Nucleic Acids Research最新文献

In human cells, the Nuclear EXosome Targeting (NEXT) and Poly(A) tail eXosome Targeting (PAXT) adaptors direct the nuclear exosome to degrade prematurely terminated RNA Polymerase II (Pol II) transcripts, ensuring nuclear RNA quality control. How these adaptors interact with transcription termination machineries remains largely unclear. Here, we leveraged in silico structure predictions of protein complexes to identify and model previously unreported interactions of NEXT- and PAXT-associated components with two transcription termination and processing machineries, the Integrator and Cleavage and Polyadenylation (CPA) complexes. Our computational models were validated through complementary in vitro biochemical approaches and single-particle cryo-EM analyses. We show that the ZC3H18 protein uses two different domains to directly recognize the INTS9/11 endonuclease module of Integrator and the mammalian Polyadenylation Specificity Factor (mPSF), a core CPA component. In turn, ZC3H18 can directly bind the scaffolding subunits of NEXT and PAXT via mutually exclusive interactions. Furthermore, we provide evidence that accessory PAXT components can be directly integrated with the mPSF core, establishing configurations that are mutually exclusive with those of canonical CPA subunits. These findings reveal a versatile interaction network capable of forming alternative structural frameworks linking transcription termination with nuclear RNA quality control.

DNMT1 is a methyltransferase that restores 5-methylcytidine marks on newly replicated DNA and is required for maintaining epigenetic inheritance. Using Halo-tagged DNMT1 and highly inclined thin illumination (HiLo) microscopy, we show that DNMT1 mobility in living human cells changes under a variety of conditions. DNMT1 molecules become increasingly bound to chromatin in the S phase of the cell cycle, but surprisingly only ∼ 12% chromatin-bound DNMT1 is sufficient to maintain DNA methylation. Upon treatment with small molecule inhibitors, GSK-3484862 (GSK), 5-azacytidine (5-azaC) and decitabine (5-aza-deoxyC), in vivo DNMT1 dynamics are greatly altered. Unexpectedly, treatment of cells with GSK, a non-covalent inhibitor, causes binding of DNMT1 to chromatin similar to that observed upon treatment with 5-azaC and decitabine, covalent inhibitors. 5-azaC inhibition of DNMT1 dynamics occurs during the S phase of the cell cycle. Unexpectedly, mutations in the disordered, Asp- and Glu-rich N-terminal region of DNMT1 dramatically decrease its mobility and increase chromatin binding. Collectively, our work using live cell single molecule imaging quantifies the molecular dynamics of DNMT1 and how this relates to its function under physiological conditions and upon drug treatment. Understanding the dynamics of DNMT1 in vivo provides a framework for developing better therapeutics that target DNMT1.

HNRNPU is an RNA-binding protein with diverse roles in transcriptional and post-transcriptional regulation. Pathogenic genetic variants of HNRNPU cause a severe neurodevelopmental disorder (NDD), but the underlying molecular mechanisms are unclear. Here, we comprehensively investigate the HNRNPU molecular interactome by integrating protein-protein interaction (PPI) mapping, RNA target identification, and genome-wide DNA methylation profiling in human neuroepithelial stem cells and differentiating neural cells. We identified extensive HNRNPU-centered networks, including an association with the mammalian SWI/SNF chromatin-remodeling complex, and uncovered a previously unrecognized role in translation. We present evidence that HNRNPU associates with messenger RNAs (mRNAs) encoding proteins important for neuronal development, including several linked to NDDs. Silencing HNRNPU reprogrammed methylation dynamics at regulatory regions, particularly at active and bivalent promoters of neurodevelopmental transcription factors. Integrative analysis across PPI, RNA, and methylome datasets identified 19 converging genes at all three molecular levels, including NDD genes within the SWI/SNF complex, SMARCA4 and SMARCC2, and RNA-processing machinery such as SYNCRIP. Together, these data showcase HNRNPU as a central coordinator of RNA metabolism and epigenetic remodeling during neural differentiation, linking RNA-binding, chromatin organization, and DNA methylation to the pathogenesis of HNRNPU-related NDDs.

Despite the many advances in single cell genomics, detecting structural rearrangements in single cells, particularly error-free sister-chromatid exchanges, remains challenging. Here we describe sci-L3-Strand-seq, a combinatorial indexing method with linear amplification for DNA template strand sequencing that cost-effectively scales to millions of single cells, as a platform for mapping mitotic crossover (CO) and resulting genome instability events. We provide a computational framework to fully leverage the throughput, as well as the relatively sparse but multifaceted genotype information within each cell that includes strandedness, digital counting of copy numbers, and haplotype-aware chromosome segmentation, to systematically distinguish seven possible types of mitotic CO outcomes. We showcase the power of sci-L3-Strand-seq by quantifying the rates of error-free and mutational COs in thousands of cells, enabling us to explore enrichment patterns of genomic and epigenomic features. The throughput of sci-L3-Strand-seq also gave us the ability to measure subtle phenotypes, opening the door for future large mutational screens. Furthermore, mapping clonal lineages provided insights into the temporal order of certain genome instability events, showcasing the potential to dissect cancer evolution. Altogether, we show the wide applicability of sci-L3-Strand-seq to the study of DNA repair and structural variations.

The ribosomal RNA gene cluster (rDNA) in Saccharomyces cerevisiae consists of about 150 tandem copies, making it a fragile site prone to copy number changes through recombination among the repeat. While extensive research has been conducted to understand the mechanisms for rDNA stability maintenance, the relationship between the stability maintenance of rDNA and other genomic regions remains unclear. In this study, we identified a mutant, sic1, that exhibited instability in both rDNA and chromosome IV (chr.IV). We revealed that Ty element-mediated ectopic recombination leads to partial duplication and elongation of chr.IV. Furthermore, we found that rDNA instability is caused by an increased SIR4 gene dosage resulting from this partial duplication. These findings suggest a link between the stability of rDNA and other genomic regions.

Chronic hepatitis B virus (HBV) persistence relies on the chromatin plasticity of covalently closed circular DNA (cccDNA), a viral minichromosome resistant to current therapies. Using proximity labeling (TurboID-dCas9), ChIP-seq and DNA pull-down assays, we identified SMARCC2-a BAF scaffolding subunit-bound to cccDNA enhancer-promoter regions (EnhⅠ/XP, CP/EnhII), where it sustains nucleosome-depleted regions (NDRs) and recruits RNA polymerase II. Genetic or pharmacological BAF inhibition compacted cccDNA chromatin, reduced histone acetylation (AcH3/AcH4), and enhanced SMC5/6-mediated silencing to suppress transcription, with the BAF ATPase inhibitor FHT-2344 reducing serum HBV DNA by 50% (P <.05) and intrahepatic HBV RNA by 70% (P <.01) without cccDNA loss, indicating epigenetic silencing. Mechanistically, BAF maintains NDRs by counteracting nucleosome retention and recruiting host transcription factors such as HNF4α. This work concludes that BAF safeguards cccDNA chromatin plasticity to enable viral persistence, and targeting BAF (e.g. FHT-2344) epigenetically silences cccDNA, offering a novel strategy for functional cure.

Temperature stress is a fundamental challenge for all organisms. While two-component systems (TCSs) are known to transduce environmental signals in microbes, their role in thermal sensing remains underexplored. Here, we unveil a novel thermosensitive TCS, DhqSR, in the thermophile Thermus thermophilus HB27. We demonstrate that the histidine kinase DhqS perceives thermal cues and autophosphorylates at His327. Its cognate response regulator, DhqR, is activated through a unique tyrosine-phosphorylation mechanism: phosphorylation at a unique Tyr84 residue, rather than the canonical aspartate. This atypical DhqSR system orchestrates cellular thermoadaptation by directly regulating a key enzyme in the shikimate pathway, type II 3-dehydroquinate dehydratase (DHQase). Our findings reveal a novel molecular mechanism of temperature sensing and adaptation, providing a new paradigm for microbial environmental adaptation and offering a unique toolbox for engineering thermotolerance.

DNA origami has become a ubiquitous platform because it enables straightforward design of nanostructures that self-assemble with high yield. The interactions between the cooperative effects involved in its assembly are currently not well understood. Fortunately, the nearly infinite number of choices available to the origami designer provides a rich environment in which to explore cooperativity. The DNA domains comprising origami have predictable energetics, and the sources of cooperativity are conceptually straightforward, and the difficulty in predicting assembly comes from their large number of cooperative interactions. We are able to probe cooperativity by using design variations and measuring their effect on assembly yield. We employ an accelerated assembly protocol that increases the sensitivity of structural perfection, or lack thereof, to design variation, and apply this approach to survey a broad set of design features. Using the resulting dataset, we develop metrics to correlate thermal stability, beneficial cooperativity from short folds, and detrimental cooperativity from long folds, with defectivity. Surprisingly, these metrics can be combined to create a single parameter with a clear correlation to yield, which serves as a useful starting place for a predictive understanding of the interplay between cooperativity and design. In doing so, we also identify qualitative trends that provide useful insight into design best practice.

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.