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 Idand TAL1/E2A bHLHgenes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is uncertain whether these observations reflect normal hematopoiesis. We have investigated the expression pattern of Id2and TAL1/E2Agenes in liquid suspension culture of purified hematopoietic progenitor cell (HPCs) undergoing erythroid or granulopoietic differentiation in the first culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2mRNA is detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas TAL1and E2AmRNAs are not. At the onset of erythroid differentiation, Id2mRNA is downregulated, while E2Aand TAL1mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1and E2Aare only transiently induced in the first week of granulopoietic differentiation. The expression 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, analyzed by Western blot, shows a pattern consistent with gel-shift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2or TAL1mRNA. The results are strictly consistent with the expression studies: anti-ld2oligomer (α-ld2)causes a significant dose-dependent increase of erythroid colony formation, whereas α-TAL1induces a selective dose-related inhibitory effect on erythroid colonies, as compared with untreated or scrambled oligomer-treated control HPCs. Finally, murine and human glutathione-S-transferase (GST)-ld2 polypeptides compete the TAL1/E2A-specific DNA binding activity when added to the nuclear extracts derived from erythroid culture cells, thus indicating biochemical and suggesting functional interaction of Id2 with the TAL1/E2A complex. These novel observations indicate a coordinate expression and function of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH heterodimer (TAL1/E2A) in normal erythroid differentiation.