Multi-species genome-wide 6mA distribution and possible sources

Since the rediscovery of the N6-deoxyadenosine (6mA) modification on eukaryotic DNA, researchers are excited about
whether it can become a new epigenetic marker.
As research progresses and new methods are developed, researchers claim that 6mA is present in a variety of eukaryotes, including plants, vertebrates, and even mammals
.
However, the shortcomings of existing methods limit the precise positioning and functional interpretation
of 6mA.
In recent years, there has been widespread debate
about whether 6mA is present in mammals and whether it can become a new genetic marker.
Therefore, it is critical to develop a sensitive and reliable detection method to determine the presence of 6 mA and the precise localization of true 6 mA sites in the genome, especially for organisms
with very low 6 mA levels.

Professor Luo Guanzheng of our institute has been focusing on epigenetics-related research for a long time, and his preliminary work has found that 6mA is a possible new epigenetic marker in eukaryotes, and summarized and prospected
its potential biological functions in higher organisms 。 Recently, Professor Luo Guanzheng's team reported a new genome-wide 6mA detection method MM-seq (Modification-induced Mismatch Sequencing) based on a new detection principle, and used this method to evaluate the genomic position of 6mA in bacteria, green algae and mammalian cells, revealing that different human cell types have very limited 6mA loci, which are scattered on
different genomic mapping 。 After knocking out the RNA m6A methyltransferase METTL3, the detectable 6 mA site in genomic DNA is further reduced
.

The study found that in double-stranded DNA, 6mA-specific antibodies can recognize modified adenine
embedded in the DNA double-strand.
After studying the chemistry of 6mA, the researchers speculated that the presence of 6mA weakened the accumulation of bases and made local DNA double-strands unstable
.
The researchers demonstrated by potassium permanganate footprint that N6-deoxyadenosine on double-stranded DNA promotes the production of mismatch-like structures, possibly through DNA respiration or base flipping
.
This unique mismatch-like structure allows 6mA to be captured by 6mA-specific antibodies and subsequently recognized
by specific nucleases or chemical lysis reactions.
Subsequently, a new 6mA positioning method MM-seq (modified-induced Mismatch Sequencing)
was developed based on this principle.

Given the high accuracy of MM-seq, the researchers then applied the method to
mammalian cells with low 6mA levels or undetectable by conventional sequencing methods.
It was found that a variety of human cell types contained very limited 6mA sites on their genomic DNA, which were randomly distributed in different genomic locations, and the reproducibility of sites in different cells was extremely low
.
Consider that although these single-base 6mA sites are very rare, these limited sites may be enriched in specific genomic regions and play an important regulatory role
.
Therefore, whether the 6mA site has a tendency to aggregate in specific regions, especially on
repeat elements (REs) of genomic or mitochondrial DNA, is further investigated.
It was found that the 6mA peak identified by traditional IP was enriched at REs, however, the site identified by MM-seq was not enriched here, suggesting that the 6mA enrichment in duplicate elements found in the previous study was probably caused by false positives of the method
.

In view of the characteristics of extremely low 6mA content in mammals, recent studies have proposed the idea that
6mA is randomly integrated into DNA by DNA polymerase due to digestion and metabolism of modified bases on RNA.
To test this idea, the researchers further knocked out the RNA m6A methyltransferase METTL3 before performing MM-seq detection
.
The results showed a further reduction in detectable 6 mA sites in genomic DNA, and the findings support that the limited 6 mA in the mammalian genome may be derived from metabolic byproducts
of RNA m6A.

Taken together, the study not only provides a completely new way to comprehensively and accurately map the distribution of 6 mA across multiple species, but will also deepen the understanding
of rare DNA modifications 。 Recently, the research results High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome published in the journal Cell Discovery, Dr.
Chen Liqian (now a postdoctoral fellow of Guangdong Provincial People's Hospital) and Associate Professor Zhang Zhang as co-first authors, Professor Luo Guanzheng as the corresponding author, and team members Hongxuan Chen, Jianfei Xi, Liu Xuehong, Ma Dongxiao, Zhong Yuhao, Huang Wenhui and Chen Tao participated in the work
.
Daniel W.
Mak of the University of Hong Kong, Chen Qi and Chen Yaoqing of the School of Public Health of Sun Yat-sen University (Shenzhen) contributed to the paper
.
The method involved in this article has been patented
for invention.

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