Nucleic Acids Research最新文献

An ultrasound-responsive transgene circuit can provide non-invasive, spatiotemporally precise remote control of gene expression and cellular behavior in synthetic biology applications. However, current ultrasound-based systems often rely on nanoparticles or harness ultrasound's thermal effects, posing risks of tissue damage and cellular stress that limit their therapeutic potential. Here, we present Spatiotemporal Ultrasound-induced Protein Expression Regulator (SUPER), a novel gene switch enabling mediator-free, non-invasive and direct regulation of protein expression via ultrasound in mammalian cells. SUPER leverages the mammalian reactive oxygen species (ROS) sensing system, featuring KEAP1 (Kelch-like ECH-associated protein 1), NRF2 (nuclear factor erythroid 2-related factor 2), and antioxidant response element (ARE) as its core components. We demonstrate that low-intensity (1.5 W/cm2, ∼45 kHz), brief (40 s) ultrasound exposure generates non-toxic levels of ROS, activating the KEAP1/NRF2 pathway in engineered cells and leading to the controlled expression of target gene(s) via a synthetic ARE promoter. The system exhibits robust expression dynamics, excellent reversibility, and functionality in various cell types, including human mesenchymal stem cell-derived lines (hMSC-TERT). In a proof-of-concept study, ultrasound stimulation of subcutaneously implanted microencapsulated engineered cells stably expressing the sonogenetic circuit in a type 1 diabetic mouse model triggered sufficient insulin production to restore normoglycemia. Our work highlights ultrasound's potential as a precise and non-invasive tool for advancing cell and gene therapies in personalized medicine.

The Philadelphia chromosome, the translocation between BCR and ABL genes, is seen in 95% of chronic myeloid leukemia (CML) patients. Although discovered >60 years ago, the molecular mechanism of BCR fragility is unclear. Here, we have identified several G4 DNA motifs at the BCR fragile region of CML patients. Various lines of experimentation revealed that the breakpoint regions could fold into multiple intramolecular G-quadruplex structures. The sodium bisulfite modification assay revealed single strandedness in the fragile region when present on a plasmid and human genome. Circular dichroism spectroscopy revealed the parallel G4 DNA formation, leading to polymerase arrest at the BCR breakpoints. Intracellular recombination assay revealed that DNA breakage at the BCR fragile region could join with the break generated by ISceI endonuclease. Finally, purified AID could bind and deaminate cytosines when present on single-stranded DNA generated due to G4 DNA, both in vitro and inside the cells. Therefore, our results suggest that AID binds to G4 DNA present at the BCR fragile region, resulting in the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, which can later get converted into a double-strand break, leading to t(9;22) chromosomal translocation.

In eukaryotic genomes, regulated access and communication between cis-regulatory elements (CREs) are necessary for enhancer-mediated transcription of genes. The molecular framework of the chromatin organization underlying such communication remains poorly understood. To better understand it, we develop a multiscale modeling pipeline to build near-atomistic models of the 200 kb Nanog gene locus in mouse embryonic stem cells comprising nucleosomes, transcription factors, co-activators, and RNA polymerase II-mediator complexes. By integrating diverse experimental data, including protein localization, genomic interaction frequencies, cryo-electron microscopy, and single-molecule fluorescence studies, our model offers novel insights into chromatin organization and its role in enhancer-promoter communication. The models equilibrated by high-performance molecular dynamics simulations span a scale of ∼350 nm, revealing an experimentally consistent local and global organization of chromatin and transcriptional machinery. Our models elucidate that the sequence-regulated chromatin accessibility facilitates the recruitment of transcription regulatory proteins exclusively at CREs, guided by the contrasting nucleosome organization compared to other regions. By constructing an experimentally consistent near-atomic model of chromatin in the cellular environment, our approach provides a robust framework for future studies on nuclear compartmentalization, chromatin organization, and transcription regulation.

The DNA-dependent protein kinase (DNA-PK) complex plays a critical role in nonhomologous end-joining (NHEJ), a template-independent pathway for repairing DNA double-strand breaks (DSBs). The association of Ku70/80 with DSB ends facilitates the assembly of the DNA-PK holoenzyme. However, key mechanisms underlying the attachment and stabilization of DNA-PK at broken DNA ends remain unclear. Here, we identify PRRX1, a homeodomain-containing protein, as a mediator of chromatin localization and subsequent activation of DNA-PK. PRRX1 oligomerizes to simultaneously bind to double-strand DNA and the SAP (SAF-A/B, Acinus, and PIAS) domain of Ku70, thereby enhancing Ku anchoring at DSBs and stabilizing DNA-PK for efficient NHEJ repair. Reduced expression or pathogenic mutations of PRRX1 are associated with genomic instability and impaired NHEJ repair. Furthermore, a peptide that disrupts PRRX1 oligomerization compromises NHEJ efficiency and reduces cell survival following irradiation. These findings provide new insights into the activation of the NHEJ machinery and offer potential strategies for optimizing cancer therapies.

There is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats), coupled with light's unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here, we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs, BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR, including indels, CRISPRa, and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing, achieving optogenetic gene editing in T lymphocytes in vivo.

DNA lesions are a threat to genome stability. To cope with them during DNA replication, cells have evolved lesion bypass mechanisms: Translesion Synthesis (TLS), which allows the cell to insert a nucleotide directly opposite the lesion, with the risk of introducing a mutation, and error-free damage avoidance (DA), which uses homologous recombination to retrieve the genetic information from the sister chromatid. In this study, we investigate the timing of lesion bypass in yeast and its implications for the accuracy of the process. Our findings reveal that DNA polymerase η can bypass common, UV-induced cyclobutane pyrimidine dimers at the fork, immediately after encountering the blocking lesion. In contrast, TLS at (6-4) photoproducts and bulky G-AAF adducts, mediated by Rev1 and Pol ζ, takes place behind the fork, at post-replicative gaps that are generated downstream of the lesion after repriming. We show that in this latter situation, TLS competes with the DA pathway, thus reducing overall mutagenicity of damage bypass. Additionally, our study demonstrates that Exo1 nuclease influences the balance between TLS and DA by modulating the size of the post-replicative gaps.

Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, and viral infections. In prokaryotes, regulation at the level of the epitranscriptome is still poorly understood. Here, we used nanopore direct RNA sequencing of Escherichia coli to study RNA modifications and their changes under heat stress. With a single sequencing reaction, we detected most known modification types in ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). RNA sequencing was complemented by a multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide polymerase chain reaction, and in vitro methylation. Known 5-methylcytidine (m5C) and N6-methyladenosine (m6A) sites in the rRNA were confirmed, but these types of modifications could not be localized in the mRNA. In response to heat stress, levels of m5C, m6A, and N6,N6-dimethyladenosine increased in the 16S rRNA. Sequencing and mass spectrometry data demonstrated a decrease in tRNA modification abundance in the anticodon loop at 45°C. In general, mRNA modifications at 37°C were enriched in the coding regions of genes associated with general metabolism and RNA processing, which shifted to genes involved in cell wall synthesis and membrane transport under heat stress. This study provides new insights into the complexity of post-transcriptional regulation in bacteria.