SINGLE CELL RNA-SEQ REVEALED CLONAL REPOPULATION DYNAMICS BY MOUSE HEMATOPOIETIC STEM CELLS

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 ciselements 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 (CUT&RUN), we did not observe GATA1 binding at the mutations.However, GATA1 binding at two consensus motifs at positions -172 and -186 was observed.Functional validation of the motifs is underway.To assess the role of the proximal CCAAT box in reactivating HBG1/2, we deleted the CCAAT motif in clones with the 13nt HPFH mutation.HPLC analysis revealed that disruption of the proximal CCAAT box significantly reduced HbF levels.Together, our data suggest: 1) HPFH mutations are associated with recruitment of GATA1, not to the site of the mutation, but to preexisting motifs upstream of the mutation; 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.