The Role of HDAC10 in γ-Globin Gene Regulation

HDACs (histone deacetylases) are enzymes that cause chromatin modifications through deacetylation of histones and the recruitment of repressor complexes to mediate gene silencing. To explore this mechanism further, experiments were performed to determine if HDACs are involved in drug-mediated γ-globin gene induction. When human K562 cells were treated with the γ-globin inducer sodium butyrate, we observed 1.8-fold increase in HDAC10 transcription compared to untreated cells. This provided indirect evidence that HDAC10 may be involved in γ-globin gene regulation. To further understand the mechanism, enforced expression experiments using 10–50 μg of the expression plasmid pCMX-HDAC10 and the pCMX empty plasmid were performed by transient transfection of K562 cells via electroporation. Total RNA was isolated and subjected to reverse transcription followed by real time quantitative PCR using gene-specific primers to measure endogenous γ-globin gene levels. Enforced expression of HDAC10 resulted in dose-dependent silencing of γ-globin gene expression. To gain further evidence for a role of HDAC10 in regulating γ-globin gene expression, we performed siRNA knockdowns using SMARTpool-siHDAC10 (Dharmacon) at four concentrations (80nM–320nM) using Oligofectamine (Invitrogen). The γ-globin gene levels were not changed significantly by the siRNA treatment. We next performed enforced expression of HDAC10 in a K562 stable line established using the pGL4.17-Luc2-neo as a base vector in which the expression of luciferase reporter was driven by the Gγ-globin promoter (−1500 to +36). Control stable lines were also established with the empty vector. Preliminary studies of HDAC10 siRNA treatment of the KGγ-CRE stable lines produced a 1.2 fold increase in γ-globin gene activity. These results suggest that HDAC10 may play a role in γ-globin gene regulation during the adult development. Understanding novel mechanisms of γ-gene regulation will expand capabilities to develop therapeutics for sickle cell patients.