Klonizakis, AntoniosAlcoverro-Bertran, MarcMassó, PereThomas, JoannaAndrés Aguayo, Luisa deWei, XiaoVaramogianni-Mamatsi, VassilikiNikolaou, ChristoforosGraf, T. (Thomas)2025-05-142025-05-142025Klonizakis A, Alcoverro-Bertran M, Massó P, Thomas J, de Andrés-Aguayo L, Wei X, et al. Synergistic and antagonistic activities of IRF8 and FOS enhancer pairs during an immune-cell fate switch. EMBO J. 2025 Apr;44(7):2025-55. DOI: 10.1038/s44318-025-00380-w0261-4189http://hdl.handle.net/10230/70383Cell 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.application/pdfeng© 2025 The Author(s). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.info:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s44318-025-00380-wCell reprogrammingEnhancer clustersEnhancer interactionsEnhancer synergyTranscription factorsinfo:eu-repo/semantics/openAccess