Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation

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 protein expression patterns during the g-globin to b-globin (g/b) switch observed throughout in vitro erythroid differentiation. 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 g-globin 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 down-regulated >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 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 a good model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation. Peripheral blood mononuclear cells were isolated from three normal donors using Histapaque-1.077. The mononuclear cells were grown in three independent cultures using the one-phase protocol as previously published. Cells were cultured in alphaMEM containing 30% fetal bovine serum, 1% deionized BSA with penicillin (100 U/mL) and streptomycin (0.1 mg/mL) at 37°C and 5% CO2. The following growth factors were added on day 0: stem cell factor (50 ng/mL), interleukin-3 (10 ng/mL) and erythropoietin (4 U/mL). Approximately 1.5 million cells were harvested from each culture (triplicate samples) on day 7, 14, 21 and 28 for microarray analysis on the whole-genome Illumina HumanWG-6 V2 Expression BeadChip.