Identification and characterization of class II histone deacetylases in gamma-globin gene regulation

Fetal hemoglobin (HbF) synthesis for the treatment of beta-hemoglobinopathies most likely involves chromatin modification in the presence of histone deacetylase (HDAC)/protein complexes to promote gamma-globin gene expression. However, the role of various HDACs in gamma-globin transcription is not very well understood therefore; the objective of my thesis project was to identify HDACs involved in gamma-gene regulation. Initial screening studies was performed in K562 erythroleukemia cells (expresses only epsilon- and gamma-globin genes) to determine endogenous transcription levels for class II HDAC genes in the absence or presence of HbF induction, which included the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB). Treatment with butyrate (2 mM), and TSA (0.5 microm) and the non-HDAC inhibitor control hemin (50 microM) significantly reduced mRNA levels of HDAC9 and its splice variant HDRP (histone deacetylase related protein) lending indirect evidence for their involvement in drug-mediated gamma-globin gene transcription. A subsequent study was performed to delineate whether HDAC9 can directly modulate gamma-globin gene transcription since a possible role for HDAC9 in hematopoiesis was previously demonstrated. To start we performed siRNA knockdown using Oligofectamine (Invitrogen) in K562 cells for 48 hrs and measured gamma-globin levels by real time quantitative PCR analysis. Treatment with siHDAC9 (Dharmacon) produced dose-dependent gamma-globin gene silencing over an 80--320 nM range; control siRNA molecules (scramble) had no effect. To observe a reverse regulatory effect when HDAC9 was over-expressed in pTarT-HDAC9 at 10--40microg concentrations, a dose dependent 2.5-fold increase in gamma-globin mRNA (p<0.05) was produced when compared to the untreated and the empty vector (pTatT) effects were subtracted. These data support a positive regulatory role for HDAC9 in gamma-gene regulation. To confirm the physiological relevance of HDAC9, similar studies were performed in human primary erythroid progenitors using a two-phase liquid culture system. The 320 nM siHDAC9 concentration produced 48% and 60% decrease in gamma-globin mRNA at day 11 (early progenitors) and day 28 (late progenitors) respectively. Enforced HDAC9 expression increased gamma-globin by 2.5-fold (p<0.05) at both days. ELISA was performed to quantify HbF levels and cytospin preps were made to visualized hemoglobin by fluorescent staining with anti-gamma-FITC antibody. HDAC9 enforced expression for 72 hrs produced a 7-fold increase in HbF and gamma-FITC positive cells increased >50%. Collectively these data support a positive role for HDAC9 in gamma-globin regulation. The molecular mechanisms of how HDAC9 maintains an active chromatin domain in the gamma-globin promoter was investigated by chromatin immunoprecipitation. We noticed that HDAC9 and HDAC1 showed 4.2 and 3.1 fold chromatin enrichment in the specific Ggamma-globin promoter in the region -1714 to -1331 relative to cap site. Collectively, these data have increased our understanding of mechanisms involved in gamma-globin regulation at a molecular level. Identification of HDACs and interacting partners/factors will help in developing better drugs for HbF reactivation. This will also provide a useful tool for efforts to understand the mechanisms of globin gene switching and identifying molecular targets for the development of more potent therapeutics for the treatment of beta-hemoglobinopathies.