Multiple ETS family proteins regulate PF4 gene expression by binding to the same ETS binding site

PLoS One. 2011;6(9):e24837. doi: 10.1371/journal.pone.0024837. Epub 2011 Sep 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Differentiation / genetics
  • Cell Differentiation / physiology
  • Chromatin Immunoprecipitation
  • Electrophoretic Mobility Shift Assay
  • Flow Cytometry
  • GA-Binding Protein Transcription Factor / genetics
  • GA-Binding Protein Transcription Factor / metabolism
  • Hep G2 Cells
  • Humans
  • Megakaryocytes / metabolism
  • Microfilament Proteins / genetics
  • Microfilament Proteins / metabolism
  • Platelet Factor 4 / genetics
  • Platelet Factor 4 / metabolism*
  • Protein Binding
  • Proto-Oncogene Proteins c-ets / genetics
  • Proto-Oncogene Proteins c-ets / metabolism*
  • RNA, Small Interfering
  • Rats
  • Real-Time Polymerase Chain Reaction
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism

Substances

  • FLII protein, human
  • GA-Binding Protein Transcription Factor
  • Microfilament Proteins
  • Proto-Oncogene Proteins c-ets
  • RNA, Small Interfering
  • Receptors, Cytoplasmic and Nuclear
  • Platelet Factor 4