Analysis of chromatin structure of the BCL11A locus in HUDEP-2 cells by Capture-Pore-C (CPC)

CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as a gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. We describe an evolutionarily conserved, enhancer-dependent chromatin architecture, the enhancer-CTCF rosette, essential for chromatin insulation and the developmental expression of BCL11A in hematopoiesis. CRISPR-mediated disruption of lineage-specific BCL11A enhancers impairs enhancer RNA transcription and NIPBL-cohesin loading, leading to destabilized chromatin configuration, impaired chromatin insulation, and epigenetic silencing of BCL11A. Antisense oligonucleotide-mediated depletion of enhancer RNA silences BCL11A by disrupting chromatin insulation in adult erythroid cells. Overall design: Capture-Pore-C data were generated from wild-type HUDEP-2 cells, sg1617-edited cells, and enhancer knockout single-cell clones enriched using biotin-labeled probes targeting CTCF binding sites or erythroid enhancers E+55, E+58, and E+62.