NEW YORK – In a study published in Science Advances on Wednesday, researchers at Linköping University in Sweden revealed that previous studies showing the presence of a bacterial epigenetic mark in mammalian cells were most likely contaminated, and that there is no sufficient evidence to prove the presence of this modification in mammals.
N6-methyladenine (6mdA) is a widespread DNA modification in bacteria, but it has also recently been characterized in mammalian DNA. However, the researchers wrote, studies have disagreed on 6mdA profiles and measurements of abundance, even when performed on DNA from identical mammalian cell types.
To further elucidate the role of 6mdA in lineage specification in mammals, the researchers leveraged the ability to differentiate human naïve T helper (NTH) cells into TH cell subsets in vitro. But in contrast to previous studies, they found no global changes in 6mdA abundance during TH differentiation, and observed that 6mdA levels in differentiating T cells were identical to that of unmethylated whole-genome-amplified DNA, suggesting that 6mdA was not present at detectable levels in human T cells.
They also analyzed 6mdA in DNA isolated from six human cell lines, and identified two cell lines with 6mdA levels above what was considered background noise. Unexpectedly, two separate cultures of the 293T cell line had different 6mdA levels, suggesting that the 6mdA signal the researchers observed was not intrinsic to the cell type itself. Indeed, when they looked further, they found that the two cell lines with elevated 6mdA levels were contaminated with Mycoplasma spp., a common bacterial cell culture contaminant rich in 6mdA. When they treated the cell lines with the mycoplasma-specific antibiotic Plasmocin, the 6mdA signal was reduced to that of the negative WGA control.
"We realized that the 6mdA 'signals' detected by these techniques were simply noise. However, as a consequence of several complex technical problems the background noise in several of the methods was not random, but appeared to be a true signal," senior author Colm Nestor said in a statement.
The researchers analyzed previous efforts to map or detect 6mdA in mammalian cells by DNA immunoprecipitation sequencing, by mass spectrometry, and by single-molecule real-time sequencing. Each method was found to have limitations or flaws that returned high false-positive discovery rates for the epigenetic mark, seeming to show evidence of a phenomenon that the Linköping researchers believe does not exist.
"We show that a combination of RNA and bacterial contaminations, antibody cross-reactivity, and other technical limitations have resulted in the repeated misidentification of 6mdA in mammalian DNA," the authors wrote. "As the family of DNA and RNA modifications continues to grow and the abundance of previously unknown modifications becomes increasingly rare, we highlight the potential for false discovery even with the use of multiple complementary approaches. We suggest the use of modified and unmodified genomic DNA standards as a minimum requirement in future studies of rare DNA modifications, as well as more thorough validation of antibody specificity before their use."
Researchers must be very careful of what methods they choose to use when studying very rare phenomena, and consider whether they're truly measuring something that exists, Nestor added. "Now we can say without a doubt that 6mdA is not present in mammals", he said. "With respect to 6mdA, a lot of time and money can be saved, and a lot of disappointment avoided, if researchers stop studying something that is simply not there."