Durdu, S.Iskar, M.Isbel, L.Hoerner, L.Wirbelauer, C.Burger, L.Hess, D.Iesmantavicius, V.Schübeler, D.2025-10-232025-10-232025Molecular Cell, 2025; 85(15):2900-29181097-27651097-4164https://hdl.handle.net/2440/147935STAR Methods: e1-e16Transcription factors (TFs) recognizing DNA motifs within regulatory regions drive cell identity. Despite recent advances, their specificity remains incompletely understood. Here, we address this by contrasting two TFs, Neurogenin-2 (NGN2) and MyoD1, which recognize ubiquitous E-box motifs yet drive distinct cell fates toward neurons and muscles, respectively. Upon induction in mouse embryonic stem cells, we monitor binding across differentiation, employing an interpretable machine learning approach that integrates preexisting DNA accessibility. This reveals a chromatin-dependent motif syntax, delineating both common and factorspecific binding, validated by cellular and in vitro assays. Shared binding sites reside in open chromatin, locally influenced by nucleosomes. In contrast, factor-specific binding in closed chromatin involves NGN2 and MyoD1 acting as pioneer factors, influenced by motif variant frequencies, motif spacing, and interaction partners, which together account for subsequent lineage divergence. Transferring our methodology to other models demonstrates how a combination of opportunistic binding and context-specific chromatin-opening underpin TF specificity, driving differentiation trajectories.en© 2025 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).transcription factor specificity; motif syntax; motif variants; pioneer factors; chromatin accessibility; gene regulation; cell differentiation; machine learning; predictive models; E-boxAnimalsBasic Helix-Loop-Helix Transcription FactorsBinding SitesCell DifferentiationCell LineageChromatinHumansMiceMouse Embryonic Stem CellsMyoD ProteinNerve Tissue ProteinsNeuronsNucleosomesNucleotide MotifsProtein BindingJournal article10.1016/j.molcel.2025.07.005747496Isbel, L. [0000-0002-5270-4347]