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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE35102 | GEO | 2012/01/14

SECONDARY ACCESSION(S): PRJNA150891

REPOSITORIES: GEO

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Project description: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.

Project description:Basak A, Munschauer M, Lareau CA, Montbleau KE, Ulirsch JC, Hartigan CR, Schenone M, Lian J, Wang Y, Huang Y, Wu X, Gehrke L, Rice CM, An X, Christou HA, Mohandas N, Carr SA, Orkin SH, Chen JJ, Lander ES, and Sankaran VG. Increased production of the beta-like gamma-globin genes that form fetal hemoglobin can ameliorate the severity of sickle cell disease and beta-thalassemia, the major hemoglobin disorders. BCL11A is a key repressor of the gamma-globin genes and is expressed in a developmental stage-specific manner to regulate the physiologic fetal-to-adult hemoglobin switch. Despite extensive studies, the upstream mechanisms underlying the developmental expression of BCL11A and hemoglobin switching in humans have remained mysterious. Here we show that BCL11A is regulated at the level of mRNA translation during human hematopoietic development. While BCL11A mRNA is comparably expressed at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A. While BCL11A protein is not well synthesized at these earlier stages of development, its mRNA curiously continues to be associated with ribosomes. Through unbiased proteomic analyses in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We show that the observed suppression of BCL11A protein translation is mediated by LIN28B through a direct interaction with BCL11A mRNA and independent of its role in let-7 microRNA biogenesis. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching and illuminate opportunities for developing improved treatments for sickle cell disease and beta-thalassemia.

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

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