COORDINATE EXPRESSION AND DEVELOPMENTAL ROLE OF ID2 PROTEIN AND TAL1 E2A HETERODIMER IN ERYTHROID PROGENITOR DIFFERENTIATION/

The Id proteins and basic helix-loop-helix (bHLH) proteins play major roles in specifying cell fate decisions in diverse biologic settings. A potential role for Id and TAL 1/E2A bHLH genes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is u ncertain whether these observations reflect normal hematopoiesis. We h ave investigated the expression pattern of Id2 and TAL1/E2A genes in l iquid suspension culture of purified hematopoietic progenitor cell (HP Cs) undergoing erythroid or granulopoietic differentiation in the firs t culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2 mRNA is detected by reverse tra nscriptase-polymerase chain reaction (RT-PCR), whereas TAL1 and E2A mR NAs are not. At the onset of erythroid differentiation, Id2 mRNA is do wnregulated, while E2A and TAL1 mRNAs are concomitantly upregulated: t heir expression is further increased at erythroblast level. Conversely , Id2 is not downmodulated in granulopoietic culture, except for a lat e decline at day 10 to 12, while TAL1 and E2A are only transiently ind uced in the first week of granulopoietic differentiation. The expressi on pattern of the TAL1/E2A heterodimer, as evaluated by mobility shift assay, is consistent with RT-PCR results (except for lower levels of the heterodimer in late erythroid maturation). TAL1 protein level, ana lyzed by Western blot, shows a pattern consistent with gel-shift resul ts. Functional experiments were performed on purified HPCs treated wit h phosphorothioate antisense oligodeoxynucleotides to Id2 or TAL1 mRNA . The results are strictly consistent with the expression studies: ant i-Id2 oligomer (alpha-Id2) causes a significant dose-dependent increas e of erythroid colony formation, whereas alpha-TAL1 induces a selectiv e dose-related inhibitory effect on erythroid colonies, as compared wi th untreated or scrambled oligomer-treated control HPCs. Finally, muri ne and human glutathione-S-transferase (GST)-Id2 polypeptides compete the TAL1/E2A-specific DNA binding activity when added to the nuclear e xtracts derived from erythroid culture cells, thus indicating biochemi cal and suggesting functional interaction of Id2 with the TAL1/E2A com plex. These novel observations indicate a coordinate expression and fu nction of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH h eterodimer (TAL1/E2A) in normal erythroid differentiation. (C) 1995 by The American Society of Hematology.