@article{21509, abstract = {Chromatin remodeling complexes mobilize nucleosomes and promote transcription factor (TF) binding. Using ensemble and single-molecule assays combined with cryo-electron microscopy (cryo-EM), we studied the interaction between pioneer TFs OCT4–SOX2 and the human BRG1/BRM-associated factor (BAF) complex on nucleosomes. BAF engages TF-bound substrates in two orientations, placing OCT4–SOX2 at either the remodeler ENTRY or EXIT site. At the ENTRY site, OCT4–SOX2 initially coexists with BAF without structural interference. However, continued DNA translocation is expected to cause collisions with bound TFs, which can trigger remodeling direction reversals or may induce TF dissociation. To accommodate TFs at the EXIT site, BAF undergoes structural rearrangements, and ensemble assays reveal a nucleosome subpopulation translocating away from TF-binding sites. Moreover, single-molecule experiments show that nucleosome-bound BAF frequently changes remodeling direction, and we identify an ADP-bound remodeler conformation as a potential intermediate. Together, these findings reveal key aspects of the conformational dynamics and remodeling outcomes underlying BAF processing of TF-bound nucleosomes.}, author = {Weiss, Joscha and Vecchia, Luca and Domjan, David and Cavadini, Simone and Sabantsev, Anton and Kempf, Georg and Pathare, Ganesh R. and Brackmann, Klaus and Michael, Alicia and Kater, Lukas and Hietter-Pfeiffer, Eric and Haddawi, Mina and Kuber, Urja P. and Mühlhäusser, Sandra and Grand, Ralph S. and Stadler, Michael B. and Deindl, Sebastian and Thomä, Nicolas H.}, issn = {1097-2765}, journal = {Molecular Cell}, number = {4}, pages = {625--639.e8}, publisher = {Elsevier}, title = {{The human BAF chromatin remodeler processes nucleosomes bound by pioneer transcription factors OCT4–SOX2}}, doi = {10.1016/j.molcel.2026.01.021}, volume = {86}, year = {2026}, } @article{20374, abstract = {Pioneer transcription factors (TFs) engage chromatinized DNA motifs. However, it is unclear how the resultant TF-nucleosome complexes are decoded by co-factors. In humans, the TF p53 regulates cell-cycle progression, apoptosis, and the DNA damage response, with a large fraction of p53-bound sites residing in nucleosome-harboring inaccessible chromatin. We examined the interaction of chromatin-bound p53 with co-factors belonging to the ubiquitin proteasome system (UPS). At two distinct motif locations on the nucleosome (super-helical location [SHL]−5.7 and SHL+5.9), the E3 ubiquitin ligase E6-E6AP was unable to bind nucleosome-engaged p53. The deubiquitinase USP7, on the other hand, readily engages nucleosome-bound p53 in vitro and in cells. A corresponding cryo-electron microscopy (cryo-EM) structure shows USP7 engaged with p53 and nucleosomes. Our work illustrates how chromatin imposes a co-factor-selective barrier for p53 interactors, whereby flexibly tethered interaction domains of co-factors and TFs govern compatibility between co-factors, TFs, and chromatin.}, author = {Chakraborty, Deyasini and Sandate, Colby R. and Isbel, Luke and Kempf, Georg and Weiss, Joscha and Cavadini, Simone and Kater, Lukas and Seebacher, Jan and Kozicka, Zuzanna and Stoos, Lisa and Grand, Ralph S. and Schübeler, Dirk and Michael, Alicia and Thomä, Nicolas H.}, issn = {1097-2765}, journal = {Molecular Cell}, number = {15}, pages = {2919--2936.e12}, publisher = {Elsevier}, title = {{Nucleosomes specify co-factor access to p53}}, doi = {10.1016/j.molcel.2025.06.027}, volume = {85}, year = {2025}, } @article{19443, abstract = {In cryo-electron microscopy, accurate particle localization and classification are imperative. Recent deep learning solutions, though successful, require extensive training datasets. The protracted generation time of physics-based models, often employed to produce these datasets, limits their broad applicability. We introduce FakET, a method based on neural style transfer, capable of simulating the forward operator of any cryo transmission electron microscope. It can be used to adapt a synthetic training dataset according to reference data producing high-quality simulated micrographs or tilt-series. To assess the quality of our generated data, we used it to train a state-of-the-art localization and classification architecture and compared its performance with a counterpart trained on benchmark data. Remarkably, our technique matches the performance, boosts data generation speed 750x, uses 33x less memory, and scales well to typical transmission electron microscope detector sizes. It leverages GPU acceleration and parallel processing. The source code is available at https://github.com/paloha/faket/.}, author = {Harar, Pavol and Herrmann, Lukas and Grohs, Philipp and Haselbach, David}, issn = {1878-4186}, journal = {Structure}, number = {4}, pages = {820--827.e4}, publisher = {Elsevier}, title = {{FakET: Simulating cryo-electron tomograms with neural style transfer}}, doi = {10.1016/j.str.2025.01.020}, volume = {33}, year = {2025}, } @article{20924, abstract = {Pioneer transcription factors (TFs) possess the ability to read out DNA motifs embedded within nucleosomes, driving changes in gene expression during cellular differentiation and reprogramming. Here, we present selected engagement on nucleosome sequencing (SeEN-seq), a protocol designed to systematically identify potential TF-binding sites on the nucleosome. We describe steps for nucleosome library assembly, SeEN-seq assay, and cryoelectron microscopy (cryo-EM) sample preparation. This protocol facilitates the preparation of homogeneous pioneer TF-nucleosome complexes for cryo-EM structure determination using single-particle analysis. For complete details on the use and execution of this protocol, please refer to Michael et al.1}, author = {Kobayashi, Wataru and Michael, Alicia and Ruangroengkulrith, Siwat and Kümmecke, Maximilian and Tachibana, Kikuë}, issn = {2666-1667}, journal = {STAR Protocols}, publisher = {Elsevier}, title = {{Protocol for integrative analysis of transcription factor-nucleosome interactions using SeEN-seq and cryo-EM structure determination}}, doi = {10.1016/j.xpro.2025.104295}, volume = {7}, year = {2025}, } @article{20935, abstract = {In situ cryo-electron tomography (cryo-ET) has emerged as the method of choice to investigate the structures of biomolecules in their native context. However, challenges remain for the efficient production and sharing of large-scale cryo-ET datasets. Here, we combined cryogenic plasma-based focused ion beam (cryo-PFIB) milling with recent advances in cryo-ET acquisition and processing to generate a dataset of 1,829 annotated tomograms of the green alga Chlamydomonas reinhardtii, which we provide as a community resource to drive method development and inspire biological discovery. To assay data quality, we performed subtomogram averaging of both soluble and membrane-bound complexes ranging in size from >3 MDa to ∼200 kDa, including 80S ribosomes, Rubisco, nucleosomes, microtubules, clathrin, photosystem II, and mitochondrial ATP synthase. The majority of these density maps reached sub-nanometer resolution, demonstrating the potential of this C. reinhardtii dataset as well as the promise of modern cryo-ET workflows and open data sharing to empower visual proteomics.}, author = {Kelley, Ron and Khavnekar, Sagar and Righetto, Ricardo D. and Heebner, Jessica and Obr, Martin and Zhang, Xianjun and Chakraborty, Saikat and Tagiltsev, Grigory and Michael, Alicia and Van Dorst, Sofie and Waltz, Florent and Mccafferty, Caitlyn L. and Lamm, Lorenz and Zufferey, Simon and Van Der Stappen, Philippe and Van Den Hoek, Hugo and Wietrzynski, Wojciech and Harar, Pavol and Wan, William and Briggs, John A.G. and Plitzko, Jürgen M. and Engel, Benjamin D. and Kotecha, Abhay}, issn = {1097-4164}, journal = {Molecular Cell}, publisher = {Elsevier}, title = {{Toward community-driven visual proteomics with large-scale cryo-electron tomography of Chlamydomonas reinhardtii}}, doi = {10.1016/j.molcel.2025.11.029}, year = {2025}, }