FUNCTIONALLY DISTINCT UTILIZATION OF CIS-REGULATORY ELEMENTS IN HEREDITARY PERSISTENCE OF FETAL HEMOGLOBIN

Abstract

Hereditary persistence of fetal hemoglobin (HPFH) mutations are rare, naturally-occurring variations which reduce the severity of beta-hemoglobinopathies by increasing postnatal expression of the gamma-globin genes (HBG1/2). Genome editing to inhibit erythroid transcriptional silencers or alter cis-elements in the HBG1/2 promoters recapitulates HPFH and should be tractable clinically. To define mechanisms involved with reactivation of fetal hemoglobin (HbF), we performed Cas9-mediated homology-directed repair to introduce HPFH mutations at the -115 distal CCAAT box cluster of the HBG1/2 promoters in an immortalized human erythroid cell line. HPFH mutations at positions -117, -114, -113, -110, and a 13-nucleotide (nt) deletion significantly elevated HbF levels compared to controls. Previous data suggest HPFH mutations either block repressor binding or create new binding sites for activators, such as GATA1. Using Cleavage Under Targets and Release Using Nuclease (CUTR and 2) an intact proximal CCAAT box is required for HBG1/2 de-repression by HPFH mutations in the distal CCAAT box. Elucidating the HBG1/2 cis-elements that control the perinatal gamma-to-beta globin switch should facilitate the development of optimal precision medicine-based treatments for beta-hemoglobinopathies.