ERYTHROID-SPECIFIC ACTIVATION OF THE DISTAL (TESTIS) PROMOTER OF GATA1 DURING DIFFERENTIATION OF PURIFIED NORMAL MURINE HEMATOPOIETIC STEM-CELLS

To understand the molecular mechanisms of erythroid differentiation, w e analyzed by semiquantitative RT-PCR the expression of the transcript ion factor GATA1, the erythropoietin receptor (EpoR), and erythroid (b eta-globin) differentiation markers in purified hematopoietic stem cel ls (HSCs) after in-vitro-induced differentiation. Whether GATA1 transc ription 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 either do or do not retain the dye rhodamine-123 (Rho-bright and Rho-dull, respectively), were pu rified. Rho-dull, but not Rho-bright, cells permanently reconstitute l ymphomyelopoiesis in W/W-V and severe-combined-immunodeficiency mice a nd, therefore, contain HSCs. Both Rho-dull and Rho-bright cells give r ise to progenitor and differentiated cells (peak values at days 15 and 5, respectively) in liquid culture. Multilineage, erythroid-restricte d or myeloid-restricted differentiation is observed when the cultures are stimulated with stem cell factor (SCF) + interleukin (IL)-3, SCF IL-3 + Epo, or SCF + IL-3 + granulocyte-colony-stimulating factor, re spectively. Rho-dull cells have barely detectable reconstitution poten tial 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 w hich 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 cultu re (maximal expression at days 10-15). Activation of GATA1 and EpoR wa s 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, beta-globin mRNA was detected only in the pres ence of Epo. The transcription of GATA1 was exclusively from the proxi mal promoter in the absence of Epo but both proximal and distal transc ripts were observed in its presence. Maximum transcription from the di stal promoter (approximately equal to 0.2% of total GATA1 mRNA) coinci ded with maximal globin mRNA levels (day 5 or day 15 for Rho-bright an d Rho-dull cells, respectively). These results indicate that GATA1 is activated at the transition point between HSCs and pluripotent progeni tor cells and erythroid-specific GATA1 regulation involves activation of the distal GATA1 promoter.