| Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms beta-hemoglobinopathy patients. While most studies have focused on induction of gamma-globin gene expression as an approach to increase HbF, we hypothesized that post-transcriptional regulation of HbF plays an important but underappreciated role in controlling HbF levels. In a parallel approach, we investigated two cellular factors as potential mediators of post-transcriptional HbF induction.;First, we determined whether increasing eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation, a key regulator of protein translation, could enhance HbF. Treatment with salubrinal, an inhibitor of eIF2alpha dephosphorylation, enhanced HbF production without changing globin mRNA expression, proliferation, or cell differentiation. These results were further supported by the post-transcriptional induction of HbF by other pharmacologic activators of the eIF2alpha pathway and by genetic inactivation of the negative regulators, GADD34 and CReP. We then identified the mechanism of post-transcriptional HbF induction and showed that gamma-globin mRNA was selectively translated during stress recovery. Additionally, we found that this novel mechanism of increasing HbF could be combined with clinically relevant transcriptional activators of gamma-globin gene expression to additively enhance HbF. These findings identify eIF2alpha phosphorylation as a post-transcriptional regulator of HbF induction that may be pharmacologically targeted, either alone or in combination, in beta-hemoglobinopathy patients.;Second, we investigated the mechanism of post-transcriptional HbF induction by 5-azacytidine, a methyltransferase inhibitor and potent HbF inducer. We found that low doses of 5-azacytidine reduced DNMT2-mediated tRNA methylation, which increased proliferation and differentiation of progenitors cells. Additionally, genetic loss of DNA Methyltransferase 2 (DNMT2) increased HbF without changing gamma-globin mRNA. These results were specific to DNMT2, since reduction of NSUN2, an alternative tRNA methyltransferase, decreased cell growth and total hemoglobin levels without enhancing HbF production. Our study identifies DNMT2 as another post-transcriptional regulator of HbF and highlights tRNA methylation as a component of 5-azacytidine's mechanism. These findings have direct application to beta-hemoglobinopathy patients as well as the potential to impact other diseases, such as myelodysplastic syndrome, where 5-azacytidine also has demonstrated therapeutic benefit. |
