Epigenetic profiling is an emerging clinical tool for tumor profiling and liquid biopsies. Here, we developed Fragment Ligation EXclusive methylation sequencing (FLEXseq), a genome-wide methylation profiler that enriches and sequences the DNA fragments flanking CCGG motifs to cover a broad range of regulatory regions. FLEXseq strongly correlates with whole genome bisulfite sequencing (WGBS; Pearson's r = 0.97) yet requires only three- to seven-fold less sequencing depth than whole genome approaches to cover cell type-specific markers and 4- to 20-fold less sequencing to cover key regulatory regions like promoters. DNA dilutions down to 250 pg decreased CpG coverage, but bias in methylation remained low (Pearson's r ≥ 0.90). To demonstrate the broad applicability of FLEXseq, including highly fragmented DNA found in clinical specimens, we verified its usage across cells, body fluids, and formalin-fixed paraffin-embedded (FFPE) tissues. Across 106 cerebrospinal fluids, a specimen type challenged by low-input and fragmented DNA, FLEXseq enabled cell type deconvolution to distinguish between different tumor types and negative controls with an accuracy of 97%. FLEXseq offers a cost-efficient, single-nucleotide resolution approach to profile the methylome even with fragmented, low-input DNA.

While it is well established that the Zα domains of ADAR1 and ZBP1 proteins bind Z-form-prone nucleic acids (Z-NAs), it has also been shown that the Zα domain of ADAR1 binds DNA G-quadruplexes (GQs). However, no binding partner of the structurally homologous Zβ domain of ADAR1 has been identified to date. Based on AlphaFold and molecular dynamics simulations, it has recently been suggested that Zβ of ADAR1 targets its substrate by recognizing GQs. Here, we provide the first experimental evidence for Zβ binding to select GQ RNA and DNA in vitro, with structural specificity and nanomolar affinity. We also demonstrate that the Zα domains of ZBP1 and ADAR1 bind to both DNA and RNA GQs with similar affinity. These findings extend the range of potential functional roles for these proteins and open new hypotheses for testing in cells.

ChIP-Atlas (https://chip-atlas.org/) is a data-mining platform that systematically processes and integrates public epigenomic profiling data, including ChIP-seq, ATAC-seq, DNase-seq, and Bisulfite-seq, together with curated sample metadata, currently encompassing >460 000 experiments across multiple model organisms. In this 2025 update, an experiment-level quality control framework was embedded in every experiment page, enabling intuitive assessment of data reliability and representativeness. In addition, a parametric analysis of gene set enrichment-based module was implemented, allowing enrichment analysis directly from continuous RNA-seq count tables without relying on threshold-defined, discrete gene lists, thereby helping elucidate underlying gene regulatory mechanisms. Combined with sustained data expansion, these updates advance ChIP-Atlas into a more quantitative and flexible platform for exploring the epigenomic landscape in multicellular organisms, marking its 10th anniversary.

Epigenetic regulation of chromatin structure is strongly influenced by histone variants and post-translational modifications. The conserved histone variant H2A.Z has been functionally linked to the pioneer factors Sox2 and Oct4, which open chromatin and activate cell fate-specific transcriptional programs. However, the molecular basis of this interaction is not well understood. Here, we combine biochemistry, NMR spectroscopy, and molecular dynamics (MD) simulations to investigate how H2A.Z nucleosome dynamics influence pioneer factor binding. We find that H2A.Z enhances Sox2 and Oct4 association at distinct positions within 601 nucleosomes, correlating with increased DNA accessibility and altered H3 N-terminal tail dynamics. We show that the H3 tail competes with Sox2 for DNA binding and is more efficiently displaced with H2A.Z, while also allowing for unique Sox2-H3 tail interactions. MD simulations reveal that H2A.Z promotes DNA unwrapping, increases inter-gyre spacing, and enhances H3 tail flexibility, while simultaneously reducing contacts with DNA and with the H2A.Z C-terminal tail. This destabilizing effect is DNA-sequence dependent and prominent in the less stable Lin28B nucleosome, which Sox2 appears to substantially reshape. Together, our results suggest that H2A.Z promotes pioneer factor binding by increasing DNA accessibility and reducing histone tail competition, with broad implications for epigenetic regulation and chromatin recognition.

Mitochondrial DNA replication occurs at contact sites between the endoplasmic reticulum (ER) and mitochondria (ERMCS). Beyond the known role of the tubular ER protein RTN4, the factors regulating this process are poorly defined. Here, we show that repressing the ER protein ERLIN2 in human fibroblasts depletes ER-mitochondrial contact sites and inhibits mitochondrial DNA replication, as does silencing RTN4 or the ER-mitochondrial tether GRP75. GRP75 or RTN4 scarcity also decreases the level of the mitochondrial calcium uniporter (MCU), whose inhibition blocks mitochondrial DNA synthesis. Because ERMCS depletion did not diminish mitochondrial calcium, and MCU complex can transport manganese, we tested whether manganese could bypass these defects. Manganese supplementation restored mitochondrial DNA replication in cells lacking ERMCS or with inhibited MCU, identifying manganese as a critical mediator. We then considered mitochondrial transcription as a potential manganese target, since it provides both transcripts for gene expression and primers for DNA replication.In vitro, manganese inhibits transcription re-start and stimulates RNA synthesis at the light-strand origin of replication. These findings support a model in which ER-mitochondrial contact sites, in conjunction with MCU, deliver manganese from the ER to mitochondria to promote DNA replication, potentially by modulating mitochondrial RNA polymerase activity.

RNA secondary structure plays a critical role in gene regulation, yet existing computational and experimental tools for structure analysis are often fragmented across prediction, ensemble modeling, and functional interpretation workflows. Here, we present ShapeRNA, a user-friendly web server for integrated RNA secondary structure prediction, ensemble inference, and structure-aware regulatory annotation. ShapeRNA supports three complementary analytical workflows, including sequence-based structure prediction, reactivity-guided modeling using SHAPE or DMS data, and sequencing-guided ensemble inference from high-throughput probing experiments. The platform integrates multiple established prediction algorithms and provides standardized data processing, ensemble clustering, and visualization. In addition, ShapeRNA enables mapping of RNA modification sites, microRNA target regions, and RNA-binding protein interaction motifs onto predicted RNA structures and representative ensemble conformations. We demonstrate the utility of ShapeRNA through applications including analysis of mutation-associated structural changes in MAPT exon 10, characterization of conformational heterogeneity in the HIV-1 Rev Response Element, and regulatory annotation of the oncogenic long non-coding RNA HULC. ShapeRNA provides an accessible and extensible platform for investigating RNA structural heterogeneity and regulatory mechanisms. This website is free and open to all users, and there is no login requirement. The server is accessible at https://shaperna.com.

DNA ends generated by double-strand breaks are vulnerable intermediates that must be rapidly recognized, protected, and resolved to preserve genome integrity. We present optical tweezers (OT)-Curtains, a single-molecule method inspired by DNA curtains that uses a custom branched DNA substrate containing multiple accessible ends for simultaneous observation on dual-trap OT coupled to confocal fluorescence microscopy. Eliminating DNA surface anchoring, facilitating rapid protein and buffer exchange, and offering the possibility for force-free experiments, OT-Curtains overcomes common limitations of flow-stretch-based methods. OT-Curtains allows real-time visualization and quantification of end recognition, protection, resection, and cleavage at several DNA ends in parallel. We demonstrate compatibility with well-studied DNA-binding systems by monitoring Ku-mediated DNA break recognition, AddAB-mediated DNA break resection, ParB-mediated DNA condensation, and KpnI-mediated DNA cleavage. We show that kinetic and mechanistic parameters can be extracted from the data under defined forces and solution conditions. OT-Curtains offers an accessible and multiplexed route to interrogate DNA-end transactions central to double-stranded DNA break repair pathways and telomere biology, as well as a general framework for benchmarking proteins acting at DNA ends.

Small RNAs regulate numerous biological processes, including intercellular communication and inter-kingdom relationships, yet their tissue-specific functions remain poorly understood. Dissecting these roles requires tools that selectively eliminate small RNAs in defined cell types. Virus-encoded proteins such as P19 have been widely used to neutralize small RNAs owing to their sequestration capacities. However, their restricted binding spectrum to 21-22-nt small RNAs and lack of discrimination between miRNAs and siRNAs limit their utility. Here we show that constitutive expression of RNASE THREE-LIKE1 (RTL1) in Arabidopsis thaliana, previously shown to degrade siRNA precursors but not miRNA precursors, also degrades siRNA duplexes, irrespective of their size, sequence, or sub-cellular localization. Grafting experiments combined with small RNA sequencing revealed that shoot-derived siRNAs accumulate in dcl2 dcl3 dcl4 mutant roots, but are eliminated upon ectopic RTL1 expression, demonstrating that RTL1 efficiently degrades mobile siRNAs in vivo. Because the Arabidopsis endogenous RTL1 is epigenetically silenced in wild-type plants, its ectopic expression under cell-type-specific promoters enables targeted depletion of siRNAs in selected tissues. As a proof of concept, we show that expression of RTL1 in phloem companion cells delays systemic post-transcriptional transgene silencing (PTGS), indicating that mobile siRNAs transit through these cells. Together, these results establish RTL1 as a powerful tool to dissect siRNA mobility pathways and tissue-specific functions.

While nanopore direct RNA sequencing has substantially advanced transcriptomics, its detection of RNA modifications remains primarily focused on abundant biological base modifications. However, therapeutic RNAs employ a diverse catalog of modifications, including base, sugar, and backbone modifications, to enhance stability and pharmacological properties. To address this gap, we systematically evaluated a set of therapeutically relevant modifications [phosphorothioate (PS)], sugar [2'-O-methylation (2'OMe), 2'-Fluoro (2'F), locked nucleic acid (LNA), 2'-O-(2'-methoxyethyl) (2'MOE)], and base [N1-methylpseudouridine (m1Ψ), 5-methylcytidine (m5C), 5-methoxyuridine (5moU), and 5-iodocytidine (5iodoC)] using direct RNA nanopore sequencing. Modifications were systematically analyzed using basecall errors, raw current signals, and modification-aware basecalling models. Ribose modifications, m1Ψ, and 5moU induced significant error rate increases and noticeable current alterations, whereas 2'OMe and 2'MOE affected dwell time adjacent to the pore. In contrast, PS linkages produced only slight current alterations without increasing basecalling errors. We further evaluated modification-aware basecallers for 2'OMe and m5C. While these tools can distinguish modification types, they are limited by poor quantification accuracy and high local error rates, especially for 2'OMe. This study establishes a critical performance baseline, clarifying the current capability and limitations of nanopore technology for the analysis of therapeutically relevant RNA modifications.