A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site

Abstract

b-hemoglobinopathies, such as sickle cell disease and b-thalassemia, result from mutations in the adult b-globin gene. Reactivating the developmentally silenced fetal g-globin gene elevates fetal hemoglobin levels and ameliorates symptoms of b-hemoglobinopathies. The continued expression of fetal g-globin into adulthood occurs naturally in a genetic condition termed hereditary persistence of fetal hemoglobin (HPFH). Point mutations in the fetal g-globin proximal promoter can cause HPFH. The 2113A>G HPFH mutation falls within the 2115 cluster of HPFH mutations, a binding site for the fetal globin repressor BCL11A. We demonstrate that the 2113A>G HPFH mutation, unlike other mutations in the cluster, does not disrupt BCL11A binding but rather creates a de novo binding site for the transcriptional activator GATA1. Introduction of the 2113A>G HPFH mutation into erythroid cells using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system increases GATA1 binding and elevates fetal globin levels. These results reveal the mechanism by which the 2113A>G HPFH mutation elevates fetal globin and demonstrate the sensitivity of the fetal globin promoter to point mutations that often disrupt repressor binding sites but here create a de novo site for an erythroid activator.