bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2025–09–14
twelve papers selected by
Connor Rogerson, University of Cambridge
  1. Real-time visualization of reconstituted transcription reveals RNAPII activation mechanisms at single promoters.
  2. Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase.
  3. Native nucleosome-positioning elements as alternatives to the 601 sequence for nucleosome repositioning studies.
  4. Epigenomic landscape of single vascular cells reflects developmental origin and disease risk loci.
  5. Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1.
  6. Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the mouse anterior brainstem.
  7. Dynamic association of H3K36me3 with pericentromeric heterochromatin regulates its replication time.
  8. RNA-binding proteins mediate the maturation of chromatin topology during differentiation.
  9. E. coli transcription factors regulate promoter activity by a universal, homeostatic mechanism.
  10. Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity.
  11. Protocol for multiplexed transcription factor activity detection using optimized barcoded reporters and an automated computational pipeline.
  12. Computational design of sequence-specific DNA-binding proteins.
  1. Cell Rep. 2025 Sep 05. pii: S2211-1247(25)01022-8. [Epub ahead of print]44(9): 116251
    Real-time visualization of reconstituted transcription reveals RNAPII activation mechanisms at single promoters.
    Megan Palacio, Dylan J Taatjes.
      RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and Mediator. TFs, Mediator, and RNAPII contain intrinsically disordered regions (IDRs) and form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a real-time in vitro fluorescence transcription (RIFT) assay for second-by-second visualization of transcription at hundreds of promoters simultaneously. Our results establish IDRs as essential for rapid RNAPII activation, without condensate formation. For example, HSF1 condensates and single molecules function identically, whereas MED1-IDR can functionally replace HSF1 but activates RNAPII with slower kinetics. Through their IDRs, Mediator and TFs rapidly and synergistically activate RNAPII bursting and re-initiation, and surprisingly, Mediator drives TF-promoter recruitment without TF-DNA binding. Importantly, RIFT directly addresses questions largely intractable with cell-based methods, yielding mechanistic insights about condensates, IDRs, enhancer-promoter communication, and RNAPII bursting that complement live-cell imaging data.
    Keywords:  CP: Molecular biology; HSF1; Mediator; PIC scaffold; RNA polymerase II; TFIID; burst size; bursting; condensates; fluorescence microscopy; intrinsically disordered regions; phase separation; re-initiation; smTIRF; transcription; transcription factor
    DOI:  https://doi.org/10.1016/j.celrep.2025.116251
  2. Nat Methods. 2025 Sep 11.
    Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase.
    Heejin Roh, Simon P Shen, Yan Hu, Hui Si Kwok, Allison P Siegenfeld, Ceejay Lee, Marcanthony U Zepeda, Chun-Jie Guo, Shelby A Roseman, Caroline Comenho, Vijay G Sankaran, Jason D Buenrostro, Brian B Liau.
      Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34+ hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34+ HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.
    DOI:  https://doi.org/10.1038/s41592-025-02811-2
  3. Nucleic Acids Res. 2025 Sep 05. pii: gkaf822. [Epub ahead of print]53(17):
    Native nucleosome-positioning elements as alternatives to the 601 sequence for nucleosome repositioning studies.
    Ruo-Wen Chen, Shane D Stoeber, Ilana M Nodelman, Hengye Chen, Wenxuan Yang, Gregory D Bowman, Lu Bai, Michael G Poirier.
      Nucleosome repositioning is essential for establishing nucleosome-depleted regions to initiate transcription. This process has been extensively studied using structural, biochemical, and single-molecule approaches, which require homogeneously positioned nucleosomes. This is often achieved using the Widom 601 sequence, a highly efficient nucleosome-positioning element (NPE) selected for its unusually strong binding to the H3-H4 histone tetramer. Due to the artificial nature of 601, native NPEs are needed to explore the role of DNA sequence in nucleosome repositioning. Here, we characterize the position distributions and nucleosome formation free energies for a set of yeast native nucleosomes from Saccharomyces cerevisiae. We show these native NPEs can be used in biochemical studies of nucleosome repositioning by transcription factors (TFs) and the chromatin remodeler Chd1. TFs could directly reposition a fraction of nucleosomes containing native NPEs, but not 601-containing nucleosomes. In contrast, partial unwrapping was similar for 601 and native NPE sequences, and the rate of ATP-dependent remodeling by Chd1 was within the range of the fast and slow directions of the 601 nucleosomes. This set of native NPEs provides an alternative to the 601 NPE that can be used for probing the repositioning of nucleosomes that contain native DNA sequences.
    DOI:  https://doi.org/10.1093/nar/gkaf822
  4. Mol Syst Biol. 2025 Sep 10.
    Epigenomic landscape of single vascular cells reflects developmental origin and disease risk loci.
    Chad S Weldy, Soumya Kundu, João Monteiro, Wenduo Gu, Albert J Pedroza, Alex R Dalal, Matthew D Worssam, Daniel Li, Brian Palmisano, Quanyi Zhao, Disha Sharma, Trieu Nguyen, Ramendra Kundu, Michael P Fischbein, Jesse Engreitz, Anshul B Kundaje, Paul P Cheng, Thomas Quertermous.
      Vascular sites have distinct susceptibility to atherosclerosis and aneurysm, yet the epigenomic and transcriptomic underpinning of vascular site-specific disease risk is largely unknown. Here, we performed single-cell chromatin accessibility (scATACseq) and gene expression profiling (scRNAseq) of mouse vascular tissue from three vascular sites. Through interrogation of epigenomic enhancers and gene regulatory networks, we discovered key regulatory enhancers to not only be cell type, but vascular site-specific. We identified epigenetic markers of embryonic origin including developmental transcription factors such as Tbx20, Hand2, Gata4, and Hoxb family members and discovered transcription factor motif accessibility to be vascular site-specific for smooth muscle, fibroblasts, and endothelial cells. We further integrated genome-wide association data for aortic dimension, and using a deep learning model to predict variant effect on chromatin accessibility, ChromBPNet, we predicted variant effects across cell type and vascular site of origin, revealing genomic regions enriched for specific TF motif footprints-including MEF2A, SMAD3, and HAND2. This work supports a paradigm that cell type and vascular site-specific enhancers govern complex genetic drivers of disease risk.
    Keywords:  Development; Epigenomics; Genomics; Single-Cell Transcriptomics; Vascular Biology
    DOI:  https://doi.org/10.1038/s44320-025-00140-2
  5. Cell Rep. 2025 Sep 04. pii: S2211-1247(25)01018-6. [Epub ahead of print]44(9): 116247
    Paraspeckle protein NONO regulates active chromatin by allosterically stimulating NSD1.
    Chen-I Hsu, Shenglin Mei, Justin Demmerle, Anil Prakash, Sarah Ruttenberg, Mahnue Sahn, Jia-Ray Yu.
      Nuclear receptor binding set domain protein 1 (NSD1) is a key histone methyltransferase that catalyzes di-methylation of lysine 36 of histone H3 (H3K36me2), essential for active chromatin domains. While the loss of NSD1 activity halts embryonic development and its aberrant gain drives oncogenesis in leukemia and glioma, the regulatory mechanisms remain poorly understood. Here, we uncover that NSD1 requires allosteric activation through the aromatic pocket of its Pro-Trp-Trp-Pro 2 (PWWP2) domain. Surprisingly, NSD1-PWWP2 binds to the non-canonical target, nuclear paraspeckle protein non-POU-domain-containing octamer binding protein (NONO), and this protein-protein interaction allosterically stimulates NSD1. Mouse embryonic stem cells engineered with mutations in the aromatic pocket of NSD1-PWWP2 cannot differentiate into neural progenitor cells, and genetic depletion of NONO partially phenocopies this defect, potentially explaining the neurodevelopmental disorder phenotypes in NSD1- and NONO-deficient diseases. Our work uncovers a mechanism driving active chromatin domain formation, an implication in the interplay between nuclear paraspeckles and active chromatin, and a vulnerability of NSD1 for therapeutic interventions.
    Keywords:  CP: Molecular biology; CP: Neuroscience; MRXS34 syndrome; Sotos syndrome; active chromatin; epigenetics; neural differentiation; neurodevelopmental disorders; nuclear paraspeckles; stem cells
    DOI:  https://doi.org/10.1016/j.celrep.2025.116247
  6. Elife. 2025 Sep 12. pii: RP105867. [Epub ahead of print]14
    Gene regulatory mechanisms guiding bifurcation of inhibitory and excitatory neuron lineages in the mouse anterior brainstem.
    Sami Kilpinen, Lassi Virtanen, Silvana Bodington Celma, Amos Bonsdorff, Heidi Heliölä, Kaia Achim, Juha Partanen.
      Selector transcription factors (TFs) control choices of alternative cellular fates during development. The ventral rhombomere 1 of the embryonic mouse (Mus musculus) brainstem produces neuronal precursors that can differentiate into either inhibitory GABAergic or excitatory glutamatergic neurons important for the control of behaviour. TFs Tal1, Gata2, and Gata3 are required for adopting the GABAergic neuronal identity and inhibiting the glutamatergic identity. Here, we asked how these selector TFs are activated and how they control the identity of the developing brainstem neurons. We addressed these questions by analysing chromatin accessibility at putative gene regulatory elements active during GABAergic and glutamatergic neuron lineage bifurcation, combined with studies of TF expression and DNA binding. Our results show that the Tal1, Gata2, and Gata3 genes are activated by highly similar mechanisms, with connections to regional patterning, neurogenic cell cycle exit and general course of neuronal differentiation. After activation, Tal1, Gata2, and Gata3 are linked by auto- and cross-regulation as well as regulatory interactions with TFs of the glutamatergic branch. Predicted targets of these selector TFs include genes expressed in GABAergic neurons, glutamatergic neurons, or both. Unlike genes specific to the glutamatergic branch, the genes expressed in GABAergic neurons appear to be under combinatorial control of Tal1, Gata2, and Gata3. Understanding gene regulatory interactions affecting the anterior brainstem GABAergic and glutamatergic neuron differentiation may give genetic and mechanistic insights into neurodevelopmental traits and disorders.
    Keywords:  brainstem; cell biology; developmental biology; differentiation; mouse; neurobiology
    DOI:  https://doi.org/10.7554/eLife.105867
  7. EMBO Rep. 2025 Sep 08.
    Dynamic association of H3K36me3 with pericentromeric heterochromatin regulates its replication time.
    Sunil Kumar Pradhan, Hui Zhang, Ksenia G Kolobynina, Alexander Rapp, Maria Arroyo, M Cristina Cardoso.
      The flexibility of the spatio-temporal genome replication program during development and disease highlights the regulatory role of plastic epigenetic mechanisms over genetic determinants. Histone post-translational modifications are broadly implicated in replication timing control, yet the specific mechanisms through which individual histone marks influence replication dynamics, particularly in heterochromatin, remain unclear. Here, we demonstrate that H3K36me3 dynamically enriches at pericentromeric heterochromatin, composed of major satellite DNA repeats, prior to replication during mid S phase in mouse embryonic stem cells. By knocking down lysine 36-specific methyltransferases or by targeting the H3K36M oncohistone to pericentromeric heterochromatin, we reduce global or local H3K36me3 levels, respectively, revealing its essential role in preserving the replication timing of constitutive heterochromatin. Loss of H3K36me3 accompanies increased RNA polymerase II serine-5 phosphorylation and lowered major satellite RNA levels, indicating transcriptional dysregulation. Notably, we identify a strand-specific contribution of major satellite forward transcripts in regulating the replication timing of constitutive heterochromatin and maintaining chromatin stability, highlighting the importance of non-coding RNAs as critical regulators of replication timing.
    Keywords:  H3K36me3; MajSat RNA; Oncohistone; Pericentromeric Heterochromatin; Replication Timing
    DOI:  https://doi.org/10.1038/s44319-025-00575-6
  8. Nat Cell Biol. 2025 Sep;27(9): 1510-1525
    RNA-binding proteins mediate the maturation of chromatin topology during differentiation.
    Bondita Dehingia, Małgorzata Milewska-Puchała, Marcin Janowski, Mahmoud-Reza Rafiee, Misbah Abbas, Aleksandra Piotrowska, Jan Senge, Piotr Blaut, Dietrich Walsh, Jacqueline Severino, Debadeep Chaudhury, Sajjad Iqbal, Rogelio Montiel-Manriquez, Sylwia Jankowska, Peyman Zare, Wolfgang Huber, Jianliang Xu, Rafael Casellas, Timo Zimmermann, Paweł Dłotko, Jeroen Krijgsveld, Aleksandra Pękowska.
      Topologically associating domains (TADs) and chromatin architectural loops impact promoter-enhancer interactions, with CCCTC-binding factor (CTCF) defining TAD borders and loop anchors. TAD boundaries and loops progressively strengthen upon embryonic stem (ES) cell differentiation, underscoring the importance of chromatin topology in ontogeny. However, the mechanisms driving this process remain unclear. Here we show a widespread increase in CTCF-RNA-binding protein (RBP) interactions upon ES to neural stem (NS) cell differentiation. While dispensable in ES cells, RBPs reinforce CTCF-anchored chromatin topology in NS cells. We identify Pantr1, a non-coding RNA, as a key facilitator of CTCF-RBP interactions, promoting chromatin maturation. Using acute CTCF degradation, we find that, through its insulator function, CTCF helps maintain neuronal gene silencing in NS cells by acting as a barrier to untimely gene activation during development. Altogether, we reveal a fundamental mechanism driving developmentally linked chromatin structural consolidation and the contribution of this process to the control of gene expression in differentiation.
    DOI:  https://doi.org/10.1038/s41556-025-01735-5
  9. Science. 2025 Sep 11. 389(6765): eadv2064
    E. coli transcription factors regulate promoter activity by a universal, homeostatic mechanism.
    Vinuselvi Parisutham, Sunil Guharajan, Melina Lian, Md Zulfikar Ali, Hannah Rogers, Shannon Joyce, Mariana Noto Guillen, Robert C Brewster.
      Transcription factors (TFs) may activate or repress gene expression through an interplay of different mechanisms, including RNA polymerase (RNAP) recruitment, exclusion, and initiation. However, depending on the regulated promoter identity, TF function can vary, and the principles underlying this context dependence remain unclear. We demonstrate an inverse scaling relationship between the promoter's basal activity and its regulation by a given TF. Specifically, activation is weaker and repression is stronger on stronger promoters. This scaling applies to both activators and repressors, which suggests a common underlying mechanism where TFs regulate expression by stabilizing RNAP binding at the promoter. The consequence of this relationship is that TFs buffer expression by affecting constant regulated expression levels across promoters of different basal activity, ensuring homeostatic control despite genetic or environmental changes.
    DOI:  https://doi.org/10.1126/science.adv2064
  10. Elife. 2025 Sep 10. pii: RP101578. [Epub ahead of print]13
    Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity.
    Khund Sayeed, Sreeja Parameswaran, Matthew J Beucler, Lee E Edsall, Andrew VonHandorf, Audrey Crowther, Omer A Donmez, Matthew R Hass, Scott Richards, Carmy R Forney, Hayley K Hesse, Sydney H Jones, Katelyn A Dunn, Jay Wright, Merrin Man Long Leong, Laura A Murray-Nerger, Vijay Yechoor, Ben E Gewurz, Kenneth M Kaufman, John B Harley, Bo Zhao, William E Miller, Leah C Kottyan, Matthew T Weirauch.
      Human cytomegalovirus (HCMV) infects up to 80% of the world's population. Here, we show that HCMV infection leads to widespread changes in human chromatin accessibility and chromatin looping, with hundreds of thousands of genomic regions affected 48 hr after infection. Integrative analyses reveal HCMV-induced perturbation of Hippo signaling through drastic reduction of TEAD1 transcription factor activity. We confirm extensive concordant loss of TEAD1 binding, active H3K27ac histone marks, and chromatin looping interactions upon infection. Our data position TEAD1 at the top of a hierarchy involving multiple altered important developmental pathways. HCMV infection reduces TEAD1 activity through four distinct mechanisms: closing of TEAD1-bound chromatin, reduction of YAP1 and phosphorylated YAP1 levels, reduction of TEAD1 transcript and protein levels, and alteration of TEAD1 exon 6 usage. Altered TEAD1-based mechanisms are highly enriched at genetic risk loci associated with eye and ear development, providing mechanistic insight into HCMV's established roles in these processes.
    Keywords:  chromosomes; cytomegalovirus; epigenetics; functional genomics; gene expression; gene regulation; human; infectious disease; microbiology; virus infection; viruses
    DOI:  https://doi.org/10.7554/eLife.101578
  11. STAR Protoc. 2025 Sep 10. pii: S2666-1667(25)00472-1. [Epub ahead of print]6(3): 104066
    Protocol for multiplexed transcription factor activity detection using optimized barcoded reporters and an automated computational pipeline.
    Max Trauernicht, Vinícius H Franceschini-Santos, Hatice Yücel, Bas van Steensel.
      Transcription factors (TFs) regulate the genome in response to signaling events. Detecting their activity is crucial to deciphering the regulatory networks of cells. Here, we present a protocol for multiplexed TF activity detection using a barcoded plasmid library of optimized "prime" TF reporters in cultured cells. We describe steps for library transfection, RNA processing for barcode sequencing, and a computational pipeline for analyzing differential TF activity, enabling high-throughput and quantitative TF profiling. For complete details on the use and execution of this protocol, please refer to Trauernicht et al.1.
    Keywords:  Bioinformatics; Cell Biology; Cell culture; Gene Expression; Genomics; Molecular Biology; RNAseq; Sequence analysis; Sequencing; Systems biology
    DOI:  https://doi.org/10.1016/j.xpro.2025.104066
  12. Nat Struct Mol Biol. 2025 Sep 12.
    Computational design of sequence-specific DNA-binding proteins.
    Cameron J Glasscock, Robert J Pecoraro, Ryan McHugh, Lindsey A Doyle, Wei Chen, Olivier Boivin, Beau Lonnquist, Emily Na, Yuliya Politanska, Hugh K Haddox, David Cox, Christoffer Norn, Brian Coventry, Inna Goreshnik, Dionne Vafeados, Gyu Rie Lee, Raluca Gordân, Barry L Stoddard, Frank DiMaio, David Baker.
      Sequence-specific DNA-binding proteins (DBPs) have critical roles in biology and biotechnology and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize short specific target sequences through interactions with bases in the major groove and use this method to generate binders for five distinct DNA targets with mid-nanomolar to high-nanomolar affinities. The individual binding modules have specificity closely matching the computational models at as many as six base-pair positions and higher-order specificity can be achieved by rigidly positioning the binders along the DNA double helix using RFdiffusion. The crystal structure of a designed DBP-target site complex is in close agreement with the design model and the designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method provides a route to small and, hence, readily deliverable sequence-specific DBPs for gene regulation and editing.
    DOI:  https://doi.org/10.1038/s41594-025-01669-4
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