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siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)-dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures.
siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)-dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures.
- Source :
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Experimental hematology [Exp Hematol] 2011 Jan; Vol. 39 (1), pp. 26-36.e1. Date of Electronic Publication: 2010 Oct 23. - Publication Year :
- 2011
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Abstract
- Objective: These studies were performed to test the hypothesis that DNMT1 is required for maintenance of DNA methylation and repression of the γ-globin gene in adult-stage erythroid cells.<br />Materials and Methods: DNMT1 levels were reduced by nucleofection of small interfering RNA targeting DNMT1 in chemical inducer of dimerization-dependent multipotential mouse bone marrow cells containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary cultures of erythroid progenitor cells derived from CD34(+) baboon bone marrow cells. The effect of reduced DNMT1 levels on globin gene expression was measured by real-time polymerase chain reaction and the effect on globin chain synthesis in primary erythroid progenitor cell cultures was determined by biosynthetic radiolabeling of globin chains followed by high-performance liquid chromatography analysis. The effect on DNA methylation was determined by bisulfite sequence analysis.<br />Results: Reduced DNMT1 levels in cells treated with siDNMT1 were associated with increased expression of γ-globin messenger RNA, an increased γ/γ+β chain ratio in cultured erythroid progenitors, and decreased DNA methylation of the γ-globin promoter. Similar effects were observed in cells treated with decitabine, a pharmacological inhibitor of DNA methyltransferase inhibitor.<br />Conclusions: DNMT1 is required to maintain DNA methylation of the γ-globin gene promoter and repress γ-globin gene expression in adult-stage erythroid cells.<br /> (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)
- Subjects :
- Animals
Antigens, CD34 immunology
Blotting, Western
Cells, Cultured
Chromosomes, Artificial, Yeast
DNA (Cytosine-5-)-Methyltransferase 1
DNA (Cytosine-5-)-Methyltransferases genetics
DNA Methylation
Dimerization
Erythroid Precursor Cells immunology
Humans
Mice
Papio
RNA, Small Interfering genetics
Transfection
Bone Marrow Cells metabolism
DNA (Cytosine-5-)-Methyltransferases physiology
Erythroid Precursor Cells metabolism
gamma-Globins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1873-2399
- Volume :
- 39
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Experimental hematology
- Publication Type :
- Academic Journal
- Accession number :
- 20974210
- Full Text :
- https://doi.org/10.1016/j.exphem.2010.10.003