Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells

The mechanisms by which the fetal-type β-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease (SCD) and β-thalassemia. Here, we identify via a CRISPR-Cas9 genetic screen two members of the NFI transcription factor family – NFIA and NFIX – as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared to fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing, and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in β-globin production.