Synergistic and antagonistic activities of IRF8 and FOS enhancer pairs during an immune-cell fate switch

Cell fate instructive genes tend to be regulated by large clusters of enhancers. Whether and how individual enhancers within such clusters cooperate in regulating gene expression is poorly understood. We have previously developed a computational method, SEGCOND, which identifies hubs that we termed Putative Transcriptional Condensates (PTCs), consisting of enhancer clusters and associated target genes. Here, we use SEGCOND to identify PTCs in a CEBPA-induced B-cell-to-macrophage transdifferentiation system. We find that PTCs are enriched for highly expressed, lineage-restricted genes and associate with BRD4, a component of transcriptional condensates. Further, we performed single and combinatorial deletions of enhancers within two PTCs active during induced transdifferentiation, harboring IRF8 and FOS. Two enhancers within the IRF8 PTC were found to provide a backup mechanism when combined, safeguarding IRF8 expression and efficient transdifferentiation. Unexpectedly, two individual enhancers within the FOS PTC antagonize each other on day 1 of transdifferentiation, delaying the conversion of B-cells into macrophages and reducing FOS expression, while on day 7, they cooperate to increase FOS levels induced cells. Our results reveal complex, differentiation-stage-specific interactions between individual enhancers within enhancer clusters.