ABSTRACT
OBJECTIVES: Erythroid differentiation is a dynamic process in which a pluripotent stem cell undergoes a series of developmental changes that commit it to a specific lineage. These alterations involve changes in gene expression profiles. In this study, gene expression profiles during differentiation of human erythroid cells of a normal blood donor were evaluated using SAGE. MATERIALS AND METHODS: Global gene expression was evaluated in cells collected immediately before addition of erythropoietin (0 h) and 192 and 336 h after addition of this hormone. Real-time PCR was used to evaluate activation of differentially expressed genes. RESULTS: The data indicate that global aspects of the transcriptome were similar during differentiation of the majority of the genes and that a relatively small set of genes is probably involved in modification of erythroid cells during differentiation. We have identified 93 differentially expressed genes during erythroid development, and expression of some of these was confirmed by qPCR. Various genes including EYA3, ERH, HES6, TIMELESS and TRIB3 were found to be homologous to those of Drosophila melanogaster and here are described for the first time during erythroid development. An important and unique carboxypeptidase inhibitor described in mammalians, LXN, was also identified. CONCLUSIONS: The results of this study amplify previously published data and may contribute to comprehension of erythroid differentiation and identification of new target genes involved in some erythroid concerning diseases.
Subject(s)
Cell Differentiation/genetics , Erythroid Cells/metabolism , Gene Expression Profiling , Gene Expression Regulation/genetics , Antigens , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Cycle Proteins/genetics , Cells, Cultured , Erythroid Cells/cytology , Erythropoietin/pharmacology , Genome/genetics , Glycoproteins/genetics , Humans , Intracellular Signaling Peptides and Proteins/genetics , Nuclear Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Protein Tyrosine Phosphatases/genetics , RNA, Messenger/genetics , Repressor Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/geneticsABSTRACT
The JAK2 V617F mutation, present in the majority of polycythemia vera (PV) patients, causes constitutive activation of JAK2 and seems to be responsible for the PV phenotype. However, the transcriptional changes triggered by the mutation have not yet been totally characterized. In this study, we performed a large-scale gene expression study using serial analysis of gene expression in bone marrow cells of a newly diagnosed PV patient harboring the JAK2 V617F mutation and in normal bone marrow cells of healthy donors. JUNB was one of the genes upregulated in PV, and we confirmed, by quantitative real-time PCR, an overexpression of JUNB in hematopoietic cells of other JAK2 V617F PV patients. Using Ba/F3-EPOR cell lines and primary human erythroblast cultures, we found that JUNB was transcriptionally induced after erythropoietin addition and that JAK2 V617F constitutively induced JunB protein expression. Furthermore, JUNB knockdown reduced not only the growth of Ba/F3 cells by inducing apoptosis, but also the clonogenic and proliferative potential of human erythroid progenitors. These results establish a role for JunB in normal erythropoiesis and indicate that JunB may play a major role in the development of JAK2 V617F myeloproliferative disorders.
