Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation
MetadataShow full item record
AbstractBackground: The fetal and adult globin genes in the human b-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal g-globin (HBG) to replace abnormal adult sickle bS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of g-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the g-globin to b-globin (g/b) switch observed throughout in vitro erythroid differentiation
Results: We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the g/b-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal gglobin expression at day 7 with a switch to a predominance of b-globin expression by day 28 and the g/b-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes downregulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kb, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in g-and b-globin regulation were identified. The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the g/b-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways.
Conclusions: The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation.
CitationBMC Genomics. 2012 Apr 26; 13:153
- Characterization of transcription factor networks involved in umbilical cord blood CD34+ stem cells-derived erythropoiesis.
- Authors: Li B, Ding L, Yang C, Kang B, Liu L, Story MD, Pace BS
- Issue date: 2014
- Plastrum testudinis induces γ-globin gene expression through epigenetic histone modifications within the γ-globin gene promoter via activation of the p38 MAPK signaling pathway.
- Authors: Qian X, Chen J, Zhao D, Guo L, Qian X
- Issue date: 2013 Jun
- Mi2β-mediated silencing of the fetal γ-globin gene in adult erythroid cells.
- Authors: Amaya M, Desai M, Gnanapragasam MN, Wang SZ, Zu Zhu S, Williams DC Jr, Ginder GD
- Issue date: 2013 Apr 25
- Globin gene expression in correlation with G protein-related genes during erythroid differentiation.
- Authors: Čokić VP, Smith RD, Biancotto A, Noguchi CT, Puri RK, Schechter AN
- Issue date: 2013 Feb 20
- Multiple physical stresses induce γ-globin gene expression and fetal hemoglobin production in erythroid cells.
- Authors: Schaeffer EK, West RJ, Conine SJ, Lowrey CH
- Issue date: 2014 Apr