Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding

Abstract

β-hemoglobinopathies such as sickle cell disease (SCD) and β-thalassemia result from mutations in the adult HBB (β-globin) gene. Reactivating the developmentally silenced fetal HBG1 and HBG2 (γ-globin) genes is a therapeutic goal for treating SCD and β-thalassemia . Some forms of hereditary persistence of fetal hemoglobin (HPFH), a rare benign condition in which individuals express the γ-globin gene throughout adulthood, are caused by point mutations in the γ-globin gene promoter at regions residing ~115 and 200 bp upstream of the transcription start site. We found that the major fetal globin gene repressors BCL11A and ZBTB7A (also known as LRF) directly bound to the sites at -115 and -200 bp, respectively. Furthermore, introduction of naturally occurring HPFH-associated mutations into erythroid cells by CRISPR-Cas9 disrupted repressor binding and raised γ-globin gene expression. These findings clarify how these HPFH-associated mutations operate and demonstrate that BCL11A and ZBTB7A are major direct repressors of the fetal globin gene.

Hit and Run Transcriptional Repressors Are Difficult to Catch in the Act

“…Indeed, our own work and that of others on BCL11A and its direct repression of the fetal γ‐globin ( HBG1 and HBG2 ) gene promoters may be instructive . Despite BCL11A being established as a potent repressor of the fetal γ‐globin genes a decade ago, direct promoter binding was not detected until recently . While conventional ChIP had failed to demonstrate the interaction, CUT&RUN proved sufficiently sensitive .…”

“…While conventional ChIP had failed to demonstrate the interaction, CUT&RUN proved sufficiently sensitive . Similarly, we detected BCL11A at the fetal γ‐globin gene promoters using a molecule tagged with an inducible estrogen receptor ligand‐binding domain as well as a V5 epitope tag . The tagging appeared to interfere with the repression function of the fusion protein such that the fetal globin genes were not completely silenced when the binding of BCL11A was readily detected.…”

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

“…Apart from β- or γ-globin gene addition in hematopoietic stem cells by traditional lentiviral vectors, several other protocols aiming to increase or reactivate fetal hemoglobin (HbF) have been explored because this outcome would be applicable for amelioration of all β-globin disorders, irrespective of the genetic mutation. Tested strategies for HbF reactivation involve either forced chromatin looping, mediated by a lentiviral vector expressing the looping factor LDB1 linked to a zinc-finger protein binding the γ-globin promoter, 5 the re-creation of γ-globin promoter mutations leading to hereditary persistence of HbF (HPFH) conditions,6, 7 or downregulation or inhibition of trans -factors involved in γ-globin silencing 8 or disruption of their binding regions 9, 10, 11. Attempts to faithfully recreate HPFH γ-globin promoter mutations through gene editing have been associated with low efficiency in normal adult CD34+ cells ex vivo because less efficient homology- or microhomology-mediated repair is required 6, 7.…”

Adenovirus vectors in hematopoietic stem cell genome editing

“…Through genome-wide association study, BCL11A was found to be a master suppressor responsible for c-globin dormancy [207,208]. Alternative strategies include the disruption of binding sites of repressors within the HBG promoter region [212][213][214], the repair of the sickle mutation [215], and the forced modification of chromatin structure [216]. However, reports demonstrated that partially knockdown of BCL11A by targeting some critical motifs in its erythroid enhancer led to efficient de-repression of c-globin while not affecting animal viability [209,210].…”