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PubMed2025

Setdb2 Regulates Inflammatory Trigger-Induced Trained Immunity of Macrophages Through Two Different Epigenetic Mechanisms.

"Trained immunity" of innate immune cells occurs through a sequential two-step process where an initial pathogenic or sterile inflammatory trigger is followed by an amplified response to a later un-related secondary pathogen challenge. The memory effect is mediated at least in part through epigenetic modifications of the chromatin landscape. Here, we investigated the role of the epigenetic modifier Setdb2 in microbial (β-glucan) or sterile trigger (Western-diet-WD/oxidized-LDL-oxLDL)-induced trained immunity of macrophages. Using genetic mouse models and genomic analysis, we uncovered a critical role of Setdb2 in regulating proinflammatory and metabolic pathway reprogramming. We further show that Setdb2 regulates trained immunity through two different complementary mechanisms: one where it positively regulates glycolytic and inflammatory pathway genes via enhancer-promoter looping, and is independent of its enzymatic activity; while the second mechanism is associated with both increased promoter associated H3K9 methylation and repression of interferon response pathway genes. Interestingly, while both mechanisms occur in response to pathogenic training, only the chromatin-looping mechanism operates in response to the sterile inflammatory stimulus. These results reveal a previously unknown bifurcation in the downstream pathways that distinguishes between pathogenic and sterile inflammatory signaling responses associated with the innate immune memory response and may provide potential therapeutic opportunities to target cytokine vs. interferon pathways to limit complications of chronic inflammation.

Setdb2↗

PubMed2026

Twisting the End Game: How Telomere Chromatin Modifications Shape Telomere Maintenance.

Cell division inevitably shortens telomeric DNA owing to the end-replication problem. Eukaryotic chromosomes possess specialized telomere structures to maintain genomic stability. In most proliferative cells, telomerase adds telomeric repeats during S-phase. In differentiated cells where telomerase is silenced, telomeres shorten progressively, thereby compromising genomic integrity. Consequently, cancer cells universally activate alternative telomere maintenance mechanisms during malignant transformation: ~80% reactivate telomerase, while a portion of the rest rely on BIR (break-induced replication)-mediated homologous recombination-based ALT (alternative lengthening of telomeres). Although these mechanisms are stable once established, the initial determinants influencing a cancer cell's choice remain poorly understood. This review discusses recent molecular insights into how telomeric chromatin properties profoundly impact this choice. After briefly introducing telomere chromatin characteristics and key players in its maintenance and dynamics, we discuss the mechanisms by which cancer cells acquire distinct telomere replication capabilities. In particular, we present an in-depth analysis linking telomere heterochromatin status to ALT. Furthermore, based on recent advances, we propose a coupled feedforward loop model explaining how the ALT state becomes "locked in" once initiated. Finally, we offer novel perspectives on rational, telomere-centric therapeutic interventions for ALT-positive cancers, focusing on strategies designed to disrupt such feedforward loops by manipulating telomeric chromatin structure.

Humans↗

PubMed2025

The CGG triplet repeat binding protein 1 counteracts R-loop induced transcription-replication stress.

The CGG triplet repeat binding protein 1 (CGGBP1) binds to CGG repeats and has several important cellular functions, but how this DNA sequence-specific binding factor affects transcription and replication processes is an open question. Here, we show that CGGBP1 binds human gene promoters containing short (< 5) CGG-repeat tracts prone to R-loop formation. Loss of CGGBP1 leads to deregulated transcription, transcription-replication-conflicts (TRCs) and accumulation of Serine-5 phosphorylated RNA polymerase II (RNAPII), indicative of promoter-proximal stalling and a defect in transcription elongation. Consistently, an episomal CGG-repeat-containing model locus as well as endogenous genes show deregulated transcription, R-loop accumulation and increased RNAPII chromatin occupancy in CGGBP1-depleted cells. We identify the DEAD-box RNA:DNA helicases DDX41 and DHX15 as interaction partners specifically recruited by CGGBP1. Co-depletion experiments show that DDX41 and CGGBP1 work in the same pathway to unwind R-loops and avoid TRCs. Together, our work shows that short trinucleotide repeats are a source of genome-destabilizing secondary structures, and cells rely on specific DNA-binding factors to maintain proper transcription and replication coordination at short CGG repeats.

Humans↗

PubMed2025

3D chromatin structures precede genome activation in Drosophila embryogenesis.

3D chromatin structure is critical for the regulation of gene expression during development. Here we used Micro-C assays at 100-bp resolution to map genome organization in Drosophila melanogaster throughout the first half of embryogenesis. These high-resolution contact maps reveal fine-scale features such as loops and boundaries delineating topologically associating domains. Notably, we observe that 3D chromatin structures form prior to zygotic genome activation and persist during successive mitotic cycles. Integrative analysis with 149 public chromatin immunoprecipitation sequencing (ChIP-seq) datasets identifies four classes of chromatin structuring elements, including a distinct group enriched for GAGA-associated factor (GAF) and Zelda binding, associated with developmental-gene regulation. These elements are mitotically retained and exhibit sequence and structure similarity between D. melanogaster and D. virilis. We propose that 3D chromatin organization in the pre-cellular embryo facilitates deployment of developmentally regulated genes during Drosophila embryogenesis.

Animals↗

PubMed1977

Chromatin ultrastructure of lower vertebrates.

Meiotic and mitotic chromosomes from amphibians and snakes were studied by electron microscopy. By using water spreading, preceded by a mild NaCl pretreatment, we showed: 1. 'Beads on a string' arrangement of the chromatin fibres; 2. The presence of loops at pachytene chromomeres as well as during metaphase of both mitosis and first meiosis; 3. Transcriptional activity for non-ribosomal RNA on peripheral loops during the middle pachytene.

Animals↗

PubMed1979

Injected histone antibodies interfere with transcription of lampbrush chromosome loops in oocytes of Pleurodeles.

Antibodies to calf thymus histone H2B were purified by chromatography on DEAE-cellulose and injected into oocyte nuclei of Pleurodeles waltlii. As shown by indirect immunofluorescence these antibodies cross-reacted strongly with corresponding histones associated with lampbrush chromosomes. Shortly after injection the lateral loops of the chromosomes retracted into the chromomeres and by 3 h postinjection the 'lampbrush' appearance was completely lost and the chromosomes appeared in light-microscopic preparations as rod-like structures consisting of longitudinally coalesced chromomeres. In control oocytes injected with non-immune immunoglobulins or antibodies against a ubiquitous transcript-associated protein no morphological alterations of the lampbrush chromosomes could be observed. Electron microscopic spreads of chromosomes prepared at various times after injection of anti-H2B revealed a progressive loss of transcriptional complexes from the loop axes. Finally, higher-order chromatin configurations, like supranucleosomal globules ('superbeads') or cable-like chromatin strands 50-60 nm thick predominated, indicating complete transcriptional inactivation of all chromosomal regions. The results indicate that H2B antibodies react specifically with histones associated with the transcribed DNA of lateral loops in their native state. The resulting antigen-antibody complexes seem to inhibit progression of the RNA polymerases along the template, thus causing the premature release of transcripts, a process analogous to the stripping effect of actinomycin D. The demonstration of histones associated with heavily transcribed regions, which are not compacted into nucleosomes but largely extended, supports the current concept that unfolding of nucleosomes to allow transcription of the DNA does not involve dissociation of histones. In contrast, amplified ribosomal RNA genes are unaffected by injected H2B antibodies. This does not necessarily indicate absence of histones from nucleolar chromatin, since we do not know whether it is accessible in vivo to antibodies or whether the histone antigenic determinants are masked by the presence of other proteins. The technique of injecting specific antibodies should be widely applicable when analysing the in vivo distribution of chromosomal components at the electron-microscopic level and when studying complex metabolic processes, like the cleavage and modification of RNA, by selective inhibition of defined enzymic steps.

Animals↗

PubMed2026

Bridging-driven condensation by eukaryotic SMC complexes is a conserved feature of genome organization.

The Structural Maintenance of Chromosome (SMC) protein family plays a central role in higher-order genome organization through ATP-dependent DNA loop extrusion by cohesin and condensin and other processes. Whether these activities fully account for the complexity of chromosome architecture remains unknown. Here, we uncover a conserved ATP-independent mechanism of chromatin condensation by SMC complexes, occurring via biomolecular condensation. Using single-molecule fluorescence imaging, we show that a variety of SMCs form dynamic DNA-bound condensates that exhibit key features of biomolecular condensates, including droplet coalescence, fluorescence recovery after photobleaching, and rapid exchange with free SMC complexes. Atomic force microscopy analysis of human cohesin-DNA assemblies reveals DNA-length-dependent clustering, providing evidence for bridging-driven condensation. Analyses of in vivo super-resolution imaging and high-throughput chromosome conformation capture (Hi-C) data indicate that these condensates form chromatin-associated clusters with multi-loop structures. Together, our results establish that SMC complexes employ ATP-independent phase condensation as well as ATP-dependent activities to shape genome architecture. This work reveals a broadly conserved principle of chromosomal organization across eukaryotes.

Chromosomal Proteins, Non-Histone↗

PubMed2026

Reduced R-loop abundance at proinflammatory loci: a shared epigenetic mechanism in inflammatory and metabolic diseases.

INTRODUCTION: R-loops, RNA-DNA hybrid structures with a displaced single-stranded DNA loop, are key regulators of transcriptional control, chromatin architecture, and genome stability and have emerging roles in inflammatory signaling. However, the relationship between R-loop abundance and strongly modulated inflammatory effector genes in metabolic inflammation and influenza virus infection remains underexplored. METHODS: We performed a locus-centric integrative analysis combining robust differentially expressed genes (DEGs) from multiple inflammatory and infection-related murine and human transcriptomic disease models with experimentally validated multi-cell R-loop annotations from the reference atlas RLoopBase. Our correlation framework evaluated the directional relationship between R-loop abundance and inflammatory gene expression rather than assuming disease-sample-matched R-loop measurements. We further analyzed R-loop regulatory proteins, NRF2-associated R-loop regulators, and overlaps between R-loop regulators and CRISPRi-identified mitochondrial and cellular reactive oxygen species (ROS) regulators. RESULTS: In angiotensin II-infused apolipoprotein E-deficient (ApoE-/-) mice, a model of abdominal aortic aneurysm (AAA), genomic regions encoding the top significantly upregulated genes exhibited significantly fewer R-loops than those encoding downregulated genes at days 14 and 28. Similarly, in atherosclerotic ApoE-/- mice fed a high-fat diet for 32 and 78 weeks, upregulated genes were associated with fewer R-loops than downregulated genes. Reduced R-loop abundance was also observed in genomic regions encoding the top significantly upregulated genes in liver tissues from patients with non-alcoholic steatohepatitis (NASH), as well as in monosodium urate (MSU)-stimulated lymphatic endothelial cells (LECs) and influenza virus-infected human umbilical vein endothelial cells (HUVECs). R-loop regulatory proteins upregulated during metabolic inflammation were enriched in immune and inflammatory pathways. NRF2 was identified as a regulator of 27 R-loop regulatory proteins, including 10 positively and 17 negatively regulated proteins. Furthermore, 54 R-loop regulatory proteins overlapped with CRISPRi-identified mitochondrial and cellular ROS regulators, suggesting potential reciprocal regulation between R-loop homeostasis and ROS signaling. Disease-associated changes in pro-ROS and anti-ROS R-loop regulatory proteins further linked R-loop regulation to inflammatory and oxidative stress pathways. DISCUSSION: These findings identify reduced R-loop abundance at genomic regions encoding strongly upregulated inflammatory genes as a shared feature across multiple models of metabolic inflammation and influenza virus infection. The results further suggest that immune-associated R-loop regulatory proteins and the NRF2-ROS axis may contribute to R-loop remodeling during inflammatory disease. This integrative framework provides new insight into the potential role of R-loops and ROS-sensitive R-loop regulators in inflammatory and metabolic diseases and identifies candidate pathways for future mechanistic investigation and therapeutic targeting.

R-loop regulatory proteins↗

PubMed1976

Formation of packaging units in circket late spermatid nuclei as visualized by spreading techniques (1).

The nuclear organization of a particular step of cricket late spermiogenesis was examined by an electron microscope study of spermatids dispersed by air-liquid surface tension. Cell spreading techniques facilitate a display of condensing spermatid nuclei sufficient to allow interpretation of chromatin packaging processes. Results indicate that nuclei of late developing cricket spermatids are integrated by multiple packaging units as the result of an orderly aggregation of individual chromatin fibers. Each packaging unit consists of a thick fasicle, formed by the alignment of smooth chromatin fibers, which frays out into tassels of looped fibers.

Animals↗

PubMed2025

Rad53 regulates RNase H1, which promotes DNA replication through sites of transcription-replication conflict.

RNA-DNA hybrids and R-loops can lead to extensive DNA damage and loss of genomic integrity if not regulated in a timely manner. Although RNase H1 overexpression is frequently used as a tool to resolve R-loops, the regulation of RNase H1, overexpressed or endogenous, remains poorly characterized. We reveal that in yeast, overexpressed RNase H1 (RNH1) has no effect on gene expression, cell growth, or RNA-DNA hybrid resolution in wild-type cells. Overexpressed RNase H1 does, however, remove RNA-DNA hybrids in mutants where hybrids have become dysregulated. Endogenous RNase H1 becomes up-regulated and chromatin-associated in the absence of Sen1 in a DNA replication checkpoint-dependent manner. Rnh1 gets recruited to genomic loci where RNA-DNA hybrids accumulate following the loss of Sen1. Rnh1, together with Sen1, promotes DNA replication at sites of transcription-replication conflict. Hence, RNase H1, overexpressed or endogenous, responds to unscheduled, stress-inducing RNA-DNA hybrids.

Ribonuclease H↗

PubMed1978

The organization of transcription on lampbrush chromosomes.

The meiotic lampbrush chromosomes of amphibian oocytes display readily distinguishable regions of transcription (lateral loops) which extend from axial condensates of chromatin (chromomeres). The chromomeres contain most of the chromosomal ENA which, along with histone, is tightly compacted as regular arrays of DNP. Many RNA transcripts are generated on the lateral loops, and heterogeneous non-histone proteins associate with these transcripts, forming periodic condensates of 20--30 nm ribonucleoprotein (RNP) particles. These unit particles aggregate in various ways and to varying degrees and thereby confer distinctive gross morphologies to particular loops. There are about 10(4) lateral loops per haploid complement of newt chromosomes and this figure is similar to the experimentally derived number of different messenger RNA sequences found in oocytes. From cytological and biochemical studies it is now possible to consider individual lateral loops from various aspects: as morphologically distinct units; as units of inheritance; as units of functional activity; as units of transcription; as units of transcribed repetitive sequences; and as units containing one coding sequence. The difficulties in arriving at a simple explanation of the organization of transcription in lampbrush chromosomes are discussed.

Animals↗

PubMed2025

Phasing single-molecule nano-NOMe-seq reveals chromatin state heterogeneity in the context of transcription and long-range interactions.

A central challenge in molecular biology is determining how 3D chromatin architecture, particularly enhancer-promoter looping and insulating CTCF-mediated interactions, influences gene transcription in individual cells, which has significant implications for healthy and diseased states. To overcome current limitations in imaging and genomic technologies, we developed a cluster-based phasing strategy using long read nano-NOMe-seq to link distinct CTCF binding states-captured at the single molecule level-to the transcriptional status of genes. By stitching partially overlapping long reads and clustering them by shared GpC-accessibility patterns, we stratify CTCF into graded binding states on individual molecules, classify RNA polymerase states at promoters/gene bodies, and infer when spatially separated loci are coordinately activated and occupy loop-competent configurations on the same molecules. When applied to Sox2, Hoxa, and Klf1 regions, cluster-based nano-NOMe-seq phasing reveals how specific topologies bias polymerase behavior and multi-locus activity in ways that bulk assays or locus-engineered imaging cannot fully capture.

Journal Article↗

PubMed2025

Pre-established ATF4 occupancy and chromatin organization instruct selective transcription activation during integrated stress response.

Cells rapidly and extensively remodel their transcriptome in response to stress to restore homeostasis, but the underlying mechanisms are not fully understood. Here, we characterize the dynamic changes in transcriptome, epigenetics, and 3D genome organization during the integrated stress response (ISR). ISR induction triggers widespread transcriptional changes within 6 h, coinciding with increased binding of ATF4, a key transcriptional effector. Notably, ATF4 binds to hundreds of genes even under non-stress conditions, priming them for stronger activation upon stress. The transcriptional changes at ATF4-bound sites during ISR do not rely on increased H3K27 acetylation, chromatin accessibility, or rewired enhancer-promoter looping. Instead, ATF4-mediated gene activation is linked to the redistribution of CEBPγ from non-ATF4 sites to a subset of ATF4-bound regions, likely by forming an ATF4/CEBPγ heterodimer. CEBPγ preferentially targets the sites pre-occupied by ATF4, as well as genomic regions exhibiting a unique higher-order chromatin structure signature. Thus, the transcriptional responses during ISR are largely pre-wired by intrinsic chromatin properties. These findings provide critical insights into transcriptional remodeling during ISR with broader implications for other stress responses.

Activating Transcription Factor 4↗

PubMed2025

Crosstalk between chromatin state and ATM signalling in DNA damage-induced transcription stress.

The DNA Damage Response (DDR) is a highly regulated process that safeguards genomic integrity against DNA lesions. Increasing evidence supports a reciprocal relationship between damaged chromatin architecture and the signalling pathways that coordinate the DDR. However, the mechanisms underlying this interplay in response to transcription-blocking DNA lesions remain largely unexplored. Here, we show that stalling of RNA polymerase II (RNAPII) at such lesions induces local chromatin acetylation, mediated primarily by the histone acetyltransferase p300. The resulting chromatin relaxation stimulates the dissociation of mature co-transcriptional spliceosomes from nascent RNA and promotes RNA:DNA hybrid (R-loop) formation, leading to ATM activation. In turn, activated ATM modulates chromatin conformation by phosphorylating histone H2A.X and triggering p38MAPK/MSK1-dependent histone H3S10 phosphorylation. Our findings highlight the cross-regulation between chromatin state and ATM signalling as a key component of the cellular response to transcription stress.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2026

Assessing reproducibility of Hi-C chromatin interactions using stratum-adjusted irreproducible discovery rate.

MOTIVATION: Hi-C is a powerful technology for mapping chromatin interactions genome-wide. However, interaction loops identified from Hi-C contact maps often vary across replicate experiments due to experimental noise, making reproducibility assessment essential. A major challenge lies in the genomic distance dependence of interaction strength, which systematically affects reproducibility but is overlooked by existing methods for reproducibility assessment. RESULTS: We introduce Stratum-Adjusted Irreproducible Discovery Rate (SIDR), a novel statistical model that integrates distance stratification into the widely-used Irreproducible Discovery Rate (IDR) framework. SIDR explicitly models the confounding effect of genomic distance, enabling global control of irreproducibility across interaction ranges. Through simulations and real Hi-C datasets, we demonstrate that SIDR improves discriminative power and recovers more biologically meaningful interactions than existing approaches, making it a valuable tool for robust and reproducible Hi-C analysis. AVAILABILITY: The R package SIDR is freely available on GitHub https://github.com/qunhualilab/SIDR.

Chromatin↗

PubMed2025

Interpreting the CTCF-mediated sequence grammar of genome folding with AkitaV2.

Interphase mammalian genomes are folded in 3D with complex locus-specific patterns that impact gene regulation. CTCF (CCCTC-binding factor) is a key architectural protein that binds specific DNA sites, halts cohesin-mediated loop extrusion, and enables long-range chromatin interactions. There are hundreds of thousands of annotated CTCF-binding sites in mammalian genomes; disruptions of some result in distinct phenotypes, while others have no visible effect. Despite their importance, the determinants of which CTCF sites are necessary for genome folding and gene regulation remain unclear. Here, we update and utilize Akita, a convolutional neural network model, to extract the sequence preferences and grammar of CTCF contributing to genome folding. Our analyses of individual CTCF sites reveal four predictions: (i) only a small fraction of genomic sites are impactful; (ii) impact is highly dependent on sequences flanking the core CTCF binding motif; (iii) core and flanking nucleotides contribute largely additively to the overall impact of a site; (iv) sites created as combinations of different core and flanking sequences have impacts proportional to the product of their average impacts, i.e. they are broadly compatible. Our analysis of collections of CTCF sites make two predictions for multi-motif grammar: (i) insulation strength depends on the number of CTCF sites within a cluster, and (ii) pattern formation is governed by the orientation and spacing of these sites, rather than any inherent specialization of the CTCF motifs themselves. In sum, we present a framework for using neural network models to probe the sequences instructing genome folding and provide a number of predictions to guide future experimental inquiries.

CCCTC-Binding Factor↗

PubMed2026

R-loops and D-loops: a delicate balance in genomic stability and instability.

R-loops and D-loops are three-stranded nucleic acid structures that have emerged as central regulators of genome stability, gene expression, and DNA metabolism. R-loops form co-transcriptionally or post-transcriptionally when nascent RNA re-anneals with the template DNA strand, generating an RNA: DNA hybrid that displaces the non-template strand into a single-stranded state. These structures are enriched at CpG island promoters, transcription termination sites, and immunoglobulin class-switch regions, where they coordinate transcription regulation, chromatin remodeling, and DNA damage signaling. D-loops are formed when a single-stranded DNA segment pairs with one strand of a duplex and displaces the other, arising through context-dependent mechanisms that include RAD51- or DMC1-mediated strand invasion in homologous recombination, shelterin-assisted invasion at telomeres, and replication-coupled strand displacement at the mitochondrial DNA origin. They serve as indispensable intermediates in double-strand break repair, telomere maintenance, and mitochondrial DNA replication. Recent cryo-electron microscopy studies have resolved the stepwise RAD51-mediated strand exchange mechanism at near-atomic resolution, substantially advancing structural understanding of D-loop biogenesis. Despite their differences in molecular composition, both structures remodel Watson-Crick base pairing and, when dysregulated, are associated with replication fork stalling, transcription-replication conflicts, and aberrant recombination. This review systematically compares the structural features, formation mechanisms, regulatory networks, and biological functions of R-loops and D-loops, with emphasis on their convergent roles in safeguarding genome integrity. We further discuss rapidly evolving detection technologies and emerging therapeutic strategies targeting these structures in cancer and neurodegeneration, identifying key unresolved questions for future investigation.

Genomic Instability↗

PubMed2026

Genome assembly of Astatotilapia latifasciata uncovers B chromosome-linked chromatin reorganization.

B chromosomes (Bs) are supernumerary genomic elements found in many eukaryotes, yet their full sequence composition, functional potential, and regulatory impact on the host genome remain unclear. Here, we present a chromosome-level genome assembly of the cichlid fish Astatotilapia latifasciata, integrating PacBio long reads, Illumina short reads, and Hi-C chromatin contact maps to resolve both A and B chromosomes. The 0.93 Gb assembly (N50 = 36.2 Mb) includes a 34 Mb B chromosome containing 789 predicted protein-coding genes and a markedly higher density of transposable elements (TEs), especially long terminal repeats (LTR) retrotransposons. Transcriptome profiling revealed that B-linked genes are predominantly transcriptionally repressed relative to their A chromosome paralogs. Hi-C-based chromatin modeling uncovered distinct 3D structural configurations associated with the B chromosome, including fewer topologically associating domains (TADs), reduced loop formation, and altered compartmentalization. These changes are linked to long-range chromatin interactions and genomic rearrangements, suggesting that the B chromosome reshapes the nuclear architecture of the host genome. Our study proposes a potential regulatory role of Bs in genome and provides a genomic resource for investigating chromosome evolution in cichlids.

Animals↗