title: Transcription activation function of C/EBPalpha is required for induction of granulocytic differentiation.
abstract: The CCAAT/enhancer binding protein-alpha (C/EBPalpha) is a transcription factor required for differentiation of myeloid progenitors. In addition to specific DNA binding, C/EBPalpha is also involved in protein-protein interactions, some of which (p21, Cdk2/Cdk4, E2F) appear to be required for inhibition of proliferation and possibly differentiation. To investigate the mechanisms of C/EBPalpha-induced granulocytic differentiation, we generated C/EBPalpha mutants reportedly defective in DNA binding, transactivation, and Cdk2/Cdk4 and E2F interaction and assessed their effects in a myeloid precursor cell line, primary bone marrow and C/EBPalpha knockout fetal liver precursor cells. We show here that the DNA binding-deficient Lys298Glu mutant, the E2F binding-deficient basic region mutant 2 (BRM-2) carrying the Ile294Ala and Arg297Ala substitutions, and the transactivation-deficient N-terminus truncated p30 mutant all fail to promote differentiation on ectopic expression in myeloid precursor cells. By contrast, ectopic expression of the Cdk2/Cdk4 interaction-deficient Delta177-191 mutant promotes differentiation and induces gene expression as effectively as wild-type C/EBPalpha. Thus, the integrity of the transactivation and DNA binding domains, but not of the Cdk2/Cdk4 interaction region, is necessary for C/EBPalpha-induced differentiation. Since the E2F binding-deficient BRM-2 mutant interacted with E2F-1 but failed to activate gene expression, our results lend support to the hypothesis that activation of gene transcription is the determining factor in C/EBPalpha-dependent differentiation.
authors.affiliation: Thomas Jefferson Medical College, Kimmel Cancer Institute, Philadelphia, PA 19107, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Keeshan
authors.forename: Giorgia
authors.initials: G
authors.lastname: Santilli
authors.forename: Francesca
authors.initials: F
authors.lastname: Corradini
authors.forename: Danilo
authors.initials: D
authors.lastname: Perrotti
authors.forename: Bruno
authors.initials: B
authors.lastname: Calabretta
mesh_terms: D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology*; D002453:Cell Cycle / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics / Q000502:physiology; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006098:Granulocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics / Q000502:physiology*
chemical_list: D022763:CCAAT-Enhancer-Binding Protein-alpha; D004268:DNA-Binding Proteins; D016179:Granulocyte Colony-Stimulating Factor