Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch

Abstract

Fetal hemoglobin (HbF, αγ) level is genetically controlled and modifies severity of adult hemoglobin (HbA, αβ) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

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 .…”

“…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 . 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 .…”

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].…”

“…Using an HDAd5/35++ vector, significant c-globin reactivation was observed by CRISPR/Cas9 targeting the critical GATAA motif in the BCL11A enhancer [55,211]. 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]. These strategies would be benefited considerably from recent advancement of gene editing efficiency.…”