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

Hdl Handle:
http://hdl.handle.net/10675.2/790
Title:
Characterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturation
Authors:
Li, Biaoru; Ding, Lianghao; Li, Wei; Story, Michael D; Pace, Betty S.
Abstract:
Background: 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.
Citation:
BMC Genomics. 2012 Apr 26; 13:153
Issue Date:
26-Apr-2012
URI:
http://hdl.handle.net/10675.2/790
DOI:
10.1186/1471-2164-13-153
PubMed ID:
22537182
PubMed Central ID:
PMC3353202
Type:
Article
ISSN:
1471-2164
Appears in Collections:
Department of Pediatrics: Faculty Research and Presentations

Full metadata record

DC FieldValue Language
dc.contributor.authorLi, Biaoruen_US
dc.contributor.authorDing, Lianghaoen_US
dc.contributor.authorLi, Weien_US
dc.contributor.authorStory, Michael Den_US
dc.contributor.authorPace, Betty S.en_US
dc.date.accessioned2012-10-26T20:30:44Z-
dc.date.available2012-10-26T20:30:44Z-
dc.date.issued2012-04-26en_US
dc.identifier.citationBMC Genomics. 2012 Apr 26; 13:153en_US
dc.identifier.issn1471-2164en_US
dc.identifier.pmid22537182en_US
dc.identifier.doi10.1186/1471-2164-13-153en_US
dc.identifier.urihttp://hdl.handle.net/10675.2/790-
dc.description.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 differentiationen_US
dc.description.abstractResults: 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.en_US
dc.description.abstractConclusions: 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.en_US
dc.rightsCopyright ©2012 Li et al; licensee BioMed Central Ltd.en_US
dc.subjectResearch Articleen_US
dc.titleCharacterization of the transcriptome profiles related to globin gene switching during in vitro erythroid maturationen_US
dc.typeArticleen_US
dc.identifier.pmcidPMC3353202en_US
dc.contributor.corporatenameDepartment of Pediatrics-

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