Highlights d Loss of b-globin leads to upregulation of g-globin via altered ATF4 signaling d Reduced ATF4 decreases the levels of MYB and BCL11A d ATF4 induces MYB through binding at the HBS1L-MYB intergenic enhancer

Fetal development and anemias such as β-hemoglobinopathies trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin, which has suggested strategies to treat hemoglobinopathies such as thalassemia and sickle cell disease. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here we use CRISPR-Cas genome editing and CRISPRi transcriptional repression to model the stress caused by reduced levels of adult β-globin. We find that loss of β-globin is sufficient to induce widespread globin compensation, including robust re-expression of γ-globin. Timecourse RNA-seq of differentiating isogenic erythroid precursors identified the ATF4 transcription factor as a causal regulator of this response. ChIP-seq of multiple erythroid precursor genotypes and differentiation states revealed that β-globin knockout leads to reduced engagement of ATF4 targets involved in the unfolded protein response. This ATF4 program indirectly regulates the levels of BCL11A, a key repressor of γ-globin. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could be relevant for proposed gene editing strategies to treat hemoglobinopathies.