NEW YORK (GenomeWeb) – Researchers have uncovered a type of DNA modification in the human genome previously thought to be absent from eukaryotes.
DNA N6-methyladenine (6mA) modification is common among prokaryotes, and though it has recently been found among some eukaryotes, it had never before been found in humans.
Now, however, a team led by researchers at the Third Affiliated Hospital of Guangzhou Medical University has found 6mA modifications within the human genome, especially the mitochondrial genome, using Pacific Biosciences sequencing data. As they reported today in Molecular Cell, the researchers also found that decreased 6mA genomic modification promotes tumorigenesis.
"The discovery of 6mA in human cells sheds light on epigenetic regulation during human diseases such as cancer," Guangzhou's Guang-Rong Yan and his colleagues wrote in their paper.
Using PacBio sequencing data from the HuaXia1 human genome, the researchers uncovered 881,240 6mA modification sites. These modifications covered about 0.051 percent of the adenines in the human genome — less than what has been found in some fungi and C. elegans, but similar to what has been observed in Drosophila and pigs.
They validated their finding of this type of DNA modification using both 6mA immunoprecipitation sequencing and liquid chromatography-tandem mass spectrometry. The 6mA sites uncovered by 6mA-IP seq overlapped with those found through SMRT sequencing, and the ratio of modified adenosines uncovered by LC-MS/MS was similar to the SMRT sequencing results.
Altogether, this suggested to the researchers that 6mA modification is indeed present in the human genome.
In particular, they noted that 6mA modification density on the sex chromosomes was about half that of the autosomes, affecting 0.023 percent of sites on the X chromosome and 0.024 percent on the Y chromosome. The mitochondrial genome, meanwhile had a higher density of 6mA modifications.
The researchers also noted 6mA modifications were enriched within exonic regions — especially of actively transcribed genes — and tend to occur at [G/C]AGG[C/T] motifs.
Through in vitro and in vivo assays, the researchers also found N6AMT1 to be the methyltransferase responsible for the 6mA modification in the human genome, and ALKBH1 to be its demethyltransferase.
In addition, Yan and his colleagues compared genomic 6mA modification levels in human cancers and matched normal tissues, and found that both genomic 6mA and N6AMT1 methyltransferase levels were downregulated in tumor tissue. At the same time, ALKBH1 demethyltransferase levels were upregulated.
In a set of human gastric and liver cancer tissues, the researchers found N6AMT1 — but not ALKBH1 — gene copy number to be decreased, as compared to normal tissues. This suggested to the researchers that its downregulation could be due to gene loss in cancer cells. Reduced 6mA levels in liver cancer were associated with tumor size, histological grade, AFP level, tumor recurrence, and disease stage.
Further, silencing N6AMT1 in cancer cells promoted tumorigenic behaviors, while silencing ALKBH1 inhibited cancer cell growth, colony formation, and more, the researchers reported. Overexpressing ALKBH1 then reversed those effects, while overexpressing N6AMT1 inhibited cancer cell growth.
"These data indicated that genomic DNA 6mA modification plays a crucial role in human diseases such as cancer," the authors wrote.
Yan and his colleagues also noted that as DNA 5mC methylation is being explored as a cancer biomarker that can be assessed via liquid biopsy, so too could DNA 6mA modifications represent biomarkers for cancer diagnosis and therapy.