Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation
<p>Abstract</p> <p>Background</p> <p>The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (<it>HB...
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| Format: | Article |
| Language: | English |
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BMC
2012-04-01
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| Series: | BMC Genomics |
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| Online Access: | http://www.biomedcentral.com/1471-2164/13/153 |
| _version_ | 1811295667694338048 |
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| author | Li Biaoru Ding Lianghao Li Wei Story Michael D Pace Betty S |
| author_facet | Li Biaoru Ding Lianghao Li Wei Story Michael D Pace Betty S |
| author_sort | Li Biaoru |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (<it>HBG</it>) to replace abnormal adult sickle β<sup>S</sup>-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-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 γ-globin to β-globin (γ/β) switch observed throughout <it>in vitro </it>erythroid differentiation.</p> <p>Results</p> <p>We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including <it>GATA1, GATA2, KLF1 </it>and <it>NFE2 </it>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-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified.</p> <p>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 <it>ATM </it>(Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included <it>KLF1, GATA1 </it>and <it>NFE2 </it>among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways.</p> <p>Conclusions</p> <p>The transcriptome analysis completed with erythroid progenitors grown <it>in vitro </it>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 <it>in vitro </it>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.</p> |
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| publishDate | 2012-04-01 |
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| spelling | doaj.art-88304eddb3874d0d937e8e8dc680c8fd2022-12-22T03:00:15ZengBMCBMC Genomics1471-21642012-04-0113115310.1186/1471-2164-13-153Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturationLi BiaoruDing LianghaoLi WeiStory Michael DPace Betty S<p>Abstract</p> <p>Background</p> <p>The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological induction of fetal γ-globin (<it>HBG</it>) to replace abnormal adult sickle β<sup>S</sup>-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-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 γ-globin to β-globin (γ/β) switch observed throughout <it>in vitro </it>erythroid differentiation.</p> <p>Results</p> <p>We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including <it>GATA1, GATA2, KLF1 </it>and <it>NFE2 </it>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-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified.</p> <p>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 <it>ATM </it>(Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included <it>KLF1, GATA1 </it>and <it>NFE2 </it>among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways.</p> <p>Conclusions</p> <p>The transcriptome analysis completed with erythroid progenitors grown <it>in vitro </it>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 <it>in vitro </it>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.</p>http://www.biomedcentral.com/1471-2164/13/153Gene profilingErythroid maturationγ-globinβ-globinHemoglobin switchingFetal hemoglobin |
| spellingShingle | Li Biaoru Ding Lianghao Li Wei Story Michael D Pace Betty S Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation BMC Genomics Gene profiling Erythroid maturation γ-globin β-globin Hemoglobin switching Fetal hemoglobin |
| title | Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation |
| title_full | Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation |
| title_fullStr | Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation |
| title_full_unstemmed | Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation |
| title_short | Characterization of the transcriptome profiles related to globin gene switching during <it>in vitro </it>erythroid maturation |
| title_sort | characterization of the transcriptome profiles related to globin gene switching during it in vitro it erythroid maturation |
| topic | Gene profiling Erythroid maturation γ-globin β-globin Hemoglobin switching Fetal hemoglobin |
| url | http://www.biomedcentral.com/1471-2164/13/153 |
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