Pharmacological Inhibition of Histone Deacetylases 1 and 2 (HDAC1/2) Induces Fetal Hemoglobin (HbF) through Activation of Gata2

Induction of HbF is an established therapeutic strategy for the treatment of sickle cell disease, and could also be effective in treating beta-thalassemia. Genetic ablation of HDAC1 or HDAC2, but not HDAC3, results in the induction of the fetal beta-like globin gene (HbG) transcript (Bradner JE, Proc Natl Acad Sci, 2010). We have previously shown that selective chemical inhibitors of HDAC1/2 elicit a dose and time dependent induction of HbG mRNA and HbF protein in cultured human CD34+ bone marrow cells undergoing erythroid differentiation (Shearstone JS, ASH Annual Meeting Abstracts, 2012). In this work, we have utilized our proof of concept molecule ACY-957, a selective inhibitor of HDAC1/2, to discover a novel role for Gata2 in the activation of HbG.

To identify genes affected by HDAC1/2 inhibition, CD34+ bone marrow cells undergoing erythroid differentiation were treated with ACY-957 or vehicle, followed by mRNA expression profiling. Among the genes differentially regulated by both pharmacological inhibition and genetic ablation of HDAC1/2 were Bcl11a and Sox6, known HbG repressors, and Gata2, a potential HbG activator. Quantitative real time PCR (QRT-PCR) time course experiments confirmed that ACY-957 treatment leads to a 2-fold and 10-fold decrease in Bcl11A and Sox6, respectively, and an 8-fold increase in Gata2 mRNA. Unlike Bcl11a and Sox6, Gata2 induction by ACY-957 was highly correlated with HbG induction, suggesting a possible role for this transcription factor in the direct activation of HbG.

To investigate this possibility, lentiviral infection was utilized to overexpress full length Gata2 transcript in differentiating primary erythroblasts. After 5 days of differentiation, Gata2 overexpression resulted in a 2.5-fold increase in HbG mRNA, while the level of the major adult beta-like globin chain (HbB) mRNA was unaffected. HbG mRNA remained elevated by Gata2 overexpression at day 7 of differentiation, while HbB was reduced by 1.6-fold. Gata2 overexpression appeared to have minimal effect on cell differentiation, as determined by the cell surface markers CD71 and GlycophorinA, a finding consistent with observations in ACY-957 treated cells with elevated Gata2. Furthermore, lentiviral delivery of short hairpin RNA (shRNA) targeting Gata2, attenuated HbG induction by ACY-957. These data suggest that elevated levels of Gata2 resulting from HDAC1/2 inhibition is sufficient to induce HbG at early stages of erythroid cell differentiation.

To understand how HDAC1/2 inhibition drives Gata2 activation, chromatin immunoprecipitation coupled with either next generation sequencing (ChIP-seq) or QRT-PCR was performed in ACY-957 and vehicle treated cells. HDAC1 and HDAC2 were present throughout the Gata2 gene body and promoter regions, and HDAC1/2 binding levels were highly correlated, suggesting co-occupancy of these enzymes at this locus. ACY-957 treatment led to elevated histone acetylation at previously described Gata2 gene regulatory regions (Bresnick et. al. 2010, J Biol Chem). Specifically, the -1.8 kb and -2.8 kb regulatory regions showed a 6-fold increase in histone H3K9 and H2BK5 acetylation, while the +9.5 kb and -3.9 kb regions showed a 3-fold increase. The Gata2 protein showed increased binding at these regulatory regions in response to ACY-957 treatment, with a maximum increase of 3-fold at the -1.8 kb region. This finding is consistent with the known positive autoregulation of the Gata2 gene. Taken together, these data suggest that selective inhibition of HDAC1/2 leads to elevated Gata2 through acetylation-induced activation of a positive autoregulatory loop.

The tight temporal correlation between Gata2 and HbG activation following HDAC1/2 inhibition argues that Gata2 may affect the beta-globin locus directly. ChIP-seq data across the 70-kb beta-globin locus demonstrated that ACY-957 treatment altered Gata2 binding only at a single region, lying within the promoter for delta globin. This region is suspected in playing a role in switching from fetal to adult globin during development, as naturally occurring deletions of this region are associated with elevated fetal hemoglobin in adults (Sankaran et. al. 2011, NEJM). Whether the change in GATA2 binding to this region is responsible for the increased expression of HbG in cells treated with HDAC1/2-selective inhibitors is under investigation.