Nucleic Acids Research最新文献

Cancer cells are highly plastic, favoring adaptation to changing conditions. Genes related to basic cellular processes evolved in ancient species, while more specialized genes appeared later with multicellularity (metazoan genes) or even after mammals evolved. Transcriptomic analyses have shown that ancient genes are up-regulated in cancer, while metazoan-origin genes are inactivated. Despite the importance of these observations, the underlying mechanisms remain unexplored. Here, we study local and global epigenomic mechanisms that may regulate genes from specific evolutionary periods. Using evolutionary gene age data, we characterize the epigenomic landscape, gene expression regulation, and chromatin organization in several cell types: human embryonic stem cells, normal primary B-cells, primary chronic lymphocytic leukemia malignant B-cells, and primary colorectal cancer samples. We identify topological changes in chromatin organization during differentiation observing patterns in Polycomb repression and RNA polymerase II pausing, which are reversed during oncogenesis. Beyond the non-random organization of genes and chromatin features in the 3D epigenome, we suggest that these patterns lead to preferential interactions among ancient, intermediate, and recent genes, mediated by RNA polymerase II, Polycomb, and the lamina, respectively. Our findings shed light on gene regulation according to evolutionary age and suggest this organization changes across differentiation and oncogenesis.

Anthropogenic evolvable genetic information systems (AEGIS) are DNA-like molecules that can be copied, support laboratory in vitro evolution (LIVE), and evolve to give AegisBodies, analogs of antibodies. However, unlike DNA aptamers built from four different nucleotides, AegisBodies are currently built from six different nucleotides. Thus, six-letter AEGIS-LIVE delivers AegisBodies with greater stability in biological mixtures, more folds, and enhanced binding and catalytic power. Unlike DNA however, AEGIS has not benefited from 4 billion years of biological evolution to create AEGIS-specialized enzymes, but only a decade or so of human design. To learn whether AEGIS can nevertheless perform as well as natural DNA as a platform to create functional molecules, we compared two six-letter AegisBodies (LZH5b and LZH8) with a single standard four-letter aptamer, both evolved to bind specific cancer cells with ∼10 cycles of LIVE. Both evolved ∼50 nM affinities. Both discovered proteins on their cancer cell surfaces thought to function only inside of cells. Both can be internalized. Internalizing of LZH5b attached to an AEGIS nanotrain brings attached drugs into the cell. These data show that AEGIS-LIVE can do what four-letter LIVE can do at its limits of performance after 4 billion years of evolution of DNA-specialized enzymes, and better by a few metrics. As synthetic biologists continue to improve enzymology and analytical chemistry to support AEGIS-LIVE, this technology shoud prove increasingly useful as a tool, especially in cancer research.

Protein intrinsically disordered regions (IDRs) are critical gene-regulatory components and aberrant fusions between IDRs and DNA-binding/chromatin-associating domains cause diverse human cancers. Despite this importance, how IDRs influence gene expression, and how aberrant IDR fusion proteins provoke oncogenesis, remains incompletely understood. Here we develop a series of synthetic dCas9-IDR fusions to establish that locus-specific recruitment of IDRs can be sufficient to stimulate endogenous gene expression. Using dCas9 fused to the paradigmatic leukemogenic NUP98 IDR, we also demonstrate that IDRs can activate transcription via localized biomolecular condensation and in a manner that is dependent upon overall IDR concentration, local binding density, and amino acid composition. To better clarify the oncogenic role of IDRs, we construct clinically observed NUP98 IDR fusions and show that, while generally non-overlapping, oncogenic NUP98-IDR fusions convergently drive a core leukemogenic gene expression program in donor-derived human hematopoietic stem cells. Interestingly, we find that this leukemic program arises through differing mechanistic routes based upon IDR fusion partner; either distributed intragenic binding and intrachromosomal looping, or dense binding at promoters. Altogether, our studies clarify the gene-regulatory roles of IDRs and, for the NUP98 IDR, connect this capacity to pathological cellular programs, creating potential opportunities for generalized and mechanistically tailored therapies.

Messenger RNA (mRNA) homeostasis is a critical aspect of cellular function, involving the dynamic interplay between transcription and decay processes. Recent advances have revealed that the ribosome plays a central role in coordinating mRNA decay, challenging the traditional view that free mRNA is the primary substrate for degradation. This review examines the mechanisms whereby ribosomes facilitate both the licensing and execution of mRNA decay. This involves factors such as the Ccr4-Not complex, small MutS-related domain endonucleases, and various quality control pathways. We discuss how translational fidelity, as well as the presence of nonoptimal codons and ribosome collisions, can trigger decay pathways such as nonstop decay and no-go decay. Furthermore, we highlight the direct association of canonical exonucleases, such as Xrn1 and the Ski-exosome system, with the ribosome, underscoring the ribosome's multifaceted role as a platform for regulatory processes governing mRNA stability. By integrating recent findings, this review offers a comprehensive overview of the structural basis of how ribosomes not only facilitate translation but also serve as critical hubs for mRNA decay coordination.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Overexpression of CENP-A is observed in many cancers and this correlates with poor prognosis. Here, we show that altered stoichiometry of histone H3 and expression of oncohistone mutation H3 E97K contributes to mislocalization of Cse4 and CIN. Oncohistone mutations in the globular domain of histone H3 such as H3 E97K occur in several cancers; however, their functional effects remain unexplored. We demonstrated that strains with reduced gene dosage of histone H3 (hht1Δ and hht2Δ) or oncohistone H3 E97K mutation exhibit enhanced Cse4-H4 interaction, an in vivo change in the conformational state of Cse4, and this contributes to mislocalization of Cse4. Oncohistone H3 E97K mutant protein was unstable and exhibited defects in interaction with histone H4. Notably, mislocalization of Cse4 and CIN phenotypes were observed in hht1Δ and oncohistone H3 E97K mutants expressing endogenous Cse4. In summary, our studies highlight the importance of histone H3 stoichiometry in preventing mislocalization of Cse4 for chromosomal stability and suggest that oncohistone H3 mutations may contribute to CIN in human cancers.

Repairing DNA double-strand breaks is crucial for maintaining genome integrity, which occurs primarily through homologous recombination (HR) in Saccharomyces cerevisiae. Nucleosomes, composed of DNA wrapped around a histone octamer, present a natural barrier to end resection to initiate HR, but the impact on the downstream HR steps of homology search, DNA strand invasion, and repair synthesis remain to be determined. Displacement loops (D-loops) play a pivotal role in HR, yet the influence of chromatin dynamics on D-loop metabolism remains unclear. Using the physical D-loop capture and D-loop extension (DLE) assays to track HR intermediates, we employed genetic analysis to reveal that H2B mono-ubiquitylation (H2Bubi) affects multiple steps during HR repair. We infer that H2Bubi modulates chromatin structure, not only promoting histone degradation for nascent D-loop formation but also stabilizing extended D-loops through nucleosome assembly. Furthermore, H2Bubi regulates DNA resection via Rad9 recruitment to suppress a feedback control mechanism that dampens D-loop formation and DLE at hyper-resected ends. Through physical and genetic assays to determine repair outcomes, we demonstrate that H2Bubi plays a crucial role in preventing break-induced replication and thus promoting genomic stability.

In contrast to traditional RNA regulatory approaches that modify the 2'-OH group, this study explores strategic base modifications using 5-carboxylcytosine (ca5C). We developed a technique where ca5C is transformed into dihydrouracil via treatment with borane-pyridine complex or 2-picoline borane complex, leading to base mutations that directly impact RNA functionality. This innovative strategy effectively manages CRISPR-Cas9 system activities, significantly minimizing off-target effects. Our approach not only demonstrates a significant advancement in RNA manipulation but also offers a new method for the precise control of gene editing technologies, showcasing its potential for broad application in chemical biology.

Genetic instability is a hallmark of cancer, and mutation hotspots in human cancer genomes co-localize with alternative DNA structure-forming sequences (e.g. H-DNA), implicating them in cancer etiology. H-DNA has been shown to stimulate genetic instability in mammals. Here, we demonstrate a new paradigm of genetic instability, where a cancer-associated H-DNA-forming sequence accumulates more oxidative lesions than B-DNA under conditions of oxidative stress (OS), often found in tumor microenvironments. We show that OS results in destabilization of the H-DNA structure and attenuates the fold increase in H-DNA-induced mutations over control B-DNA in mammalian cells. Furthermore, the mutation spectra revealed that the damaged H-DNA-containing region was processed differently compared to H-DNA in the absence of oxidative damage in mammalian cells. The oxidatively modified H-DNA elicits differential recruitment of DNA repair proteins from both the base excision repair and nucleotide excision repair mechanisms. Altogether, these results suggest a new model of genetic instability whereby H-DNA-forming regions are hotspots for DNA damage in oxidative microenvironments, resulting in its altered mutagenic processing. Our findings provide valuable insights into the role of OS in DNA structure-induced genetic instability and may establish H-DNA-forming sequences as promising genomic biomarkers and potential therapeutic targets for genetic diseases.

In all three domains of life, genes with related functions can be organized into specific genomic regions known as gene clusters. In eukaryotes, histone, piRNA (Piwi-interacting RNA), and rDNA (ribosomal DNA) clusters are among the most notable clusters which play fundamental roles in chromatin formation, genome integrity, and translation, respectively. These clusters have long been thought to be regulated by distinct transcriptional mechanisms. In this study, using Caenorhabditis elegans as a model system we identify ATTF-6, a member of the AT-hook family, as a key factor for the expression of histone, piRNA, and 5S rDNA-SL1 (spliced leader 1) clusters. ATTF-6 is essential for C. elegans viability. It forms distinct nuclear foci at both piRNA and 5S rDNA-SL1 clusters. Loss of ATTF-6 leads to a depletion of histone mRNAs, SL1 transcripts, and piRNAs. Additionally, we demonstrate that ATTF-6 is required for the recruitment of USTC (Upstream Sequence Transcription Complex) to piRNA clusters, which is necessary for piRNA production. Collectively, our findings reveal a unifying role for an AT-hook transcription factor in promoting the expression of fundamental gene clusters.