@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{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}, } @article{18072, abstract = {The individualization of chromosomes during early mitosis and their clustering upon exit from cell division are two key transitions that ensure efficient segregation of eukaryotic chromosomes. Both processes are regulated by the surfactant-like protein Ki-67, but how Ki-67 achieves these diametric functions has remained unknown. Here, we report that Ki-67 radically switches from a chromosome repellent to a chromosome attractant during anaphase in human cells. We show that Ki-67 dephosphorylation during mitotic exit and the simultaneous exposure of a conserved basic patch induce the RNA-dependent formation of a liquid-like condensed phase on the chromosome surface. Experiments and coarse-grained simulations support a model in which the coalescence of chromosome surfaces, driven by co-condensation of Ki-67 and RNA, promotes clustering of chromosomes. Our study reveals how the switch of Ki-67 from a surfactant to a liquid-like condensed phase can generate mechanical forces during genome segregation that are required for re-establishing nuclear-cytoplasmic compartmentalization after mitosis.}, author = {Hernandez-Armendariz, Alberto and Sorichetti, Valerio and Hayashi, Yuki and Koskova, Zuzana and Brunner, Andreas and Ellenberg, Jan and Šarić, Anđela and Cuylen-Haering, Sara}, issn = {1097-4164}, journal = {Molecular Cell}, number = {17}, pages = {P3254--3270.E9}, publisher = {Cell Press}, title = {{A liquid-like coat mediates chromosome clustering during mitotic exit}}, doi = {10.1016/j.molcel.2024.07.022}, volume = {84}, year = {2024}, } @article{18553, abstract = {Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates DNA damage that impedes gene transcription by RNA polymerase II (RNA Pol II). TC-NER is initiated by the recognition of lesion-stalled RNA Pol II by CSB, which recruits the CRL4CSA ubiquitin ligase and UVSSA. RNA Pol II ubiquitylation at RPB1-K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing RNA Pol II stability and initiating DNA damage excision by TFIIH recruitment. However, the precise regulatory mechanisms of CRL4CSA activity and TFIIH recruitment remain elusive. Here, we reveal human serine/threonine-protein kinase 19 (STK19) as a TC-NER factor, which is essential for correct DNA damage removal and subsequent transcription restart. Cryogenic electron microscopy (cryo-EM) studies demonstrate that STK19 is an integral part of the RNA Pol II-TC-NER complex, bridging CSA, UVSSA, RNA Pol II, and downstream DNA. STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient RNA Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core TC-NER component.}, author = {Ramadhin, Anisha R. and Lee, Shun-Hsiao and Zhou, Di and Testa Salmazo, Anita P and Gonzalo-Hansen, Camila and van Sluis, Marjolein and Blom, Cindy M.A. and Janssens, Roel C. and Raams, Anja and Dekkers, Dick and Bezstarosti, Karel and Slade, Dea and Vermeulen, Wim and Pines, Alex and Demmers, Jeroen A.A. and Bernecky, Carrie A and Sixma, Titia K. and Marteijn, Jurgen A.}, issn = {1097-2765}, journal = {Molecular Cell}, number = {24}, pages = {4740--4757.e12}, publisher = {Elsevier}, title = {{STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment}}, doi = {10.1016/j.molcel.2024.10.030}, volume = {84}, year = {2024}, } @article{15129, abstract = {Type I CRISPR-Cas systems employ multi-subunit Cascade effector complexes to target foreign nucleic acids for destruction. Here, we present structures of D. vulgaris type I-C Cascade at various stages of double-stranded (ds)DNA target capture, revealing mechanisms that underpin PAM recognition and Cascade allosteric activation. We uncover an interesting mechanism of non-target strand (NTS) DNA stabilization via stacking interactions with the “belly” subunits, securing the NTS in place. This “molecular seatbelt” mechanism facilitates efficient R-loop formation and prevents dsDNA reannealing. Additionally, we provide structural insights into how two anti-CRISPR (Acr) proteins utilize distinct strategies to achieve a shared mechanism of type I-C Cascade inhibition by blocking PAM scanning. These observations form a structural basis for directional R-loop formation and reveal how different Acr proteins have converged upon common molecular mechanisms to efficiently shut down CRISPR immunity.}, author = {O’Brien, Roisin E. and Bravo, Jack Peter Kelly and Ramos, Delisa and Hibshman, Grace N. and Wright, Jacquelyn T. and Taylor, David W.}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {5}, pages = {746--758.e5}, publisher = {Elsevier}, title = {{Structural snapshots of R-loop formation by a type I-C CRISPR Cascade}}, doi = {10.1016/j.molcel.2023.01.024}, volume = {83}, year = {2023}, } @article{12143, abstract = {MicroRNA (miRNA) and RNA interference (RNAi) pathways rely on small RNAs produced by Dicer endonucleases. Mammalian Dicer primarily supports the essential gene-regulating miRNA pathway, but how it is specifically adapted to miRNA biogenesis is unknown. We show that the adaptation entails a unique structural role of Dicer’s DExD/H helicase domain. Although mice tolerate loss of its putative ATPase function, the complete absence of the domain is lethal because it assures high-fidelity miRNA biogenesis. Structures of murine Dicer⋅miRNA precursor complexes revealed that the DExD/H domain has a helicase-unrelated structural function. It locks Dicer in a closed state, which facilitates miRNA precursor selection. Transition to a cleavage-competent open state is stimulated by Dicer-binding protein TARBP2. Absence of the DExD/H domain or its mutations unlocks the closed state, reduces substrate selectivity, and activates RNAi. Thus, the DExD/H domain structurally contributes to mammalian miRNA biogenesis and underlies mechanistical partitioning of miRNA and RNAi pathways.}, author = {Zapletal, David and Taborska, Eliska and Pasulka, Josef and Malik, Radek and Kubicek, Karel and Zanova, Martina and Much, Christian and Sebesta, Marek and Buccheri, Valeria and Horvat, Filip and Jenickova, Irena and Prochazkova, Michaela and Prochazka, Jan and Pinkas, Matyas and Novacek, Jiri and Joseph, Diego F. and Sedlacek, Radislav and Bernecky, Carrie A and O’Carroll, Dónal and Stefl, Richard and Svoboda, Petr}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {21}, pages = {4064--4079.e13}, publisher = {Elsevier}, title = {{Structural and functional basis of mammalian microRNA biogenesis by Dicer}}, doi = {10.1016/j.molcel.2022.10.010}, volume = {82}, year = {2022}, } @article{15140, abstract = {Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, we present a 3.9-Å-resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.}, author = {Bravo, Jack Peter Kelly and Dangerfield, Tyler L. and Taylor, David W. and Johnson, Kenneth A.}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {7}, pages = {1548--1552.e4}, publisher = {Elsevier}, title = {{Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication}}, doi = {10.1016/j.molcel.2021.01.035}, volume = {81}, year = {2021}, } @article{9526, abstract = {DNA methylation and histone H1 mediate transcriptional silencing of genes and transposable elements, but how they interact is unclear. In plants and animals with mosaic genomic methylation, functionally mysterious methylation is also common within constitutively active housekeeping genes. Here, we show that H1 is enriched in methylated sequences, including genes, of Arabidopsis thaliana, yet this enrichment is independent of DNA methylation. Loss of H1 disperses heterochromatin, globally alters nucleosome organization, and activates H1-bound genes, but only weakly de-represses transposable elements. However, H1 loss strongly activates transposable elements hypomethylated through mutation of DNA methyltransferase MET1. Hypomethylation of genes also activates antisense transcription, which is modestly enhanced by H1 loss. Our results demonstrate that H1 and DNA methylation jointly maintain transcriptional homeostasis by silencing transposable elements and aberrant intragenic transcripts. Such functionality plausibly explains why DNA methylation, a well-known mutagen, has been maintained within coding sequences of crucial plant and animal genes.}, author = {Choi, Jaemyung and Lyons, David B. and Kim, M. Yvonne and Moore, Jonathan D. and Zilberman, Daniel}, issn = {1097-4164}, journal = {Molecular Cell}, number = {2}, pages = {310--323.e7}, publisher = {Elsevier}, title = {{DNA methylation and histone H1 jointly repress transposable elements and aberrant intragenic transcripts}}, doi = {10.1016/j.molcel.2019.10.011}, volume = {77}, year = {2020}, } @article{7395, abstract = {The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between “closed” and “open” conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs.}, author = {Letts, James A and Fiedorczuk, Karol and Degliesposti, Gianluca and Skehel, Mark and Sazanov, Leonid A}, issn = {1097-2765}, journal = {Molecular Cell}, number = {6}, pages = {1131--1146.e6}, publisher = {Cell Press}, title = {{Structures of respiratory supercomplex I+III2 reveal functional and conformational crosstalk}}, doi = {10.1016/j.molcel.2019.07.022}, volume = {75}, year = {2019}, } @article{15155, abstract = {The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.}, author = {Gustafson, Chelsea L. and Parsley, Nicole C. and Asimgil, Hande and Lee, Hsiau-Wei and Ahlbach, Christopher and Michael, Alicia Kathleen and Xu, Haiyan and Williams, Owen L. and Davis, Tara L. and Liu, Andrew C. and Partch, Carrie L.}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {4}, pages = {447--457.e7}, publisher = {Elsevier}, title = {{A slow conformational switch in the BMAL1 transactivation domain modulates circadian rhythms}}, doi = {10.1016/j.molcel.2017.04.011}, volume = {66}, year = {2017}, } @article{15160, abstract = {The circadian clock orchestrates global changes in transcriptional regulation on a daily basis via the bHLH-PAS transcription factor CLOCK:BMAL1. Pathways driven by other bHLH-PAS transcription factors have a homologous repressor that modulates activity on a tissue-specific basis, but none have been identified for CLOCK:BMAL1. We show here that the cancer/testis antigen PASD1 fulfills this role to suppress circadian rhythms. PASD1 is evolutionarily related to CLOCK and interacts with the CLOCK:BMAL1 complex to repress transcriptional activation. Expression of PASD1 is restricted to germline tissues in healthy individuals but can be induced in cells of somatic origin upon oncogenic transformation. Reducing PASD1 in human cancer cells significantly increases the amplitude of transcriptional oscillations to generate more robust circadian rhythms. Our results describe a function for a germline-specific protein in regulation of the circadian clock and provide a molecular link from oncogenic transformation to suppression of circadian rhythms.}, author = {Michael, Alicia Kathleen and Harvey, Stacy L. and Sammons, Patrick J. and Anderson, Amanda P. and Kopalle, Hema M. and Banham, Alison H. and Partch, Carrie L.}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {5}, pages = {743--754}, publisher = {Elsevier}, title = {{Cancer/Testis antigen PASD1 silences the circadian clock}}, doi = {10.1016/j.molcel.2015.03.031}, volume = {58}, year = {2015}, } @article{11127, abstract = {Nuclear formation in Xenopus egg extracts requires cytosol and is inhibited by GTPγS, indicating a requirement for GTPase activity. Nuclear envelope (NE) vesicle fusion is extensively inhibited by GTPγS and two mutant forms of the Ran GTPase, Q69L and T24N. Depletion of either Ran or RCC1, the exchange factor for Ran, from the assembly reaction also inhibits this step of NE formation. Ran depletion can be complemented by the addition of Ran loaded with either GTP or GDP but not with GTPγS. RCC1 depletion is only complemented by RCC1 itself or by RanGTP. Thus, generation of RanGTP by RCC1 and GTP hydrolysis by Ran are both required for the extensive membrane fusion events that lead to NE formation.}, author = {HETZER, Martin W and Bilbao-Cortés, Daniel and Walther, Tobias C and Gruss, Oliver J and Mattaj, Iain W}, issn = {1097-2765}, journal = {Molecular Cell}, keywords = {Cell Biology, Molecular Biology}, number = {6}, pages = {1013--1024}, publisher = {Elsevier}, title = {{GTP hydrolysis by Ran is required for nuclear envelope assembly}}, doi = {10.1016/s1097-2765(00)80266-x}, volume = {5}, year = {2000}, }