bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2025–04–20
25 papers selected by
Connor Rogerson, University of Cambridge
  1. TFE3 fusion oncoprotein condensates drive transcriptional reprogramming and cancer progression in translocation renal cell carcinoma.
  2. PP1/PNUTS phosphatase binds the restrictor complex and stimulates RNA Pol II transcription termination.
  3. Rewriting regulatory DNA to dissect and reprogram gene expression.
  4. A specific pluripotency-associated eRNA controls Nanog locus by shaping the epigenetic landscape and stabilizing enhancer-promoter interaction.
  5. pC-SAC: A method for high-resolution 3D genome reconstruction from low-resolution Hi-C data.
  6. Low overlap of transcription factor DNA binding and regulatory targets.
  7. Tripartite phosphorylation of SPT5 by CDK9 times pause release and tunes elongation rate of RNA polymerase II.
  8. Integrating genetic variation with deep learning provides context for variants impacting transcription factor binding during embryogenesis.
  9. An oncohistone-driven H3.3K27M/CREB5/ID1 axis maintains the stemness and malignancy of diffuse intrinsic pontine glioma.
  10. High-resolution dynamic imaging of chromatin DNA communication using Oligo-LiveFISH.
  11. Med12 cooperates with multiple differentiation signals to facilitate efficient lineage transitions in embryonic stem cells.
  12. Dynamics of transcriptional programs and chromatin accessibility in mouse spermatogonial cells from early postnatal to adult life.
  13. Unified molecular approach for spatial epigenome, transcriptome, and cell lineages.
  14. A boundary-defining protein facilitates megabase-scale regulatory chromosomal loop formation in Drosophila neurons.
  15. TRAPT: a multi-stage fused deep learning framework for predicting transcriptional regulators based on large-scale epigenomic data.
  16. Unveiling chromatin dynamics with virtual epigenome.
  17. Cryo-EM Structures of Native Chromatin Units From Human Cells.
  18. High-resolution mapping of sigma factor DNA-binding sequences using artificial promoters, RNA aptamers, and deep sequencing.
  19. The genetic architecture of cell type-specific cis regulation in maize.
  20. Histone chaperones coupled to DNA replication and transcription control divergent chromatin elements to maintain cell fate.
  21. Synthetic deconvolution of an auxin-dependent transcriptional code.
  22. Systematic genetic perturbation reveals principles underpinning robustness of the epigenetic regulatory network.
  23. scMultiSim: simulation of single-cell multi-omics and spatial data guided by gene regulatory networks and cell-cell interactions.
  24. Mutant CEBPA promotes tolerance to inflammatory stress through deficient AP-1 activation.
  25. Integrator loss leads to dsRNA formation that triggers the integrated stress response.
  1. Cell Rep. 2025 Apr 11. pii: S2211-1247(25)00310-9. [Epub ahead of print]44(4): 115539
    TFE3 fusion oncoprotein condensates drive transcriptional reprogramming and cancer progression in translocation renal cell carcinoma.
    Choon Leng So, Ye Jin Lee, Bujamin H Vokshi, Wanlu Chen, Binglin Huang, Emily De Sousa, Yangzhenyu Gao, Marie Elena Portuallo, Sumaiya Begum, Kasturee Jagirdar, W Marston Linehan, Vito W Rebecca, Hongkai Ji, Eneda Toska, Danfeng Cai.
      Translocation renal cell carcinoma (tRCC) presents a significant clinical challenge due to its aggressiveness and limited treatment options. It is primarily driven by fusion oncoproteins (FOs), yet their role in oncogenesis is not fully understood. Here, we investigate TFE3 fusions in tRCC, focusing on NONO::TFE3 and SFPQ::TFE3. We demonstrate that TFE3 FOs form liquid-like condensates with increased transcriptional activity, localizing to TFE3 target genes and promoting cell proliferation and migration. The coiled-coil domains (CCDs) of NONO and SFPQ are essential for condensate formation, prolonging TFE3 FOs' chromatin binding time and enhancing transcription. Compared with wild-type TFE3, TFE3 FOs bind to new chromatin regions, alter chromatin accessibility, and form new enhancers and super-enhancers at pro-growth gene loci. Disruption of condensate formation via CCD modification abolishes these genome-wide changes. Altogether, our integrated analyses underscore the critical functions of TFE3 FO condensates in driving tumor cell growth, providing key insights for future therapeutic strategies.
    Keywords:  CP: Cancer; CP: Molecular biology; TFE3 fusion; biomolecular condensates; cancer; chromatin accessibility; fusion oncoprotein; gene regulation; single-particle tracking; translocation renal cell carcinoma
    DOI:  https://doi.org/10.1016/j.celrep.2025.115539
  2. Cell Rep. 2025 Apr 16. pii: S2211-1247(25)00335-3. [Epub ahead of print]44(5): 115564
    PP1/PNUTS phosphatase binds the restrictor complex and stimulates RNA Pol II transcription termination.
    Benjamin Erickson, Roman Fedoryshchak, Nova Fong, Ryan Sheridan, Keira Y Larson, Anthony J Saviola, Stephane Mouilleron, Kirk C Hansen, Richard Treisman, David L Bentley.
      The restrictor ZC3H4/WDR82 terminates antisense transcription from bidirectional promoters, but its mechanism is poorly understood. We report that ZC3H4/WDR82 immunoprecipitates with PP1 phosphatase and its nuclear targeting subunit, PP1 phosphatase nuclear targeting subunit (PNUTS), which binds to WDR82. AlphaFold predicts a complex of PP1/PNUTS with the restrictor where both PNUTS and ZC3H4 contact WDR82. A substrate trap, PP1H66K-PNUTS, comprising inactive PP1 fused to the PNUTS C terminus, antagonizes restrictor-mediated termination, whereas PP1WT-PNUTS has less of an effect, suggesting that phosphatase activity is required for termination. One PP1/PNUTS substrate implicated in termination by the restrictor is RNA polymerase II (RNA Pol II) CTD Ser5-P. PP1H66K-PNUTS induces Ser5-P hyperphosphorylation at 5' ends, presumably by inhibiting dephosphorylation. NET-seq analysis suggests that CTD Ser5 dephosphorylation would promote termination by increasing RNA Pol II pausing. Both inhibition of termination and CTD hyperphosphorylation require the WDR82 binding domain of PP1H66K-PNUTS, which mediates restrictor binding. In summary, the PP1/PNUTS phosphatase associated with the restrictor via WDR82 promotes efficient transcription termination.
    Keywords:  CP: Molecular biology; CTD dephosphorylation; PP1 phosphatase; Pol II CTD Ser5 phosphorylation; WDR82; ZC3H4; restrictor; transcription elongation; transcription termination
    DOI:  https://doi.org/10.1016/j.celrep.2025.115564
  3. Cell. 2025 Apr 14. pii: S0092-8674(25)00352-6. [Epub ahead of print]
    Rewriting regulatory DNA to dissect and reprogram gene expression.
    Gabriella E Martyn, Michael T Montgomery, Hank Jones, Katherine Guo, Benjamin R Doughty, Johannes Linder, Deepa Bisht, Fan Xia, Xiangmeng S Cai, Ziwei Chen, Kelly Cochran, Kathryn A Lawrence, Glen Munson, Anusri Pampari, Charles P Fulco, Nidhi Sahni, David R Kelley, Eric S Lander, Anshul Kundaje, Jesse M Engreitz.
      Regulatory DNA provides a platform for transcription factor binding to encode cell-type-specific patterns of gene expression. However, the effects and programmability of regulatory DNA sequences remain difficult to map or predict. Here, we develop variant effects from flow-sorting experiments with CRISPR targeting screens (Variant-EFFECTS) to introduce hundreds of designed edits to endogenous regulatory DNA and quantify their effects on gene expression. We systematically dissect and reprogram 3 regulatory elements for 2 genes in 2 cell types. These data reveal endogenous binding sites with effects specific to genomic context, transcription factor motifs with cell-type-specific activities, and limitations of computational models for predicting the effect sizes of variants. We identify small edits that can tune gene expression over a large dynamic range, suggesting new possibilities for prime-editing-based therapeutics targeting regulatory DNA. Variant-EFFECTS provides a generalizable tool to dissect regulatory DNA and to identify genome editing reagents that tune gene expression in an endogenous context.
    Keywords:  CRISPR; RNA FlowFISH; enhancers; gene regulation; high-throughput screening; non-coding variants; predictive models; prime editing; sequence design; transcription factors
    DOI:  https://doi.org/10.1016/j.cell.2025.03.034
  4. Nucleic Acids Res. 2025 Apr 10. pii: gkaf274. [Epub ahead of print]53(7):
    A specific pluripotency-associated eRNA controls Nanog locus by shaping the epigenetic landscape and stabilizing enhancer-promoter interaction.
    Mariella Cuomo, Davide Costabile, Rosa Della Monica, Michela Buonaiuto, Federica Trio, Giulia De Riso, Roberta Visconti, Lorenzo Chiariotti.
      Despite a plethora of studies exploring the transcriptional regulation of the Nanog gene, the role of the enhancer RNAs (eRNAs) derived from Nanog-interacting super-enhancers (SEs) remains under-investigated. In the present study, we examined the functional role of the eRNAs transcribed from the -5 kb Nanog SE in mouse embryonic stem cells (mESCs) and found that an eRNA, here defined as -5KNAR, was essential to maintain the Nanog locus in an epigenetically active configuration, thereby ensuring pluripotency. We found that the here identified -5KNAR functionally interacts with the RAD21 protein, suggesting a role in stabilizing a cohesin complex at the Nanog locus, ensuring the generation and maintenance of an enhancer-promoter loop. Silencing of -5KNAR caused a cascade of events, including the generation of a DNA methylation wave (likely spreading from a single methylated CpG site), substantial chromatin remodeling, and loss of the enhancer-promoter loop, inducing Nanog silencing and mESC differentiation. Under these conditions, exogenous re-expression of Nanog was unable to restore either the endogenous Nanog expression or the enhancer-promoter interaction, suggesting that, at hierarchical level, the expression of the -5KNAR plays a prominent role in maintaining the pluripotency in mESCs.
    DOI:  https://doi.org/10.1093/nar/gkaf274
  5. Nucleic Acids Res. 2025 Apr 10. pii: gkaf289. [Epub ahead of print]53(7):
    pC-SAC: A method for high-resolution 3D genome reconstruction from low-resolution Hi-C data.
    J Carlos Angel, Narjis El Amraoui, Gamze Gürsoy.
      The three-dimensional (3D) organization of the genome is crucial for gene regulation, with disruptions linked to various diseases. High-throughput Chromosome Conformation Capture (Hi-C) and related technologies have advanced our understanding of 3D genome organization by mapping interactions between distal genomic regions. However, capturing enhancer-promoter interactions at high resolution remains challenging due to the high sequencing depth required. We introduce pC-SAC (probabilistically Constrained Self-Avoiding Chromatin), a novel computational method for producing accurate high-resolution Hi-C matrices from low-resolution data. pC-SAC uses adaptive importance sampling with sequential Monte Carlo to generate ensembles of 3D chromatin chains that satisfy physical constraints derived from low-resolution Hi-C data. Our method achieves over 95% accuracy in reconstructing high-resolution chromatin maps and identifies novel interactions enriched with candidate cis-regulatory elements (cCREs) and expression quantitative trait loci (eQTLs). Benchmarking against state-of-the-art deep learning models demonstrates pC-SAC's performance in both short- and long-range interaction reconstruction. pC-SAC offers a cost-effective solution for enhancing the resolution of Hi-C data, thus enabling deeper insights into 3D genome organization and its role in gene regulation and disease. Our tool can be found at https://github.com/G2Lab/pCSAC.
    DOI:  https://doi.org/10.1093/nar/gkaf289
  6. Nature. 2025 Apr 16.
    Low overlap of transcription factor DNA binding and regulatory targets.
    Lakshmi Mahendrawada, Linda Warfield, Rafal Donczew, Steven Hahn.
      DNA sequence-specific transcription factors (TFs) modulate transcription and chromatin architecture, acting from regulatory sites in enhancers and promoters of eukaryotic genes1,2. How multiple TFs cooperate to regulate individual genes is still unclear. In yeast, most TFs are thought to regulate transcription via binding to upstream activating sequences, which are situated within a few hundred base pairs upstream of the regulated gene3. Although this model has been validated for individual TFs and specific genes, it has not been tested in a systematic way. Here we integrated information on the binding and expression targets for the near-complete set of yeast TFs and show that, contrary to expectations, there are few TFs with dedicated activator or repressor roles, and that most TFs have a dual function. Although nearly all protein-coding genes are regulated by one or more TFs, our analysis revealed limited overlap between TF binding and gene regulation. Rapid depletion of many TFs also revealed many regulatory targets that were distant from detectable TF binding sites, suggesting unexpected regulatory mechanisms. Our study provides a comprehensive survey of TF functions and offers insights into interactions between the set of TFs expressed in a single cell type and how they contribute to the complex programme of gene regulation.
    DOI:  https://doi.org/10.1038/s41586-025-08916-0
  7. Mol Cell. 2025 Apr 10. pii: S1097-2765(25)00265-5. [Epub ahead of print]
    Tripartite phosphorylation of SPT5 by CDK9 times pause release and tunes elongation rate of RNA polymerase II.
    Rui Sun, Robert P Fisher.
      The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by protein phosphorylation. Previously, two regions phosphorylated by cyclin-dependent kinase 9 (CDK9) in elongation factor SPT5-the linker between Kyrpides-Ouzounis-Woese (KOW) x-4 and 5 domains and carboxy-terminal repeat (CTR) 1-were implicated in promoter-proximal pausing and termination, respectively. Here, we show that phosphorylations in the linker, CTR1, and a third region, CTR2, coordinately control pause release, elongation speed, and termination in HCT116 human colon cancer cells. Pausing was unaffected or increased by mutations preventing CTR1 or CTR2 phosphorylation, respectively, but attenuated when both CTRs were mutated. Whereas loss of CTR1 phosphorylation slowed elongation and repressed nascent transcription, simultaneous CTR2 mutation partially reversed both effects. Nevertheless, mutating both CTRs had additive effects on splicing, termination, steady-state mRNA levels, and cell proliferation. Therefore, tripartite SPT5 phosphorylation times pause release and tunes RNAPII elongation rate to ensure productive transcription and cell viability.
    Keywords:  CDK9; DRB sensitivity-inducing factor; DSIF; P-TEFb; RNA polymerase II; RNAPII; SPT5; chemical genetics; cyclin-dependent kinase 9; phosphorylation; positive transcription elongation factor b; pre-mRNA splicing; promoter-proximal pausing; transcription; transcription elongation; transcription termination
    DOI:  https://doi.org/10.1016/j.molcel.2025.03.021
  8. Genome Res. 2025 Apr 15.
    Integrating genetic variation with deep learning provides context for variants impacting transcription factor binding during embryogenesis.
    Olga M Sigalova, Mattia Forneris, Frosina Stojanovska, Bingqing Zhao, Rebecca R Viales, Adam Rabinowitz, Fayrouz Hammal, Benoît Ballester, Judith B Zaugg, Eileen E M Furlong.
      Understanding how genetic variation impacts transcription factor (TF) binding remains a major challenge, limiting our ability to model disease-associated variants. Here, we used a highly controlled system of F1 crosses with extensive genetic diversity to profile allele-specific binding of four TFs at several time points during Drosophila embryogenesis. Using a combined haplotype test, we identified 9%-18% of TF-bound regions impacted by genetic variation even for essential regulators. By expanding WASP (a tool for allele-specific read mapping) to examine indels, we increased detection of allelically imbalanced peaks by 30%-50%. This fine-grained "mutagenesis" can reconstruct functionalized binding motifs for all factors. To prioritize causal variants, we trained a convolutional neural network (Basenji) to accurately predict binding from DNA sequence. The model can also predict measured allelic imbalance for strong effect variants, providing a mechanistic interpretation for how the variant impacts binding. This reveals unexpected relationships between TFs, including potential cooperative pairs, and mechanisms of tissue-specific recruitment of the ubiquitous factor CTCF.
    DOI:  https://doi.org/10.1101/gr.279652.124
  9. Nat Commun. 2025 Apr 17. 16(1): 3675
    An oncohistone-driven H3.3K27M/CREB5/ID1 axis maintains the stemness and malignancy of diffuse intrinsic pontine glioma.
    Wei Zhou, Cheng Xu, Shuangrui Yang, Haocheng Li, Changcun Pan, Zhuang Jiang, Luyang Xie, Xiaohan Li, Huimin Qiao, Da Mi, Yujie Tang, Liwei Zhang, Qiaoran Xi.
      Diffuse intrinsic pontine glioma (DIPG), a lethal pediatric cancer driven by H3K27M oncohistones, exhibits aberrant epigenetic regulation and stem-like cell states. Here, we uncover an axis involving H3.3K27M oncohistones, CREB5/ID1, which sustains the stem-like state of DIPG cells, promoting malignancy. We demonstrate that CREB5 mediates elevated ID1 levels in the H3.3K27M/ACVR1WT subtype, promoting tumor growth; while BMP signaling regulates this process in the H3.1K27M/ACVR1MUT subtype. Furthermore, we reveal that H3.3K27M directly enhances CREB5 expression by reshaping the H3K27me3 landscape at the CREB5 locus, particularly at super-enhancer regions. Additionally, we elucidate the collaboration between CREB5 and BRG1, the SWI/SNF chromatin remodeling complex catalytic subunit, in driving oncogenic transcriptional changes in H3.3K27M DIPG. Intriguingly, disrupting CREB5 super-enhancers with ABBV-075 significantly reduces its expression and inhibits H3.3K27M DIPG tumor growth. Combined treatment with ABBV-075 and a BRG1 inhibitor presents a promising therapeutic strategy for clinical translation in H3.3K27M DIPG treatment.
    DOI:  https://doi.org/10.1038/s41467-025-58795-2
  10. Cell. 2025 Apr 11. pii: S0092-8674(25)00350-2. [Epub ahead of print]
    High-resolution dynamic imaging of chromatin DNA communication using Oligo-LiveFISH.
    Yanyu Zhu, Ashwin Balaji, Mengting Han, Leonid Andronov, Anish R Roy, Zheng Wei, Crystal Chen, Leanne Miles, Sa Cai, Zhengxi Gu, Ariana Tse, Betty Chentzu Yu, Takeshi Uenaka, Xueqiu Lin, Andrew J Spakowitz, W E Moerner, Lei S Qi.
      Three-dimensional (3D) genome dynamics are crucial for cellular functions and disease. However, real-time, live-cell DNA visualization remains challenging, as existing methods are often confined to repetitive regions, suffer from low resolution, or require complex genome engineering. Here, we present Oligo-LiveFISH, a high-resolution, reagent-based platform for dynamically tracking non-repetitive genomic loci in diverse cell types, including primary cells. Oligo-LiveFISH utilizes fluorescent guide RNA (gRNA) oligo pools generated by computational design, in vitro transcription, and chemical labeling, delivered as ribonucleoproteins. Utilizing machine learning, we characterized the impact of gRNA design and chromatin features on imaging efficiency. Multi-color Oligo-LiveFISH achieved 20-nm spatial resolution and 50-ms temporal resolution in 3D, capturing real-time enhancer and promoter dynamics. Our measurements and dynamic modeling revealed two distinct modes of chromatin communication, and active transcription slows enhancer-promoter dynamics at endogenous genes like FOS. Oligo-LiveFISH offers a versatile platform for studying 3D genome dynamics and their links to cellular processes and disease.
    Keywords:  CRISPR imaging; DNA communication; Oligo-LiveFISH; dynamic tracking; enhancer-promoter interaction; high-resolution imaging; live DNA imaging; non-repetitive genome imaging; polymer modeling; primary cell
    DOI:  https://doi.org/10.1016/j.cell.2025.03.032
  11. J Cell Sci. 2025 Apr 16. pii: jcs.263794. [Epub ahead of print]
    Med12 cooperates with multiple differentiation signals to facilitate efficient lineage transitions in embryonic stem cells.
    Max Fernkorn, Christian Schröter.
      Cell differentiation results from coordinated changes in gene transcription in response to combinations of signals. FGF, Wnt, and mTOR signals regulate the differentiation of pluripotent mammalian cells towards embryonic and extraembryonic lineages, but how these signals cooperate with general transcriptional regulators is not fully resolved. Here, we report a genome-wide CRISPR screen that reveals both signaling components and general transcriptional regulators for differentiation-associated gene expression in mESCs. Focusing on the Mediator subunit Med12 as one of the strongest hits in the screen, we show that it regulates gene expression in parallel to FGF and mTOR signals. Loss of Med12 is compatible with differentiation along both the embryonic epiblast and the extraembryonic primitive endoderm lineage, but impairs pluripotency gene expression and slows down transitions between pluripotency states. These findings suggest that Med12 helps pluripotent cells to efficiently execute transcriptional changes during differentiation, thereby modulating the effects of a broad range of signals.
    Keywords:  CRISPR screening; Differentiation; Embryonic stem cells; Lineage transitions; Mediator; Primitive endoderm
    DOI:  https://doi.org/10.1242/jcs.263794
  12. Elife. 2025 Apr 15. pii: RP91528. [Epub ahead of print]12
    Dynamics of transcriptional programs and chromatin accessibility in mouse spermatogonial cells from early postnatal to adult life.
    Irina Lazar-Contes, Rodrigo G Arzate-Mejia, Deepak K Tanwar, Leonard C Steg, Kerem Uzel, Olivier Ulrich Feudjio, Marion Crespo, Pierre-Luc Germain, Isabelle M Mansuy.
      In mammals, spermatogonial cells (SPGs) are undifferentiated male germ cells in testis that are quiescent until birth and then self-renew and differentiate to produce spermatogenic cells and functional sperm from early postnatal life throughout adulthood. The transcriptome of SPGs is highly dynamic and timely regulated during postnatal development. We examined if such dynamics involves changes in chromatin organization by profiling the transcriptome and chromatin accessibility of SPGs from early postnatal stages to adulthood in mice using deep RNA-seq, ATAC-seq and computational deconvolution analyses. By integrating transcriptomic and epigenomic features, we show that SPGs undergo massive chromatin remodeling during postnatal development that partially correlates with distinct gene expression profiles and transcription factors (TF) motif enrichment. We identify genomic regions with significantly different chromatin accessibility in adult SPGs that are marked by histone modifications associated with enhancers and promoters. Some of the regions with increased accessibility correspond to transposable element subtypes enriched in multiple TFs motifs and close to differentially expressed genes. Our results underscore the dynamics of chromatin organization in developing germ cells and complement existing datasets on SPGs by providing maps of the regulatory genome at high resolution from the same cell populations at early postnatal, late postnatal and adult stages collected from single individuals.
    Keywords:  Chromatin; RNA; development; genetics; genomics; mouse; spermatogonial cells; transcription
    DOI:  https://doi.org/10.7554/eLife.91528
  13. Proc Natl Acad Sci U S A. 2025 Apr 22. 122(16): e2424070122
    Unified molecular approach for spatial epigenome, transcriptome, and cell lineages.
    Yung-Hsin Huang, Julia A Belk, Ruochi Zhang, Natasha E Weiser, Zachary Chiang, Matthew G Jones, Paul S Mischel, Jason D Buenrostro, Howard Y Chang.
      Spatial epigenomics and multiomics can provide fine-grained insights into cellular states but their widespread adoption is limited by the requirement for bespoke slides and capture chemistries for each data modality. Here, we present SPatial assay for Accessible chromatin, Cell lineages, and gene Expression with sequencing (SPACE-seq), a method that utilizes polyadenine-tailed epigenomic libraries to enable facile spatial multiomics using standard whole transcriptome reagents. Applying SPACE-seq to a human glioblastoma specimen, we reveal the state of the tumor microenvironment, extrachromosomal DNA copy numbers, and identify putative mitochondrial DNA variants.
    Keywords:  cell lineages; chromatin accessibility; spatial genomics; spatial multiomics
    DOI:  https://doi.org/10.1073/pnas.2424070122
  14. Genes Dev. 2025 Apr 16.
    A boundary-defining protein facilitates megabase-scale regulatory chromosomal loop formation in Drosophila neurons.
    Marion Mouginot, Sahar Hani, Pascal Cousin, Julien Dorier, Arianna Ravera, Maria Cristina Gambetta.
      Regulatory elements, such as enhancers and silencers, control transcription by establishing physical proximity to target gene promoters. Neurons in flies and mammals exhibit long-range three-dimensional genome contacts, proposed to connect genes with distal regulatory elements. However, the relevance of these contacts for neuronal gene transcription and the mechanisms underlying their specificity necessitate further investigation. Here, we precisely disrupt several long-range contacts in fly neurons, demonstrating their importance for megabase-range gene regulation and uncovering a hierarchical process in their formation. We further reveal an essential role for the chromosomal boundary-forming protein Cp190 in anchoring many long-range contacts, highlighting a mechanistic interplay between boundary and loop formation. Finally, we develop an unbiased proteomics-based method to systematically identify factors required for specific long-range contacts. Our findings underscore the essential role of architectural proteins such as Cp190 in cell type-specific genome organization in enabling specialized neuronal transcriptional programs.
    Keywords:  CTCF; Cp190; Drosophila; TAD; genome folding; genome organization; long-range gene regulation; meta-domain; meta-loop; neuron; transcription
    DOI:  https://doi.org/10.1101/gad.352646.125
  15. Nat Commun. 2025 Apr 16. 16(1): 3611
    TRAPT: a multi-stage fused deep learning framework for predicting transcriptional regulators based on large-scale epigenomic data.
    Guorui Zhang, Chao Song, Mingxue Yin, Liyuan Liu, Yuexin Zhang, Ye Li, Jianing Zhang, Maozu Guo, Chunquan Li.
      It is challenging to identify regulatory transcriptional regulators (TRs), which control gene expression via regulatory elements and epigenomic signals, in context-specific studies on the onset and progression of diseases. The use of large-scale multi-omics epigenomic data enables the representation of the complex epigenomic patterns of control of the regulatory elements and the regulators. Herein, we propose Transcription Regulator Activity Prediction Tool (TRAPT), a multi-modality deep learning framework, which infers regulator activity by learning and integrating the regulatory potentials of target gene cis-regulatory elements and genome-wide binding sites. The results of experiments on 570 TR-related datasets show that TRAPT outperformed state-of-the-art methods in predicting the TRs, especially in terms of forecasting transcription co-factors and chromatin regulators. Moreover, we successfully identify key TRs associated with diseases, genetic variations, cell-fate decisions, and tissues. Our method provides an innovative perspective on identifying TRs by using epigenomic data.
    DOI:  https://doi.org/10.1038/s41467-025-58921-0
  16. Nat Commun. 2025 Apr 12. 16(1): 3491
    Unveiling chromatin dynamics with virtual epigenome.
    Ming-Yu Lin, Yu-Cheng Lo, Jui-Hung Hung.
      The three-dimensional organization of chromatin is essential for gene regulation and cellular function, with epigenome playing a key role. Hi-C methods have expanded our understanding of chromatin interactions, but their high cost and complexity limit their use. Existing models for predicting chromatin interactions rely on limited ChIP-seq inputs, reducing their accuracy and generalizability. In this work, we present a computational approach, EpiVerse, which leverages imputed epigenetic signals and advanced deep learning techniques. EpiVerse significantly improves the accuracy of cross-cell-type Hi-C prediction, while also enhancing model interpretability by incorporating chromatin state prediction within a multitask learning framework. Moreover, EpiVerse predicts Hi-C contact maps across an array of 39 human tissues, which provides a comprehensive view of the complex relationship between chromatin structure and gene regulation. Furthermore, EpiVerse facilitates unprecedented in silico perturbation experiments at the "epigenome-level" to unveil the chromatin architecture under specific conditions. EpiVerse is available on GitHub: https://github.com/jhhung/EpiVerse .
    DOI:  https://doi.org/10.1038/s41467-025-58481-3
  17. Genes Cells. 2025 May;30(3): e70019
    Cryo-EM Structures of Native Chromatin Units From Human Cells.
    Suguru Hatazawa, Yoshiyuki Fukuda, Yuki Kobayashi, Lumi Negishi, Masahide Kikkawa, Yoshimasa Takizawa, Hitoshi Kurumizaka.
      In eukaryotic cells, genomic DNA is compacted by nucleosomes, as basic repeating units, into chromatin. The nucleosome arrangement in chromatin fibers could be an important determinant for chromatin folding, by which genomic DNA is regulated in the nucleus. To study the structures of chromatin units in cells, we have established a method for the structural analysis of native mono- and poly-nucleosomes prepared from HeLa cells. In this method, the chromatin in isolated nuclei was crosslinked to preserve the proximity information between nucleosomes, followed by chromatin fragmentation by micrococcal nuclease treatment. The mono- and poly-nucleosomes were then fractionated by sucrose gradient ultracentrifugation, and their structures were analyzed by cryo-electron microscopy. Cryo-electron microscopy single particle analysis and cryo-electron tomography visualized a native nucleosome structure and secondary nucleosome arrangements in cellular chromatin. This method provides a complementary strategy to fill the gap between in vitro and in situ analyses of chromatin structure.
    Keywords:  chromatin; cryo‐EM; cryo‐electron tomography; nucleosome; single particle analysis
    DOI:  https://doi.org/10.1111/gtc.70019
  18. Nucleic Acids Res. 2025 Apr 10. pii: gkaf306. [Epub ahead of print]53(7):
    High-resolution mapping of sigma factor DNA-binding sequences using artificial promoters, RNA aptamers, and deep sequencing.
    Essa Ahsan Khan, Christian Rückert-Reed, Gurvinder Singh Dahiya, Lisa Tietze, Maxime Fages-Lartaud, Tobias Busche, Jörn Kalinowski, Victoria Shingler, Rahmi Lale.
      The variable sigma (σ) subunit of the bacterial RNA polymerase holoenzyme determines promoter specificity and facilitates open complex formation during transcription initiation. Understanding σ-factor binding sequences is therefore crucial for deciphering bacterial gene regulation. Here, we present a data-driven high-throughput approach that utilizes an extensive library of 1.54 million DNA templates providing artificial promoters and 5' untranslated region sequences for σ-factor DNA-binding motif discovery. This method combines the generation of extensive DNA libraries, in vitro transcription, RNA aptamer, and deep DNA and RNA sequencing. It allows direct assessment of promoter activity, identification of transcription start sites, and quantification of promoter strength based on mRNA production levels. We applied this approach to map σ54 DNA-binding sequences in Pseudomonas putida. Deep sequencing of the enriched RNA pool revealed 64 966 distinct σ54 binding motifs, significantly expanding the known repertoire. This data-driven approach surpasses traditional methods by directly evaluating promoter function and avoiding selection bias based solely on binding affinity. This comprehensive dataset enhances our understanding of σ-factor binding sequences and their regulatory roles, opening avenues for new research in biology and biotechnology.
    DOI:  https://doi.org/10.1093/nar/gkaf306
  19. Science. 2025 Apr 18. 388(6744): eads6601
    The genetic architecture of cell type-specific cis regulation in maize.
    Alexandre P Marand, Luguang Jiang, Fabio Gomez-Cano, Mark A A Minow, Xuan Zhang, John P Mendieta, Ziliang Luo, Sohyun Bang, Haidong Yan, Cullan Meyer, Luca Schlegel, Frank Johannes, Robert J Schmitz.
      Gene expression and complex phenotypes are determined by the activity of cis-regulatory elements. However, an understanding of how extant genetic variants affect cis regulation remains limited. Here, we investigated the consequences of cis-regulatory diversity using single-cell genomics of more than 0.7 million nuclei across 172 Zea mays (maize) inbreds. Our analyses pinpointed cis-regulatory elements distinct to domesticated maize and revealed how historical transposon activity has shaped the cis-regulatory landscape. Leveraging population genetics principles, we fine-mapped about 22,000 chromatin accessibility-associated genetic variants with widespread cell type-specific effects. Variants in TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR-binding sites were the most prevalent determinants of chromatin accessibility. Finally, integrating chromatin accessibility-associated variants, organismal trait variation, and population differentiation revealed how local adaptation has rewired regulatory networks in unique cellular contexts to alter maize flowering.
    DOI:  https://doi.org/10.1126/science.ads6601
  20. Genes Dev. 2025 Apr 16.
    Histone chaperones coupled to DNA replication and transcription control divergent chromatin elements to maintain cell fate.
    Reuben Franklin, Brian Zhang, Jonah Frazier, Meijuan Chen, Brian T Do, Sally Padayao, Kun Wu, Matthew G Vander Heiden, Christopher R Vakoc, Jae-Seok Roe, Maria Ninova, Jernej Murn, David B Sykes, Sihem Cheloufi.
      The manipulation of DNA replication and transcription can be harnessed to control cell fate. Central to the regulation of these DNA-templated processes are histone chaperones, which in turn are emerging as cell fate regulators. Histone chaperones are a group of proteins with diverse functions that are primarily involved in escorting histones to assemble nucleosomes and maintain the chromatin landscape. Whether distinct histone chaperone pathways control cell fate and whether they function using related mechanisms remain unclear. To address this, we performed a screen to assess the requirement of diverse histone chaperones in the self-renewal of hematopoietic stem and progenitor cells. Remarkably, all candidates were required to maintain cell fate to differing extents, with no clear correlation with their specific histone partners or DNA-templated process. Among all the histone chaperones, the loss of the transcription-coupled histone chaperone SPT6 most strongly promoted differentiation, even more than the major replication-coupled chromatin assembly factor complex CAF-1. To directly compare how DNA replication- and transcription-coupled histone chaperones maintain stem cell self-renewal, we generated an isogenic dual-inducible system to perturb each pathway individually. We found that SPT6 and CAF-1 perturbations required cell division to induce differentiation but had distinct effects on cell cycle progression, chromatin accessibility, and lineage choice. CAF-1 depletion led to S-phase accumulation, increased heterochromatic accessibility (particularly at H3K27me3 sites), and aberrant multilineage gene expression. In contrast, SPT6 loss triggered cell cycle arrest, altered accessibility at promoter elements, and drove lineage-specific differentiation, which is in part influenced by AP-1 transcription factors. Thus, CAF-1 and SPT6 histone chaperones maintain cell fate through distinct mechanisms, highlighting how different chromatin assembly pathways can be leveraged to alter cell fate.
    Keywords:  DNA replication; histone chaperones; lineage choice; stem cells; transcription; transcription factors
    DOI:  https://doi.org/10.1101/gad.352316.124
  21. Cell. 2025 Apr 14. pii: S0092-8674(25)00346-0. [Epub ahead of print]
    Synthetic deconvolution of an auxin-dependent transcriptional code.
    Raquel Martin-Arevalillo, Bruno Guillotin, Jonas Schön, Alice Hugues, Marie-France Gerentes, Kun Tang, Jérémy Lucas, Emmanuel Thévenon, Marianne Dreuillet, Graeme Vissers, Mohammed Mohammed Ateequr, Carlos S Galvan-Ampudia, Guillaume Cerutti, Jonathan Legrand, Coralie Cance, Annick Dubois, François Parcy, Kenneth D Birnbaum, Matias D Zurbriggen, Renaud Dumas, François Roudier, Teva Vernoux.
      How developmental signals program gene expression in space and time is still poorly understood. Here, we addressed this question for the plant master regulator, auxin. Transcriptional responses to auxin rely on a large multigenic transcription factor family, the auxin response factors (ARFs). We deconvoluted the complexity of ARF-regulated transcription using auxin-inducible synthetic promoters built from cis-element pair configurations differentially bound by ARFs. We demonstrate using cellular systems that ARF transcriptional properties are not only intrinsic but also depend on the cis-element pair configurations they bind to, thus identifying a bi-layer ARF/cis-element transcriptional code. Auxin-inducible synthetic promoters were expressed differentially in planta showing at single-cell resolution how this bi-layer code patterns transcriptional responses to auxin. Combining cis-element pair configurations in synthetic promoters created distinct patterns, demonstrating the combinatorial power of the auxin bi-layer code in generating diverse gene expression patterns that are not simply a direct translation of auxin distribution.
    Keywords:  ARF; DNA binding; TF; auxin; expression pattern; plant development; specificity; synthetic biology; synthetic deconvolution; transcriptional code
    DOI:  https://doi.org/10.1016/j.cell.2025.03.028
  22. Nucleic Acids Res. 2025 Apr 10. pii: gkaf297. [Epub ahead of print]53(7):
    Systematic genetic perturbation reveals principles underpinning robustness of the epigenetic regulatory network.
    Thomas Stuart Wilson, Roberta Noberini, Eirini Moysidou, Ifeyinwa Ojukwu, Marta Milan, Ming Jiang, Gavin Kelly, Michael Howell, Tiziana Bonaldi, Paola Scaffidi.
      The molecular control of epigenetic information relies on hundreds of proteins of diverse function, which cooperate in defining chromatin structure and DNA methylation landscapes. While many individual pathways have been characterized, how different classes of epigenetic regulators interact to build a resilient epigenetic regulatory network (ERN) remains poorly understood. Here, we show that most individual regulators are dispensable for somatic cell fitness, and that robustness emerges from multiple layers of functional cooperation and degeneracy among network components. By disrupting 200 epigenetic regulator genes, individually or in combination, we generated network-wide maps of functional interactions for representative regulators. We found that paralogues represent only a first layer of functional compensation within the ERN, with intra- or inter-class interactions buffering the effects of perturbation in a gene-specific manner: while CREBBP cooperates with multiple acetyltransferases to form a subnetwork that ensures robust chromatin acetylation, ARID1A interacts with regulators from across all functional classes. When combined with oncogene activation, the accumulated epigenetic disorder exposes a synthetic fragility and broadly sensitizes ARID1A-deficient cells to further perturbation. Our findings reveal homeostatic mechanisms through which the ERN sustains somatic cell fitness and uncover how the network remodels as the epigenome is progressively deregulated in disease.
    DOI:  https://doi.org/10.1093/nar/gkaf297
  23. Nat Methods. 2025 Apr 17.
    scMultiSim: simulation of single-cell multi-omics and spatial data guided by gene regulatory networks and cell-cell interactions.
    Hechen Li, Ziqi Zhang, Michael Squires, Xi Chen, Xiuwei Zhang.
      Simulated single-cell data are essential for designing and evaluating computational methods in the absence of experimental ground truth. Here we present scMultiSim, a comprehensive simulator that generates multimodal single-cell data encompassing gene expression, chromatin accessibility, RNA velocity and spatial cell locations while accounting for the relationships between modalities. Unlike existing tools that focus on limited biological factors, scMultiSim simultaneously models cell identity, gene regulatory networks, cell-cell interactions and chromatin accessibility while incorporating technical noise. Moreover, it allows users to adjust each factor's effect easily. Here we show that scMultiSim generates data with expected biological effects, and demonstrate its applications by benchmarking a wide range of computational tasks, including multimodal and multi-batch data integration, RNA velocity estimation, gene regulatory network inference and cell-cell interaction inference using spatially resolved gene expression data. Compared to existing simulators, scMultiSim can benchmark a much broader range of existing computational problems and even new potential tasks.
    DOI:  https://doi.org/10.1038/s41592-025-02651-0
  24. Nat Commun. 2025 Apr 12. 16(1): 3492
    Mutant CEBPA promotes tolerance to inflammatory stress through deficient AP-1 activation.
    Maria Cadefau-Fabregat, Gerard Martínez-Cebrián, Lucía Lorenzi, Felix D Weiss, Anne-Katrine Frank, José Manuel Castelló-García, Eric Julià-Vilella, Andrés Gámez-García, Laura Yera, Carini Picardi Morais de Castro, Yi-Fang Wang, Felix Meissner, Alejandro Vaquero, Matthias Merkenschlager, Bo T Porse, Sergi Cuartero.
      The CEBPA transcription factor is frequently mutated in acute myeloid leukemia (AML). Mutations in the CEBPA gene, which are typically biallelic, result in the production of a shorter isoform known as p30. Both the canonical 42-kDa isoform (p42) and the AML-associated p30 isoform bind chromatin and activate transcription, but the specific transcriptional programs controlled by each protein and how they are linked to a selective advantage in AML is not well understood. Here, we show that cells expressing the AML-associated p30 have reduced baseline inflammatory gene expression and display altered dynamics of transcriptional induction in response to LPS, consequently impacting cytokine secretion. This confers p30-expressing cells an increased resistance to the adverse effects of prolonged exposure to inflammatory signals. Mechanistically, we show that these differences primarily arise from the differential regulation of AP-1 family proteins. In addition, we find that the impaired function of the AP-1 member ATF4 in p30-expressing cells alters their response to ER stress. Collectively, these findings uncover a link between mutant CEBPA, inflammation and the stress response, potentially revealing a vulnerability in AML.
    DOI:  https://doi.org/10.1038/s41467-025-58712-7
  25. Cell. 2025 Apr 10. pii: S0092-8674(25)00343-5. [Epub ahead of print]
    Integrator loss leads to dsRNA formation that triggers the integrated stress response.
    Apoorva Baluapuri, Nicole ChenCheng Zhao, Ryan J Marina, Kai-Lieh Huang, Anastasia Kuzkina, Maria E Amodeo, Chad B Stein, Lucie Y Ahn, Jordan S Farr, Ashleigh E Schaffer, Vikram Khurana, Eric J Wagner, Karen Adelman.
      Integrator (INT) is a metazoan-specific complex that targets promoter-proximally paused RNA polymerase II (RNAPII) for termination, preventing immature RNAPII from entering gene bodies and functionally attenuating transcription of stress-responsive genes. Mutations in INT subunits are associated with many human diseases, including cancer, ciliopathies, and neurodevelopmental disorders, but how reduced INT activity contributes to disease is unknown. Here, we demonstrate that the loss of INT-mediated termination in human cells triggers the integrated stress response (ISR). INT depletion causes upregulation of short genes such as the ISR transcription factor activating transcription factor 3 (ATF3). Further, immature RNAPII that escapes into genes upon INT depletion is prone to premature termination, generating incomplete pre-mRNAs with retained introns. Retroelements within retained introns form double-stranded RNA (dsRNA) that is recognized by protein kinase R (PKR), which drives ATF4 activation and prolonged ISR. Critically, patient cells with INT mutations exhibit dsRNA accumulation and ISR activation, thereby implicating chronic ISR in diseases caused by INT deficiency.
    Keywords:  IR-Alu; Integrator; RNA polymerase II pausing; double-stranded RNA; gene regulation; integrated stress response; premature cleavage and polyadenylation; premature termination; protein kinase R
    DOI:  https://doi.org/10.1016/j.cell.2025.03.025
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