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Unraveling DNA Methylation Patterns Eases Introduction of Foreign DNA Into Host Cells

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DNA methylation, an epigenetic mechanism that involves the addition of a methyl group to DNA, regulates gene expression, allows cells to distinguish between native DNA and foreign, "alien" DNA, and marks old DNA strands during replication. Methylation is catalyzed by a group of enzymes known as methyltransferases, which strategically decorate DNA with methyl groups in specific patterns.

When analyzing methylation patterns, it has proven difficult for scientists to address which enzyme is the artist behind each specific pattern. Now, researchers from The Novo Nordisk Foundation Center for Biosustainability (DTU Biosustain) have successfully coupled enzymes with specific methylation patterns in two strains of bacteria, Moorella thermoacetica (M. thermoacetica) and Acetobacterium woodii (A. woodii). Their findings are published in the journal Nature Communications

Foreign DNA can't hide

Methylation patterns help a cell to identify DNA that is considered foreign to its own DNA. This can be problematic for scientists when they are actively choosing to insert foreign DNA into a host organism. For example, in the development of biopharmaceuticals, a host cell (typically a bacterium or yeast) is often used as a "factory" to produce a molecule or compound that it wouldn't synthesize naturally, such as a medicine.

In many cases, the host cell will reject the inserted DNA and destroy it, as the methylation patterns on the DNA highlight the fact it is foreign.

Thus, it is advantageous for researchers to know precisely which methyltransferases are producing specific methylation patterns, as Torbjørn Ølshøj Jensen from DTU Biosustain explains: "With this knowledge, you can construct model organisms with artificial methylomes, mimicking the methylation pattern of the strain you want to introduce DNA to. In this way you can ensure "survival" of introduced DNA."

He continues: "Working in other bacteria than Escherichia coli, you often have to do a lot of trial and error when it comes to DNA transformation, but that's just not good enough. You need knowledge and tools. With this, you have a systematic and rational way of fixing the problems."

Creating a "library" of enzyme-to-motif couplings

The researchers created plasmids that contained one of the methyltransferases and "cassettes" that hold multiple copies of certain DNA patterns, known as motifs. These motifs are the targets for methyltransferases, and so by coupling the two together, the methyltransferase expressed by the plasmid marks the DNA in a specific way, and voila, reveals the enzyme's methylation pattern.

The team repeated this for all methyltransferases, and the plasmids were sequenced to reveal the methyl groups. As a result, they created a library of specific enzyme-to-motif couplings.

The scientists then chose to validate their method, named "MetMap" by analyzing the genomes of two temperature-resistant bacteria, M. thermoacetica and A. woodii. These bacteria were chosen specifically on the basis that they hold great promise for industrial applications.

The two bacterial species possess 23 methyltransferase genes in total, but only demonstrate modifications on 12 different DNA-motifs; meaning that not all the methyltransferases are active. The researchers were able to couple 11 of the 12 motifs with specific methyltransferase genes.

Designing hosts with an unambiguous "methylome"

Using this relatively quick method of identifying methylation patterns, the scientists look to design hosts that have an "unambiguous methylome". This would simplify the introduction of foreign DNA, as the host organism would only possess wanted methyltransferases. The authors anticipate that their method will be useful both when building cell factories using more novel, less well-researched hosts, and in the study of gene expression regulation and cell differentiation. 

Reference: Jensen et al. 2019. Genome-wide systematic identification of methyltransferase recognition and modification patterns. Nature Communications. DOI: https://doi.org/10.1038/s41467-019-11179-9.