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Rockefeller Researchers Identify Sixth Nucleotide

NEW YORK (GenomeWeb News) – Two researchers from Rockefeller University have identified a new nucleotide, according to a paper appearing in the online, advanced issue of Science yesterday.

While evaluating 5-methylcytosine levels in two types of mouse brain cells, the team detected a nucleotide that they could not identify. When they looked more closely at this nucleotide using thin layer chromatography, high pressure liquid chromatography, mass spectrometry, and other approaches, the researchers discovered that they were dealing with 5-hydroxymethylcytosine, a form of methylated cytosine found stably in bacterial viruses.

Their subsequent experiments suggest the nucleotide is enriched in brain cells but apparently absent from several other cell types. Based on these findings, the researchers speculated that 5-hydroxymethylcytosine may contribute to epigenetic regulation, particularly in neurons.

"This is another mechanism for regulation of gene expression and nuclear structure that no one has had any insight into," senior author Nathaniel Heintz, a Howard Hughes Medical Institute investigator and molecular biologist at Rockefeller University, said in a statement. "The results are discrete and crystalline clear; there is no uncertainty. I think this finding will electrify the field of epigenetics."

Along with the four best known DNA bases, researchers have known about a modified form of cytosine — 5-methylcytosine — for some time. Sometimes referred to as the fifth nucleotide, 5-methylcytosine often contributes to epigenetic regulation in cells. The new paper suggests that there is actually another stable methylated cytosine, 5-hydroxymethylcytosine.

The duo identified this new nucleotide serendipitously while evaluating 5-methylcytosine levels in two types of mouse brain cells: a type of small neurons called granule cells and large neurons called Purkinje cells. "We didn't go looking for this modification," lead author Skirmantas Kriaucionis, a post-doctoral associate in Heintz's lab, said in a statement. "We just found it."

The first clue came when the duo looked 5-methylcytosine levels in nuclei isolated from mouse Purkinje and granule cells. The amount of 5-methylcytosine was lower than expected in the Purkinje cells. Instead, their TLC analysis revealed an unknown spot subsequently shown to be 5-hydroxymethylcytosine.

5-hydroxymethylcytosine levels were not the same in all cell types tested. About 40 percent of methylated cytosines in Purkinje cells were 5-hydroxymethylcytosine. In granule cells, the new nucleotide accounted for just 10 percent of methylated cytosine. And whereas the new nucleotide accounts for roughly 0.6 percent of bases in Purkinje cells, it represents just 0.2 percent of nucleotides in granule cells and was not found at all in four mouse and human cell lines.

Overall, the researchers reported that 5-hydroxymethylcytosine was enriched in brain tissues tested, occurring most often in cortex and brainstem areas. Consequently, the researchers speculated that it may contribute to epigenetic regulation of neurons.

"It is notable that [5-hydroxymethylcytosine] is nearly 40 percent as abundant as [5-methylcytosine] in Purkinje cell DNA," Kriaucionis and Heintz concluded. "Given the critical role of [5-methylcytosine] in epigenetic regulation of the genome, we believe that [5-hydroxymethylcytosine] may also have an important biological role in vivo."

A separate paper, also appearing online in Science yesterday, suggests an enzyme called TET1 converts 5-methylcytosine to 5-hydroxymethylcytosine in mammalian cells.

A paper from the early 1970s hinted that 5-hydroxymethylcytosine could be present in mammalian genomes. But those initial results could not be replicated. In addition, methods currently used for evaluating methylation patterns may miss the nucleotide, the researchers noted, explaining that bisulfite treatment seems to convert 5-hydroxymethylcytosine to a chemical structure resembling the better known 5-methylcytosine after, which means it is easily missed by bisulfite sequencing methods.

That may be important for large-scale epigenetic studies such as the Human Epigenome Project, Kriaucionis cautioned. "If it turns out that [5-hydroxymethylcytosine and 5-methylcytosine] have different stable biological meanings, which we believe very likely, then epigenome mapping experiments will have to be repeated with the help of new tools that would distinguish the two," he said.

Kriaucionis has already started mapping 5-hydroxymethylcytosine in the genome, and down the road the researchers plan to look at the effects of increasing or decreasing 5-hydroxymethylcytosine levels in mice.