Induction of human gamma globin gene expression by histone deacetylase inhibitors

Abstract

We investigated the induction of human gamma globin gene activity by 3 classes of histone deacetylase inhibitors: amide analogues of trichostatin A, hydroxamic acid analogues of trapoxin, and scriptaid and its analogues. The screening consisted of measuring the effects of these compounds on gamma and beta human gene promoter activity by using cultures of GM979 cells stably transfected with a construct containing a gamma promoter linked to firefly luciferase and a beta promoter linked to renilla luciferase. Compounds belonging to all 3 classes induced gamma gene promoter activity in the screening assay in low micromolar concentrations. Histone deacetylase (HDAC) inhibitors increased acetylation of histone H4 and induced the expression of endogenous murine embryonic genes. They also increased the levels of gamma mRNA and the frequency of fetal hemoglobin-containing erythroblasts in erythroid burst-forming unit (BFUe) cultures from healthy adult individuals. Compounds that displayed very similar degrees of inhibition of the HDAC activity in an HDAC enzymatic assay differed strikingly on their effects on gamma gene promoter activity, raising the possibility of selectivity of HDACs that interact with the gamma globin gene chromatin.

Figure 1. Induction of γ gene promoter by amide analogues of trichostatin A

Four compounds were tested: M344 (■), M360 (), MD85 (●), and M355 (). Each data point is based on triplicate determinations done in 3 separate experiments. For M344 and M360, concentrations greater than those presented in the figure were severely toxic to the cells. Vertical bars indicate SEM.

Figure 2. Induction of γ globin gene promoter by hydroxamic acid analogues of trapoxin

(A) Data from compounds M232 (■), M183 (), SW187 (▲), SW188 (●), and SW189 (). (B) Data from compounds SW68 (■), SW70 (), SW99 (●), and SW86 (). Each data point is based on triplicate determinations done in 3 separate experiments. Except for M232, concentrations greater than those presented in the figure were toxic to the cells. Error bars indicate SEM.

Figure 3. Induction of γ globin gene promoter by scriptaid and analogues

Three compounds were tested: scriptaid (HR13) (■), HR11 (), and HR10 (△). Each data point is based on triplicate determinations done in 3 separate experiments. Except for HR10, concentrations greater than those presented in the figure were toxic to the cells. Error bars indicate SEM.

Figure 4. Effects on histone acetylation

(A) Electrophoretic separation of histones isolated from GM979 cells treated with 15 μM compound SW68 (lanes marked 1), 15 μM compound SW70 (lanes marked 2), or control GM979 cells cultured in the absence of HDAC inhibitors (lanes marked C). Histone preparations (5 μL, 8 μL, or 10 μL) were loaded to the gels. (B) Ratio of acetylated to nonacetylated histone H4. Notice that SW68 and SW70 increase the ratio by at least 3-fold. Error bars indicate SD.

Figure 5. Assessment of effects on globin RNA levels as determined by RNAse protection

RNA from erythroblasts from BFUe’s cultured in the presence of increasing concentrations of M344 was used in this experiment. Notice the increase of γ mRNA as the concentration of the compound increases in culture and the reciprocal decrease in the β mRNA. Also notice the increase in α mRNA, indicating that the compound has also nonspecific effects on globin gene expression related to the induction of terminal maturation. It is the decrease in the γ/γ + β ratio that indicates that M344 is a specific γ globin gene inducer.

Figure 6. Flow cytometric analysis of erythroblasts stained with a specific anti-HbF fluoresceinated monoclonal antibody

Peripheral blood mononuclear cells from a healthy individual were cultured in the presence of increasing concentrations of SW68. At day 14 of culture, BFUe-derived colonies were collected and stained with a specific PE-conjugated anti-HbF antibody, and the percentage of HbF-positive erythroblasts was determined. Notice the consistent increase of HbF-positive erythroblasts as the concentration of the compound increases in culture. Solid lines indicate results obtained in cultures done in the presence of SW68. Gray area represents the baseline measurements in a BFUe culture done in the absence of SW68.

Figure 7. HDAC inhibitors induce γ-globin gene expression in human BFUe cultures

Cultures done in the presence of 8 compounds that induced the γ globin gene promoter in the dual luciferase assay. ● indicates levels of γ mRNA (expressed at percentage of γ/γ + β ratios). ○ indicates frequencies of HbF-positive erythroblasts. The data represent means ± SEMs derived from 3 to 5 independent BFUe cultures done with mononuclear cells of different adult individuals. Notice that both the level of γ mRNA and the frequency of F-positive erythroblasts increase in response to these compounds.

Figure 8. Studies of 4 HDAC inhibitors that failed to induce γ gene expression

(A) Effects on cell numbers. Notice that all compounds are cytotoxic in the concentrations that failed to induce the γ gene promoter. SW183 (■), SW187 (), SW188 (●), and SW189 (). (B) Induction of erythroblast maturation as detected by measurements of benzidine-positive GM979 cells. SW183 (■), SW187 (), SW188 (●), and SW189 (). Error bars indicate SEM.