Lineage-determining transcription factors constrain cohesin to drive multi-enhancer oncogene regulation

Multiple enhancers, often separated by vast genomic distances, regulate key genes. However, how the folding of individual chromatin fibres enables cell-type-restricted multi-enhancer regulation remains unclear. Here, using acute protein degradation and time-resolved chromatin conformation capture in mantle cell lymphoma, we found that the B cell-lineage-determining factor EBF1 organizes multiple enhancers around sparsely distributed genes essential for B cell identity and oncogenesis. Time-resolved sub-diffraction optical tracing of more than 100,000 chromatin fibres further revealed diverse topological conformations that facilitate multi-enhancer interactions. Mechanistically, we discovered that enhancer positioning at local topological centres is required for promoter engagement, with EBF1 acting as a permeable barrier to loop-extruding cohesin at enhancers. Extending these findings to T cell leukaemia, we show that lineage-determining transcription factors such as EBF1 and TCF1 radially position enhancers within gene loci to enable multi-enhancer regulation of key oncogenes at the single-allele level.