A unique approach to studying changes in chemical markers on DNA

A new way to study Specific changes in DNA after replication have been published as a technical report in Nature Cell Biology. The researchers developed a highly sensitive mass spectrometry-based approach, called iDEMS (DNA Isolation by EdU Labeling for Mass Spectrometry).

says d. Stuart Morgan, co-first author of the report, is from Groth’s lab at the Novo Nordisk Foundation Center for Protein Research (CPR) at the University of Copenhagen.

This unique approach is the result of a joint project with Hajkova’s lab at the MRC London Institute of Medical Sciences (LMS). “In Groth’s lab we have expertise in replication and Hajkova’s lab has expertise in studying DNA methylation by mass spectrometry. I think this interdisciplinary collaboration is a big part of the reason the project is so successful,” explains Dr. Stewart Morgan. “The results of our research using iDEMS are definitive and open up new avenues for future research.”

DNA modifications and cell stability

The genome is the complete set of DNA instructions found in a cell. Almost all cells of an organism contain the same genetic information – but which genes are expressed depends on the function of the cell. This cell-specific gene expression is regulated by the cell’s epigenome, which consists of DNA-binding proteins, as well as direct chemical modifications to the DNA. One of the most important regulators of epigenetic inheritance is DNA methylation – a chemical marker that turns off regions of the genome that should not be expressed. The pattern of these markers is very important in maintaining cell stability and identity: for example, the DNA methylation pattern in a liver cell will differ from the DNA methylation pattern in a blood cell.

When DNA is transcribed during cell division, epigenetic marks associated with DNA, including DNA methylation, are attenuated. Newly generated DNA strands need to re-establish the level and pattern of methylation to maintain control over gene expression, genomic stability, and epigenetic memory of cell identity.

However, much about this process is not known, and loss of DNA methylation is a common feature in cells that have divided many times, such as highly proliferating cancer cells and senescent cells that have multiplied many times over the course of a person’s life. In recent years, several groups have attempted to investigate this process using sequencing methods, however the exact kinetics of methylation maintenance after replication has remained unclear.

Re-methylation

Using iDEMS, the researchers found that DNA methylation levels increased steadily after replication, and after 4 hours the levels in replication DNA and genomic DNA were equal. This indicates that this process is proceeding at a steady and slow pace. However, it is bypassed by cell division.

“Over time, cells do not have enough time to re-establish their methylation after replication, and genome methylation is eventually attenuated. This is the first time that very clear kinetics of re-methylation have been shown. Moreover, we saw an absolute quantification of DNA methylation levels, which enables us to distinguish between newly generated methylation markers. This gave us confidence in our kinetic measurements,” Stuart Morgan reports.

A second chemical sign

The researchers also used iDEMS to study a second marker — DNA hydroxymethylation — which is a genomic marker that is much rarer than methylation. Their results confirmed previous research, says Stuart Morgan: “We found that one strand of DNA, the ‘paternal’ template or strand, always contained more hydroxymethylation than the other ‘daughter’ strand, supporting previous work, suggesting that this marker DNA distinguishes strands based on age,” she says.

“However, we also discovered that there is no point at which hydroxymethylation levels are equal between parental and daughter strands throughout the cell cycle. This opens up new questions about how cells use this difference between strands, for example during DNA repair.”

iDEMS capabilities

By direct quantification of DNA modifications on replicating DNA, iDEMS resolves DNA methylation and hydroxymethylation kinetics after DNA replication. “iDEMS is a dynamic and informative tool for addressing important questions in epigenome maintenance and DNA modification biology,” says Stewart Morgan.

Looking in the future, the iDEMS system will be useful for characterizing methylation and hydroxymethylation-binding dynamics in different cellular contexts, including aging and cancer development. Compared to sequencing data, mass spectrometry provides a simple and rapid readout, and thus iDEMS can be useful where efficiency is key, such as in medical settings and drug discovery studies.

“Our results highlight how important new approaches are to understanding biology through more than one lens. iDEMS is very flexible, as it can be combined with other methods used in molecular biology to look at the epigenome. This method thus adds an important tool to the suite of techniques that are looking at stability of the epigenome,” concludes Stuart Morgan.

– This press release was provided by the University of Copenhagen

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