NFI factors repress fetal hemoglobin in adult erythorid cells [ATAC-seq]

The mechanisms by which the fetal type b-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells is a basic biology question in human development. Reversal of such mechanisms is beneficial for b hemoglobinopathies, such as sickle cell disease (SCD). A CRISPR-Cas9 genetic screen uncovered two members of the NFI transcription factor family – NFIA and NFIX – as novel HBG1/2 repressors. Both factors are expressed at elevated levels in adult erythroid cells, and their single or combined depletion revealed cooperativity in HBG1/2 regulation in cultured cells and human-to-mouse xenotransplant experiments, as well as in preventing sickling of SCD-derived erythroblasts. Genomic profiling, gene editing and in vitro binding assays demonstrated that the potent concerted activity of NFIA and NFIX factors is explained in part by their ability to activate expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by repressing the HBG1/2 genes via two direct binding sites. NFI factors emerge as versatile regulators of the fetal-to-adult switch in b-globin production. Overall design: Open chromation regions were analyzed by ATAC-seq in undiffereniateed HUDEP2 cells targeted with CRISPR-Cas12a control sgRNA or NFIA&NFIX sgRNA.