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SMRT Sequencing Contributes to Detection of DNA Methylation in C. elegans

Tuesday, July 14, 2015

A recent paper in the journal Cell presents novel findings of DNA methylation in C. elegans, an organism previously believed not to have such epigenetic marks. Scientists used several approaches to analyze the adenine N6-methylation (6mA) found in C. elegans, including SMRT® Sequencing to directly observe base modifications across the genome.

From lead authors Eric Greer and Mario Blanco with senior author Yang Shi at Harvard Medical School, “DNA Methylation on N6-Adenine in C. elegans” describes a range of technological methods deployed to assess methylation across the worm’s genome. The team queried the nematode with specific antibodies for 6mA; immunofluorescence; ultra-high-performance liquid chromatography combined with triple-quadrupole tandem mass spectrometry; SMRT Sequencing; and MeDIPseq, an antibody-based immunoprecipitation paired with DNA sequencing.

PacBio® sequencing was chosen for its ability to directly interrogate base modifications without using antibodies. “In this analysis, SMRT sequencing detected 6mA on 225,586 adenines—0.7% of the total adenines in the worm genome—which is equivalent to 0.3% bulk adenine methylation, as some adenines were methylated 10% of the time, whereas others were methylated 90% of the time,” the authors report. That value supported what was seen in the mass spec analysis. “Similar to the MeDIP-seq results, the SMRT sequencing analysis identified a broad distribution of 6mA across all chromosomes of the worm genome, with no one genomic feature being significantly enriched or depleted for 6mA,” they add.

Beyond finding this evidence of methylation in C. elegans, the team also showed that these marks are heritable, with a modification increasing over several generations. In this project, the scientists found a DNA demethylase as well as a potential methyltransferase. “Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes,” the scientists report. They also found indications of crosstalk between histone methylation and 6mA.

According to the paper, scientists have mostly studied 6mA in prokaryotes. In various organisms, its function has been linked to identifying invasive DNA, influencing gene expression, affecting RNA splicing, and more. “At the present time, the molecular function of 6mA is still unclear,” the researchers write. “Our study identifies a new DNA modification in C. elegans, as well as regulators that control the dynamics of this modification, and advances 6mA as a potential carrier of non-genetic information across generations.”

This paper is one of three in the Cell issue focused on the presence of m6A in eukaryotes; the other two demonstrate the presence of this form of methylation in Chlamydomonas reinhardtii and Drosophila melanogaster. For a nice perspective on the overall trend of newly detected m6A, check out “An Adenine Code for DNA: A Second Life for N6-Methyladenine” from the same issue.

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