Regulated expression and functional role of the transcription factor CHOP (GADD153) in erythroid growth and differentiation.
The hematopoietic growth factor erythropoietin (Epo) triggers changes in the expression of genes that encode important regulators of erythroid cell growth and differentiation. We now report that Epo markedly upregulates chop (gadd153) expression and that this transcription factor plays a role in erythropoiesis. Using a differential hybridization assay, we isolated a full-length cDNA of chop as an Epo upregulated gene in Rauscher murine erythroleukemia cells. RNase protection assays demonstrated that Epo or dimethyl sulfoxide induction increased steady-state mRNA levels 10- to 20-fold after 24 to 48 hours. Western blot analysis confirmed a marked increase in CHOP protein. Among the other c/ebp family members, only c/ebp beta was also upregulated during erythroid differentiation. Among normal hematopoietic cells examined, steady-state mRNA levels were highest in erythroid cells, with levels peaking during terminal differentiation. Transient overexpression of chop in Rauscher cells resulted in a significant increase in Epo- or dimethyl sulfoxide (DMSO)-induced hemoglobinization, further linking chop upregulation to erythroid differentiation. Artificial downregulation of chop in normal murine bone marrow cells with antisense oligodeoxynucleotides inhibited colony-forming unit-erythroid (CFU-E)-derived colony growth in a concentration-dependent manner. Burst-forming unit-erythroid (BFU-E)-derived colony growth was not affected. Using a Far Western type of analysis, we detected several potential CHOP binding partners among the nuclear proteins of Rauscher cells. Importantly, the number and relative abundance of these proteins changed with differentiation. The results strongly suggest that CHOP plays a role in erythropoiesis, possibly through interactions with both C/EBP and non-C/EBP family members.
Coutts
M
M
Laboratory for Cell and Molecular Biology, Division of Hematology and Oncology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
Cui
K
K
Davis
K L
KL
Keutzer
J C
JC
Sytkowski
A J
AJ
D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology; D022762:CCAAT-Enhancer-Binding Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D015672:Erythroid Precursor Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D005260:Female; D015723:Gene Library; D007700:Kinetics; D004915:Leukemia, Erythroblastic, Acute; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology; D051742:Transcription Factor CHOP; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; C497250:Ddit3 protein, mouse; D009687:Nuclear Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D051742:Transcription Factor CHOP; D004121:Dimethyl Sulfoxide