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New Study Uses SMRT-ChIP Method to Find Novel Methylation in Mouse Embryonic Stem Cells

Wednesday, March 30, 2016

In a new Nature publication, scientists from Yale and other institutions report the discovery of N6-methyladenine (N6-mA) in mouse embryonic stem cells (ESCs), contrary to the conventional wisdom that the only form of methylation in mammals is 5-methylcytosine. Through the project, the team also developed a new method for pairing chromatin immunoprecipitation (ChIP) with SMRT Sequencing. Both of these developments have significant implications for the genomics community.

DNA methylation on N6-adenine in mammalian embryonic stem cells” comes from lead author Tao Wu and senior author Andrew Xiao, both at Yale School of Medicine. The team also included collaborators from the University of Arkansas for Medical Sciences, the University of North Carolina, the Icahn School of Medicine at Mount Sinai, and PacBio. “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,” Wu et al. write.

To conduct this study, the team developed a SMRT-ChIP method to study DNA modifications at specific histone variant regions. The SMRT Sequencing data demonstrate the presence of N6-mA at nearly 400 sites in the genomic regions studied, a finding that was confirmed with mass spec analysis. The team focused on the H2A.X deposition, which has been associated with cell fate transitions, sequencing the enriched, unamplified DNA from those regions. They also compared SMRT-ChIP results to those from DIP-seq, an orthogonal method, and found strong concordance.

The scientists identified Alkbh1 as the demethylase that regulates adenine methylation and went on to create cell lines with this gene knocked out, showing that N6-mA levels increased by a significant degree without the demethylase. The team also used Alkbh1 to shed light on how these N6-mA sites function; in the knockout cells, the expression of 550 genes was downregulated compared to the original cell line. That contrasts with other recent discoveries of N6-mA in organisms including C. elegans and D. melanogaster.  “Intriguingly, [those] studies implicated N6-mA in gene activation, instead of repression, as is the case for 5mC repression,” the scientists write.

They also report a strong location bias for the methylated sites, with the greatest enrichment on the X chromosome. “N6-methyladenine deposition is inversely correlated with the evolutionary age of LINE-1 transposons; its deposition is strongly enriched at young (<1.5 million years old) but not old (>6 million years old) L1 elements,” the authors write, noting that young L1s are important in the beginning of embryogenesis. “We favour the view that N6-mA-mediated silencing plays an important role in safeguarding active L1 elements in mammalian genomes. The levels of N6-mA are controlled precisely by Alkbh1 in ES cells such that they favour L1 transcription while preventing it from succumbing to overactivation and genomic instability.”

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