Erythroid-Specific Activation of the Distal (TESTIS) Promoter of GATA1 During Differentiation of Purified Normal Murine Hematopoietic Stem Cells

  • Anna Rita Migliaccio
  • Giovanni Migliaccio
  • Eishi Ashihara
  • Emanuela Moroni
  • Barbara Giglioni
  • Sergio Ottolenghi

Abstract

To understand the molecular mechanisms of erythroid differentiation, we analyzed by semiquantitative RT-PCR the expression of the transcription factor GATA1, the erythropoietin receptor (EpoR), and erythroid (ß-globin) differentiation markers in purified hematopoietic stem cells (HSCs) after in-vitro-induced differentiation. Whether GATA1 transcription was from the proximal (with respect to the AUG, also known as erythroid) or the distal (also known as testis) promoter was analyzed as well. Low-density marrow cells which bind to wheat germ agglutinin, but not to the antibody 15.1.1, and which do or do not retain the dye rhodamine 123 (Rho-bright and Rho-dull, respectively), were purified. Rho-dull, but not Rho-bright, cells permanently reconstitute lymphomyelopoiesis in W/Wv and severe-combined-immunodeficiency mice and, therefore, contain HSCs. Both Rho-dull and Rho-bright cells give rise to progenitor and differentiated cells (peak values at days 15 and 5, respectively) in liquid culture. Multilineage, erythroid-restricted or myeloid-restricted differentiation is observed when cultures are stimulated with stem cell factor (SCF) + interleukin (IL)-3, SCF + IL-3 + Epo, or SCF + IL-3 + granulocyte-colony-stimulating factor, respectively. Rho-dull cells have barely detectable reconstitution potential at day 5 of culture. None of the genes examined were expressed in purified Rho-bright or Rho-dull cells. The only exception was GATA1 which was expressed at maximal levels in Rho-bright cells at the onset of culture. Rho-dull cells did not express GATA1 before day 3 of culture (maximal expression at days 10 – 15). Activation of GATA1 and EpoR was observed in all growth factor combinations. There was a significant correlation between the amount of mRNA for the two genes expressed by the cells. In contrast, β-globin mRNA was detected only in the presence of Epo. The transcription of GATA1 was exclusively from the proximal promoter in the absence of Epo but both proximal and distal transcripts were observed in itd presence. Maximum transcription from the distal promoter (approximately equal to 0.2% of total GATA1mRNA) coincided with maximal globin mRNA levels (day 5 or day 15 for rho-bright and Rho-dull cells, respectively). These results indicate that GATA1 is activated at the transition point between HSC and pluripotent progenitor cells and erythroid specific GATA1 regulation involves activation of the distal GATA1 promoter.

Keywords

Agarose Electrophoresis Sorting Rhodamine Erythropoietin 

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Copyright information

© Plenum Press, New York 1996

Authors and Affiliations

  • Anna Rita Migliaccio
    • 1
    • 2
    • 5
  • Giovanni Migliaccio
    • 1
    • 2
  • Eishi Ashihara
    • 1
  • Emanuela Moroni
    • 3
  • Barbara Giglioni
    • 3
  • Sergio Ottolenghi
    • 4
  1. 1.The New York Blood CenterNew YorkUSA
  2. 2.Istituto Superiore di SanitàRomeItaly
  3. 3.Centro di Studio sulla Patologia CellulareCNRMilanItaly
  4. 4.Dipartimento di Genetica e Biologia dei MicroorganismiUniversity of MilanMilanItaly
  5. 5.Laboratory of Hematopoietic Growth FactorsNew York Blood CenterNew YorkUSA

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