Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation Free

The transcription factor BCL11A is a validated target for reactivating fetal hemoglobin (HbF) to treat major hemoglobin disorders, including sickle cell disease (SCD) and β-thalassemia. CRISPR/Cas9-mediated genome editing with a single guide RNA (sgRNA) targeting the BCL11A +58 enhancer， located 58kb downstream of its transcriptional start site (TSS), effectively suppresses BCL11A and induces fetal hemoglobin (HBG) gene expression. This strategy serves as the basis of the first CRISPR-based therapy for β-hemoglobinopathies. Despite the clinical successes, the molecular basis for the remarkable efficacy of CRISPR-mediated enhancer ablation in silencing BCL11A remains poorly understood.

In this study, we systemically analyzed the enhancer landscape, epigenetic states, and mRNA expression of BCL11Aduring human and mouse hematopoiesis. BCL11A is expressed in multiple hematopoietic cell types, including hematopoietic stem and progenitor cells (HSPCs), erythroid, B-lymphoid, and myeloid cells, but is absent in T-lymphoid and NK cells. We identified putative enhancer elements associated with lineage-specific chromatin accessibility and activating histone marks at the BCL11A locus. Notably, this locus also harbors Polycomb-mediated repressive chromatin domains demarcated by discrete CTCF-binding sites (CBS) associated with constitutive or lineage-specific CTCF binding. To elucidate the role of 3D genome configuration in regulating lineage-specific BCL11A transcription, we developed Capture Pore-C, a long read-sequencing-based chromatin conformation capture assay, using probes targeting enhancer and/or CTCF-binding sites in BCL11A-expressing erythroid cells. This approach revealed lineage-specific assemblies of high-order chromatin structures, including both pairwise and multi-way interactions among BCL11A enhancers, the promoter, and CTCF sites. These cis-regulatory elements, together with CTCF, segregate the BCL11A locus into distinct chromatin domains characterized by specific epigenetic states. Importantly, CRISPR-mediated disruption of lineage-specific BCL11A enhancers destabilized this 3D chromatin configuration, impaired enhancer-promoter interactions, and led to the spreading of Polycomb-mediated repressive chromatin at the BCL11Alocus in erythroid cells. Furthermore, we identified lineage-specific enhancer RNAs (eRNAs) transcribed from the BCL11A +58 enhancer that facilitate promote promoter-enhancer looping via NIPBL-mediated cohesin loading. Antisense oligonucleotide (ASO)-mediated depletion of BCL11A eRNAs triggers BCL11A silencing by disrupting epigenetic insulation, leading to HbF reactivation in adult erythroid cells.

Taken together, our findings demonstrate the critical role of enhancer-dependent 3D chromatin configuration in regulating BCL11A transcription during hematopoiesis. This mechanism underlies the remarkable efficiency of CRISPR-based editing of the BCL11A enhancer for the treatment of hemoglobinopathies. Further investigations into enhancer-regulating transcription factors and associated pathways will not only uncover the mechanisms for lineage-specific BCL11A expression but also provide new insights into understanding the role of enhancer-dependent epigenetic insulation in developmental gene regulation. Moreover, the discovery of enhancer-dependent epigenetic insulation in BCL11Aregulation reveals new, targetable vulnerabilities, presenting unexpected opportunities for therapeutic intervention.