title: The gamma-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: 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.
  
  
  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.
authors.affiliation: Jesse Brown VA Medical Center, Chicago, IL 60612, USA. mpsingh@uic.edu
authors.forename: Mahipal
authors.initials: M
authors.lastname: Singh
authors.forename: Donald
authors.initials: D
authors.lastname: Lavelle
authors.forename: Kestis
authors.initials: K
authors.lastname: Vaitkus
authors.forename: Nadim
authors.initials: N
authors.lastname: Mahmud
authors.forename: Maria
authors.initials: M
authors.lastname: Hankewych
authors.forename: Joseph
authors.initials: J
authors.lastname: DeSimone
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002454:Cell Differentiation / Q000235:genetics*; D019070:Cell Lineage / Q000235:genetics*; D002478:Cells, Cultured; D019175:DNA Methylation; D004900:Erythroblasts; D041905:Erythroid Cells / Q000166:cytology*; D004920:Erythropoiesis / Q000235:genetics; D005333:Fetus / Q000166:cytology; D005914:Globins / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D008099:Liver / Q000166:cytology; D010215:Papio; D011401:Promoter Regions, Genetic / Q000235:genetics*
chemical_list: D005914:Globins