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- W2012158392 abstract "ObjectiveThe aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34+ cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the α-globin locus in the developing erythron.Materials and MethodsA basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34+ cells. Expression patterns of the α- and β-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the α-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis.ResultsHuman CD34+ cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the α-globin locus during erythroid differentiation of CD34+ cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the α-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and θ gene, and were conspicuously low at the α-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the α-globin locus as CD34+ cells differentiated into erythroid pathway.ConclusionOur results indicate that remodeling of the upstream elements may be the primary event in activation of α-globin gene expression. Activation of α-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1. The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34+ cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the α-globin locus in the developing erythron. A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34+ cells. Expression patterns of the α- and β-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the α-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis. Human CD34+ cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the α-globin locus during erythroid differentiation of CD34+ cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the α-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and θ gene, and were conspicuously low at the α-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the α-globin locus as CD34+ cells differentiated into erythroid pathway. Our results indicate that remodeling of the upstream elements may be the primary event in activation of α-globin gene expression. Activation of α-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1." @default.
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- W2012158392 date "2009-10-01" @default.
- W2012158392 modified "2023-10-17" @default.
- W2012158392 title "Dynamics of α-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34+ cells in culture" @default.
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- W2012158392 doi "https://doi.org/10.1016/j.exphem.2009.07.001" @default.
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