Abstract

Elevated levels of fetal hemoglobin (HbF) reduce the symptoms of sickle cell disease and increase the life span of patients. Future pharmacologic therapies to increase HbF will depend on increased knowledge of the mechanism(s) regulating γ-globin gene expression. To investigate the relationship between DNA methylation, chromatin structure, and γ-globin gene regulation, DNA methylation of 5 CpG sites within γ-globin gene promoter, levels of acetyl-histone H3 and H4 and histone H3 lys4 trimethyl throughout the β-globin gene complex, and the pattern of γ-globin polypeptide chain synthesis were analyzed in

  1. primary baboon fetal liver (FL) erythroid cells,

  2. primary baboon bone marrow (ABM) erythroid cells from phlebotomized adults expressing low levels of HbF,

  3. ABM erythroid cells from adults expressing high levels of HbF following treatment in vivo with the DNA demethylating drug decitabine, and

  4. erythroid cells expressing high levels of HbF generated from CD34+ baboon BM erythroid progenitors in a liquid culture system.

High levels of histone acetylation were associated with the ε- and γ-globin genes and low levels with the β-globin gene in FL erythroid cells while in ABM erythroid cells expressing low levels of HbF (6.69±1.93%) high levels were associated with the β-globin gene and low levels with the γ-globin gene. Histone H3 lys 4 trimethyl was enriched near the γ-globin gene in FL and the β-globin gene in ABM. The γ-globin gene was not methylated in FL. The level of methylation was similar in bled (75.1±8.26%) and normal (82.1±7.51%) ABM erythroid cells. The ratio of expression of 5′ Iγ- and 3′ Vγ-globin genes in the fetus (1.85) differed from bled adults (0.65). Expression of γ-globin was reactivated to similar levels in decitabine-treated baboons (51.5±4.50%) and in erythroid progenitor cell cultures (45.3±12.1%; d14). The Iγ- and Vγ-globin chains were expressed at the characteristic fetal ratio in erythroid progenitor cell cultures (1.76±0.21), but not in decitabine-treated baboons (0.76±0.32). Distribution of histone acetylation and histone H3 lys4 trimethyl was nearly identical in erythroid cells expressing high levels of HbF from decitabine-treated animals and from erythroid progenitor cultures and was characterized by high levels of histone H3 lys4 trimethyl associated with both the active γ- and β-globin genes and enrichment of histone H3 and H4 acetylation near the ε-, γ-, and β-globin genes. HbF reactivation in decitabine-treated baboons was associated with a reduction in the level of γ-globin gene methylation (33.5±9.43% dmC). Surprisingly, the level of γ-globin gene methylation in erythroid cells purified from erythroid progenitor cell cultures (79.5±9.80% dmC; d11) synthesizing high levels of γ-globin was not significantly different than in ABM erythroid cells expressing <2% HbF. These results demonstrate that reactivation of γ-globin expression in erythroid progenitor cell cultures is uniquely characterized by the Iγ/Vγ-globin chain ratio of fetal development. In contrast to the absence of γ-globin gene methylation in FL and reduced levels associated with reactivation of HbF following decitabine treatment, the level of γ-globin gene methylation in erythroid progenitor cell cultures was the same as in ABM. Our data strongly suggests that reactivation of γ-globin gene expression in erythroid progenitor cell cultures is achieved through a novel mechanism not dependent on loss of DNA methylation.

Author notes

Disclosure: No relevant conflicts of interest to declare.