Frömel, RobertRühle, JuliaBernal, AinaSzu-Tu, ChelseaPacheco Pastor, FelixMartínez Corral, Rosa, 1991-Velten, Lars2025-09-082025-09-082025Frömel R, Rühle J, Bernal Martinez A, Szu-Tu C, Pacheco Pastor F, Martinez-Corral R, et al. Design principles of cell-state-specific enhancers in hematopoiesis. Cell. 2025 Jun 12;188(12):3202-18.e21. DOI: 10.1016/j.cell.2025.04.0170092-8674http://hdl.handle.net/10230/71147During cellular differentiation, enhancers transform overlapping gradients of transcription factors (TFs) to highly specific gene expression patterns. However, the vast complexity of regulatory DNA impedes the identification of the underlying cis-regulatory rules. Here, we characterized 64,400 fully synthetic DNA sequences to bottom-up dissect design principles of cell-state-specific enhancers in the context of the differentiation of blood stem cells to seven myeloid lineages. Focusing on binding sites for 38 TFs and their pairwise interactions, we found that identical sites displayed both repressive and activating function as a consequence of cell state, site combinatorics, or simply predicted occupancy of a TF on an enhancer. Surprisingly, combinations of activating sites frequently neutralized one another or gained repressive function. These negative synergies convert quantitative imbalances in TF expression into binary activity patterns. We exploit this principle to automatically create enhancers with specificity to user-defined combinations of hematopoietic progenitor cell states from scratch.application/pdfeng© 2025 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).info:eu-repo/semantics/articlehttp://dx.doi.org/10.1016/j.cell.2025.04.017MPRACellular differentiationFunctional genomicsGene regulationHematopoiesisSynthetic enhancersTranscription factorsinfo:eu-repo/semantics/openAccess