siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)-dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures.
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.


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.


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.


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.
Banzon
Virryan
V
Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA.
Ibanez
Vinzon
V
Vaitkus
Kestis
K
Ruiz
Maria Armila
MA
Peterson
Kenneth
K
DeSimone
Joseph
J
Lavelle
Donald
D
D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism*; D002478:Cells, Cultured; D018244:Chromosomes, Artificial, Yeast; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000502:physiology*; D019175:DNA Methylation; D019281:Dimerization; D015672:Erythroid Precursor Cells / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010215:Papio; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection; D055545:gamma-Globins / Q000235:genetics*
D018952:Antigens, CD34; D034741:RNA, Small Interfering; D055545:gamma-Globins; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; C000619380:DNMT1 protein, human; C000619381:Dnmt1 protein, mouse