bims-crepig 2026-03-01 papers

bims-crepig

Biomed News

on Chromatin regulation and epigenetics in cell fate and cancer

Issue of 2026–03–01
twenty-six papers selected by
Connor Rogerson, University of Cambridge

High-resolution map of chromatin accessibility - insights into the focused binding of a large number of transcription factors.
Chromatin accessibility landscapes define stromal cell identities across tissues.
Nascent transcriptome of embryonic genome activation reveals a regulatory axis linking transcriptional priming to early lineage specification in mouse embryos.
Three-dimensional genome reorganization foreshadows zygotic genome activation in Drosophila.
Nucleosome spacing regulates linker methylation by DNMT3A2/3B3.
Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.
Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila.
Bromodomain protein IBD1 bridges histone acetylation and H2A.Z deposition to fine-tune transcription.
Massively parallel reporter assay for mapping gene-specific regulatory regions at single-nucleotide resolution.
Chromatin spatial analysis by METALoci unveils sex-determining 3D regulatory hubs.
CFP1-mediated H3K4me3 broad domains control early B cell lineage fate determination.
Single-cell ultra-high-throughput multiplexed chromatin accessibility and gene expression sequencing (SUM-seq).
Dynamic landscape of transcription initiation by yeast RNA polymerase I.
LBR and LAP2 mediate heterochromatin tethering to the nuclear periphery to preserve genome homeostasis.
CIRCE: a scalable Python package to predict cis-regulatory DNA interactions from single-cell chromatin accessibility data.
Functional dissection of complex trait variants at single-nucleotide resolution.
Nuclear speckles enable processing of RNA from GC-rich isochores.
Mechanisms of DNA methyltransferase 3A1 mediated DNA methylation of nucleosomes.
A robust cis-regulatory network ensures Otx2 expression during retinal development.
Pervasive transcription in the human genome exceeds background noise.
Global maps of transcription factor properties reveal threshold-based formation of DNA-bound and mobile clusters.
Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response.
Genome-wide dynamic nascent transcript profiles reveal that most paused RNA polymerases terminate.
Directing stem cell differentiation by chromatin state approximation.
A de novo H3.2K9me2 deposition pathway establishes heterochromatin for suppressing transposon mobilization during fly somatic development.
Hi-C for genome-wide detection of enhancer-hijacking rearrangements in routine lymphoid cancer biopsies.

Epigenetics Chromatin. 2026 Feb 22.

High-resolution map of chromatin accessibility - insights into the focused binding of a large number of transcription factors.

Iris Zhu, David Landsman.

   BACKGROUND: Emerging evidence has shown the common occupancy of dozens to hundreds of transcription factors (TFs) on cis-regulatory elements (CREs), yet the underlying details are largely unknown.
RESULTS: In this study, leveraging extensive collections of TF ChIP-seq data of more than 1000 TFs in human HepG2 and K562 cells, we located highly focused TF binding sites (FBSs) within CREs as single-nucleosome depleted regions, which accommodate the majority of the total TF binding events. Approximately 25,000 strong FBSs were identified in each cell type. For more than 90% of TFs, including some pioneer factors such as GATA1 and JUN, their binding sites out of FBSs barely show nucleosome depletion. Essential cellular function related motifs and phenotypically causal variants are strongly enriched in the FBSs, but not in their immediate flanking regions within CREs. Most TFs bind to FBSs not containing their canonical motifs.
CONCLUSION: Our study revealed the critical connection between highly focused TF binding and the nucleosome depleted status of DNA in vivo. Meanwhile, we constructed high-resolution maps of chromatin accessibility at distal CREs in the two human cells. We propose a model of TF co-binding in vivo and suggest that a short DNA residence time of most TFs underlies the requirement of a large number of TFs for sustained nucleosome depletion at CREs.

Keywords:  Chromatin accessibility; Focused binding of a large number of TFs; Nucleosome depletion

DOI:  https://doi.org/10.1186/s13072-026-00665-2
Commun Biol. 2026 Feb 25.

Chromatin accessibility landscapes define stromal cell identities across tissues.

Amin Nooranikhojasteh, Ghazaleh Tavallaee, Nicholas Khuu, Shu Yi Shen, Shaida Ouladan, Ayush Raman, Elias Orouji.

Chromatin accessibility is crucial in regulating gene expression and maintaining cellular identity. While single-cell RNA sequencing has revolutionized transcriptomic profiling, the understanding of chromatin dynamics across diverse tissues remains limited. Here, we use single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) to explore chromatin accessibility landscapes across murine tissues. We profile chromatin accessibility in 51,248 cells from nine mouse tissues, identifying 28 major cell types with distinct accessibility signatures. Our data reveal both conserved and tissue-specific cis-regulatory elements, highlighting the dynamic interplay between transcription factors and chromatin states in cell differentiation and tissue function. Motif enrichment analyses uncover transcription factor motifs driving these regulatory landscapes. Notably, we demonstrate that chromatin accessibility profiles enable tracing stromal cells, including endothelial cells, fibroblasts, and macrophages back to their tissue of origin. Using a metacell approach, we identify specific chromatin modules reflecting tissue-specific epigenomic landscapes, underscoring the role of chromatin accessibility in defining stromal cell identities. Our study provides a comprehensive atlas of chromatin accessibility across murine tissues, offering insights into the regulatory architecture that governs tissue and cell-type specificity. The ability to trace stromal cells to their tissue of origin through chromatin signatures holds important implications for diagnostics and therapeutic interventions in disease contexts.

DOI: https://doi.org/10.1038/s42003-026-09720-w
Nucleic Acids Res. 2026 Feb 24. pii: gkag173. [Epub ahead of print]54(5):

Nascent transcriptome of embryonic genome activation reveals a regulatory axis linking transcriptional priming to early lineage specification in mouse embryos.

Yue Hu, Yuxiang Wang, Maosheng Ye, Yuanlin He, Zhangyi Ouyang, Chao Ren, Tianyin Miao, Siqi Wang, Ou Zhong, Li Liu, Wenjie Shu, Ran Huo.

Embryonic genome activation (EGA) marks a critical developmental transition, yet its regulatory architecture remains incompletely defined. Here, we employed optimized low-input SLAM-seq (thiol(SH)-linked alkylation for the metabolic sequencing) to map the temporal hierarchy of nascent transcription during mouse EGA. We uncovered patterns of transcriptional priming characterized by pre-activated genes (PAGs) with permissive chromatin states, followed by pronounced accumulation of PAGs-encoded proteins in blastocysts, suggesting that EGA memory propagates from early transcriptional activation to later lineage commitment. Furthermore, Integrative analysis nominated two-cell nascent transcription factors (TFs) as candidate regulators of the first lineage specification. Functional investigations demonstrated KLF17 as a key TF linking EGA to the first lineage specification via regulation of PAGs transcription. KLF17 deficiency led to the failure of transcriptional activation in approximately half of PAGs at the two-cell stage. Our work provides a detailed framework for decoding mammalian EGA and offers insights into how embryonic transcriptional priming is coordinated with early cell fate specification.

DOI: https://doi.org/10.1093/nar/gkag173
Nat Genet. 2026 Feb 24.

Three-dimensional genome reorganization foreshadows zygotic genome activation in Drosophila.

Noura Maziak, Yuchen Zhang, Fabian Groll, Haley E Brown, Alla Madich, Yadwinder Kaur, Melissa M Harrison, Jian Zhou, Juan M Vaquerizas.

How chromatin conformation relates to chromatin state remains a central challenge in genome regulation. Here we present Pico-C, a low-input Micro-C approach that enables high-resolution, temporally resolved three-dimensional genome mapping during early Drosophila embryogenesis. Contrary to a prevailing view of a disorganized genome before zygotic genome activation (ZGA), we uncover a dynamic and ordered emergence of chromatin loops during pre-ZGA nuclear cycles. Spatial autocorrelation analysis points to context-dependent regulatory influences on chromatin. Notably, inhibition of transcriptional elongation has site-specific effects, retaining some early loops while weakening insulation at active promoters, suggesting distinct regulatory dependencies. Machine learning models trained on sequence features identify orthogonal, motif-specific contributions to architecture. Co-depletion of the pioneer factors Zelda and GAF leads to factor-specific perturbations in chromatin architecture, further highlighting a modular regulatory logic in genome establishment. Together, our findings reveal that early genome organization is orchestrated by an interplay of overlapping yet separable regulatory inputs.

DOI: https://doi.org/10.1038/s41588-026-02503-3
Mol Cell. 2026 Feb 24. pii: S1097-2765(26)00094-8. [Epub ahead of print]

Nucleosome spacing regulates linker methylation by DNMT3A2/3B3.

Xiaoyan Xie, Minmin Liu, Gabriella N L Chua, X Edward Zhou, Michelle L Dykstra, Shixin Liu, Peter A Jones, Evan J Worden.

  De novo CpG methylation (mCpG) is deposited by DNMT3A and DNMT3B, which target DNA linkers between nucleosomes. Cells contain millions of unique linkers, but the rules dictating which linkers get targeted by DNMT3 enzymes are not understood. We show that nucleosome spacing controls linker DNA methylation and H3K36me2 recognition by human DNMT3A2/3B3, linking de novo methylation to chromatin architecture. We present structures of DNMT3A2/3B3 bound to dinucleosomes, revealing that short linkers promote dinucleosome bridging, blocking access to linker DNA and suppressing methylation, whereas long linkers allow DNMT3A2/3B3 to engage each nucleosome separately and methylate linker DNA. Finally, we show that DNMT3A2/3B3 positions proline-tryptophan-tryptophan-proline (PWWP) domains to scan for H3K36me2. However, H3K36me2 recognition is blocked when DNMT3A2/3B3 bridges dinucleosomes with short linkers, imposing an additional structural constraint on DNMT3A2/3B3 function. Together, these findings uncover the mechanisms that govern de novo methylation in chromatin and explain how DNMT3 enzymes target linkers in cells.

Keywords:  DNA methylation; DNA methyltransferase; cancer; chromatin; nucleosome

DOI:  https://doi.org/10.1016/j.molcel.2026.01.030
Cell Stem Cell. 2026 Feb 24. pii: S1934-5909(26)00038-X. [Epub ahead of print]

Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.

Ming Zhu, Junsong Yuan, Qiuchen Meng, Jiawei Yu, Xiaoqing Xu, Mingyue Xu, Xiaoyu Ren, Yanyan Hu, Guoan Wei, Zeran Jia, Guohua Yuan, Lu Zang, Shaorui Liu, Yuanyuan Yang, Yuan Zheng, Jiacheng Wang, Tingting Cong, Wei Xie, Xun Lan, Le Cong, Tianhua Ma, Sheng Ding, Weixiang Guo, Xuegong Zhang, Yinqing Li.

  Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.

Keywords:  chromatin architecture; chromatin condensate; chromatin perturbation; genome editing; neural stem cell; premature differentiation; somatic stem cell

DOI:  https://doi.org/10.1016/j.stem.2026.01.015
Sci Adv. 2026 Feb 27. 12(9): eady7568

Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila.

Raku Saito, Yusuke Umemura, Shiho Makino, Takashi Fukaya.

The zinc finger-associated domain (ZAD)-containing C2H2 zinc finger proteins (ZAD-ZnFs) represent the most abundant class of transcription factors that emerged during insect evolution, yet their molecular diversity and biological functions remain largely unclear. Here, we established a systematic CRISPR-based protein-tagging approach that enables direct, unambiguous comparison of nuclear localization and genome-wide binding profiles of endogenous ZAD-ZnFs in developing Drosophila embryos. Evidence is provided that a subset of ZAD-ZnFs forms nuclear condensates through the stacking of the N-terminal ZAD dimerization surface. Disruption of condensation activity leads to misregulation of genome-wide binding profiles and lethality, underscoring its functional and physiological significance in development. Integrative chromatin immunoprecipitation sequencing and Micro-C analyses reveal that many ZAD-ZnFs colocalize with core insulator proteins such as CCCTC-binding factor and Centrosomal protein 190 kD to control the formation of topological boundaries. We suggest that the diverse molecular functions of ZAD-ZnFs have evolutionarily arisen from their ancestral role as insulator-binding proteins.

DOI: https://doi.org/10.1126/sciadv.ady7568
Nucleic Acids Res. 2026 Feb 05. pii: gkag148. [Epub ahead of print]54(4):

Bromodomain protein IBD1 bridges histone acetylation and H2A.Z deposition to fine-tune transcription.

Zhe Zhang, Haicheng Li, Aili Ju, Fei Ye, Fan Wei, Yongqiang Liu, Junhua Niu, Hongzhen Jiang, Yuanyuan Wang, Shan Gao.

Eukaryotic gene expression is dynamically regulated through the interplay between histone modifications and chromatin remodeling, yet how these processes are coordinated remains incompletely understood. Here, we uncover IBD1 as a critical adaptor that bridges histone acetylation and SWR-mediated H2A.Z deposition. Mechanistically, IBD1's bromodomain recognizes histone acetylation, specifically H3K9/K14 di-acetylation, to recruit the SWR complex subunit ARP6, ensuring precise H2A.Z incorporation into chromatin. H3K9Q mutation and genetic disruption of IBD1, either by deletion or bromodomain mutation, significantly reduce H2A.Z occupancy at target loci. In contrast, disruption of IBD1 has little effect on H3K9/K14 acetylation levels, confirming the directional hierarchy of the acetylation-IBD1-H2A.Z regulatory axis. Intriguingly, perturbation of this axis, through IBD1 loss or bromodomain impairment, leads to widespread transcriptional upregulation, particularly at genes co-enriched for IBD1, H3K9/K14ac, and H2A.Z, with the strongest effects at hyperacetylated loci. This transcriptional imbalance coincides with reduced growth rates, underscoring the functional significance of IBD1-mediated H2A.Z deposition. Given that H2A.Z enrichment is classically correlated with transcriptional levels, this observation highlights a dual role for H2A.Z: sustaining basal transcription and constraining overactivation at highly active genes. Together, our findings define a novel regulatory mechanism in which IBD1 bridges acetyl-mark decoding with SWR-dependent H2A.Z deposition, establishing transcriptional homeostasis.

DOI: https://doi.org/10.1093/nar/gkag148
Elife. 2026 Feb 25. pii: RP107565. [Epub ahead of print]14

Massively parallel reporter assay for mapping gene-specific regulatory regions at single-nucleotide resolution.

Alastair J Tulloch, Ryan Nicholas Delgado, Rinaldo Catta-Preta, Constance L Cepko.

  Precise gene regulation is essential for tissue development and function, yet mapping cis-regulatory modules (CRMs) at high resolution and in specific cell types remains challenging. We introduce two complementary strategies-a locus-specific massively parallel reporter assay (LS-MPRA) and a degenerate MPRA (d-MPRA)-designed to overcome limitations in throughput, resolution, and prior knowledge requirements. LS-MPRA uses BAC-based libraries to densely sample genomic regions, enabling unbiased interrogation of millions of DNA fragments for CRM activity. D-MPRA applies systematic mutagenesis to resolve CRM architecture at single-nucleotide resolution, nominating essential bases that may function as TF binding sites or other regulatory elements. We applied these methods to retinal genes expressed in mature rods and bipolar interneurons using in vivo and ex vivo mouse (Mus musculus) tissue. LS-MPRA recapitulated known CRMs and identified previously uncharacterized CRMs, including those embedded in neighboring genes. Applied to Olig2, a dynamically expressed gene in retinal progenitors, LS-MPRA identified three CRM regions, which d-MPRA and motif analyses further dissected. CUT&RUN confirmed direct binding of candidate TFs. Extending LS-MPRA to chick (Gallus gallus) retina and spinal cord demonstrated cross-species and cross-tissue applicability. Together, these approaches provide a rapid, scalable, inexpensive, and accessible platform for CRM discovery that can be carried out without prior element annotation and with tunable (small) fragment sizes.

Keywords:  Olig2; chicken; chromosomes; cis-regulatory module; developmental biology; gene expression; gene regulation; massively parallel reporter assay; mouse; retinal progenitor cell

DOI:  https://doi.org/10.7554/eLife.107565
Nat Struct Mol Biol. 2026 Feb 24.

Chromatin spatial analysis by METALoci unveils sex-determining 3D regulatory hubs.

Irene Mota-Gómez, Juan Antonio Rodríguez, Shannon Dupont, Alicia Hurtado, Vanessa Cadenas, Leo Zuber, Iago Maceda, Oscar Lao, Johanna Jedamzick, Ralf Kühn, Scott Lacadie, Sara Alexandra García-Moreno, Miguel Torres, Francisca M Real, Rafael D Acemel, Blanche Capel, Marc A Marti-Renom, Darío G Lupiáñez.

Mammalian sex is determined by opposing networks of ovarian and testicular genes that are well characterized; however, its epigenetic regulation is still largely unknown. Here we explore the 3D chromatin landscape of sex determination in vivo by profiling fluorescence-activated cell-sorted embryonic mouse gonadal populations in both sexes before and after sex determination. Through conventional Hi-C analyses, we show that chromatin structures, particularly topologically associating domains, remain largely unchanged during sex determination, suggesting a preformed configuration. We further integrate Hi-C data with ChIP-seq experiments using METALoci, a spatial autocorrelation analysis that identifies three-dimensional (3D) regulatory hubs across the genome. We uncover a prominent rewiring of chromatin interactions during sex determination, affecting the 3D regulatory hubs of hundreds of genes that display time-specific and sex-specific expression. By combining predictive approaches and validations in transgenic mice, we identify a 3D regulatory hub for the protesticular gene Fgf9. The deletion of this gonad-specific hub allows mutant mice to survive through development, overcoming lung lethality associated with Fgf9 loss of function while exhibiting male-to-female sex reversal. Through the reconstruction of gene regulatory networks, we identify a function for Meis genes, which act redundantly to specify sexual identity during ovarian and testicular development. Our results underscore the dynamic role of the 3D genome during sex determination, highlighting the potential of epigenomic approaches to uncover regulators of developmental processes.

DOI: https://doi.org/10.1038/s41594-026-01749-z
Nucleic Acids Res. 2026 Feb 05. pii: gkag125. [Epub ahead of print]54(4):

CFP1-mediated H3K4me3 broad domains control early B cell lineage fate determination.

Wanyu Bai, Yalin Yang, Yanan Zhao, Shuoxu Gong, Yimiao Lu, Zhonghui Xue, Lie Wang, Fei-Long Meng, Chun Chen, Junchao Dong.

The histone H3K4me3 broad domain (BD) is a unique epigenetic feature marking cell identity-associated genes, but its physiological role in lineage differentiation remains unclear. Here we show that CFP1, an integral component of the Setd1A/B methyltransferase complex, is critically required for functional H3K4me3-BD installation and B cell fate determination. Cfp1 deletion impairs H3K4me3-BDs on a subset of B lineage genes and redistributes H3K4me3 from active genes to bivalent promoters. Transcription of subsets of the H3K4me3-BD-bound genes is diminished in part due to reduced RNA polymerase II at the promoter and gene body. Specifically, CFP1 ablation abolishes H3K4me3-BD at the recombination center and distal PAIR elements in the immunoglobulin heavy chain (IgH) gene, severely compromises its locus contraction for efficient V-to-DJ recombination, and consequently arrests cells at the pro-B stage. Moreover, Cfp1-deficient pro-B cells up-regulate a panel of progenitor and myeloid-specific genes with elevated H3K4me3 marks and can trans-differentiate into various myeloid cell types. Therefore, our study reveals pivotal roles for CFP1-mediated H3K4me3-BDs in the transcriptional regulation of cell lineage fate specification and commitment.

DOI: https://doi.org/10.1093/nar/gkag125
Nat Protoc. 2026 Feb 26.

Single-cell ultra-high-throughput multiplexed chromatin accessibility and gene expression sequencing (SUM-seq).

Umut Yildiz, Sara Lobato-Moreno, Annique Claringbould, Hanke Gwendolyn Bauersachs, Nila H Servaas, Evi P Vlachou, Christian Arnold, Víctor Campos-Fornés, Karin D Prummel, Judith B Zaugg, Kyung Min Noh, Mikael Marttinen.

Single-cell epigenome and transcriptome profiling enables the dissection of gene regulatory networks, offering a powerful approach to characterize cellular heterogeneity and regulatory landscapes of cell states. Here we describe a single-cell ultra-high-throughput multiplexed sequencing (SUM-seq) assay for scalable and cost-effective simultaneous profiling of chromatin accessibility and gene expression in single nuclei. SUM-seq combines sample-specific accessible DNA and mRNA in situ barcoding with droplet-based microfluidic barcoding, introducing sample multiplexing and means to resolve multinucleated droplets for multiomic single-cell library preparation. In comparison with existing methods for multimodal profiling of chromatin accessibility and gene expression from the same cell, SUM-seq offers increased throughput and an unmatched multiplexing capability. This permits substantial scaling of the number of samples and nuclei assayed in one experiment, adhering to the needs of large-scale atlas projects, time-course experiments and perturbation screens while considerably reducing costs. We provide guidelines for experimental design and sample handling to accommodate various settings and sample types. Moreover, we discuss potential applications and provide guidelines for data processing. From sample collection to library preparation, the assay can be completed in 2-3 days, followed by sequencing and 1 day of data processing. Although the protocol can be implemented by researchers with general molecular biology skills, prior experience with single-cell assays is recommended.

DOI: https://doi.org/10.1038/s41596-025-01310-0
Nucleic Acids Res. 2026 Feb 24. pii: gkag153. [Epub ahead of print]54(5):

Dynamic landscape of transcription initiation by yeast RNA polymerase I.

Olena Parilova, Piia Bartos, Anssi M Malinen.

RNA polymerase I (Pol I) synthesizes precursor ribosomal RNA, a key step in ribosome biogenesis. Elevated Pol I activity supports rapid cell growth-a hallmark of cancer-making Pol I a therapeutic target. The initial step in synthesis involves assembly of the Pol I transcription initiation complex on the gene promoter; however, its quantitative and dynamic parameters remain poorly defined. Here, we integrate biochemical, biophysical, and molecular dynamics approaches to dissect promoter and transcription start site (TSS) recognition by the Saccharomyces cerevisiae Pol I machinery. We show that core factor (CF) identifies the promoter through a two-step mechanism: a rapid encounter is followed by a slower conformational transition that establishes stabilizing interactions. In contrast, CF binds nonpromoter DNA in a single step without such transitions, forming nonspecific complexes that dissociate quickly. Correct promoter binding allows CF to recruit and position Pol I near the TSS, inducing DNA bending, helix distortion, and melting as the preinitiation complex converts into an active state. These functional and dynamic parameters contribute to a quantitative framework for elucidating the molecular mechanisms of Pol I regulation and inhibition.

DOI: https://doi.org/10.1093/nar/gkag153
Nat Cell Biol. 2026 Feb 24.

LBR and LAP2 mediate heterochromatin tethering to the nuclear periphery to preserve genome homeostasis.

Renard Lewis, Virginia Sinigiani, Noura Maziak, Krisztian Koos, Cristiana Bersaglieri, Ivo Zemp, Caroline Ashiono, Constance Ciaudo, Peter Horvath, Juan M Vaquerizas, Raffaella Santoro, Puneet Sharma, Ulrike Kutay.

In most eukaryotic cells, euchromatin is localized in the nuclear interior, whereas heterochromatin is enriched at the nuclear envelope (NE). This conventional chromatin organization is established by heterochromatin tethering to the NE; however, its importance for cellular homeostasis is largely unexplored. One tether is constituted by the lamin B receptor (LBR) in mammals, but the enigmatic nature of other redundant tethers has hampered functional analyses. Here we demonstrate that downregulation of abundant, ubiquitous NE proteins can induce the global detachment of heterochromatin from the NE and its repositioning to the nuclear interior. We identify LBR and lamina-associated polypeptide 2 (LAP2) as key factors for peripheral heterochromatin positioning in differentiated and pluripotent mammalian cells. Their long-term loss leads to changes in three-dimensional chromatin organization and a reduction in repressive epigenetic marks, especially H3K27me3. These changes are associated with massive deregulation of gene expression, activation of antiviral innate immunity, and defects in cell fate determination.

DOI: https://doi.org/10.1038/s41556-025-01822-7
Bioinformatics. 2026 Feb 24. pii: btag092. [Epub ahead of print]

CIRCE: a scalable Python package to predict cis-regulatory DNA interactions from single-cell chromatin accessibility data.

Trimbour Rémi, Saez-Rodriguez Julio, Cantini Laura.

MOTIVATION: Chromatin 3D folding creates numerous DNA interactions, participating in gene expression regulation. Single-cell chromatin-accessibility assays now profile hundreds of thousands of cells, challenging existing methods for mapping cis-regulatory interactions.
RESULTS: We present CIRCE, a fast and scalable Python package to predict cis-regulatory DNA interactions from single-cell chromatin accessibility data. CIRCE re-implements the Cicero workflow to analyse single-cell atlases, cutting runtime and memory use by several orders of magnitude. We also provide new options to compute metacells, grouping similar cells to reduce data sparsity.We benchmarked CIRCE against Cicero on two datasets of different sizes and demonstrated the improvement from CIRCE's metacells' strategy with promoter capture Hi-C data. We also evaluated how DNA interaction predictions are impacted by different pre-processing. We observed a negative impact of Cicero's count normalization, and the best performance was obtained with the single-cell count matrix directly. Finally, we demonstrated the scalability of CIRCE by processing a dataset of more than 700000 cells and 1 million DNA regions in less than an hour. CIRCE should greatly facilitate the prediction of DNA region interactions for scverse and Python users, while providing new and up-to-date pre-processing insights.
AVAILABILITY: CIRCE is released as an open-source software under the AGPL-3.0 license. The package source code is available on GitHub at https://github.com/cantinilab/CIRCE, and its documentation is accessible at https://circe.readthedocs.io.The code to reproduce the presented results is available as a Snakemake pipeline at https://github.com/cantinilab/circe_reproducibility.s.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btag092
Nature. 2026 Feb 25.

Functional dissection of complex trait variants at single-nucleotide resolution.

Layla Siraj, Rodrigo I Castro, Hannah B Dewey, Susan Kales, John C Butts, Thanh Thanh L Nguyen, Masahiro Kanai, Daniel Berenzy, Kousuke Mouri, Qingbo S Wang, Petko P Fiziev, Kristin Tsuo, Zachary R McCaw, Sager J Gosai, François Aguet, Ran Cui, Irfahan Kassam, Jeremy McRae, Christopher M Vockley, Caleb A Lareau, Sergey Abramov, Alexandr Boystov, Jeff Vierstra, Yukinori Okada, Alexander Gusev, Thouis R Jones, Eric S Lander, Pardis C Sabeti, Hilary K Finucane, Steven K Reilly, Jacob C Ulirsch, Ryan Tewhey.

Identifying the causal variants and mechanisms that drive complex traits and diseases remains a core problem in human genetics1-5. Most of these variants individually have weak effects6 and lie in non-coding gene-regulatory elements7-10, for which we lack a complete understanding of how single-nucleotide alterations modulate transcriptional processes to affect human phenotypes5,11-15. To address this problem, we measured the activity of 221,412 fine-mapped trait-associated variants using a massively parallel reporter assay16-20 in 5 diverse cell types. We show that this assay effectively discriminates between likely causal variants and controls, and identified 13,121 regulatory variants with high precision. Although the effects of these variants largely agree with orthogonal measures of function, only 69% of them can plausibly be explained by the disruption of a known transcription factor binding motif. We investigated the mechanisms of 136 variants using saturation mutagenesis and assigned affected transcription factors for 91% of variants without a clear canonical mechanism. Finally, we detected regulatory epistasis at 11% of tested regulatory variants in close proximity and identified multiple functional variants on the same haplotype at a small, but important, subset of trait-associated loci. Overall, our study provides a systematic functional characterization of likely causal common variants that underlie complex and molecular human traits, enabling new insights into the regulatory grammar underlying disease risk.

DOI: https://doi.org/10.1038/s41586-026-10121-6
Cell. 2026 Feb 25. pii: S0092-8674(26)00098-X. [Epub ahead of print]

Nuclear speckles enable processing of RNA from GC-rich isochores.

Michał Małszycki, Lisa Martina, İbrahim Avşar Ilık, Daniela Salgado Figueroa, Nirmalya Dasgupta, Menşura Feray Çoşar, Keun-Tae Kim, Gil Carraco, Beatrix Fauler, David Meierhofer, Thorsten Mielke, Hiroo Imai, Cantaş Alev, Ferhat Ay, Tuğçe Aktaş.

  Nuclear speckles are conserved, membrane-less organelles linked to various post-transcriptional processes. Here, we examined their roles in human cells by engineered, acute removal of SON and SRRM2, two conserved speckle core components characterized by intrinsically disordered regions (IDRs). Their removal results in a significant downregulation of GC-rich genes with short introns clustered within GC-rich isochores, caused by inefficient and chaotic splicing; in contrast, the expression or splicing of genes outside these isochores remains unaffected. Comparative analysis across eukaryotes, from fungi to mammals, reveals that both GC-rich isochores and speckles are found exclusively in amniotes; moreover, the IDRs of SON have undergone notable expansion in the latter. Together, these findings suggest that the expansion of IDRs in vertebrates facilitated an increase in GC content by creating a condensate essential for splicing the by-products of this process: GC-rich, leveled exon-intron architectures.

Keywords:  RNA splicing; biological condensates; genome evolution; nuclear organization; nuclear speckles

DOI:  https://doi.org/10.1016/j.cell.2026.01.011
J Biol Chem. 2026 Feb 20. pii: S0021-9258(26)00173-0. [Epub ahead of print] 111303

Mechanisms of DNA methyltransferase 3A1 mediated DNA methylation of nucleosomes.

Ethan Ward, Drew McDonald, Tyler Holl, Norbert Reich.

  DNA methyltransferase 3A1 (DNMT3A1) plays a crucial role in establishing DNA methylation patterns that regulate gene expression and drive cellular differentiation. In this study, we investigated the biochemical mechanisms underlying DNMT3A1's interactions with nucleosomes, the fundamental units of chromatin. Using radiochemical activity assays, along with fluorescence anisotropy and AlphaLISA binding assays, we demonstrated that DNMT3A1 has multivalent interactions with nucleosomes, binding linker DNA and nucleosome cores. Nanopore-based 5mC sequencing revealed that DNMT3A1 interactions with the nucleosome stimulate methylation of linker DNA up to 24 bp away from the nucleosome core. Additionally, our results indicate that DNMT3A1 binding is restricted to a single nucleosome, suggesting that its activity is not allosterically regulated by unattached nucleosomes. Together, these findings provide new mechanistic insights into how DNMT3A1 engages nucleosomes.

Keywords:  DNA enzyme; DNA methylation; enzyme kinetics; enzyme mechanism; epigenetics; multivalent; nucleosome

DOI:  https://doi.org/10.1016/j.jbc.2026.111303
Development. 2026 Feb 25. pii: dev.204881. [Epub ahead of print]

A robust cis-regulatory network ensures Otx2 expression during retinal development.

Ian J Purvis, Omar E Ochoa Olmos, Ko Uoon Park, Michael L Kaufman, Charles M Henry, Chris Schaaf, Olivia J Clise, Corey D Tesdahl, Ami Haas, Joseph A Brzezinski.

  The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand how Otx2 expression is regulated, we explored the activity and function of three of its enhancers (DHS2, DHS4, & DHS15). Enhancer reporter assays and lineage tracing show that DHS4 initiates Otx2 expression while DHS2 and DHS15 maintain expression in photoreceptors. Matched CRISPR/Cas9 and CRISPR interference (CRISPRi) systems were used to mutate or epigenetically silence enhancers, respectively. CRISPR reduced OTX2 expression acutely, but failed to significantly alter cell fate choice over the long-term. In contrast, CRISPRi of these enhancers caused permanent OTX2 loss and corresponding cell fate changes. While these data suggest that each enhancer is needed for normal Otx2 expression, it also highlights that the enhancers can interact and substitute for each other during development. This cis-regulatory element flexibility likely promotes Otx2 expression robustness. Such robustness may enable complex genes, like Otx2, to resist environmental stressors and regulatory disruptions to promote reproducible developmental outcomes.

Keywords:   Otx2 ; Bipolar Cells; CRISPRi; Cell Fate Specification; Enhancers; Mouse; Photoreceptors; Promoters; Retinal Development

DOI:  https://doi.org/10.1242/dev.204881
Genome Biol Evol. 2026 Feb 25. pii: evag042. [Epub ahead of print]

Pervasive transcription in the human genome exceeds background noise.

Brett N Adey, Danielle J Maddock, Sylvie Hermann-Le Denmat, Marcel E Dinger, Paul P Gardner, Anthony M Poole, Austen R D Ganley.

  Large genomes such as the human genome are pervasively transcribed yet encode relatively few unambiguously functional elements. This has led to debate over whether pervasive transcription is indicative of large suites of uncharacterized functional elements or is simply background noise. Here we used a deep-learning model to estimate background transcription in the human genome as a way of distinguishing between these two hypotheses. We applied the model to randomised (reversed or shuffled) versions of the human genome and found that transcription is predicted to be sparse across all randomisation methods, initiating with at least four-fold lower frequencies than in the native human genome. This relatively low level of background transcription from the human genome suggests that most transcription is not a consequence of background noise, thus it requires other explanations. We find that randomizing only interspersed repeats in human genome has little impact on predicted transcription, suggesting that transcription of mobile elements does not explain the excess transcription in the human genome. Instead, most transcriptional events may derive from functional noncoding RNA transcripts, some general requirement for extensive transcription initiation/elongation, and/or mutational biases leading to the frequent appearance of transcription initiation sites by chance.

Keywords:  Background noise; Deep-learning; Junk DNA; Noncoding RNA; Pervasive transcription; Random DNA

DOI:  https://doi.org/10.1093/gbe/evag042
Sci Adv. 2026 Feb 27. 12(9): eady3909

Global maps of transcription factor properties reveal threshold-based formation of DNA-bound and mobile clusters.

Sadia Siddika Dima, Meghan A Turner, Hernan G Garcia, Gregory T Reeves.

The relationship between bulk transcription factor concentration and DNA binding has been a central question in gene regulation for decades. Recent studies propose that DNA-bound transcription factor hubs, or clusters, aid in fast and precise transcriptional interpretation. Using live imaging techniques, we quantify the concentration, binding, and mobility of the morphogen Dorsal (Dl), both in bulk and in clusters, in Drosophila blastoderm embryo. Our experiments encompass multiple length and timescales, allowing us to obtain a nucleus-wide view of the mechanism connecting hub formation to bulk Dl concentration. Our results show that previously unobserved, slowly moving clusters of Dl are present, in addition to the expected populations of freely mobile and DNA-bound Dl. Furthermore, both mobile clusters and DNA-bound Dl appear only once a threshold concentration in the nucleus is surpassed, a behavior consistent with liquid-liquid phase separation. Thus, our work elucidates how bulk transcription factor concentration dictates the formation and spatiotemporal changes of different populations needed for gene regulation.

DOI: https://doi.org/10.1126/sciadv.ady3909
Nucleic Acids Res. 2026 Feb 05. pii: gkag118. [Epub ahead of print]54(4):

Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response.

Helen E King, Savannah O'Connell, Daisy Kavanagh, Sofia Mason, Cerys McCool, Javier Fernandez-Chamorro, Christine L Chaffer, Susan J Clark, Helaine Graziele S Vieira, Timothy Sterne-Weiler, Robert J Weatheritt.

CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed Isoform-Specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 51.6% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.

DOI: https://doi.org/10.1093/nar/gkag118
Nucleic Acids Res. 2026 Feb 05. pii: gkag128. [Epub ahead of print]54(4):

Genome-wide dynamic nascent transcript profiles reveal that most paused RNA polymerases terminate.

Rudradeep Mukherjee, Michael J Guertin.

We present a simple model for analyzing and interpreting data from kinetic experiments that measure engaged RNA polymerase occupancy. The framework represents the densities of nascent transcripts within the pause region and the gene body as steady-state values determined by four key transcriptional processes: initiation, pause release, premature termination, and elongation. We validate the model's predictions using data from experiments that rapidly inhibit initiation and pause release. The model successfully classified factors based on the steps in early transcription that they regulate, confirming TBP and ZNF143 as initiation factors and heat shock factor and glucocorticoid receptor as pause release factors. We found that most paused polymerases terminate and paused polymerases are short-lived with half lives less than a minute. We make this model available as software to serve as a quantitative tool for determining the kinetic mechanisms of transcriptional regulation.

DOI: https://doi.org/10.1093/nar/gkag128
Nucleic Acids Res. 2026 Feb 24. pii: gkag124. [Epub ahead of print]54(5):

Directing stem cell differentiation by chromatin state approximation.

Luis F Montano-Gutierrez, Sophie Müller, Ana P Kutschat, Igor Adameyko, Davide Seruggia, Florian Halbritter.

A prime goal of regenerative medicine is to replace dysfunctional cells in the body. To design protocols for producing target cells in the laboratory, one may need to consider exponentially large combinations of culture components. Here, we investigated the potential of iteratively approximating the target phenotype by quantifying the distance between chromatin profiles (ATAC-seq) of differentiating cells in vitro and their in vivo counterparts. We tested this approach on the well-studied generation of erythroblasts from haematopoietic stem cells, evaluating a fixed number of components over two sequential differentiation rounds (8 × 8 protocols). We found that the most erythroblast-like cells upon the first round yielded the most erythroblast-like cells at the second round, suggesting that greedy selection by chromatin approximation can be a viable optimisation strategy. Furthermore, by analysing regulatory sequences in incompletely reprogrammed chromatin regions, we uncovered transcriptional regulators linked to roadblocks in differentiation and made a data-driven selection of ligands that further improved erythropoiesis. In future, our methodology can help craft notoriously difficult cells in vitro, such as B cells.

DOI: https://doi.org/10.1093/nar/gkag124
Nucleic Acids Res. 2026 Feb 24. pii: gkag146. [Epub ahead of print]54(5):

A de novo H3.2K9me2 deposition pathway establishes heterochromatin for suppressing transposon mobilization during fly somatic development.

Yi Ni Luo, Yazi Deng, Yu Liang, Wei Wu, Wei Wu, Lu Wang.

Histone variants along with their associated chaperones have been considered as one of the major complexes to provide versatility in organizing chromatin structure. Post-translational modifications (PTMs) of H3 variants serve as very important factors in promoting heterochromatin assembly, protecting telomere stability, and suppressing transposon activity. However, the precise mechanism by which specific PTMs on H3 variants suppress transposons remains elusive. Here, by monitoring retrotransposon mobilization during Drosophila hindgut development, we identified the DNA synthesis-coupled (DSC) H3.2K9me2 deposition pathway as a pivotal mechanism for transposon suppression. Depleting the factors in the DSC H3.2 complex, but not in the DNA synthesis-independent (DSI) H3.3 chaperone pathway, unleashed massive retrotransposon activation. DSC chaperones specifically establish dimethylation at the H3.2K9 site in heterochromatic regions by directly interacting with and recruiting the histone methyltransferase, G9a. Intriguingly, the cross-talk between DSC H3.2K9me2 and DSI H3.3K9me3 in heterochromatin is dynamically regulated and properly balanced. Although DSI H3.3K9me3 could efficiently be incorporated into transposon loci when the DSC H3.2K9me2 deposition pathway was disrupted, H3.3K9me3 alone was insufficient to establish functional heterochromatin required for transposon silencing during development. Altogether, our discoveries provide a framework to understand how cells employ specific histone variant modifications to construct and maintain heterochromatin, thereby ensuring transposon repression and safeguarding genome integrity.

DOI: https://doi.org/10.1093/nar/gkag146
Cell Genom. 2026 Feb 20. pii: S2666-979X(26)00028-5. [Epub ahead of print] 101166

Hi-C for genome-wide detection of enhancer-hijacking rearrangements in routine lymphoid cancer biopsies.

Jamin Wu, Shih-Chun A Chu, Jang Cho, Misha Movahed-Ezazi, Kristyn Galbraith, Camila S Fang, Yiying Yang, Chanel Schroff, Kristin Sikkink, Michelle Perez-Arreola, Logan Van Meter, Savanna Gemus, Jon-Matthew Belton, Xue Song, Aishwarya Gurumurthy, Hong Xiao, Valentina Nardi, Abner Louissant, Raju K Pillai, Joo Y Song, Dennis Shasha, Aristotelis Tsirigos, Anamarija Perry, Noah Brown, Tatyana Gindin, Lina Shao, Marcin P Cieslik, Minji Kim, Anthony D Schmitt, Matija Snuderl, Russell J H Ryan.

  Standard techniques for detecting genomic rearrangements in formalin-fixed paraffin-embedded (FFPE) biopsies have important limitations. We performed FFPE-compatible Hi-C on 44 clinical biopsies comprising large B cell lymphomas (n = 18), plasma cell neoplasms (n = 14), and other diverse lymphoid cancers, identifying consistent topological differences between malignant B cell and plasma cell states. Hi-C detected expected oncogene rearrangements at high concordance with fluorescence in situ hybridization (FISH) and supported enhancer hijacking in recurrent rearrangements of BCL2, CCND1, and MYC plus unanticipated variants involving homologous loci. Hi-C identified unanticipated non-coding rearrangements involving PD-1 ligand genes and other loci of potential therapeutic relevance, distinguished between functionally divergent classes of BCL6 rearrangements, and provided topological information supporting interpretation of variant MYC rearrangements. Hi-C revealed disease-selective MYC locus topological features that correlated with disease-selective MYC locus enhancers and rearrangement breakpoint distributions. FFPE-compatible Hi-C detects oncogene rearrangements and their topological consequences at genome-wide scale, finding clinically relevant drivers missed by standard approaches.

Keywords:  Hi-C; enhancer hijacking; epigenomics; genomic rearrangement; leukemia; lymphoma; molecular diagnostics; myeloma; precision medicine; translational research

DOI:  https://doi.org/10.1016/j.xgen.2026.101166