Since the discovery of the nucleotide 5-hydroxymethylcytosine (5hmC) scientists have been trying to figure out what it does and how it shapes the way cells differentiate and develop. To do so, they first need to determine where 5hmC sits in segments of DNA. Now, a team of researchers from the University of Cambridge and the Babraham Institute has developed a technique to map the nucleotide and its relative, 5-methylcytosine (5mC), at individual sites within a genome.
"This is the first method to quantitatively sequence 5hmC and 5mC at single-base resolution," said study author and Cambridge chemist Shankar Balasubramanian. He and his colleagues describe the process in an April 26 Science Express article (1).
The relationship between 5hmC and 5mC is a recent discovery, one that may influence how cell environments, or epigenetic factors, control and shape the way stem cells differentiate and develop. Because of this importance, the nucleotides, and the ability to map them on a genome, are of "clear importance to cancer, neurobiology, and indeed other aspects of biology and medicine," said Balasubramanian.
While bisulfite sequencing is the current standard for identifying 5mC in a genome, the process alone cannot differentiate between 5mC and 5hmC. So Balasubramanian and colleagues looked for chemical reactions that would change one of the nucleotides so that it would be read as a different type of molecule during sequencing.
To test how well the oxidation reaction targeted only 5hmC, the scientists exposed three synthetic, double-stranded DNAs made of either cytosine, 5mC, or 5hmC to potassium perruthenate (KRuO4) and then running the strands through bisulfate sequencing. The experiments showed that 5mC and cytosine nucleotides did not convert to uracil, but the 5hmC residues did turn into uracil.
Next, the team used the oxidation process to target 5hmC in the genomic DNA of mouse embryonic stem cells. Using the oxidation followed by bisulfite sequencing process, the team quantitatively mapped 5hmC with “high precision,” said Balasubramanian. By comparing the results with standard bisulfite sequencing data, the team pinpointed the exact locations of 5hmC and 5mC.
"The method is relatively simple and straightforward to implement, and it is also compatible with all sequencing platforms," said Balasubramanian. "The main cost will still be the actual sequencing, assuming it is whole genome scale.”
However, because scientists are just in the preliminary stages of their investigation into 5hmC, applications to stem cell and regenerative medicine research will not be immediate. "In due course, the field will advance to fully understand the mechanistic role of 5hmC in all these areas, and I expect the outcomes will help inform us of how this new information can usefully be applied to various areas of medicine," he said.
1. Booth, M. et. al. 2012. Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution. Science Express. 1-4.