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A cell-free pipeline for recreating methylation patterns radically enhances DNA transformation in bacteria

ABSTRACT: We report a method for bypassing restriction modification systems to increase DNA transformation in batceria and develop a high-throughput way to determine the optimal methylation pattern for DNA transformation.

ORGANISM(S): Bifidobacterium breve

PROVIDER: GSE189864 | GEO | 2024/01/01

REPOSITORIES: GEO

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A cell-free pipeline for recreating methylation patterns radically enhances DNA transformation in bacteria

Project description:The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic-biology chasses. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here we report IMPRINT, a generalized, rapid and scalable approach to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes cell-free systems to express DNA methyltransferases from the bacterial host’s restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the host DNA’s methylation pattern, circumventing restriction and enhancing transformation. Unlike established approaches, IMPRINT can be completed in under one day, readily accommodates all methyltransferase types, and avoids methylation-induced cytotoxicity. With IMPRINT, we efficiently multiplex DNA methylation and maximize plasmid transformation in gram-negative and gram-positive bacteria. We also developed a high-throughput pipeline that identifies the most consequential methyltransferases in one transformation. Overall, IMPRINT can vastly enhance DNA transformation, enabling use of increasingly sophisticated genetic manipulation tools across the bacterial world.

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A cell-free pipeline for recreating methylation patterns radically enhances DNA transformation in bacteria

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DNA methylation patterns separate senescence from malignant transformation potential in human cells [methylation]

Project description:Replicative senescent cells reportedly share similar DNA methylation changes with cancer cells, which are purported to facilitate tumorigenesis in cells bypassing senescence. However, we now report biologically critical and distinct patterns of DNA methylation evolution between replicative and oncogene-induced senescence and transformation in a classic human cell transformation model. While overall DNA methylation losses and gains occur in both replicative senescent and transformed cells, the patterns evolve more programmatically for the former and stochastically for the latter. Oncogene-induced senescence is an acute process involving minimal changes to DNA methylation. The stochastic DNA methylation alterations in transformation mainly involve a set of pro-survival promoter CpG-island methylation events targeting genes controlling development and differentiation processes, while the senescence-specific promoter changes occur in genes involved in positive regulation of cellular biosynthesis and macromolecular metabolism. The above set of pro-survival promoter CpG-island hypermethylation events, but not the senescence-specific events, are prone to occur in primary tumors and aging tissues. Importantly, cells manifest senescence-specific epigenomic patterns very early during commitment to senescence, and while these “near-senescent” cells can be immortalized, they are refractory to transformation by H-Ras oncoprotein (H-rasV12). The senescence-specific methylation is retained during immortalization and transformation attempts, suggesting it does not function to promote tumorigenesis. Thus, abnormalities in cancer-related methylation has their origins in aging and not replicative senescence, and senescence-associated methylation potentially prevents malignant transformation.

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