GSE104676
Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch
Fetal hemoglobin (HbF) level is genetically controlled and modifies severity of adult hemoglobin (HbA) disorders. Common genetic variation affects expression of BCL11A, a critical regulator of HbF silencing. Current models suggest that BCL11A acts at a distance from the gamma-globin genes via long-distance chromosomal interactions. Here we use a functional cellular assay and protein-binding microarray to establish a requirement for a zinc-finger cluster of BCL11A for globin repression, and identify a preferred DNA recognition sequence (TGACCA). The motif is present in embryonic and fetal-expressed globin promoters, and duplicated in gamma-globin promoters, yet only the distal motif is mutated in alleles of individuals with hereditary persistence of hemoglobin. Using CUT&RUN to map protein binding sites, we detected BCL11A occupancy preferentially at the distal motif, and validated its absence in HbF-expressing, promoter-edited erythroid cells. Taken together, our findings reveal that direct gamma-globin gene promoter repression by BCL11A underlies hemoglobin switching. Overall design: To assess DNA binding of BCL11A, CUT&RUN experiments with BCL11A as antibody were performed on HUDEP-2 and CD34+ differentiated cells at various differentiation days. Various pA-MNase digestion times, and controls (IgG, knockout) were included. CUT&RUN was also assessed on CRISPR promoter edited cells where the gamma-globin promoter was modified. ATAC-seq experiments were performed in HUDEP-2 cells, single clone edited cells, bulk edited cells to assess their chromatin accessibility.  ChIP-seq experiments were also performed in HUDEP-2 cells.