RESUMO
How chronic mutational processes and punctuated bursts of DNA damage drive evolution of the cancer genome is poorly understood. Here, we demonstrate a strategy to disentangle and quantify distinct mechanisms underlying genome evolution in single cells, during single mitoses and at single-strand resolution. To distinguish between chronic (reactive oxygen species (ROS)) and acute (ultraviolet light (UV)) mutagenesis, we microfluidically separate pairs of sister cells from the first mitosis following burst UV damage. Strikingly, UV mutations manifest as sister-specific events, revealing mirror-image mutation phasing genome-wide. In contrast, ROS mutagenesis in transcribed regions is reduced strand agnostically. Successive rounds of genome replication over persisting UV damage drives multiallelic variation at CC dinucleotides. Finally, we show that mutation phasing can be resolved to single strands across the entire genome of liver tumors from F1 mice. This strategy can be broadly used to distinguish the contributions of overlapping cancer relevant mutational processes.
Assuntos
Dano ao DNA , Reparo do DNA , Mitose , Mutagênese , Raios Ultravioleta , Animais , Camundongos , Reparo do DNA/genética , Raios Ultravioleta/efeitos adversos , Dano ao DNA/genética , Mitose/genética , Espécies Reativas de Oxigênio/metabolismo , Mutação , HumanosRESUMO
During the ongoing COVID-19 pandemic, PCR testing and antigen tests have proven critical for helping to stem the spread of its causative agent, SARS-CoV-2. However, these methods suffer from either general applicability and/or sensitivity. Moreover, the emergence of variant strains creates the need for flexibility to correctly and efficiently diagnose the presence of substrains. To address these needs we developed the diagnostic test ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Optimization) which employs Cas13 to diagnose patients in 1 h without sophisticated equipment. Using an extensive panel of clinical samples, we demonstrate that ADESSO correctly identifies infected individuals at a sensitivity and specificity comparable to RT-qPCR on extracted RNA and higher than antigen tests for unextracted samples. Altogether, ADESSO is a fast, sensitive and cheap method that can be applied in a point of care setting to diagnose COVID-19 and can be quickly adjusted to detect new variants.
Assuntos
COVID-19 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Pandemias , RNA Viral/análise , RNA Viral/genética , SARS-CoV-2 , Sensibilidade e EspecificidadeRESUMO
DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.
Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Desmetilação do DNA , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Mapeamento Cromossômico , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Epigênese Genética/genética , Genoma/genética , Histonas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/genética , DNA Metiltransferase 3BRESUMO
Regulation of transcription, replication, and cell division relies on differential protein binding to DNA and chromatin, yet it is unclear which regulatory components remain bound to compacted mitotic chromosomes. By utilizing the buoyant density of DNA-protein complexes after cross-linking, we here develop a mass spectrometry-based approach to quantify the chromatin-associated proteome at separate stages of the cell cycle. While epigenetic modifiers that promote transcription are lost from mitotic chromatin, repressive modifiers generally remain associated. Furthermore, while proteins involved in transcriptional elongation are evicted, most identified transcription factors are retained on mitotic chromatin to varying degrees, including core promoter binding proteins. This predicts conservation of the regulatory landscape on mitotic chromosomes, which we confirm by genome-wide measurements of chromatin accessibility. In summary, this work establishes an approach to study chromatin, provides a comprehensive catalog of chromatin changes during the cell cycle, and reveals the degree to which the genomic regulatory landscape is maintained through mitosis.
