bims-crepig Biomed News
on Chromatin regulation and epigenetics in cell fate and cancer
Issue of 2025–08–24
seventeen papers selected by
Connor Rogerson, University of Cambridge
  1. Uncovering the multi-layer cis-regulatory landscape of rice via integrative nascent RNA analysis.
  2. H3K4me3 amplifies transcription at intergenic active regulatory elements.
  3. A transcription factor toggle switch determines differentiated epidermal cell identities in Hydra.
  4. Bipartite chromatin recognition by Hop1 from two diverged Holozoa.
  5. A distinct mechanism of epigenetic reprogramming silences PAX2 and initiates endometrial carcinogenesis.
  6. A modular enhancer mediates SCRT2 repression of ISLET1 in the spinal cord.
  7. Tracing the evolution of single-cell 3D genomes in Kras-driven cancers.
  8. NANOG is repurposed after implantation to repress Sox2 and begin pluripotency extinction.
  9. RNA Pol II inhibition activates cell death independently from the loss of transcription.
  10. Genetic effects on chromatin accessibility reveal the molecular mechanisms of complex traits in maize.
  11. DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes.
  12. Single cell multiomics approach to analyze replication timing and gene expression in mouse preimplantation embryos.
  13. Kingdom-wide CRISPR guide design with ALLEGRO.
  14. Systematic decoding of functional enhancer connectomes and risk variants in human glioma.
  15. The challenge of chromatin model comparison and validation: A project from the first international 4D Nucleome Hackathon.
  16. Peripheral heterochromatin tethering is required for chromatin-based nuclear mechanical response.
  17. ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.
  1. Genome Biol. 2025 Aug 18. 26(1): 250
    Uncovering the multi-layer cis-regulatory landscape of rice via integrative nascent RNA analysis.
    Marina Goliasse, Aurore Johary, Adrian E Platts, Fabian Ortner-Krause, Patrick P Edger, Jae Young Choi, Michael D Purugganan, Zoé Joly-Lopez.
       BACKGROUND: Efforts to characterize regulatory elements in plant genomes traditionally rely on evolutionary conservation and chromatin accessibility. Recently, intergenic bi-directional nascent transcript has emerged as a putative hallmark of active enhancers. Here, we integrate these approaches to better define the cis-regulatory landscape of the rice genome.
    RESULTS: In juvenile leaf tissues of the Azucena rice variety, we analyze conserved noncoding sequences, intergenic bi-directional transcripts, and regions of open chromatin. These three features highlight distinct classes of regulatory targets, each exhibiting complexity and regulatory roles. Conserved noncoding sequences are associated with more complex regulatory interactions, while regions marked by chromatin accessibility or bi-directional nascent transcription tend to promote more stable regulatory activity. Some transcribed regulatory sites harbor elements linked to transposable element silencing, whereas others correlate with increased expression of nearby genes, pointing to candidate transcribed regulatory elements. We further identified molecular interactions between genic regions and intergenic transcribed regulatory elements using 3-dimensional chromatin contact data, we identify physical interactions between transcribed intergenic regions and genic regions. These interactions often co-localize with expression quantitative trait loci and coincide with increased transcription, further supporting a regulatory role.
    CONCLUSIONS: Our integrative analysis reveals multiple distinct classes of regulatory elements in the rice genome, with overlapping but non-identical targets and signatures. Many candidate elements share features consistent with transcriptional enhancement, though the specific criteria for defining active enhancers in plants require further characterization. These findings underscore the importance of using complementary genomic signals to discover and characterize functionally diverse regulatory elements in plant genomes.
    Keywords:   Oryza sativa ; cis-regulatory elements; ATAC-seq; Chromatin architecture; Conserved noncoding sequences; Functional genomics; Gene regulation; Methylation; PRO-seq; Pore-C; Transcribed enhancers; Transposable elements
    DOI:  https://doi.org/10.1186/s13059-025-03715-2
  2. Genes Dev. 2025 Aug 18.
    H3K4me3 amplifies transcription at intergenic active regulatory elements.
    Haoming Yu, Yongyan Zhang, Zhicong Liao, Benjamin William Walters, Bluma J Lesch.
      Mammalian genomes undergo pervasive transcription in both genic and intergenic regions. Trimethylation of histone H3 lysine 4 (H3K4me3) is a deeply conserved and functionally important histone modification enriched at transcriptionally active promoters, where it facilitates RNA polymerase activity. H3K4me3 is also commonly found in intergenic regions, where its role is poorly understood. We interrogated the role of H3K4me3 at intergenic regulatory elements by using epigenetic editing to efficiently deposit H3K4me3 at intergenic loci. We found that H3K4me3 amplifies RNA polymerase activity and is actively remodeled at intergenic regions, shedding light on these important but poorly understood regions of the genome.
    Keywords:  chromatin; enhancer; epigenetics
    DOI:  https://doi.org/10.1101/gad.352841.125
  3. Development. 2025 Aug 18. pii: dev.204725. [Epub ahead of print]
    A transcription factor toggle switch determines differentiated epidermal cell identities in Hydra.
    Jaroslav Ferenc, Marylène Bonvin, Panagiotis Papasaikas, Jacqueline Ferralli, Clara Nuninger, Charisios D Tsiairis.
      In Hydra, a simple cnidarian model, epithelio-muscular cells shape and maintain body architecture through continuous renewal. Undifferentiated cells from the mid-body region migrate passively toward the extremities, replacing shed cells and acquiring region-specific identities. This ongoing turnover, together with Hydra's stable axial organization, provides a powerful model to study how cell type specification is integrated with body patterning. Yet, the molecular mechanisms that govern epithelial identity remain largely unknown. Here, we identify a double-negative feedback loop between the transcription factors Zic4 and Gata3 that functions as a toggle switch to control terminal epidermal cell fates. Zic4, activated by Wnt signaling from the head organizer, induces battery cell specification in tentacles. In contrast, Gata3 promotes basal disk cell identity at the aboral end. These factors are mutually repressive: silencing one leads to expansion of the other's domain and ectopic cell fate induction. Notably, simultaneous knockdown of both restores normal patterning, suggesting that identity is dictated by their relative balance rather than absolute levels. Our study reveals how opposing transcriptional signals coordinate epithelial identity with axial patterning at Hydra's poles.
    Keywords:  Axial patterning; Cell fate specification; Differentiation; Homeotic transformation
    DOI:  https://doi.org/10.1242/dev.204725
  4. Life Sci Alliance. 2025 Nov;pii: e202503428. [Epub ahead of print]8(11):
    Bipartite chromatin recognition by Hop1 from two diverged Holozoa.
    Alyssa A Rodriguez, Alessandro E Cirulli, Katie Chau, Justin Nguyen, Qiaozhen Ye, Kevin D Corbett.
      In meiosis, ploidy reduction is driven by a complex series of DNA breakage and recombination events between homologous chromosomes, orchestrated by meiotic HORMA domain proteins (HORMADs). Meiotic HORMADs possess a central chromatin binding region (CBR) whose architecture varies across eukaryotic groups. Here, we determine high-resolution crystal structures of the meiotic HORMAD CBR from two diverged aquatic Holozoa, Schistosoma mansoni and Patiria miniata, which reveal tightly associated plant homeodomain (PHD) and winged helix-turn-helix (wHTH) domains. We show that PHD-wHTH CBRs bind duplex DNA through their wHTH domains, and identify key residues that disrupt this interaction. Combining experimental and predicted structures, we show that the CBRs' PHDs likely interact with the tail of histone H3, and may discriminate between unmethylated and trimethylated H3 lysine 4. Finally, we show that Holozoa Hop1 CBRs bind nucleosomes in vitro in a bipartite manner involving both the PHD and wHTH domain. Our data reveal how meiotic HORMADs with PHD-wHTH CBRs can bind chromatin and potentially discriminate between chromatin states to drive meiotic recombination to specific chromosomal regions.
    DOI:  https://doi.org/10.26508/lsa.202503428
  5. J Clin Invest. 2025 Aug 15. pii: e190989. [Epub ahead of print]135(16):
    A distinct mechanism of epigenetic reprogramming silences PAX2 and initiates endometrial carcinogenesis.
    Subhransu S Sahoo, Susmita G Ramanand, Ileana C Cuevas, Yunpeng Gao, Sora Lee, Ahmed Abbas, Xunzhi Zhang, Ashwani Kumar, Prasad Koduru, Sambit Roy, Russell R Broaddus, Victoria L Bae-Jump, Andrew B Gladden, Jayanthi Lea, Elena Lucas, Chao Xing, Akio Kobayashi, Ram S Mani, Diego H Castrillon.
      Functional inactivation of tumor suppressor genes drives cancer initiation, progression, and treatment responses. Most tumor suppressor genes are inactivated through 1 of 2 well-characterized mechanisms: DNA-level mutations, such as point mutations or deletions, and promoter DNA hypermethylation. Here, we report a distinct third mechanism of tumor suppressor inactivation based on alterations to the histone rather than DNA code. We demonstrated that PAX2 is an endometrial tumor suppressor recurrently inactivated by a distinct epigenetic reprogramming event in more than 80% of human endometrial cancers. Integrative transcriptomic, epigenomic, 3D genomic, and machine learning analyses showed that PAX2 transcriptional downregulation is associated with replacement of open/active chromatin features (H3K27ac/H3K4me3) with inaccessible/repressive chromatin features (H3K27me3) in a framework dictated by 3D genome organization. The spread of the repressive H3K27me3 signal resembled a pearl necklace, with its length modulated by cohesin loops, thereby preventing transcriptional dysregulation of neighboring genes. This mechanism, involving the loss of a promoter-proximal superenhancer, was shown to underlie transcriptional silencing of PAX2 in human endometrial cancers. Mouse and human preclinical models established PAX2 as a potent endometrial tumor suppressor. Functionally, PAX2 loss promoted endometrial carcinogenesis by rewiring the transcriptional landscape via global enhancer reprogramming. The discovery that most endometrial cancers originate from a recurring epigenetic alteration carries profound implications for their diagnosis and treatment.
    Keywords:  Mouse models; Obstetrics/gynecology; Oncology; Reproductive biology; Tumor suppressors
    DOI:  https://doi.org/10.1172/JCI190989
  6. Dev Biol. 2025 Aug 14. pii: S0012-1606(25)00225-8. [Epub ahead of print]
    A modular enhancer mediates SCRT2 repression of ISLET1 in the spinal cord.
    Vitória S Botezelli, Tatiane Y Kanno, Ee Shan Liau, Carolina P Goes, Shirley de La Cruz Anticona, Ana Paula Azambuja, Marcos Simoes-Costa, C Y Irene Yan.
      How transcriptional programs coordinate the transition from neural progenitors to lineage-committed neurons in the spinal cord remains poorly understood. While much is known about transcription factors acting in the proliferative and differentiated zones, the role of intermediate zone (IZ) factors during lineage specification is less clear. Here, we investigate the function of SCRATCH2 (SCRT2), expressed in the postmitotic cells of the IZ, during dorsal interneuron differentiation. Overexpression of SCRT2 in vivo reduced the number of ISLET1+ dorsal interneurons. Chromatin profiling revealed that SCRT2 primarily binds to intergenic, transcriptionally inactive regions near neurogenic genes. Among these, we identified a conserved regulatory element, ECR4, located between ISLET1 and PARP8. Functional assays showed that ECR4 drives neural transcription and is composed of two subregions: ECR4B, an enhancer activated by ISLET1 and POU4F1, and ECR4A, which contains SCRT2 binding motifs and mediates transcriptional repression. Mutation of the vCES-box, a predicted SCRT2-binding motif within ECR4A, abolished repression, confirming a repressive regulatory interaction. Together, these data support a model in which SCRT2 represses ISLET1 through ECR4 to modulate dI3 lineage specification. These findings identify a novel regulatory mechanism linking intermediate zone transcriptional repression to dorsal interneuron development in the spinal cord.
    Keywords:  ISLET1; SCRT2; dorsal interneurons; gene regulation; neural tube
    DOI:  https://doi.org/10.1016/j.ydbio.2025.08.008
  7. Nat Genet. 2025 Aug 18.
    Tracing the evolution of single-cell 3D genomes in Kras-driven cancers.
    Miao Liu, Shengyan Jin, Sherry S Agabiti, Tyler B Jensen, Tianqi Yang, Jonathan S D Radda, Christian F Ruiz, Gabriel Baldissera, Moein Rajaei, Fang-Yong Li, Jeffrey P Townsend, Mandar Deepak Muzumdar, Siyuan Wang.
      Although three-dimensional (3D) genome structures are altered in cancer, it remains unclear how these changes evolve and diversify during cancer progression. Leveraging genome-wide chromatin tracing to visualize 3D genome folding directly in tissues, we generated 3D genome cancer atlases of oncogenic Kras-driven mouse lung adenocarcinoma (LUAD) and pancreatic ductal adenocarcinoma. Here we define nonmonotonic, stage-specific alterations in 3D genome compaction, heterogeneity and compartmentalization as cancers progress from normal to preinvasive and ultimately to invasive tumors, discovering a potential structural bottleneck in early tumor progression. Remarkably, 3D genome architectures distinguish morphologic cancer states in single cells, despite considerable cell-to-cell heterogeneity. Analyses of genome compartmentalization changes not only showed that compartment-associated genes are more homogeneously regulated but also elucidated prognostic and dependency genes in LUAD, as well as an unexpected role for Rnf2 in 3D genome regulation. Our results highlight the power of single-cell 3D genome mapping to identify diagnostic, prognostic and therapeutic biomarkers in cancer.
    DOI:  https://doi.org/10.1038/s41588-025-02297-w
  8. EMBO J. 2025 Aug 18.
    NANOG is repurposed after implantation to repress Sox2 and begin pluripotency extinction.
    Frederick C K Wong, Man Zhang, Ella Thomson, Linus J Schumacher, Anestis Tsakiridis, James Ashmore, Tong Li, Guillaume Blin, Eleni Karagianni, Nicholas P Mullin, Ian Chambers, Valerie Wilson.
      Loss of pluripotency is an essential step in post-implantation development that facilitates the emergence of somatic cell identities essential for gastrulation. Before implantation, pluripotent cell identity is governed by a gene regulatory network that includes the key transcription factors SOX2 and NANOG. However, it is unclear how the pluripotency gene regulatory network is dissolved to enable lineage restriction. Here, we show that SOX2 is required for post-implantation pluripotent identity in the mouse, and cells that lose SOX2 expression in the posterior epiblast are no longer pluripotent. Using in vitro and in vivo analyses, we demonstrate anticorrelated expression of NANOG and SOX2 preceding gastrulation, culminating in an early disappearance of pluripotent identity from posterior NANOGhigh/SOX2low epiblast. Surprisingly, Sox2 expression is repressed by NANOG and embryos with post-implantation deletion of Nanog maintain posterior SOX2 expression. Our results demonstrate that the distinctive features of post-implantation pluripotency are underpinned by altered functionality of pluripotency transcription factors, ensuring correct spatio-temporal loss of embryonic pluripotency.
    Keywords:  Embryo; NANOG; Pluripotency; Post Implantation; SOX2
    DOI:  https://doi.org/10.1038/s44318-025-00527-9
  9. Cell. 2025 Aug 14. pii: S0092-8674(25)00856-6. [Epub ahead of print]
    RNA Pol II inhibition activates cell death independently from the loss of transcription.
    Nicholas W Harper, Gavin A Birdsall, Megan E Honeywell, Kelly M Ward, Athma A Pai, Michael J Lee.
      RNA Pol II-mediated transcription is essential for eukaryotic life. Although loss of transcription is thought to be universally lethal, the associated mechanisms promoting cell death are not yet known. Here, we show that death following the loss of RNA Pol II activity does not result from dysregulated gene expression. Instead, it occurs in response to loss of the hypophosphorylated form of Rbp1 (also called RNA Pol IIA). Loss of RNA Pol IIA exclusively activates apoptosis, and expression of a transcriptionally inactive version of Rpb1 rescues cell viability. Using functional genomics, we identify the mechanisms driving lethality following the loss of RNA Pol IIA, which we call the Pol II degradation-dependent apoptotic response (PDAR). Using the genetic dependencies of PDAR, we identify clinically used drugs that owe their lethality to a PDAR-dependent mechanism. Our findings unveil an apoptotic signaling response that contributes to the efficacy of a wide array of anti-cancer therapies.
    Keywords:  BCL2L12; DNA damage; PTBP1; RNA polymerase II; apoptosis; cancer therapy; cell death; chemotherapy; cisplatin; transcription
    DOI:  https://doi.org/10.1016/j.cell.2025.07.034
  10. Plant J. 2025 Aug;123(4): e70437
    Genetic effects on chromatin accessibility reveal the molecular mechanisms of complex traits in maize.
    Yongli Zhu, Heiyuen Ngan, Weilun Liu, Tao Zhu, Wenqiang Li, Yingjie Xiao, Lin Zhuo, Dijun Chen, Xiaoyu Tu, Kang Gao, Jianbing Yan, Silin Zhong, Ning Yang.
      Cis-regulatory elements (CREs) are critical for modulating gene expression and phenotypic diversity in maize. While genome-wide association study (GWAS) hits and expression quantitative trait loci (eQTLs) are often enriched in CREs, their molecular mechanisms remain poorly understood. Characterizing CREs within accessible chromatin regions (ACRs) offers a powerful approach to link noncoding variants to chromatin structure alterations and phenotypic variation. Here, we generated ATAC-seq profiles from seedling leaves of 214 maize inbred lines, identifying 82 174 consensus ACRs. Notably, 39.55% of these ACRs exhibited significant population-wide chromatin accessibility variation. By mapping chromatin accessibility quantitative trait loci (caQTLs), we discovered 27 004 loci, including 1398 predicted to disrupt transcription factor (TF)-binding sites. Integration with multi-omics data revealed 7405 caACR-target gene pairs and linked 56 caACRs to GWAS signals for 51 agronomic traits, with significant enrichment in flowering-related pathways. Functional candidates such as ZmZIM30 - putatively regulated by caACRs - emerged as key regulators of flowering time. At the fad7 locus associated with linolenic acid content, allelic variants overlapping a caQTL showed differential chromatin accessibility. Our study provides a high-resolution cis-elements of maize leaves, deciphers the genetic basis of chromatin accessibility variation, and bridges noncoding caQTLs to molecular mechanisms underlying GWAS hits.
    Keywords:  ATAC‐seq; caQTL; cis‐regulatory elements; maize
    DOI:  https://doi.org/10.1111/tpj.70437
  11. iScience. 2025 Aug 15. 28(8): 113119
    DAXX-dependent H3.3 deposition maintains myoblast cell identity independently of other histone chaperone complexes.
    Virginia Zoglio, Sarah Chebouti, Fayez Issa, Frédéric Relaix, Joana Esteves de Lima.
      H3.3 histone chaperone DAXX regulates heterochromatin silencing; however, its function in transcription regulation remains understudied. Here, we show that Daxx knockout (KO) myoblasts have impaired differentiation and fusion. Transcriptomic analysis revealed a loss in myogenic gene expression and broad transcription dysregulation in Daxx KO myoblasts. Chromatin immunoprecipitation followed by sequencing demonstrated a marked reduction in H3.3 deposition at myogenic loci in Daxx KO myoblasts, which was further linked to decreased H3K27ac. Intriguingly, the double KO of Daxx and Hira resulted in distinct transcriptomic alterations than those of single KOs, demonstrating that DAXX and HIRA have both overlapping and unique roles in H3.3 incorporation. Our findings establish DAXX as a critical regulator of myogenic gene expression and muscle cell identity through a distinct mechanism from that of HIRA and highlighted an unanticipated plasticity in the deposition loci for DAXX and HIRA in myoblasts.
    Keywords:  cell biology; transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2025.113119
  12. Commun Biol. 2025 Aug 19. 8(1): 1245
    Single cell multiomics approach to analyze replication timing and gene expression in mouse preimplantation embryos.
    Anala V Shetty, Clifford J Steer, Walter C Low.
      Analysis of a cell's replication timing (RT) provides insight into how genes replicate, early or late, during the S-phase of the cell cycle. RT is cell-type specific, inheritable, and has been correlated to gene expression in normal and diseased states. However, most studies have been limited to somatic cells. Very little is known about RT control in early mouse embryos, and how it correlates with the start of transcription during zygote gene activation (ZGA), at the 2-cell stage. In this study, we develop an in-house single-cell multiomics approach to simultaneously analyze RT and gene expression in individual cells of the mouse 1-cell, 2-cell, and 4-cell embryos. We detect that RT is established at the 1-cell stage prior to ZGA. Surprisingly, we observe that the coordinated RT and gene expression control is different in early totipotent embryos, compared to previously published studies in somatic cells. Late replicating regions correlate with higher gene expression and open chromatin in the early developing embryos. Lastly, we perform an integrated pseudo time trajectory analysis combining RT and gene expression information per cell.
    DOI:  https://doi.org/10.1038/s42003-025-08694-5
  13. Nucleic Acids Res. 2025 Aug 11. pii: gkaf783. [Epub ahead of print]53(15):
    Kingdom-wide CRISPR guide design with ALLEGRO.
    Amirsadra Mohseni, Reyhane Ghorbani Nia, Aida Tafrishi, Mario León López, Xin-Zhan Liu, Jason E Stajich, Stefano Lonardi, Ian Wheeldon.
      Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we introduce ALLEGRO, a combinatorial optimization algorithm designed to compose minimal, yet highly effective sgRNA libraries targeting thousands of species at the same time. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting multiple genes of interest for over 2000 species across the fungal kingdom. We experimentally validated sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, Yarrowia lipolytica, and Saccharomyces cerevisiae, confirming successful genome edits. Additionally, we employed a generalized Cas9-ribonucleoprotein delivery system to apply ALLEGRO's sgRNA libraries to untested fungal genomes, such as Rhodotorula araucariae. Our experimental findings, together with cross-validation, demonstrate that ALLEGRO facilitates efficient CRISPR genome editing, enabling the development of universal sgRNA libraries applicable to entire taxonomic groups.
    DOI:  https://doi.org/10.1093/nar/gkaf783
  14. Nat Cell Biol. 2025 Aug 19.
    Systematic decoding of functional enhancer connectomes and risk variants in human glioma.
    Jinfang Bi, Weipeng Mo, Man Liu, Yingjie Song, Qian Xiao, Sibo Fan, Wenbin Wang, Tengfei Shi, Yaoqiang Zheng, Jie Lian, Rong Liu, Bohan Chen, Xiaofeng Huang, Peng Li, Zhongfang Zhao, Jiandang Shi, Lei Zhang, Guangsong Su, Nu Zhang, Wange Lu.
      Genetic and epigenetic variations contribute to the progression of glioma, but the mechanisms underlying these effects, particularly for enhancer-associated genetic variations in non-coding regions, still remain unclear. Here we performed high-throughput CRISPR interference screening to identify pro-tumour enhancers in glioma cells. By integrating genome-wide H3K27ac HiChIP data, we identified the target genes of these pro-tumour enhancers and revealed the essential role of enhancer connectomes in promoting glioma progression. Through systematic analysis of enhancers carrying glioma risk-associated single-nucleotide polymorphisms (SNPs), we found that these SNPs can promote glioma progression through the enhancer connectome. Using CRISPR-Cas9-mediated enhancer interference and SNP editing, we demonstrated that glioma-specific enhancer carrying the risk SNP rs2297440 regulates SOX18 expression by specifically recruiting transcription factor MEIS1 binding, thereby contributing to glioma progression. Our study sheds light on the molecular mechanisms underlying glioma susceptibility and provides potential therapeutic targets to treat glioma.
    DOI:  https://doi.org/10.1038/s41556-025-01737-3
  15. PLoS Comput Biol. 2025 Aug 19. 21(8): e1013358
    The challenge of chromatin model comparison and validation: A project from the first international 4D Nucleome Hackathon.
    Jędrzej Kubica, Sevastianos Korsak, Krzysztof H Banecki, Dvir Schirman, Anurupa Devi Yadavalli, Ariana Brenner Clerkin, David Kouřil, Michał Kadlof, Ben Busby, Dariusz Plewczynski.
      The computational modeling of chromatin structure is highly complex due to the hierarchical organization of chromatin, which reflects its diverse biophysical principles, as well as inherent dynamism, which underlies its complexity. Chromatin structure modeling can be based on diverse approaches and assumptions, making it essential to determine how different methods influence the modeling outcomes. We conducted a project at the NIH-funded 4D Nucleome Hackathon on March 18-21, 2024, at The University of Washington in Seattle, USA. The hackathon provided an amazing opportunity to gather an international, multi-institutional and unbiased group of experts to discuss, understand and undertake the challenges of chromatin model comparison and validation. Here we give an overview of the current state of the 3D chromatin field and discuss our efforts to run and validate the models. We used distance matrices to represent chromatin models and we calculated Spearman correlation coefficients to estimate differences between models, as well as between models and experimental data. In addition, we discuss challenges in chromatin structure modeling that include: 1) different aspects of chromatin biophysics and scales complicate model comparisons, 2) large diversity of experimental data (e.g., population-based, single-cell, protein-specific) that differ in mathematical properties, heatmap smoothness, noise and resolutions complicates model validation, 3) expertise in biology, bioinformatics, and physics is necessary to conduct comprehensive research on chromatin structure, 4) bioinformatic software, which is often developed in academic settings, is characterized by insufficient support and documentation. We also emphasize the importance of establishing guidelines for software development and standardization.
    DOI:  https://doi.org/10.1371/journal.pcbi.1013358
  16. Nucleic Acids Res. 2025 Aug 11. pii: gkaf763. [Epub ahead of print]53(15):
    Peripheral heterochromatin tethering is required for chromatin-based nuclear mechanical response.
    Ali Göktuğ Attar, Jarosław Paturej, Ozan S Sarıyer, Edward J Banigan, Aykut Erbaş.
      The cell nucleus is a mechanically responsive structure that governs how external forces affect chromosomes. Chromatin, particularly transcriptionally inactive heterochromatin, resists nuclear deformations through its mechanical response. However, chromatin also exhibits liquid-like properties, casting ambiguity on the physical mechanisms of chromatin-based nuclear elasticity. To determine how heterochromatin strengthens nuclear mechanical response, we performed polymer physics simulations of a nucleus model validated by micromechanical measurements and chromosome conformation capture data. The attachment of peripheral heterochromatin to the lamina is required to transmit forces directly to the chromatin and elicit its elastic response. Thus, increases in heterochromatin levels increase nuclear rigidity by increasing the linkages between chromatin and the lamina. Crosslinks within heterochromatin, such as HP1α proteins, can also stiffen nuclei, but only if chromatin is peripherally tethered. In contrast, heterochromatin affinity interactions that may drive liquid-liquid phase separation do not contribute to nuclear rigidity. When the nucleus is stretched, gel-like peripheral heterochromatin can bear stresses and deform, while the more fluid-like interior euchromatin is less perturbed. Thus, heterochromatin's internal structure and stiffness may regulate nuclear mechanics via peripheral attachment to the lamina, while also enabling nuclear mechanosensing of external forces and external measurement of the nucleus' internal architecture.
    DOI:  https://doi.org/10.1093/nar/gkaf763
  17. Nucleic Acids Res. 2025 Aug 11. pii: gkaf809. [Epub ahead of print]53(15):
    ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.
    Valentina Migliori, Michaela B Bruntraeger, Ivan S Gyulev, Florence Lichou, Thomas Burgold, Daniel P Gitterman, Sho Iwama, Andrew L Trinh, Sam J Washer, Carla P Jones, Gosia Trynka, Andrew R Bassett.
      We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering.
    DOI:  https://doi.org/10.1093/nar/gkaf809
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