Objective: Treatment with the DNA demethylating drug 5-aza-2'-deoxycytidine (Dacogen; DAC) increased fetal hemoglobin and F cells to therapeutically significant levels in patients with sickle cell disease. To gain more insight into the mechanism of action of this drug and to increase our understanding of the relationship between DNA methylation and chromatin structure, we have determined the effect of DAC on covalent histone modifications of chromatin associated with the epsilon, gamma-, and beta-globin promoters in purified bone marrow erythroid cells of four baboons (P. anubis) pre- and posttreatment.
Results: Fetal hemoglobin increased from 6.45%+/-1.75% in pretreatment samples to 62.1%+/-7.94% following DAC. DNA methylation of three CpG sites within the epsilon-globin promoter and 5 CpG sites within the gamma-globin promoter decreased more than 50% following DAC treatment. Levels of RNA polymerase II, acetyl-histone H3, acetyl-histone H4, dimethyl-histone H3 (lys4), dimethyl-histone H3 (lys36), and dimethyl-histone H3 (lys79) associated with the epsilon-, gamma-, and beta-globin promoters were determined by chromatin immunoprecipitation of formaldehyde-fixed chromatin followed by real-time PCR. Dacogen treatment increased the association of RNA polymerase II, acetyl-histone H3, and acetyl-histone H4 with the gamma-globin promoter but did not significantly affect the association of dimethyl-histone H3 (lys4), dimethyl-histone H3 (lys36), and dimethyl-histone H3 (lys79) with the epsilon-, gamma-, and beta-globin gene promoters.
Conclusion: These experiments illustrate the usefulness of the baboon model to investigate the mechanism of pharmacologic reactivation of fetal hemoglobin synthesis at the molecular level.
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