Sequencing An Important Epigenetic Mark

Because access to DNA is tightly controlled, hair doesn’t grow on bone and toenails don’t appear in the spleen. Control comes from a system that includes chemical modifications to DNA bases, or epigenetic marks, that ensure the right genes are expressed in the right place at the right time. Two widely known chemical modifications are 5-hydroxymethyl­cytosine (5hmC) and 5-methylcytosine (5mC). What they do can now be better understood with the first tool to measure, with single-base resolution, their relative amounts (Science, DOI: 10.1126/science.1220671).

Researchers have known that when cytosine bases are methylated, the gene is silenced. The precise role of 5hmC is still debated, but it’s clear it has enormous biological importance, says Shankar Balasubramanian, a chemical biologist at Cambridge University.

Most researchers believe that 5hmC marks are an epigenetic “on” switch for genes. Others think that 5hmC is an intermediate in so-called active demethylation, which occurs in the first moments after a sperm fertilizes an egg. During this process, cytosine bases on the newly combined DNA are rapidly demethylated to create an embryonic stem cell.

Resolving the debate has been difficult because current genome-sequencing methods can’t distinguish between the two decorated cytosines with single-base resolution, says Skirmantas Kriaucionis, a biochemist at Oxford University. Now, Balasubramanian and colleagues have developed a technique that can do so.

The technique first uses a DNA-sequencing method that doesn’t differentiate the two. In this method, called bisulfite sequencing, both decorated cytosines are measured as cytosine, while naked cytosine bases are recorded as thymine. Next, the team repeats the sequencing after oxidizing 5hmC; this time 5hmC is recorded as uracil instead of cytosine. Because the first sequencing round tabulates both 5hmC and 5mC, while the second sequencing round tabulates only 5mC, the difference provides a quantitative measurement of 5hmC.

The new technique is “an elegant strategy,” says Chuan He, a chemist at the University of Chicago, who says his alternative method for quantitatively sequencing 5hmC will be published soon.