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GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression

V Lemarchandel1, J Ghysdael, V Mignotte

  • 1INSERM U.91, Hôpital Henri-Mondor, Creteil, France.

Molecular and Cellular Biology
|January 1, 1993
PubMed
Summary

The human glycoprotein IIB (GPIIB) gene

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • The human glycoprotein IIB (GPIIB) gene is crucial for platelet function and is specifically expressed in megakaryocytes.
  • Understanding the regulatory mechanisms of GPIIB gene expression is key to comprehending megakaryocyte development and function.

Purpose of the Study:

  • To identify and characterize the cis-acting regulatory elements responsible for the megakaryocyte-specific expression of the human GPIIB gene.
  • To elucidate the role of transcription factors GATA and Ets in regulating GPIIB gene expression.

Main Methods:

  • Site-directed mutagenesis to disrupt GATA and Ets binding sites within the GPIIB promoter.
  • Reporter gene assays (transfection) to assess promoter activity in megakaryocytic and non-megakaryocytic cells.
  • Co-transfection assays with transcription factor expression vectors.
  • Northern blot analysis to examine ets-1 mRNA levels during megakaryocyte differentiation.
  • Gel retardation assays to study transcription factor-DNA interactions.

Main Results:

  • Two critical cis-acting sequences, a GATA binding site at -55 and an Ets consensus sequence at -40, were identified in the GPIIB promoter.
  • Mutations in either the GATA or Ets binding sites significantly reduced GPIIB promoter activity, while preserving tissue specificity.
  • A 40-bp DNA fragment containing both sites conferred megakaryocytic activity to a heterologous promoter.
  • Co-transfection with GATA1 and c-Ets-1 activated the GPIIB promoter in HeLa cells, with additive effects observed.
  • ets-1 mRNA levels increased during megakaryocyte differentiation, and similar GATA-Ets associations were found in other megakaryocyte-specific gene regulatory regions.

Conclusions:

  • GATA and Ets transcription factors, binding to specific cis-acting sequences in the GPIIB promoter, are essential determinants of megakaryocyte-specific gene expression.
  • The interplay between GATA and Ets factors plays a significant role in regulating the expression of genes critical for megakaryocyte function.
  • These findings provide insights into the molecular mechanisms governing megakaryopoiesis and platelet formation.

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