The Effect of Decitabine on Covalent Histone Modifications of Chromatin Associated with the ε-,γ -, and β-Globin Gene Promoters in the Baboon (P. anubis).

Treatment with the DNA demethylating drug decitabine increased HbF and F cells to therapeutically significant levels in patients with sickle cell disease in clinical trials. 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 decitabine on DNA methylation and covalent histone modifications of chromatin associated with the e-, γ-, and β-globin gene promoters in purified erythroid bone marrow-derived cells obtained from 2 baboons (P. anubis) pre- and post-treatment. Baboons were phlebotomized for 10 days to maintain a hematocrit of 20 followed by subcutaneous decitabine administration (0.52mg/kg/day; 10–13 days). Erythroid cells (30–50 X 10 6 ) were purified from pre and post-treatment bone marrow apirates by immunomagnetic columns using an anti-baboon RBC antibody. The level of DNA methylation of 3 sites in the e-globin promoter and 5 sites in the γ-globin promoter was determined by sequence analysis of ten cloned PCR products of each sample following bisulfite modification. Levels of ac-H3, ac-H4, dimethyl H3 ly4 (H3-dimeK4), dimethyl H3 ly79 (H3-meK79, dimethyl H3 ly36 (H3-meK36), and RNA pol II associated with the e-, γ-, and β-globin promoters were determined by chromatin immunoprecipitation of sheared, formaldehyde fixed chromatin followed by real time PCR. In pre-treatment samples the level of DNA methylation of the γ-globin promoter (75%, 59.5%) was lower than the e-globin promoter (84.%, 83.3%) and was associated with a small increase in HbF (5.4%, 9.0%). Following decitabine treatment HbF increased 8–10 fold (51.1%, 76%) and the level of DNA methylation of the e-globin promoter (30.5%, 38.8%) and γ-globin promoter (24.3%, 25.0%) decreased 2–3 fold. Binding of RNA pol II to βthe -promoter was 2–3 fold higher than to the γ-promoter in pre-treatment samples. Following decitabine treatment the level of pol II associated with the γ-globin promoter was at least tenfold higher than with the β-globin promoter. In pre-treatment samples the level of ac-H3 and ac-H4 associated with the β-globin promoter was 2–4 fold higher than with the γ-globin promoter and 50 fold higher than with the e-globin promoter. Following decitabine treatment the level of ac-H3 associated with the γ-promoter was 3–4 fold higher than with the β-promoter, while the level of ac-H4 associated with the γ-promoter increased to a level equivalent to that bound to the β-promoter. While no difference in the pattern of H3-dimeK4 associated with the e-, γ-, and β-globin promoters was observed between pre- and post-treatment samples, minor changes in H3-meK79 and H3-meK36 were observed. These results demonstrate that decitabine treatment decreases DNA methylation of both the e- and γ-globin promoters, greatly increases binding of RNA pol II to the γ-globin promoter, and increases the levels of ac-H3 and ac-H4 in chromatin associated with the γ-globin promoter. These experiments illustrate the usefulness of the baboon model to investigate the mechanism of pharmacologic reactivation of HbF synthesis at the molecular level.