Transposons are among the most abundant mobile genetic elements in nature. IStrons are a unique class of transposons, encoding a transposase for transposon mobility, an RNA-guided nuclease for maintenance, and a self-splicing group I intron for element removal from host mRNA. However, it is unclear how a single polynucleotide sequence balances these distinct biochemical functions. Here, we employed pooled library mutagenesis coupled with high-throughput sequencing to systematically dissect the molecular determinants of IStron transposition, RNA-guided DNA cleavage, and self-splicing. We found that the terminal trinucleotide of the transposon right end is constrained by all three functions, identifying a molecular convergence point. Cross-assay comparisons revealed that most variants maintained or lost activity across multiple assays simultaneously. However, a subset selectively retained one activity while losing another, revealing an antagonism between DNA cleavage and splicing governed by guide RNA structural stability. Increased GC content at the base of the guide RNA 3' terminal stem-loop abolished splicing while maintaining DNA cleavage, and the stably folded guide RNA sterically occludes alternative splice sites, ensuring splicing accuracy across variable flanking contexts. Thus, IStron transcripts face an inherent trade-off between guide RNA maturation and splicing, with RNA structural stability as the primary determinant of pathway choice.

LIN28A is a conserved RNA-binding protein essential for pluripotency, let-7 miRNA regulation, and cellular metabolism. However, the mechanisms controlling its transcriptional activation during early stem cell fate transitions remain poorly defined. Using the Lin28a locus as a model, we identified multiple intragenic enhancers that generate distinct enhancer RNAs (eRNAs) exhibiting temporally ordered and stimulus-specific activation patterns. Functional perturbation demonstrated that these eRNAs, particularly antisense-derived transcripts, contribute to the regulation of Lin28a transcription and pluripotency maintenance, as their depletion reduced Lin28a expression and impaired embryoid body formation. Distinct environmental inputs induced differential temporal patterns of eRNA activation, accompanied by coordinated changes in enhancer chromatin state and transcription factor occupancy. These findings suggest that intragenic eRNAs make temporally coordinated and differential contributions to stimulus-responsive Lin28a transcriptional dynamics. Together, our findings define a temporally coordinated and context-dependent enhancer logic at the Lin28a locus, in which chromatin state, transcription factor occupancy, and intragenic eRNA induction are integrated to couple external cues with dynamic transcriptional responses during stem cell state transitions.

Given their defect in homologous recombination, BRCA1-deficient cancers use polymerase theta (POLQ) microhomology-mediated end joining (MMEJ) for repair of stressed replication forks. We found that BRCA1-deficient cells repress microRNA (miR) 4485-3p, and reconstituting expression of this miR resulted in the death of BRCA1-deficient cells. In our interrogation of the mechanism of this synthetic lethality, we discovered that miR-4485-3p suppresses expression of RPRD1B protein. Depletion of RPRD1B led to selective BRCA1-deficient cell death, decreased MMEJ, and decreased replication fork repair after replication stress. RPRD1B promotes the recruitment of the MMEJ components 53BP1, PARP1, and POLQ to the stressed replication fork. However, RPRD1B does not have a canonical DNA-binding domain, and our search for the mechanism of its recruitment to stressed replication forks discovered that this is mediated by RECQL5 and ubiquitinated (Ub) PCNA. Thus, our results unveil an essential signaling cascade at the stressed replication fork in BRCA1-deficient cancer cells, in which Ub PCNA recruits RECQL5, which recruits RPRD1B to mediate the assembly of the MMEJ repair apparatus. These data suggest that this repair scaffold may be targeted in the development of a novel synthetic therapy of BRCA1-deficient cancers.

IFNγ and IL-4, the canonical Th1 and Th2 cytokines, typically induce opposing macrophage polarization. Beyond their frequent coexistence and mutual antagonism in pathological settings, a synergistic crosstalk may also exist and remains to be defined. Through integrated transcriptomic, epigenomic, and CRISPR-based analyses, we identify that co-exposure to IL-4 and IFNγ results in an intermediate macrophage polarization state in murine macrophages, associated with a cohort of synergistically activated genes. We show that this synergistic activation is mediated by co-binding of STAT6 and STAT1 at regulatory regions marked with pronounced H3K27Ac accumulation and is dependent on the BRD4 cofactor. Deletion of either STAT factor or disruption of their binding motifs abolishes the synergistic transcriptional response. Our data support a model in which chromatin openness induced by one cytokine facilitates binding of the opposing cytokine-activated STAT transcription factor (TF), contributing to synergistic gene activation. We further show that IRF1, an IFNγ-induced TF whose expression persists in the presence of IL-4, is indispensable for a substantial subset of this program. Importantly, single-cell RNA sequencing of the PyMT murine breast cancer model reveals an in vivo tumor-associated macrophage subset showing STAT1 and STAT6 activation and is enriched for the synergistic gene signature.

Chromatin-associated RNAs (caRNAs) play critical roles in regulating chromatin organization and transcription, underscoring the importance of their study. Proximity labeling has emerged as a promising and versatile technique for profiling caRNAs with high spatiotemporal resolution. While being a powerful technique, traditional proximity labeling methods depend on complex, high-input enrichment protocols, which significantly limit their wide practical application. Here, we developed a straightforward, enrichment-free caRNA profiling strategy: proximity crosslinking-induced RNA depletion sequencing (PCRD-seq). This approach leverages the proximity crosslinking between chromatin and its surrounding RNAs induced by singlet oxygen generated by HoeDBF, a photosensitizer targeting chromatin region. The proximity crosslinking hinders the release of caRNAs during routine TRIzol extraction, consequently leading to a specific depletion of these RNAs. This method was successfully applied to investigate the role of U1 small nuclear RNA (snRNA) in RNA chromatin retention and the differences in chromatin-associated transcriptomes between two ovarian cancer cell lines with opposite metastatic capability. Moreover, our PCRD-seq exhibits potential in profiling nuclear lamina-associated RNAs, which paves the way for its application to profile RNAs associated with other chromatin subdomains. The minimal cell input and simple workflow render PCRD-seq a transformative tool for wide applications.

The flexibility of nucleic acids plays a central role in numerous biological processes, including chromatin organization, gene regulation, and ribosome assembly. While elastic network models (ENMs) have successfully captured conformational changes in proteins through harmonic normal modes (NMs), analogous approaches for nucleic acids remain limited. Here, we introduce a generalized essential dynamics-refined ENM (edENM) for both DNA, RNA, and protein-nucleic acid complexes, parametrized against a diverse set of molecular dynamics simulations and validated using experimental ensembles from nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy. edENM achieves high agreement with experimental conformational changes across a curated benchmark of ∼60 DNA, RNA, and protein-nucleic acid systems. Compared to uniform-spring parametrizations, it produces significantly more collective NMs and suppresses unphysical backbone ruptures. We further integrate edENM into eBDIMS2, an efficient Brownian Dynamics path-sampling framework, extending its applicability to nucleic acid-containing systems at the megadalton scale. This enables the exploration of complex conformational transitions, including rearrangements of RNA folds in coronaviruses, large-scale remodeling in Argonaute-RNA complexes, multi-nucleosome assemblies in chromatin, as well as ribosomal particles. Together, these results establish an accessible and scalable elastic network framework for modeling conformational changes across the full spectrum of nucleic acid-containing biological systems.

The universally conserved enzyme uracil-DNA N-glycosylase (UNG) plays a central role in maintaining genome stability. It functions as the initiating factor in uracil base excision repair (UBER) by catalyzing the removal of uracil lesions in genomic DNA, a necessary first step in restoring genome integrity after hydrolytic deamination of cytosine to uracil or misincorporation of deoxyuridine monophosphate during replication. Although methods have been developed to study UBER in vitro and in cellulo, none provide a quantitative readout of UNG activity on the chromosomal DNA of living cells. To address this gap, we created an UNG biosensor (U-report) that utilizes a modified cytosine base editor to generate a targeted genomic uracil lesion in a fluorescent reporter for C-to-U editing activity. UNG activity ablation through uracil DNA glycosylase inhibition (Ugi) or UNG-knockout results in elevated reporter fluorescence. Isoform-specific knockouts show that mitochondrial UNG1 also contributes to UBER of nuclear DNA. Our studies establish a real-time biosensor for quantification of chromosomal DNA uracil excision activity in living cells and, in support of prior studies, indicate that both UNG isoforms should be addressed in small molecule inhibitor development programs.

RNA-guided obligate mobile element guided activity systems derived from transposable elements have emerged as compact genome-editing tools that may replace clustered regularly interspaced short palindromic repeats platforms. We established a dual-mode genome regulation platform using ISDge10 TnpB effectors and engineered ωRNAs, in which modulation of the ωRNA guide length enables switching between programmable transcriptional activation and genome editing. TnpB programmed with a 10-nt guide region of the ωRNA engages target DNA without inducing double-strand breaks. Fusion of transcriptional activators with Sso7d (DNA-binding protein from Sulfolobus solfataricus) enables specific transcriptional upregulation across endogenous loci. Restoring the ωRNA guide length to 20 nt triggers DNA cleavage, thereby supporting homology-directed repair-mediated sequence correction. A catalytically inactivated TnpB-based adenine base editor enabled A-to-G base conversion at genomic targets. TnpB shows strict ωRNA-dependent mismatch sensitivity with low off-target effects, suggesting its potential as a high-fidelity genome regulation platform. Compact ISDge10 TnpB facilitates co-packaging of effector and ωRNA in a single adeno-associated virus vector and co-expression of large functional domains. Thus, this study expands RNA-guided genome-editing capabilities.

In the cell, DNA must be tightly packed to facilitate its organization into the nucleus, where recognition of homologous sequences underpins key processes such as recombination. Yet the structural basis of DNA-DNA pairing remains unknown. Here, we combine high-resolution atomic force microscopy and atomistic MD simulations to provide the first direct visualization of DNA pairing in the presence of divalent ions. We show that strongly paired DNAs often achieve groove-to-groove alignment, driven by ionic bridges connecting the minor grooves of the two duplexes. These contacts are further stabilized by sequence-specific interactions, whose strength and specificity vary with the ion type. This mechanism of ion-mediated groove alignment provides a molecular framework for the long-hypothesized 'helical alignment' model, in which homologous recognition would be facilitated by preserving structural register between the two helices. Together, our findings reveal a fundamental principle by which divalent ions promote DNA-DNA pairing, with broad implications for chromosomal condensation and genome organization.

Deciphering sequence-function relationships in long non-coding RNAs (lncRNAs) remains challenging due to rapid evolutionary turnover and limited primary sequence conservation. Alignment-based approaches often fail to detect functional domains, and fixed-length k-mer models inadequately capture variable-length regulatory elements. Here, we introduce RNA-Lexis, a non-parametric statistical framework for unbiased discovery of candidate RNA sequence elements. RNA-Lexis applies segmentation based on local conditional probabilities to identify non-random sequence extensions, enabling detection of recurrent, variable-length motifs without prior biological assumptions. Conceptually analogous to language segmentation, the framework partitions continuous RNA sequences into statistically defined units ("xmotifs" and "cores"), providing an interpretable representation of sequence architecture. RNA-Lexis reconstructs the modular organization of well-characterized lncRNAs, including XIST and NORAD. In additional case studies, RNA-Lexis prioritized recurrent GC-rich elements in SNHG14 that were tested experimentally and shown to bind histones in RNA pulldown assays. RNA-Lexis also identified recurrent LINC01001 core motifs that overlap chromatin interaction patterns detected by GRID-seq. These analyses support the use of RNA-Lexis to nominate candidate sequence elements for functional follow-up, while biological function remains dependent on orthogonal experimental validation. RNA-Lexis provides a statistically grounded and interpretable framework for motif-level analysis of lncRNAs. Rather than directly inferring function, the method identifies recurrent sequence architecture and prioritizes candidate elements for mechanistic testing.

Multidrug-resistant bacteria pose a severe threat to global health. Mutations in transcriptional regulators accelerate the emergence of multidrug resistance and may have a crucial impact on pathogen evolvability under antibiotic exposure. Here, we investigate these dynamics in the bacterial pathogen Yersinia enterocolitica. In this organism, we identified a high-frequency de novo mutation in the promoter of an AraC/XylS-family transcriptional regulator, Rob. This mutation arose independently during resistance evolution against three of six antibiotic classes. Sequence and structure alignments indicate that Rob is a previously uncharacterized, lineage-specific regulator in Y. enterocolitica, featuring a conserved promoter architecture. This promoter mutation resulted in robust rob overexpression, leading to the activation of multiple downstream efflux- and membrane-associated pathways. This regulatory mutation emerges early and shapes the acquisition of tetracycline resistance while also enhancing high-level enrofloxacin resistance in combination with canonical mutations in gyrA and parC. Despite being favored under antibiotic selection, rob overexpression is costly, resulting in counter-selection of the -57 G > A rob allele in the absence of antibiotics. Together, these findings identify Rob as a Yersinia-specific efflux regulator and demonstrate how regulatory mutations can transiently accelerate antibiotic resistance.

Separase plays central roles in chromosome separation during mitosis and in the centrosome duplication cycle. Tight control of separase activity is required to prevent unscheduled resolution of sister chromatid cohesion and centrosome aberrations, thereby maintaining genome stability. In mammals, despite their disassembly during early mitosis, some nuclear envelope components have mitotic roles, but the links with separase activity remain unexplored. Here, we uncover a novel mechanism of separase regulation involving lamin B1, a key nuclear envelope factor. We show that separase and lamin B1 associate preferentially during early mitotic stages. Importantly, lamin B1 depletion leads to an increase in separase recruitment to chromosomes together with premature chromatid separation, a phenotype reminiscent of separase overexpression. Conversely, similar to separase depletion, lamin B1 overexpression induces diplochromosome formation, together with centrosome amplification, phenotypes consistent with impaired sister chromatid separation. Importantly, increasing separase levels prevents lamin B1-induced centrosome aberrations, suggesting a separase defect at their origin. Indeed, we show that overexpression of lamin B1 leads to a decrease in the recruitment of separase to chromosomes and a delay in its activity. Taken together, this study unveils a novel mechanism of separase regulation involving the nuclear envelope factor lamin B1 that is critical for genome integrity.

Neuronal activity induces widespread changes in chromatin organization, yet the mechanisms of activity-dependent 3D genome remodelling remain incompletely understood. Here, we identify SATB2 as a key regulator of activity-dependent 3D chromatin dynamics in cortical pyramidal neurons. Using primary cultures from floxed and Satb2 conditional knockout mice, we combined Hi-C and chromatin accessibility mapping to capture rapid 3D epigenome reorganization following neuronal stimulation. Within 1 h of activation, floxed neurons exhibited enhanced chromatin interactions, increased chromatin accessibility, and the emergence of a novel activity-dependent (AD) compartment enriched for SATB2 binding sites. The AD compartment harbours metabolic and housekeeping genes that undergo transient repression upon neuronal activation, thereby prioritizing translation of long synaptic transcripts. Loss of SATB2 disrupted these activity-induced changes, including chromatin accessibility, long-range interactions, and AD compartment formation, resulting in attenuated induction and repression of key activity-regulated genes. These findings establish SATB2 as a central organizer of activity-dependent chromatin architecture dynamics, required for coordinated activation and repression of activity-regulated genes in cortical pyramidal neurons.

3D genome organization is crucial to modulate gene expression. Topologically associating domain boundaries isolate genes and their regulatory elements within the same topological neighborhood, avoiding crosstalk between regulatory elements. Perturbation of domain boundaries causes aberrant genomic contacts and gene expression misregulation. CTCF and the cohesin complex are critical to form boundaries through loop extrusion. However, we still lack a complete understanding of what makes a boundary more effective at insulating genomic contacts than others. Here, we experimentally classified boundaries according to their chromatin accessibility as a proxy of protein occupancy in K562 human cells. Highly accessible boundaries are occupied by more proteins including CTCF, are more robust contact insulators, have a more conserved CTCF DNA-binding motif, and are more conserved across cell types. By exploring proteins enriched at boundaries with different accessibility, we found that CTCF and cohesin bind together with REST forming one module, while ZNF316 and EMSY form another, both occupying boundaries very frequently. CRISPR-Cas9 deletion of the ZNF316/EMSY binding region at a robust CTCF-free boundary increases inter-domain contacts, demonstrating that this module contributes to chromatin insulation. Our results emphasize the importance of protein combination and abundance in boundary strength and identify ZNF316/EMSY as a novel chromatin insulator module.