Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin

Abstract

Butyrate is a prototype of histone deacetylase inhibitors that is believed to reactivate silent genes by inducing epigenetic modifications. Although butyrate was shown to induce fetal hemoglobin (HbF) production in patients with hemoglobin disorders, the mechanism of this induction has not been fully elucidated. Our studies of the epigenetic configuration of the beta-globin cluster suggest that DNA methylation and histone H3 acetylation are important for the regulation of developmental stage-specific expression of the beta-like globin genes, whereas acetylation of both histones H3 and H4 seem to be important for the regulation of tissue-specific expression. These studies suggest that DNA methylation may be important for the silencing of the beta-like globin genes in nonerythroid hematopoietic cells but may not be necessary for their silencing in nonhematopoietic cells. Furthermore, our studies demonstrate that butyrate exposure results in a true reversal of the normal developmental switch from gamma- to beta-globin expression. This is associated with increased histone acetylation and decreased DNA methylation of the gamma-globin genes, with opposite changes in the beta-globin gene. These studies provide strong support for the role of epigenetic modifications in the normal developmental and tissue-specific regulation of globin gene expression and in the butyrate-mediated pharmacologic induction of HbF production.

Figure 1

Effect of butyrate on γ-globin chain synthesis. Mononuclear cells from 3 patients with SCD (□, ▵, ◇) were cultured into BFU-E–derived colonies. (A) Effects of butyrate exposure in vitro on γ-globin chain synthesis in BFU-E cultured before initiation of butyrate infusions. (B) γ-Globin chain synthesis in BFU-E colonies cultured (without in vitro butyrate) from the same patients before and 24 hours after butyrate infusions. (C) Effects of butyrate exposure in vitro on γ-globin chain synthesis in BFU-E cultures initiated 24 hours after butyrate infusions in vivo. Data are expressed as percentageof γ-globin chain synthesis (ie, % γ/[γ + β]). Each data point represents the mean plus or minus 1 SD of triplicate cultures. Solid horizontal lines represent the mean value for every condition.

Figure 2

Patterns of histone acetylation at the promoters of the β-like globin genes in erythroid (K562 and BFU-E) and nonerythroid (HeLa and CFU-GM) cells. (A) Schematic map of the human β-globin gene cluster in which the 5 genes are represented by solid boxes. The regions analyzed for histone acetylation are marked by a horizontal line under the map. (B,C) Levels of acetylation of histones H3 (acH3) and H4 (acH4) and their sum (acH3 + acH4) at the promoters of ϵ-, γ-, and β-globin and α-actin genes assessed by quantitative real-time PCR-based ChIP analysis. The data are expressed in relative units. Error bars are SD. This experiment is representative of 3 independent experiments.

Figure 3

Patterns of DNA methylation at the promoters of the γ- and β-globin genes in erythroid (K562 and BFU-E) and nonerythroid (HeLa and CFU-GM) cells. (A) Example of pyrograms generated from pyrosequencing reactions at CpG sites −54 and −51 of the γ-globin promoters (shadowed area). (B,C) Levels of DNA methylation at 5 CpG dinucleotides at γ-globin promoters and 3 CpG dinucleotides at the β-globin promoter were determined by pyrosequencing. The data are expressed as percent methylation. Error bars represent SD. This experiment is representative of 3 independent experiments. T indicates thymidine; C, cytosine.

Figure 4

Effects of different concentrations of butyrate on the expression and the epigenetic modifications of γ-globin genes. (A) Mononuclear cells from a patient with SCD were cultured at 50 and 150 μM concentrations of butyrate. BFU-E–derived colonies were counted and expressed as number of colonies per 10⁶ nucleated cells (colonies/10⁶ NC). Error bars are SD. (B) γ-Globin chain synthesis was assessed by polyacrylamide gel electrophoresis and fluorography and expressed as percentage of γ-globin chain synthesis (ie, % γ/[γ + β]). (C,D) mRNA levels were measured by quantitative real-time RT-PCR and expressed as fold change relative to the control (0 μM) and as percent γ/(β + γ). (E) Acetylation of histones H3 and H4 at the γ-globin promoters was measured by quantitative real-time PCR-based ChIP assay and is expressed in relative units and is presented as the sum of acH3 and acH4 (acH3 + acH4). (F) DNA methylation at 5 CpG sites in the γ-globin promoters was determined by pyrosequencing and is expressed as the mean of all the methylation measurements at all 5 sites. Medium and large symbols indicate that the changes relative to the control and to the 50 μM butyrate were statistically significant, respectively. This experiment is representative of 3 independent experiments.

Figure 5

Effects of butyrate on the expression and epigenetic modifications of the γ- and β-globin genes. Mononuclear cells from 5 patients with SCD (+, ×, □, ▵, ○) were cultured into BFU-E–derived colonies in the absence and presence of butyrate. mRNA levels were measured by quantitative real-time RT-PCR and expressed as percentage γ/(β + γ) and percent β/(β + γ). The levels of acetylation of histones H3 and H4 are expressed in relative units and are presented as the sum of acH3 and acH4 (acH3 + acH4). The levels of DNA methylation at the promoters of γ- and β-globin genes are expressed as the mean of methylation levels at all CpG sites (ie, 5 sites for γ-globin and 3 sites for β-globin). Solid horizontal lines represent the mean values for every condition.