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Researchers Identify Methylation Mark Previously Uncharacterized in Mammalian Cells

NEW YORK (GenomeWeb) – Researchers from Yale University have identified a methylation mark that was previously not known to exist in mammalian cells and have shown that it is involved in gene repression.

The team described using single-molecule sequencing on Pacific Biosciences' RSII instrument to identify the methylation modification in a publication in Nature this week.

The epigenetic mark, N6-methyladenine, is known in bacterial genomes, but previously, the only type of DNA methylation thought to be present in mammalian genomes was 5-methylcytosine. Recently, a number of studies have shown that N6-methyladenine also exists in some insects, nematodes, and algae.

To look for epigenetic marks in mammalian genomes, the researchers developed a targeted ChIP-seq approach on the RSII to sequence DNA from mouse embryonic stem cells. They focused the sequencing specifically on histone variant deposition regions, called H2A.X, since such regions are known to be important in epigenetic regulation, and the researchers hypothesized that such regions may be more likely to contain chemical DNA modifications.

The team, led by senior author Andrew Xiao from the department of genetics at Yale, first performed targeted chromatin immunoprecipitation of the H2A.X deposition regions in mouse embryonic stem cells. They next sequenced these regions PCR-free on the RSII and used PacBio's bioinformatics pipeline to identify epigenetic modifications. They identified 398 N6-methyladenine sites deemed high confidence because they had more than 30X sequence coverage and a quality score above 30. They also identified an additional 1,108 sites with more than 25X coverage and quality scores of 20 or above. In addition, they identified DNA motifs other than the H2A.X deposition regions that were significantly associated with some of the N6-mA sites.

Previous research has found that genes in the Alkbh family play a role in demethylation. For instance, proteins encoded by Alkbh2 and Alkbh3 remove 1-methyladenine or 3-methylcytosine from DNA or RNA. For N6-mA removal, the researchers thought that Alkbh1 was "arguably the most intriguing" because Alkbh1 deficiency in mice results in an 80 percent reduction in litter size due to embryonic lethality, "indicating that Alkbh1 plays a critical role in early development."

To investigate this further, the team generated Alkbh1 homozygous knockout embryonic stem cell lines using CRISPR/Cas9 technology. Using mass spectrometry, they confirmed that N6-mA levels were elevated in these cell lines. In addition, they found that expressing wild-type Alkbh1 restored levels of  N6-mA.

They next performed RNA-seq of the Alkbh1 knockout cells, identifying 550 genes that were significantly downregulated, with the strongest effect on genes involved in development and lineage determination, while expression of pluripotency genes was unchanged. In addition, the downregulated genes were mostly located on the X chromosome, suggesting that N6-mA represses transcription on the X chromosome, the authors wrote.

To examine that further, they looked at the expression of evolutionarily young full-length long interspersed element 1 transposons (L1 elements), which are enriched on the X chromosome and known to be controlled by 5mC in mammals. The researchers found that L1 elements were more than 60-fold more repressed on the X chromosome compared to L1 elements on other chromosomes, suggesting that both genes and L1 elements on X chromosomes may be co-regulated by N6-mA.

Interestingly, while N6-mA is implicated in gene activation in other species, the Yale team's results suggest it is involved in transcriptional silencing in mammals.

"The discovery of N6-mA in mammalian ES cells sheds new light on epigenetic regulation during early embryogenesis and may have impacts in the fields of epigenetics, stem cells and developmental biology," the authors concluded.