Engineering of the endogenous HBD promoter increases HbA2.

The ß-hemoglobinopathies, such as sickle cell disease and ß-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of ß-globin subunits in adult hemoglobin. Many gene editing efforts to treat the ß-hemoglobinopathies attempt to correct ß-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to ß-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total ß-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat ß-hemoglobinopathies.

ATF4 Regulates MYB to Increase γ-Globin in Response to Loss of ß-Globin.

ß-Hemoglobinopathies can 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. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here, we use CRISPR-Cas genome editing to model the stress caused by reduced levels of adult ß-globin. We find that decreased ß-globin is sufficient to induce robust re-expression of γ-globin, and RNA sequencing (RNA-seq) of differentiating isogenic erythroid precursors implicates ATF4 as a causal regulator of this response. ATF4 binds within the HBS1L-MYB intergenic enhancer and regulates expression of MYB, a known γ-globin regulator. Overall, the reduction of ATF4 upon ß-globin knockout decreases the levels of MYB and BCL11A. 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 inform strategies to treat hemoglobinopathies.