Transcriptional activation of the γ-globin gene in baboons treated with decitabine and in cultured erythroid progenitor cells involves different mechanisms

Objective. To investigate the mechanism(s) responsible for increased gamma-globin expression in vivo in decitabine-treated baboons and in vitro in cultured erythroid progenitor cells (EPC) from adult baboon bone marrow (BM). Materials and Methods. Fetal liver, adult BM erythroid cells pre- and post-decitabine, and cultured EPCs were analyzed for distribution of RNA polymerase II , histone acetylation, and histone H3 (lys4) trimethyl throughout the gamma-globin gene complex by chromatin immunoprecipitation. DNA methylation of the gamma-globin promoter was determined by bisulfite sequencing. Expression of the baboon I gamma- and V gamma-globin chains was determined by high performance liquid chromatography (HPLC). Expression of BCL11A, a recently identified repressor of gamma-globin expression, was analyzed by Western blot. Results. Increased gamma-globin expression in decitabine-treated baboons and cultured EPC correlated with increased levels of RNA polymerase 11, histone acetylation, and histone H3 (lys4) trimethyl associated with the gamma-globin gene consistent with a transcriptional activation mechanism. Cultured EPC expressed the I gamma- and V gamma-globin chains in a pattern characteristic of fetal development. The level of DNA methylation of the gamma-globin gene promoter in EPC cultures was similar to BM crythroid cells from normal adult baboons. Different BCL11A isoforms were observed in BM erythroid cells and cultured EPC. Conclusion. The mechanism responsible for increased gamma-globin expression in cultured EPC was unexpectedly not associated with increased DNA hypomethylation of the gamma-globin gene promoter compared to normal BM erythroid cells, in contrast to BM erythroid cells of decitabine-treated baboons. Rather, increased fetal hemoglobin in EPC cultures was associated with a fetal I gamma/V gamma chain ratio and a difference in the size of the BCL11A protein compared to normal BM erythroid cells. (C) 2009 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.