Transcription factor competition at the ?-globin promoters controls hemoglobin switching [CRISPR Screen]

BCL11A, the major regulator of HbF(a2?2) level, represses ?-globin expression through direct promoter binding in adult erythroid cells in a switch to adult adult-type HbA (a2ß2) production. Yet, the mechanism remains unclear. To uncover how BCL11A initiates repression, we used CRISPR/Cas9 and dCas9 screens to dissect the ?-globin promoters and identified an apparent activator element near the BCL11A binding region. Using CUT&RUN and base editing approaches, we demonstrate that this element, the proximal CCAAT box, is the binding site of transcription activator NF-Y. BCL11A competes with NF-Y binding through steric hindrance to initiate ?-globin repression, and the distance between the two motifs is critical for direct competition. Occupancy of NF-Y is rapidly established upon BCL11A depletion, and precedes ?-globin derepression and LCR-globin loop formation. Our findings reveal that the critical fetal-to-adult hemoglobin switch is initiated by the competition between transcription factors within a discrete region in the ?-globin promoters. Overall design: We performed CRISPR/Cas9 dense perturbation throughout the g-globin gene cluster to identify previously unrecognized regulatory elements. We employed adult-type (low HbF) HUDEP-2 cells that stably express 1) Cas9 to mutate target sequences, 2) inactive Cas9 (dCas9) to bind target sequences but not introduce DNA breaks, or 3) dCas9-VP64 or dCas9-KRAB to deliver transcription (co)activators or (co)repressors. Following introduction of pooled, densely spaced gRNAs (9293 gRNAs targeting 106 kb around the b-globin gene cluster, 1 gRNA per ~11 bp), a high HbF cell population was isolated to assess enrichment or depletion of individual gRNAs. In principle, enriched gRNAs may target elements repressive for g-globin expression, whereas depleted gRNAs may pinpoint activating sequences.