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 TAL1/E2A bHLH genes 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 Id2 and TAL1/E2A genes 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, Id2 mRNA is detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas TAL1 and E2A mRNAs are not. At the onset of erythroid differentiation, Id2 mRNA is downregulated, while E2A and TAL1 mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2 is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1 and E2A are 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 gelshift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2 or TAL1 mRNA. The results are strictly consistent with the expression studies: anti-Id2 oligomer (alpha-Id2) causes a significant dose-dependent increase of erythroid colony formation, whereas alpha-TAL1 induces 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)-Id2 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.
Condorelli
G
G
Thomas Jefferson Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107-5541, USA.
Vitelli
L
L
Valtieri
M
M
Marta
I
I
Montesoro
E
E
Lulli
V
V
Baer
R
R
Peschle
C
C
D000328:Adult; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D016384:Consensus Sequence; D046508:Culture Techniques / Q000379:methods; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004920:Erythropoiesis / Q000187:drug effects; D004921:Erythropoietin / Q000494:pharmacology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D018257:Helix-Loop-Helix Motifs / Q000502:physiology*; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D007377:Interleukin-3 / Q000494:pharmacology; D008297:Male; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011487:Protein Conformation; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012097:Repressor Proteins; D019089:Stem Cell Factor; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D051820:TCF Transcription Factors; D013873:Thionucleotides / Q000494:pharmacology; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D051792:Basic Helix-Loop-Helix Proteins; D004268:DNA-Binding Proteins; D004921:Erythropoietin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D016298:Hematopoietic Cell Growth Factors; D051797:Inhibitor of Differentiation Protein 2; D007377:Interleukin-3; D016376:Oligonucleotides, Antisense; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D019089:Stem Cell Factor; D000073942:T-Cell Acute Lymphocytic Leukemia Protein 1; D051820:TCF Transcription Factors; D013873:Thionucleotides; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; C497450:ID2 protein, human; C544912:TCF7L1 protein, human; C067143:TAL1 protein, human