“In the Groth lab we have know-how in replication and the Hajkova lab has know-how in studying DNA methylation by mass spectrometry. One of the most crucial epigenetic regulators is DNA methylation– a chemical marker which turns off regions of the genome that ought to not be expressed. The pattern of these markers is very important in maintaining a cells stability and identity: for example, DNA methylation in a liver cell will differ from the DNA methylation pattern in a blood cell.
Utilizing iDEMS, the researchers discovered that DNA methylation levels increase gradually after replication, and after 4 hours the levels on reproduced DNA and the genomic DNA were equal. We saw absolute metrology of the levels of DNA methylation, enabling us to differentiate which methylation marks were newly developed.
The new technique is called iDEMS (isolation of DNA by EdU labeling for Mass Spectrometry).
A current technical report in Nature Cell Biology has presented an unique method to analyze particular modifications in DNA following duplication. Researchers have actually devised a highly delicate, quantitative method using mass spectrometry known as iDEMS (isolation of DNA by EdU labeling for Mass Spectrometry).
” The novelty in our work is that we didnt use sequencing methods extensively utilized in this field, rather we used mass spectrometry, which is the very first time this technique has actually been utilized to determine DNA modifications on purified, replicated DNA,” states Dr. Stewart-Morgan, co-first author of the report, from the Groth laboratory at the Novo Nordisk Foundation Center for Protein Research (CPR) at the University of Copenhagen.
“In the Groth laboratory we have competence in replication and the Hajkova laboratory has knowledge in studying DNA methylation by mass spectrometry. “The outcomes of our research using iDEMS are open and conclusive brand-new avenues for future research.”
DNA adjustments and cell stability
The genome is the whole set of DNA instructions found in a cell. Virtually all cells in an organism include the same genetic details– however which genes are revealed is based on the cells function. This cell-specific gene expression is regulated by the cells epigenome, which includes proteins bound to DNA, as well as direct chemical modifications to DNA. Among the most crucial epigenetic regulators is DNA methylation– a chemical marker which shuts off regions of the genome that need to not be expressed. The pattern of these markers is very important in keeping a cells stability and identity: for example, DNA methylation in a liver cell will differ from the DNA methylation pattern in a blood cell.
When DNA is reproduced throughout cell department, the epigenetic marks connected with the DNA, including DNA methylation, are watered down. The freshly developed DNA strands need to re-establish the level and pattern of methylation to preserve control of gene expression, genomic stability, and the epigenetic memory of the cells identity.
Nevertheless, much about this procedure is unidentified, and loss of DNA methylation is a typical function in cells that have actually divided lot of times, such as cancer cells which are aged and very proliferative cells that have actually duplicated often times over the course of an individuals life-span. Over the last few years numerous groups have actually tried to examine this procedure using sequencing methods, nevertheless, the exact kinetics of post-replicative methylation maintenance remained unclear.
Methylation re-establishment
Utilizing iDEMS, the researchers found that DNA methylation levels increase steadily after duplication, and after 4 hours the levels on replicated DNA and the genomic DNA were equal. This indicates that this process continues at a steady, slow rate. However, it is outmatched by cellular division.
” Over time cells dont have long enough to re-establish their methylation after duplication, and the methylation of the genome is eventually diluted. This is the very first time extremely clear kinetics for methylation re-establishment have actually been shown. We saw absolute metrology of the levels of DNA methylation, allowing us to identify which methylation marks were newly developed. This gave us confidence in our kinetic measurements,” Dr. Stewart-Morgan reports.
A 2nd chemical marker
The researchers likewise used iDEMS to study a 2nd marker– DNA hydroxymethylation– which is a much rarer genomic marker than methylation. Their outcomes proven earlier research study, says Dr Stewart-Morgan: “We found that one DNA hair, the template or adult strand, always has more hydroxymethylation than the other child hair, supporting earlier work which indicated that this marker differentiates DNA strands based on age,” she states.
” However, we likewise found that there is no point at which the levels of hydroxymethylation are equivalent between the adult and daughter hairs throughout the cell cycle. This opens new concerns about how this distinction in between strands might be utilized by cells, for instance throughout DNA repair work.”
The potential of iDEMS
By straight quantifying DNA modifications on reproduced DNA, iDEMS solves DNA methylation and hydroxymethylation kinetics following DNA replication. “iDEMS is a useful and vibrant tool for attending to crucial questions in epigenome maintenance and DNA adjustment biology,” Dr. Stewart-Morgan says.
Seeking to the future, iDEMS will work in profiling methylation and hydroxymethylation dynamics in various cellular contexts, including aging and cancer advancement. Compared to sequencing data, mass spectrometry offers a simple, quick readout, and iDEMS might therefore be helpful where effectiveness is crucial, such as in medical settings and drug discovery research studies.
” Our outcomes highlight how crucial brand-new approaches are for understanding biology through more than one lens. iDEMS is extremely flexible, as it can be combined with other recognized techniques used in molecular biology to look at the epigenome. This technique, for that reason, adds an essential tool to the suite of innovations examining epigenome stability,” concludes Dr. Stewart-Morgan.
Reference: “Quantifying propagation of DNA methylation and hydroxymethylation with iDEMS” by Kathleen R. Stewart-Morgan, Cristina E. Requena, Valentin Flury, Qian Du, Zoe Heckhausen, Petra Hajkova and Anja Groth, 12 January 2023, Nature Cell Biology.DOI: 10.1038/ s41556-022-01048-x.
By University of Copenhagen – The Faculty of Health and Medical Sciences
February 14, 2023
