Chromatin Accessibility Mapping of Primary Erythroid Cell Populations Leads to Identification and Validation of NFIX As a Novel Fetal Hemoglobin Repressor

Human 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 novel 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 discrete sorted erythroblast populations derived from bone marrow (BM) or cord blood (CB) progenitors, representing adult and fetal states, respectively. Comparison of the ATAC-seq profiles revealed enrichment of NFI DNA binding motifs and increased chromatin accessibility at the NFIX promoter in BM populations relative to CB populations, 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 of NFIX as a novel target for HbF activation has potential implications in the development of therapeutics for hemoglobinopathies. Overall design: Examination of chromatin accessibility via ATAC-seq in 1) HUDEP-1 and HUDEP-2 cell lines; 2) Primary bone marrow derived erythroblasts and cord blood derived eyrthroblasts across erythrocyte differention; and 3) Primary bone marrow derived erythroblasts transduced with NFIX shRNA or a non-targeting control.