An intergenic silencer region regulates HBG expression

• An intergenic region upstream of HBG2 termed the fetal chromatin domain binds trans-factors and undergoes DNA looping during erythropoiesis. • Knockout of the fetal chromatin domain produced partial reversal of HBG silencing, supporting a silencer role for fetal hemoglobin expression. The most common genetic disorders worldwide are the β-hemoglobinopathies, including sickle cell disease and β-thalassemia, caused by mutations in the HBB gene. Five functional β-like globin genes on chromosome 11 are expressed during development in a tissue and stage-specific manner. The HBG genes encoding the γ-globin polypeptide chains for the fetal hemoglobin (HbF) protein undergo gene silencing during the first year of life. Our study aimed to characterize an upstream DNA cis-regulatory element that can be targeted to re-activate HBG during adult development. We investigated an intergenic region between HBE1 and HBG2 , which we termed the fetal chromatin domain (FCD), and defined its role in globin gene regulation. Using erythroid progenitors generated from human CD34 + stem cells, we characterized transcription factor binding to the FCD region, and long-range chromatin looping between the FCD and the locus control region and HBG genes. Using CRISPR/Cas9 genome editing, the FCD was knocked out in KU812 and CD34 + stem cells. We observed enhanced HBG transcription and HbF expression in FCD-knockout cells. Moreover, flow cytometry revealed increased HbF-positive cells and HbF expression, as well as enhanced long-range interactions between the LCR and HBG . Subsequent single-cell RNA sequencing revealed normal erythropoiesis in FCD-KO cells and partial reversal of HBG silencing, supporting a putative silencer role for the FCD in regulating HBG transcription. This research advances our understanding of globin gene regulation and potential therapeutic targets for further investigation.