Migliaccio A R, Migliaccio G, Ashihara E, Moroni E, Giglioni B, Ottolenghi S
Laboratory of Hematopoietic Growth Factors, New York Blood Center, NY 10021, USA.
Acta Haematol. 1996;95(3-4):229-35. doi: 10.1159/000203883.
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 (beta-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 either 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 the 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 of mRNA for the two genes expressed by the cells. In contrast, beta-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 its presence. Maximum transcription from the distal promoter (approximately equal to 0.2% of total GATA1 mRNA) 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 HSCs and pluripotent progenitor cells and erythroid-specific GATA1 regulation involves activation of the distal GATA1 promoter.
为了解红细胞分化的分子机制，我们通过半定量逆转录聚合酶链反应（RT-PCR）分析了体外诱导分化后纯化的造血干细胞（HSC）中转录因子GATA1、促红细胞生成素受体（EpoR）以及红细胞（β-珠蛋白）分化标志物的表达情况。同时还分析了GATA1转录是来自近端启动子（相对于AUG，也称为红细胞型）还是远端启动子（也称为睾丸型）。纯化了与麦胚凝集素结合但不与抗体15.1.1结合、且分别保留或不保留罗丹明-123染料（分别为Rho-bright和Rho-dull）的低密度骨髓细胞。Rho-dull细胞而非Rho-bright细胞能在W/Wv和严重联合免疫缺陷小鼠中永久性重建淋巴细胞生成和髓细胞生成，因此含有造血干细胞。在液体培养中，Rho-dull细胞和Rho-bright细胞均可产生祖细胞和分化细胞（分别在第15天和第5天达到峰值）。当培养物分别用干细胞因子（SCF）+白细胞介素（IL）-3、SCF + IL-3 + Epo或SCF + IL-3 +粒细胞集落刺激因子刺激时，可观察到多系、红细胞系受限或髓细胞系受限的分化。在培养第5天时，Rho-dull细胞的重建潜能几乎检测不到。在所检测的基因中，纯化的Rho-bright细胞或Rho-dull细胞均未表达。唯一的例外是GATA1，其在培养开始时在Rho-bright细胞中以最高水平表达。Rho-dull细胞在培养第3天之前不表达GATA1（在第10 - 15天达到最大表达）。观察到细胞表达的这两个基因的所有mRNA生长过程中GATA1和EpoR均被激活。相比之下，仅在有Epo存在时才检测到β-珠蛋白mRNA。在没有Epo的情况下，GATA1的转录仅来自近端启动子，但在其存在时可观察到近端和远端转录本。远端启动子的最大转录（约占总GATA1 mRNA的0.2%）与最大珠蛋白mRNA水平同时出现（Rho-bright细胞和Rho-dull细胞分别在第5天或第15天）。这些结果表明，GATA1在造血干细胞和多能祖细胞之间的转变点被激活，并且红细胞特异性GATA1调节涉及远端GATA1启动子的激活。
