AbstractHuman genetics has validated de-repression of fetal gamma globin (HBG) in adult erythroblasts as a powerful therapeutic paradigm in diseases involving defective adult beta globin (HBB)¹. To identify factors involved in the switch from HBG to HBB expression, we performed Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq)² on sorted erythroid lineage cells derived from bone marrow (BM) or cord blood (CB), representing adult and fetal states, respectively. BM to CB cell ATAC-seq profile comparisons revealed genome-wide enrichment of NFI DNA binding motifs and increased NFIX promoter chromatin accessibility, suggesting that NFIX may repress HBG. NFIX knockdown in BM cells increased HBG mRNA and fetal hemoglobin (HbF) protein levels, coincident with increased chromatin accessibility and decreased DNA methylation at the HBG promoter. Conversely, overexpression of NFIX in CB cells reduced HbF levels. Identification and validation of NFIX as a new target for HbF activation has implications in the development of therapeutics for hemoglobinopathies.