site in the PF4 promoter
Summary
Megakaryocytic differentiation is accompanied by drastic morphological changes that are induced by endomitosis and proplatelet formation. To understand the molecular mechanism of megakaryocyte-specific gene regulation during this unique differentiation process, several megakaryocyte-specific gene promoters, including the promoters of platelet factor 4 (PF4), c-Mpl, Glycoprotein (GP) IIb, GPV, GPIX, GPVI, GPIb and platelet basic protein (PBP), have been studied (reviewed in [1]). Each of these promoters includes multiple GATA and ETS motifs. Multiple ETS family transcription factors, such as FLI-1 and PU., are known to bind to ETS motifs in each megakaryocyte-specific gene promoter. This indicates that PU. may function as a negative regulator of megakaryocyte-specific gene expression
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