Objective: 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.
Materials and methods: 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.
Results: 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.
Conclusions: 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.