Anyone who studied a little genetics in high school has heard of adenine, thymine, guanine and cytosine - the A,T,G and C that make up the DNA code.
But those are not the whole story. The rise of epigenetics in the past decade has drawn attention to a fifth nucleotide, 5-methylcytosine (5-mC), that sometimes replaces cytosine in the famous DNA double helix to regulate which genes are expressed. And now there's a sixth. In experiments to be published online Thursday by Science , researchers reveal an additional character in the mammalian DNA code, opening an entirely new front in epigenetic research.
The work, conducted in Nathaniel Heintz's Laboratory of Molecular Biology at The Rockefeller University, suggests that a new layer of complexity exists between our basic genetic blueprints and the creatures that grow out of them. "This is another mechanism for regulation of gene expression and nuclear structure that no one has had any insight into," says Heintz, who is also a Howard Hughes Medical Institute investigator. "The results are discrete and crystalline and clear; there is no uncertainty. I think this finding will electrify the field of epigenetics."
Genes alone cannot explain the vast differences in complexity among worms, mice, monkeys and humans, all of which have roughly the same amount of genetic material. Scientists have found that these differences arise in part from the dynamic regulation of gene expression rather than the genes themselves. Epigenetics, a relatively young and very hot field in biology, is the study of nongenetic factors that manage this regulation.
One key epigenetic player is DNA methylation, which targets sites where cytosine precedes guanine in the DNA code. An enzyme called DNA methyltransferase affixes a methyl group to cytosine, creating a different but stable nucleotide called 5-methylcytosine. This modification in the promoter region of a gene results in gene silencing.
Some regional DNA methylation occurs in the earliest stages of life, influencing differentiation of embryonic stem cells into the different cell types that constitute the diverse organs, tissues and systems of the body. Recent research has shown, however, that environmental factors and experiences, such as the type of care a rat pup receives from its mother, can also result in methylation patterns and corresponding behaviors that are heritable for several generations. Thousands upon thousands of scientific papers have focused on the role of 5-methylcytosine in development.
The discovery of a new nucleotide may make biologists rethink their approaches to investigating DNA methylation. Ironically, the latest addition to the DNA vocabulary was found by chance during investigations of the level of 5-methylcytosine in the very large nuclei of Purkinje cells, says Skirmantas Kriaucionis, a postdoctoral associate in the Heintz lab, who did the research. "We didn't go looking for this modification," he says. "We just found it."
Kriaucionis was working to compare the levels of 5-methylcytosine in two very different but connected neurons in the mouse brain - Purkinje cells, the largest brain cells, and granule cells, the most numerous and among the smallest. Together, these two types of cells coordinate motor function in the cerebellum. After developing a new method to separate the nuclei of individual cell types from one another, Kriaucionis was analyzing the epigenetic makeup of the cells when he came across substantial amounts of an unexpected and anomalous nucleotide, which he labeled 'x.'
The nucleotide accounted for roughly 40 percent of the methylated cytosine in Purkinje cells and 10 percent in granule neurons. He then performed a series of tests on 'x,' including mass spectrometry, which determines the elemental components of molecules by breaking them down into their constituent parts, charging the particles and measuring their mass-to-charge ratio. He repeated the experiments more than 10 times and came up with the same result: x was 5-hydroxymethylcytosine, a stable nucleotide previously observed only in the simplest of life forms, bacterial viruses. A number of other tests showed that 'x' could not be a byproduct of age, DNA damage during the cell-type isolation procedure or RNA contamination. "It's stable and it's abundant in the mouse and human brain," Kriaucionis says. "It's really exciting."