Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggered its transcriptional reactivation. This activity depended on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting SA to the fetal γ-globin promoter in primary adult human erythroblasts increased γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

Hydroxyurea represents an approved treatment for sickle cell anemia and acts as a nitric oxide donor under oxidative conditions in vitro. Electron paramagnetic resonance spectroscopy shows that hydroxyurea reacts with oxy-, deoxy-, and methemoglobin to produce 2-6% of iron nitrosyl hemoglobin. No S-nitrosohemoglobin forms during these reactions. Cyanide and carbon monoxide trapping studies reveal that hydroxyurea oxidizes deoxyhemoglobin to methemoglobin and reduces methemoglobin to deoxyhemoglobin. Similar experiments reveal that iron nitrosyl hemoglobin formation specifically occurs during the reaction of hydroxyurea and methemoglobin. Experiments with hydroxyurea analogues indicate that nitric oxide transfer requires an unsubstituted acylhydroxylamine group and that the reactions of hydroxyurea and deoxy- and methemoglobin likely proceed by inner-sphere mechanisms. The formation of nitrate during the reaction of hydroxyurea and oxyhemoglobin and the lack of nitrous oxide production in these reactions suggest the intermediacy of nitric oxide as opposed to its redox form nitroxyl. A mechanistic model that includes a redox cycle between deoxyhemoglobin and methemoglobin has been forwarded to explain these results that define the reactivity of hydroxyurea and hemoglobin. These direct nitric oxide producing reactions of hydroxyurea and hemoglobin may contribute to the overall pathophysiological properties of this drug.

Key Points Ldb1 transcription factor self-association domain fused to γ-globin promoter-specific ZF protein increases HbF, reduces HbS in hSCD cells. In vitro reactivation of HbF mediated by ZF-Ldb1 exceeds pharmacologic treatment in adult hSCD cells.

The genes of the vertebrate ␤-globin locus undergo a switch in expression during erythroid development whereby embryonic͞ fetal genes of the cluster are sequentially silenced and adult genes are activated. We describe here a role for DNA methylation and MBD2 in the silencing of the human fetal ␥-globin gene. The ␥-globin gene is reactivated upon treatment with the DNA methyltransferase inhibitor 5-azacytidine in the context of a mouse containing the entire human ␤-globin locus as a yeast artificial chromosome (␤YAC) transgene. To elucidate the mechanism through which DNA methylation represses the ␥-globin gene in adult erythroid cells, ␤YAC͞MBD2؊/؊ mice were generated by breeding ␤YAC mice with MBD2؊͞؊ mice. Adult ␤YAC͞MBD2؊/؊ mice continue to express the ␥-globin gene at a level commensurate with 5-azacytidine treatment, 10-to 20-fold over that observed with 1-acetyl-2-phenylhydrazine treatment alone. In addition, the level of ␥-globin expression is consistently higher in MBD2؊͞؊ mice in 14.5-and 16.5-days postcoitus fetal liver erythroblasts suggesting a role for MBD2 in embryonic͞fetal erythroid development. DNA methylation levels are modestly decreased in MBD2؊͞؊ mice. MBD2 does not bind to the ␥-globin promoter region to maintain ␥-globin silencing. Finally, treatment of MBD2-null mice with 5-azacytidine induces only a small, nonadditive induction of ␥-globin mRNA, signifying that DNA methylation acts primarily through MBD2 to maintain ␥-globin suppression in adult erythroid cells.DNA methylation ͉ epigenetics ͉ transcription