Genes regulated by BCL11A during erythropoiesis in vitro. (A) Gene expression changes identified by RNA-seq analysis of erythroblasts generated by in vitro differentiation of gene-targeted CD34⁺ HSPCs. Volcano plots illustrate DEGs in BCL11A^E- (top panel) or HBG^P-disrupted (bottom panel) proerythroblasts compared with CD33-disrupted ones. HBG refers to HBG1 + HBG2 transcripts. Dashed lines represent the 0.05 FDR statistical significance cutoff (y-axis) and log₂ fold change of 1 or −1 (x-axis). (B) Gene set enrichment analysis (GSEA) from BCL11A^E- vs CD33-disrupted CFU-E, proerythroblasts, and basophilic erythroblasts. Gene sets represented on the left and right halves of the graph are enriched in downregulated and upregulated genes, respectively. Gray shapes indicate that the gene set has an FDR ≥0.05 or normalized enrichment score ≤1.5. (C) Analysis of 25 genes regulated directly by BCL11A in erythroblasts. Left panel illustrates RNA expression heat map in HBG^P- or BCL11A^E-disrupted vs CD33-disrupted erythroblasts. Right panel illustrates heat map of assay for transposase-accessible chromatin using sequencing (ATAC-seq) signals reflecting changes in chromatin accessibility near BCL11A-bound regions in BCL11A^E-disrupted vs untreated erythroblasts. Asterisks indicate direct BCL11A target genes that were identified in HUDEP-2 cells.²² (D) BCL11A Cleavage Under Targets & Release Using Nuclease, ATAC-seq, and RNA-seq signals around regulatory regions in 3 selected BCL11A target genes. Ranged values in upper left of tracks represent y-axis scaling of counts per million reads. BasoE, basophilic erythroblast; CFU-E, colony-forming unit erythroid; log FC, log fold change; ProE, proerythroblast.