bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2025–01–12
twenty-six papers selected by
Connor Rogerson, University of Cambridge
  1. Mechanism of mammalian transcriptional repression by noncoding RNA.
  2. Oncogenic IDH1mut drives robust loss of histone acetylation and increases chromatin heterogeneity.
  3. DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos.
  4. Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system.
  5. Chromatin enables precise and scalable gene regulation with factors of limited specificity.
  6. A foundation model of transcription across human cell types.
  7. Specifying cellular context of transcription factor regulons for exploring context-specific gene regulation programs.
  8. Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications.
  9. Depth-corrected multi-factor dissection of chromatin accessibility for scATAC-seq data with PACS.
  10. Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.
  11. Pan-inhibition of super-enhancer-driven oncogenic transcription by next-generation synthetic ecteinascidins yields potent anti-cancer activity.
  12. Crypt density and recruited enhancers underlie intestinal tumour initiation.
  13. KLF5 loss sensitizes cells to ATR inhibition and is synthetic lethal with ARID1A deficiency.
  14. Gene regulation by convergent promoters.
  15. YAP-driven malignant reprogramming of oral epithelial stem cells at single cell resolution.
  16. Active learning of enhancers and silencers in the developing neural retina.
  17. Rapid and quantitative functional interrogation of human enhancer variant activity in live mice.
  18. The nuclear matrix stabilizes primed-specific genes in human pluripotent stem cells.
  19. Thermodynamics of nucleosome breathing and positioning.
  20. Morphogen-induced kinase condensates transduce Hh signal by allosterically activating Gli.
  21. Deaminase-mediated chromatin accessibility profiling with single-allele resolution.
  22. Ectopic expression of DNMT3L in human trophoblast stem cells restores features of the placental methylome.
  23. Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging.
  24. IRF1 cooperates with ISGF3 or GAF to form innate immune de novo enhancers in macrophages.
  25. Repurposing of a gill gene regulatory program for outer ear evolution.
  26. Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF.
  1. Nat Struct Mol Biol. 2025 Jan 06.
    Mechanism of mammalian transcriptional repression by noncoding RNA.
    Katarína Tlučková, Beata Kaczmarek, Anita Salmazo, Carrie Bernecky.
      Transcription by RNA polymerase II (Pol II) can be repressed by noncoding RNA, including the human RNA Alu. However, the mechanism by which endogenous RNAs repress transcription remains unclear. Here we present cryogenic-electron microscopy structures of Pol II bound to Alu RNA, which reveal that Alu RNA mimics how DNA and RNA bind to Pol II during transcription elongation. Further, we show how distinct domains of the general transcription factor TFIIF control repressive activity. Together, we reveal how a noncoding RNA can regulate mammalian gene expression.
    DOI:  https://doi.org/10.1038/s41594-024-01448-7
  2. Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2403862122
    Oncogenic IDH1mut drives robust loss of histone acetylation and increases chromatin heterogeneity.
    Noa Furth, Niv Cohen, Avishay Spitzer, Tomer-Meir Salame, Bareket Dassa, Tevie Mehlman, Alexander Brandis, Arieh Moussaieff, Dinorah Friedmann-Morvinski, Maria G Castro, Jerome Fortin, Mario L Suvà, Itay Tirosh, Ayelet Erez, Guy Ron, Efrat Shema.
      Malignant gliomas are heterogeneous tumors, mostly incurable, arising in the central nervous system (CNS) driven by genetic, epigenetic, and metabolic aberrations. Mutations in isocitrate dehydrogenase (IDH1/2mut) enzymes are predominantly found in low-grade gliomas and secondary high-grade gliomas, with IDH1 mutations being more prevalent. Mutant-IDH1/2 confers a gain-of-function activity that favors the conversion of a-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), resulting in an aberrant hypermethylation phenotype. Yet, the complete depiction of the epigenetic alterations in IDHmut cells has not been thoroughly explored. Here, we applied an unbiased approach, leveraging epigenetic-focused cytometry by time-of-flight (CyTOF) analysis, to systematically profile the effect of mutant-IDH1 expression on a broad panel of histone modifications at single-cell resolution. This analysis revealed extensive remodeling of chromatin patterns by mutant-IDH1, with the most prominent being deregulation of histone acetylation marks. The loss of histone acetylation occurs rapidly following mutant-IDH1 induction and affects acetylation patterns over enhancers and intergenic regions. Notably, the changes in acetylation are not predominantly driven by 2-HG, can be rescued by pharmacological inhibition of mutant-IDH1, and reversed by acetate supplementations. Furthermore, cells expressing mutant-IDH1 show higher epigenetic and transcriptional heterogeneity and upregulation of oncogenes such as KRAS and MYC, highlighting its tumorigenic potential. Our study underscores the tight interaction between chromatin and metabolism dysregulation in glioma and highlights epigenetic and oncogenic pathways affected by mutant-IDH1-driven metabolic rewiring.
    Keywords:  CyTOF; chromatin; epigenetics; mutant-IDH1; oncometabolite
    DOI:  https://doi.org/10.1073/pnas.2403862122
  3. Nat Commun. 2025 Jan 07. 16(1): 465
    DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos.
    Grigorios Fanourgakis, Laura Gaspa-Toneu, Pavel A Komarov, Panagiotis Papasaikas, Evgeniy A Ozonov, Sebastien A Smallwood, Antoine H F M Peters.
      In the germ line and during early embryogenesis, DNA methylation (DNAme) undergoes global erasure and re-establishment to support germ cell and embryonic development. While DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin are unknown. Through conditional gene deletion of the de novo DNA methyltransferases Dnmt3a and/or Dnmt3b, we observe that DNMT3A primarily safeguards against DNA hypomethylation in undifferentiated spermatogonia, while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm. To assess the impact of altered sperm chromatin in formatting embryonic chromatin, we measure H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a transposon-based tagging approach. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.
    DOI:  https://doi.org/10.1038/s41467-024-55441-1
  4. Sci Adv. 2025 Jan 10. 11(2): eadq6652
    Chromatin conformation, gene transcription, and nucleosome remodeling as an emergent system.
    Luay M Almassalha, Marcelo Carignano, Emily Pujadas Liwag, Wing Shun Li, Ruyi Gong, Nicolas Acosta, Cody L Dunton, Paola Carrillo Gonzalez, Lucas M Carter, Rivaan Kakkaramadam, Martin Kröger, Kyle L MacQuarrie, Jane Frederick, I Chae Ye, Patrick Su, Tiffany Kuo, Karla I Medina, Josh A Pritchard, Andrew Skol, Rikkert Nap, Masato Kanemaki, Vinayak Dravid, Igal Szleifer, Vadim Backman.
      In single cells, variably sized nanoscale chromatin structures are observed, but it is unknown whether these form a cohesive framework that regulates RNA transcription. Here, we demonstrate that the human genome is an emergent, self-assembling, reinforcement learning system. Conformationally defined heterogeneous, nanoscopic packing domains form by the interplay of transcription, nucleosome remodeling, and loop extrusion. We show that packing domains are not topologically associated domains. Instead, packing domains exist across a structure-function life cycle that couples heterochromatin and transcription in situ, explaining how heterochromatin enzyme inhibition can produce a paradoxical decrease in transcription by destabilizing domain cores. Applied to development and aging, we show the pairing of heterochromatin and transcription at myogenic genes that could be disrupted by nuclear swelling. In sum, packing domains represent a foundation to explore the interactions of chromatin and transcription at the single-cell level in human health.
    DOI:  https://doi.org/10.1126/sciadv.adq6652
  5. Proc Natl Acad Sci U S A. 2025 Jan 07. 122(1): e2411887121
    Chromatin enables precise and scalable gene regulation with factors of limited specificity.
    Mindy Liu Perkins, Justin Crocker, Gašper Tkačik.
      Biophysical constraints limit the specificity with which transcription factors (TFs) can target regulatory DNA. While individual nontarget binding events may be low affinity, the sheer number of such interactions could present a challenge for gene regulation by degrading its precision or possibly leading to an erroneous induction state. Chromatin can prevent nontarget binding by rendering DNA physically inaccessible to TFs, at the cost of energy-consuming remodeling orchestrated by pioneer factors (PFs). Under what conditions and by how much can chromatin reduce regulatory errors on a global scale? We use a theoretical approach to compare two scenarios for gene regulation: one that relies on TF binding to free DNA alone and one that uses a combination of TFs and chromatin-regulating PFs to achieve desired gene expression patterns. We find, first, that chromatin effectively silences groups of genes that should be simultaneously OFF, thereby allowing more accurate graded control of expression for the remaining ON genes. Second, chromatin buffers the deleterious consequences of nontarget binding as the number of OFF genes grows, permitting a substantial expansion in regulatory complexity. Third, chromatin-based regulation productively co-opts nontarget TF binding for ON genes in order to establish a "leaky" baseline expression level, which targeted activator or repressor binding subsequently up- or down-modulates. Thus, on a global scale, using chromatin simultaneously alleviates pressure for high specificity of regulatory interactions and enables an increase in genome size with minimal impact on global expression error.
    Keywords:  chromatin; crosstalk; gene regulation; optimization; regulatory networks
    DOI:  https://doi.org/10.1073/pnas.2411887121
  6. Nature. 2025 Jan 08.
    A foundation model of transcription across human cell types.
    Xi Fu, Shentong Mo, Alejandro Buendia, Anouchka P Laurent, Anqi Shao, Maria Del Mar Alvarez-Torres, Tianji Yu, Jimin Tan, Jiayu Su, Romella Sagatelian, Adolfo A Ferrando, Alberto Ciccia, Yanyan Lan, David M Owens, Teresa Palomero, Eric P Xing, Raul Rabadan.
      Transcriptional regulation, which involves a complex interplay between regulatory sequences and proteins, directs all biological processes. Computational models of transcription lack generalizability to accurately extrapolate to unseen cell types and conditions. Here we introduce GET (general expression transformer), an interpretable foundation model designed to uncover regulatory grammars across 213 human fetal and adult cell types1,2. Relying exclusively on chromatin accessibility data and sequence information, GET achieves experimental-level accuracy in predicting gene expression even in previously unseen cell types3. GET also shows remarkable adaptability across new sequencing platforms and assays, enabling regulatory inference across a broad range of cell types and conditions, and uncovers universal and cell-type-specific transcription factor interaction networks. We evaluated its performance in prediction of regulatory activity, inference of regulatory elements and regulators, and identification of physical interactions between transcription factors and found that it outperforms current models4 in predicting lentivirus-based massively parallel reporter assay readout5,6. In fetal erythroblasts7, we identified distal (greater than 1 Mbp) regulatory regions that were missed by previous models, and, in B cells, we identified a lymphocyte-specific transcription factor-transcription factor interaction that explains the functional significance of a leukaemia risk predisposing germline mutation8-10. In sum, we provide a generalizable and accurate model for transcription together with catalogues of gene regulation and transcription factor interactions, all with cell type specificity.
    DOI:  https://doi.org/10.1038/s41586-024-08391-z
  7. NAR Genom Bioinform. 2025 Mar;7(1): lqae178
    Specifying cellular context of transcription factor regulons for exploring context-specific gene regulation programs.
    Mariia Minaeva, Júlia Domingo, Philipp Rentzsch, Tuuli Lappalainen.
      Understanding the role of transcription and transcription factors (TFs) in cellular identity and disease, such as cancer, is essential. However, comprehensive data resources for cell line-specific TF-to-target gene annotations are currently limited. To address this, we employed a straightforward method to define regulons that capture the cell-specific aspects of TF binding and transcript expression levels. By integrating cellular transcriptome and TF binding data, we generated regulons for 40 common cell lines comprising both proximal and distal cell line-specific regulatory events. Through systematic benchmarking involving TF knockout experiments, we demonstrated performance on par with state-of-the-art methods, with our method being easily applicable to other cell types of interest. We present case studies using three cancer single-cell datasets to showcase the utility of these cell-type-specific regulons in exploring transcriptional dysregulation. In summary, this study provides a valuable pipeline and a resource for systematically exploring cell line-specific transcriptional regulations, emphasizing the utility of network analysis in deciphering disease mechanisms.
    DOI:  https://doi.org/10.1093/nargab/lqae178
  8. Nat Struct Mol Biol. 2025 Jan 07.
    Structural basis for the inhibition of PRC2 by active transcription histone posttranslational modifications.
    Trinity Cookis, Alexandria Lydecker, Paul Sauer, Vignesh Kasinath, Eva Nogales.
      Polycomb repressive complex 2 (PRC2) trimethylates histone H3 on K27 (H3K27me3) leading to gene silencing that is essential for embryonic development and maintenance of cell identity. PRC2 is regulated by protein cofactors and their crosstalk with histone modifications. Trimethylated histone H3 on K4 (H3K4me3) and K36 (H3K36me3) localize to sites of active transcription and inhibit PRC2 activity through unknown mechanisms. Using cryo-electron microscopy, we reveal that histone H3 tails containing H3K36me3 engage poorly with PRC2 and preclude its effective interaction with chromatin, while H3K4me3 binds to the allosteric site in the EED subunit, acting as an antagonist that competes with activators required for spreading of the H3K27me3 repressive mark. Thus, the location of the H3K4me3 and H3K36me3 modifications along the H3 tail allows them to target two requirements for efficient trimethylation of H3K27 by PRC2. We further show that the JARID2 cofactor modulates PRC2 activity in the presence of these histone modifications.
    DOI:  https://doi.org/10.1038/s41594-024-01452-x
  9. Nat Commun. 2025 Jan 05. 16(1): 401
    Depth-corrected multi-factor dissection of chromatin accessibility for scATAC-seq data with PACS.
    Zhen Miao, Jianqiao Wang, Kernyu Park, Da Kuang, Junhyong Kim.
      Single cell ATAC-seq (scATAC-seq) experimental designs have become increasingly complex, with multiple factors that might affect chromatin accessibility, including genotype, cell type, tissue of origin, sample location, batch, etc., whose compound effects are difficult to test by existing methods. In addition, current scATAC-seq data present statistical difficulties due to their sparsity and variations in individual sequence capture. To address these problems, we present a zero-adjusted statistical model, Probability model of Accessible Chromatin of Single cells (PACS), that allows complex hypothesis testing of accessibility-modulating factors while accounting for sparse and incomplete data. For differential accessibility analysis, PACS controls the false positive rate and achieves a 17% to 122% higher power on average than existing tools. We demonstrate the effectiveness of PACS through several analysis tasks, including supervised cell type annotation, compound hypothesis testing, batch effect correction, and spatiotemporal modeling. We apply PACS to datasets from various tissues and show its ability to reveal previously undiscovered insights in scATAC-seq data.
    DOI:  https://doi.org/10.1038/s41467-024-55580-5
  10. J Cell Biol. 2025 Mar 03. pii: e202405169. [Epub ahead of print]224(3):
    Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.
    Andreas Brunner, Natalia Rosalía Morero, Wanlu Zhang, M Julius Hossain, Marko Lampe, Hannah Pflaumer, Aliaksandr Halavatyi, Jan-Michael Peters, Kai S Beckwith, Jan Ellenberg.
      How cells establish the interphase genome organization after mitosis is incompletely understood. Using quantitative and super-resolution microscopy, we show that the transition from a Condensin to a Cohesin-based genome organization occurs dynamically over 2 h. While a significant fraction of Condensins remains chromatin-bound until early G1, Cohesin-STAG1 and its boundary factor CTCF are rapidly imported into daughter nuclei in telophase, immediately bind chromosomes as individual complexes, and are sufficient to build the first interphase TAD structures. By contrast, the more abundant Cohesin-STAG2 accumulates on chromosomes only gradually later in G1, is responsible for compaction inside TAD structures, and forms paired complexes upon completed nuclear import. Our quantitative time-resolved mapping of mitotic and interphase loop extruders in single cells reveals that the nested loop architecture formed by the sequential action of two Condensins in mitosis is seamlessly replaced by a less compact but conceptually similar hierarchically nested loop architecture driven by the sequential action of two Cohesins.
    DOI:  https://doi.org/10.1083/jcb.202405169
  11. Nat Commun. 2025 Jan 08. 16(1): 512
    Pan-inhibition of super-enhancer-driven oncogenic transcription by next-generation synthetic ecteinascidins yields potent anti-cancer activity.
    Max Cigrang, Julian Obid, Maguelone Nogaret, Léane Seno, Tao Ye, Guillaume Davidson, Philippe Catez, Pietro Berico, Clara Capelli, Clara Marechal, Amélie Zachayus, Clémence Elly, Marie Jose Guillen Navarro, Marta Martinez Diez, Gema Santamaria Nunez, Tsai-Kun Li, Emmanuel Compe, Pablo Avilés, Irwin Davidson, Jean-Marc Egly, Carmen Cuevas, Frédéric Coin.
      The plasticity of cancer cells facilitates their ability to adopt heterogeneous differentiation states, posing a significant challenge to therapeutic interventions. Specific gene expression programs, driven in part by super-enhancers (SEs), underlie cancer cell states. Here we successfully inhibit SE-driven transcription in phenotypically distinct metastatic melanoma cells using next-generation synthetic ecteinascidins. Through functional genomic methodologies, we demonstrate that these compounds inhibit the expression of genes encoding lineage-specific or ubiquitous transcription factors/coactivators by selectively targeting the CpG-rich sequences within their promoters and/or enhancers. This prevents the formation of transcription factor/coactivator condensates necessary for SE-dependent gene expression. Consequently, these compounds exhibit cytotoxic activity across distinct subpopulations of metastatic melanoma cells and inhibit tumor proliferation, including those resistant to current therapies. These findings extend to other cancers, like small cell lung cancer, recently approved for ecteinascidin-based treatment. Overall, our study provides preclinical proof that pan-inhibition of SE-dependent genes with synthetic ecteinascidins is a promising therapeutic approach for tumors with heterogeneous transcriptional landscapes.
    DOI:  https://doi.org/10.1038/s41467-024-55667-z
  12. Nature. 2025 Jan 08.
    Crypt density and recruited enhancers underlie intestinal tumour initiation.
    Liam Gaynor, Harshabad Singh, Guodong Tie, Krithika Badarinath, Shariq Madha, Andrew Mancini, Swarnabh Bhattacharya, Mikio Hoshino, Frederic J de Sauvage, Kazutaka Murata, Unmesh Jadhav, Ramesh A Shivdasani.
      Oncogenic mutations that drive colorectal cancer can be present in healthy intestines for long periods without overt consequence1,2. Mutation of Adenomatous polyposis coli (Apc), the most common initiating event in conventional adenomas3, activates Wnt signalling, hence conferring fitness on mutant intestinal stem cells (ISCs)4,5. Apc mutations may occur in ISCs that arose by routine self-renewal or by dedifferentiation of their progeny. Although ISCs of these different origins are fundamentally similar6,7, it is unclear if both generate tumours equally well in uninjured intestines. Also unknown is whether cis-regulatory elements are substantively modulated upon Wnt hyperactivation or as a feature of subsequent tumours. Here, we show in two mouse models that adenomas are not an obligatory outcome of Apc deletion in either ISC source but require proximity of mutant intestinal crypts. Reduced crypt density abrogates, and aggregation of mutant colonic crypts augments, adenoma formation. Moreover, adenoma-resident ISCs open chromatin at thousands of enhancers that are inaccessible in Apc-null ISCs not associated with adenomas. These cis-elements explain adenoma-selective gene activity and persist, with little further expansion of the repertoire, as other oncogenic mutations accumulate. Thus, cooperativity between neighbouring mutant crypts and new accessibility at specific enhancers are key steps early in intestinal tumourigenesis.
    DOI:  https://doi.org/10.1038/s41586-024-08573-9
  13. Nat Commun. 2025 Jan 08. 16(1): 480
    KLF5 loss sensitizes cells to ATR inhibition and is synthetic lethal with ARID1A deficiency.
    Samah W Awwad, Colm Doyle, Josie Coulthard, Aldo S Bader, Nadia Gueorguieva, Simon Lam, Vipul Gupta, Rimma Belotserkovskaya, Tuan-Anh Tran, Shankar Balasubramanian, Stephen P Jackson.
      ATR plays key roles in cellular responses to DNA damage and replication stress, a pervasive feature of cancer cells. ATR inhibitors (ATRi) are in clinical development for treating various cancers, including those with high replication stress, such as is elicited by ARID1A deficiency, but the cellular mechanisms that determine ATRi efficacy in such backgrounds are unclear. Here, we have conducted unbiased genome-scale CRISPR screens in ARID1A-deficient and proficient cells treated with ATRi. We found that loss of transcription factor KLF5 has severe negative impact on fitness of ARID1A-deficient cells while hypersensitising ARID1A-proficient cells to ATRi. KLF5 loss induced replication stress, DNA damage, increased DNA-RNA hybrid formation, and genomic instability upon ATR inhibition. Mechanistically, we show that KLF5 protects cells from replication stress, at least in part through regulating BRD4 recruitment to chromatin. Overall, our work identifies KLF5 as a potential target for eradicating ARID1A-deficient cancers.
    DOI:  https://doi.org/10.1038/s41467-024-55637-5
  14. Nat Genet. 2025 Jan 06.
    Gene regulation by convergent promoters.
    Elina Wiechens, Flavia Vigliotti, Kanstantsin Siniuk, Robert Schwarz, Katjana Schwab, Konstantin Riege, Alena van Bömmel, Ivonne Görlich, Martin Bens, Arne Sahm, Marco Groth, Morgan A Sammons, Alexander Loewer, Steve Hoffmann, Martin Fischer.
      Convergent transcription, that is, the collision of sense and antisense transcription, is ubiquitous in mammalian genomes and believed to diminish RNA expression. Recently, antisense transcription downstream of promoters was found to be surprisingly prevalent. However, functional characteristics of affected promoters are poorly investigated. Here we show that convergent transcription marks an unexpected positively co-regulated promoter constellation. By assessing transcriptional dynamic systems, we identified co-regulated constituent promoters connected through a distinct chromatin structure. Within these cis-regulatory domains, transcription factors can regulate both constituting promoters by binding to only one of them. Convergent promoters comprise about a quarter of all active transcript start sites and initiate 5'-overlapping antisense RNAs-an RNA class believed previously to be rare. Visualization of nascent RNA molecules reveals convergent cotranscription at these loci. Together, our results demonstrate that co-regulated convergent promoters substantially expand the cis-regulatory repertoire, reveal limitations of the transcription interference model and call for adjusting the promoter concept.
    DOI:  https://doi.org/10.1038/s41588-024-02025-w
  15. Nat Commun. 2025 Jan 08. 16(1): 498
    YAP-driven malignant reprogramming of oral epithelial stem cells at single cell resolution.
    Farhoud Faraji, Sydney I Ramirez, Lauren M Clubb, Kuniaki Sato, Valeria Burghi, Thomas S Hoang, Adam Officer, Paola Y Anguiano Quiroz, William M G Galloway, Zbigniew Mikulski, Kate Medetgul-Ernar, Pauline Marangoni, Kyle B Jones, Yuwei Cao, Alfredo A Molinolo, Kenneth Kim, Kanako Sakaguchi, Joseph A Califano, Quinton Smith, Alon Goren, Ophir D Klein, Pablo Tamayo, J Silvio Gutkind.
      Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells at single cell resolution. Tumor initiating cells displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal invasive gene programs. YAP-mediated tumor initiating cell programs included activation of oncogenic transcriptional networks and mTOR signaling, and recruitment of myeloid cells to the invasive front contributing to tumor infiltration. Tumor initiating cell transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention.
    DOI:  https://doi.org/10.1038/s41467-024-55660-6
  16. Cell Syst. 2024 Dec 31. pii: S2405-4712(24)00368-5. [Epub ahead of print] 101163
    Active learning of enhancers and silencers in the developing neural retina.
    Ryan Z Friedman, Avinash Ramu, Sara Lichtarge, Yawei Wu, Lloyd Tripp, Daniel Lyon, Connie A Myers, David M Granas, Maria Gause, Joseph C Corbo, Barak A Cohen, Michael A White.
      Deep learning is a promising strategy for modeling cis-regulatory elements. However, models trained on genomic sequences often fail to explain why the same transcription factor can activate or repress transcription in different contexts. To address this limitation, we developed an active learning approach to train models that distinguish between enhancers and silencers composed of binding sites for the photoreceptor transcription factor cone-rod homeobox (CRX). After training the model on nearly all bound CRX sites from the genome, we coupled synthetic biology with uncertainty sampling to generate additional rounds of informative training data. This allowed us to iteratively train models on data from multiple rounds of massively parallel reporter assays. The ability of the resulting models to discriminate between CRX sites with identical sequence but opposite functions establishes active learning as an effective strategy to train models of regulatory DNA. A record of this paper's transparent peer review process is included in the supplemental information.
    Keywords:  active learning; cis-regulatory elements; enhancers; gene regulation; machine learning; retina; silencers; transcription factors
    DOI:  https://doi.org/10.1016/j.cels.2024.12.004
  17. Nat Commun. 2025 Jan 06. 16(1): 409
    Rapid and quantitative functional interrogation of human enhancer variant activity in live mice.
    Ethan W Hollingsworth, Taryn A Liu, Joshua A Alcantara, Cindy X Chen, Sandra H Jacinto, Evgeny Z Kvon.
      Functional analysis of non-coding variants associated with congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterise gene expression in cells where the enhancer is normally and ectopically active, revealing candidate pathways that may lead to enhancer misregulation. Finally, we demonstrate the widespread utility of dual-enSERT by testing the effects of fifteen previously uncharacterised rare and common non-coding variants linked to neurodevelopmental disorders. In doing so we identify variants that reproducibly alter the in vivo activity of OTX2 and MIR9-2 brain enhancers, implicating them in autism. Dual-enSERT thus allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.
    DOI:  https://doi.org/10.1038/s41467-024-55500-7
  18. Nat Cell Biol. 2025 Jan 09.
    The nuclear matrix stabilizes primed-specific genes in human pluripotent stem cells.
    Gang Ma, Xiuling Fu, Lulu Zhou, Isaac A Babarinde, Liyang Shi, Wenting Yang, Jiao Chen, Zhen Xiao, Yu Qiao, Lisha Ma, Yuhao Ou, Yuhao Li, Chen Chang, Boping Deng, Ran Zhang, Li Sun, Guoqing Tong, Dongwei Li, Yiming Li, Andrew P Hutchins.
      The nuclear matrix, a proteinaceous gel composed of proteins and RNA, is an important nuclear structure that supports chromatin architecture, but its role in human pluripotent stem cells (hPSCs) has not been described. Here we show that by disrupting heterogeneous nuclear ribonucleoprotein U (HNRNPU) or the nuclear matrix protein, Matrin-3, primed hPSCs adopted features of the naive pluripotent state, including morphology and upregulation of naive-specific marker genes. We demonstrate that HNRNPU depletion leads to increased chromatin accessibility, reduced DNA contacts and increased nuclear size. Mechanistically, HNRNPU acts as a transcriptional co-factor that anchors promoters of primed-specific genes to the nuclear matrix with POLII to promote their expression and their RNA stability. Overall, HNRNPU promotes cell-type stability and when reduced promotes conversion to earlier embryonic states.
    DOI:  https://doi.org/10.1038/s41556-024-01595-5
  19. J Chem Phys. 2025 Jan 14. pii: 025101. [Epub ahead of print]162(2):
    Thermodynamics of nucleosome breathing and positioning.
    Kharerin Hungyo, Benjamin Audit, Cédric Vaillant, Alexandre V Morozov.
      Nucleosomes are fundamental units of chromatin in which a length of genomic DNA is wrapped around a histone octamer spool in a left-handed superhelix. Large-scale nucleosome maps show a wide distribution of DNA wrapping lengths, which in some cases are tens of base pairs (bp) shorter than the 147 bp canonical wrapping length observed in nucleosome crystal structures. Here, we develop a thermodynamic model that assumes a constant free energy cost of unwrapping a nucleosomal bp. Our model also incorporates linker DNA-short DNA segments between neighboring nucleosomes imposed by the folding of nucleosome arrays into chromatin fibers and other higher-order chromatin structures. We use this model to study nucleosome positioning and occupancy in the presence of nucleosome "breathing"-partial unwrapping and rewrapping of nucleosomal DNA due to interactions with the neighboring particles. We find that, as the unwrapping cost per bp and the chemical potential are varied, the nucleosome arrays are characterized by three distinct states, with low, intermediate, and high densities. The transition between the latter two states proceeds through an equiprobable state in which all nucleosome wrapping lengths are equally likely. We study the equiprobable state theoretically using a mean-field approach, obtaining an excellent agreement with numerical simulations. Finally, we use our model to reproduce S. cerevisiae nucleosome occupancy profiles observed in the vicinity of transcription start sites, as well as genome-wide distributions of nucleosome wrapping lengths. Overall, our results highlight the key role of partial nucleosome unwrapping in shaping the genome-wide patterns of nucleosome positioning and occupancy.
    DOI:  https://doi.org/10.1063/5.0245457
  20. Sci Adv. 2025 Jan 10. 11(2): eadq1790
    Morphogen-induced kinase condensates transduce Hh signal by allosterically activating Gli.
    Yuhong Han, Mengmeng Zhou, Bing Wang, Jin Jiang.
      Hedgehog (Hh) morphogen governs embryonic development and tissue homeostasis through the Ci/Gli family transcription factors. Here we report that Hh induces phase separation of the fused (Fu)/Ulk family kinases to allosterically regulate Ci/Gli. We find that Hh-induced phosphorylation of Fu/Ulk3 promotes SUMOylation of their inverted phosphorylation-dependent SUMOylation motifs. Subsequent interaction between SUMO and SUMO-interacting motif drives Fu/Ulk3 self-assembly to form biomolecular condensates that recruit Ci-Sufu and Gli-Sufu in the cytoplasm and primary cilium, respectively. Within the condensates, Fu/Ulk3 undergoes a conformational change to expose Ci/Gli for Fu/Ulk3-mediated phosphorylation and activation, leading to gradual accumulation of nuclear CiA/GliA transcriptional complexes in proportion to ligand dose and exposure time. Our findings provide mechanistic insights into the spatiotemporal control of Hh signal transduction, reveal previously unexplored regulatory mechanism and function for biomolecular condensation, and establish a paradigm for kinase-mediated signal transduction whereby a kinase allosterically activates its substrate through ligand-induced and condensation-driven conformational change.
    DOI:  https://doi.org/10.1126/sciadv.adq1790
  21. bioRxiv. 2024 Dec 20. pii: 2024.12.17.628768. [Epub ahead of print]
    Deaminase-mediated chromatin accessibility profiling with single-allele resolution.
    Tian Yu, Zhijian Li, Ellie Gibbs, Reina Iwase, Matthew J Francoeur, Quang Vinh Phan, Jing Zhao, Jane Rosin, Phillip A Cole, Luca Pinello, Richard I Sherwood.
      Binding of transcription factors (TFs) at gene regulatory elements controls cellular epigenetic state and gene expression. Current genome-wide chromatin profiling approaches have inherently limited resolution, complicating assessment of TF occupancy and co-occupancy, especially at individual alleles. In this work, we introduce Accessible Chromatin by Cytosine Editing Site Sequencing with ATAC-seq (ACCESS-ATAC), which harnesses a double-stranded DNA cytosine deaminase (Ddd) enzyme to stencil TF binding locations within accessible chromatin regions. We optimize bulk and single-cell ACCESS-ATAC protocols and develop computational methods to show that the increased resolution compared with ATAC-seq improves the accuracy of TF binding site prediction. We use ACCESS-ATAC to perform genome-wide allelic occupancy and co-occupancy imputation for 64 TFs each in HepG2 and K562, revealing that the propensity of a majority of TFs to co-occupy nearby motifs oscillates with a period approximating the helical turn of DNA. Altogether, ACCESS-ATAC expands the resolution and capabilities of bulk and single-cell epigenomic profiling.
    DOI:  https://doi.org/10.1101/2024.12.17.628768
  22. Cell Stem Cell. 2024 Dec 31. pii: S1934-5909(24)00447-8. [Epub ahead of print]
    Ectopic expression of DNMT3L in human trophoblast stem cells restores features of the placental methylome.
    Georgia Lea, Paula Doria-Borrell, Ana Ferrero-Micó, Anakha Varma, Claire Simon, Holly Anderson, Laura Biggins, Katrien De Clercq, Simon Andrews, Kathy K Niakan, Lenka Gahurova, Naomi McGovern, Vicente Pérez-García, Courtney W Hanna.
      The placental DNA methylation landscape is unique, with widespread partially methylated domains (PMDs). The placental "methylome" is conserved across mammals, a shared feature of many cancers, and extensively studied for links with pregnancy complications. Human trophoblast stem cells (hTSCs) offer exciting potential for functional studies to better understand this epigenetic feature; however, whether the hTSC epigenome recapitulates primary trophoblast remains unclear. We find that hTSCs exhibit an atypical methylome compared with trophectoderm and 1st trimester cytotrophoblast. Regardless of cell origin, oxygen levels, or culture conditions, hTSCs show localized DNA methylation within transcribed gene bodies and a complete loss of PMDs. Unlike early human trophoblasts, hTSCs display a notable absence of DNMT3L expression, which is necessary for PMD establishment in mouse trophoblasts. Remarkably, we demonstrate that ectopic expression of DNMT3L in hTSCs restores placental PMDs, supporting a conserved role for DNMT3L in de novo methylation in trophoblast development in human embryogenesis.
    Keywords:  DNA methylation; DNMT3L; differentiation; epigenetics; histone modifications; human trophoblast stem cells; partially methylated domains; placenta; syncytiotrophoblast
    DOI:  https://doi.org/10.1016/j.stem.2024.12.007
  23. Elife. 2025 Jan 10. pii: RP92979. [Epub ahead of print]12
    Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging.
    Liza Dahal, Thomas G W Graham, Gina M Dailey, Alec Heckert, Robert Tjian, Xavier Darzacq.
      Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.
    Keywords:  chromosomes; gene expression; gene regulatory networks; human; molecular biophysics; nuclear receptors; protein-protein interactions; single-molecule tracking; structural biology; transcription factors
    DOI:  https://doi.org/10.7554/eLife.92979
  24. Sci Signal. 2025 Jan 07. 18(868): eado8860
    IRF1 cooperates with ISGF3 or GAF to form innate immune de novo enhancers in macrophages.
    Carolina Chavez, Kelly Lin, Alexis Malveaux, Aleksandr Gorin, Stefanie Brizuela, Quen J Cheng, Alexander Hoffmann.
      Macrophages exposed to immune stimuli reprogram their epigenomes to alter their subsequent functions. Exposure to bacterial lipopolysaccharide (LPS) causes widespread nucleosome remodeling and the formation of thousands of de novo enhancers. We dissected the regulatory logic by which the network of interferon regulatory factors (IRFs) induces the opening of chromatin and the formation of de novo enhancers. We found that LPS-activated IRF3 mediated de novo enhancer formation indirectly by activating the type I interferon (IFN)-induced ISGF3. However, ISGF3 was generally needed to collaborate with IRF1, particularly where chromatin was less accessible. At these locations, IRF1 was required for the initial opening of chromatin, with ISGF3 extending accessibility and promoting the deposition of H3K4me1, marking poised enhancers. Because IRF1 expression depends on the transcription factor NF-κB, which is activated in infected but not bystander cells, IRF-regulated enhancers required activation of both the IRF3 and NF-κB branches of the innate immune signaling network. However, type II IFN (IFN-γ), which is typically produced by T cells, may also induce IRF1 expression through the STAT1 homodimer GAF. We showed that, upon IFN-γ stimulation, IRF1 was also responsible for opening inaccessible chromatin sites that could then be exploited by GAF to form de novo enhancers. Together, our results reveal how combinatorial logic gates of IRF1-ISGF3 or IRF1-GAF restrict immune epigenomic memory formation to macrophages exposed to pathogens or IFN-γ-secreting T cells but not bystander macrophages exposed transiently to type I IFN.
    DOI:  https://doi.org/10.1126/scisignal.ado8860
  25. Nature. 2025 Jan 09.
    Repurposing of a gill gene regulatory program for outer ear evolution.
    Mathi Thiruppathy, Lauren Teubner, Ryan R Roberts, Micaela Lasser, Alessandra Moscatello, Ya-Wen Chen, Christian Hochstim, Seth Ruffins, Arijita Sarkar, Jade Tassey, Denis Evseenko, Thomas P Lozito, Helen Rankin Willsey, J Andrew Gillis, J Gage Crump.
      How novel structures emerge during evolution has long fascinated biologists. A dramatic example is how the diminutive bones of the mammalian middle ear arose from ancestral fish jawbones1. In contrast, the evolutionary origin of the outer ear, another mammalian innovation, remains a mystery, in part because it is supported by non-mineralized elastic cartilage rarely recovered in fossils. Whether the outer ear arose de novo or through reuse of ancestral developmental programs is unknown. Here we show that the outer ear shares gene regulatory programs with the gills of fishes and amphibians for both its initial outgrowth and later development of elastic cartilage. Comparative single-nuclei multiomics of the human outer ear and zebrafish gills reveals conserved gene expression and putative enhancers enriched for common transcription factor binding motifs. This is reflected by transgenic activity of human outer ear enhancers in gills, and fish gill enhancers in the outer ear. Further, single-cell multiomics of the cartilaginous book gills of horseshoe crabs reveal a shared DLX-mediated gill program with vertebrates, with a book gill distalless enhancer driving expression in zebrafish gills. We propose that elements of an invertebrate gill program were reutilized in vertebrates to generate first gills and then the outer ear.
    DOI:  https://doi.org/10.1038/s41586-024-08577-5
  26. Nucleic Acids Res. 2025 Jan 07. pii: gkae1281. [Epub ahead of print]53(1):
    Single-molecule analysis reveals that IPMK enhances the DNA-binding activity of the transcription factor SRF.
    Hyoungjoon Ahn, Jeongmin Yu, Kwangmin Ryu, Jaeseung Ryu, Sera Kim, Jae Yeong Park, Ji Kwang Kim, Inhong Jung, Haejin An, Sehoon Hong, Eunha Kim, Kihyun Park, Myunghwan Ahn, Sunwoo Min, Inkyung Jung, Daeyoup Lee, Thomas Lee, Youngjoo Byun, Ji-Joon Song, Jaehoon Kim, Won-Ki Cho, Gwangrog Lee, Seyun Kim.
      Serum response factor (SRF) is a master transcription factor that regulates immediate early genes and cytoskeletal remodeling genes. Despite its importance, the mechanisms through which SRF stably associates with its cognate promoter remain unknown. Our biochemical and protein-induced fluorescence enhancement analyses showed that the binding of SRF to serum response element was significantly increased by inositol polyphosphate multikinase (IPMK), an SRF cofactor. Moreover, real-time tracking of SRF loci in live cell nuclei demonstrated that the chromatin residence time of SRF was reduced by IPMK depletion in fibroblasts. Conversely, elevated IPMK levels extended the SRF-chromatin association. We identified that IPMK binds to the intrinsically disordered region of SRF, which is required for the IPMK-induced stable interaction of SRF with DNA. IPMK-mediated conformational changes in SRF were observed by single-molecule fluorescence resonance energy transfer assays. Therefore, our findings demonstrate that IPMK is a critical factor for promoting high-affinity SRF-chromatin association and provide insights into the mechanisms of SRF-dependent transcription control via chaperone-like activity.
    DOI:  https://doi.org/10.1093/nar/gkae1281
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