The G9A Histone Methyltransferase BIX-01294 Reactivates ε-Globin Expression and Blocks Erythroid Differentiation in Primary Baboon Erythroid Progenitor Cell Cultures

Abstract

The development of new therapies to increase fetal hemoglobin (HbF) levels in patients with sickle cell disease and β-thalassemia depends on an increased understanding of the mechanism responsible for the developmental regulation of globin gene expression. A role for epigenetic modifications in the mechanism of of globin gene regulation is suggested by the presence of high levels of DNA methylation near the 5' regions of developmentally silenced ε- and γ-globin genes and the ability of pharmacological inhibitors of DNA methyltransferase (DNMTase) to reactivate ε- and γ-globin expression in adults. Whether additional epigenetic modifications associated with gene silencing and DNA methylation, such as histone H3 (lys9) dimethylation, are also involved is unknown. To investigate the hypothesis that histone H3 (lys9) dimethylation may function in the mechanism of developmental globin gene silencing, chromatin immunopreciptation assays were performed to determine the distribution of histone H3 (lys9) dimethyl and histone H3 (lys9) acetyl throughout the β-globin gene complex in purified primary baboon bone marrow (BM) erythroid cells from phlebotomized baboons expressing low levels (5–10%) of HbF and purified erythroid cells from erythroid progenitor cell cultures expressing high levels of HbF (30–50%). In BM erythroid cells, the level of histone H3 (lys9) acetyl associated with the β-globin gene was 10–20 fold higher than with the ε- and γ-globin genes, while the level of histone H3 (lys9) dimethyl associated with the ε- and γ-globin genes was 2–4 fold higher than with the β-globin gene. In erythroid cells from day 12 erythroid progenitor cell cultures, the level of histone H3 (lys9) acetyl associated with the highly expressed γ- and β-globin genes was 10–20 fold higher than with the silent ε-globin gene, while the level of histone H3 (lys9) dimethyl associated with the ε-globin gene was 2–4 fold higher than with the γ- and β-globin genes. Therefore a reciprocal relationship was observed between levels of histone H3 (lys9) acetylation and dimethylation associated with active and inactive globin genes. Experiments were performed to further investigate the role of histone H3 (lys9) dimethyl in ε-globin gene silencing by determining the effect of the G9A histone methyltransferase inhibitor BIX-01294 on ε-globin expression. Erythroid progenitor cell cultures derived from CD34+ BM cells of three individual baboons were treated with the varying doses of the DNMTase inhibitor decitabine (0.125–1.0μM), and BIX-01294 (1.25–5μM), alone and in combination. Changes in ε- globin were assessed by real time PCR using the ΔΔCT method with α-globin as the standard. Decitabine (0.5μM) increased ε-globin 25.8±7.7 fold while BIX-01294 (2.5μM) increased ε-globin 3.09±1.16 fold. Decitabine (1μM) and BIX-01294 (2.5μM) in combination increased ε-globin 55.7±24.9 fold. BIX-01294 enhanced ε-globin expression approximately twofold at all decitabine doses ranging from 0.125–1.0μM (mean increase=103± 44.7%). BIX-01294 also blocked terminal erythroid differentiation and allowed expansion of more primitive cells as evidenced by the presence of a large population of basophilic erythroblasts at late stages of culture (day 14). These results demonstrate that BIX-01294 reactivates expression of the silenced ε-globin gene and that synergistic reactivation can be achieved using combinations of BIX-01294 and decitabine. While these results are consistent with the hypothesis that epigenetic modifications are important in the mechanism of developmental globin gene silencing, the observation that BIX-01294 blocks erythroid differentiation suggests the possible involvement of a reprogramming mechanism.