The γ-globin gene promoter progressively demethylates as the hematopoietic stem progenitor cells differentiate along the erythroid lineage in baboon fetal liver and adult bone marrow

Abstract

To determine whether the difference in gamma-globin gene promoter methylation in terminal erythroblasts at the fetal and adult stages of development is a result of fetal stage-specific demethylation or adult stage-specific de novo methylation during erythropoiesis. Fetal liver- (FL, n = 2) and adult bone marrow- (ABM, n = 3) derived hematopoietic stem/progenitor cells and mature erythroblasts were purified by passage through a Miltenyi Magnetic Column followed by fluorescein-activated cell sorting (FACS) into subpopulations, defined by expression of CD34 and CD36 antigens. CD34(+)CD36(-), CD34(+)CD36(+), and CD34(-)CD36(+) subpopulations were purified by FACS and their degree of differentiation verified using the colony-forming cell assay. The methylation pattern of 5 CpG sites in the gamma-globin promoter region of these purified cell populations was determined using bisulfite sequencing. The gamma-globin promoter was highly methylated in the earliest stage of hematopoietic stem progenitor cells (CD34(+)CD36(-)) and methylation progressively decreased as erythroid differentiation progressed in FL and appears so in ABM as well. These data support a model in which differences in the methylation pattern of the gamma-globin gene in differentiating erythroblasts at different stages of development is the result of fetal stage-specific demethylation associated with transcriptional activation, rather than de novo methylation in the adults. The difference in the extent of gamma-globin gene demethylation in FL and ABM is correlated with the difference in gamma-globin expression at these developmental stages.