[tt] New nucleotide could revolutionize epigenetics | Science Blog

[Brian Atkins]

http://www.scienceblog.com/cms/new-nucleotide-could-revolutionize-epigenetics-20334.html

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.'

It 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."

What this nucleotide does is not yet clear. Initial tests suggested that it may 
play a role in demethylating DNA, but Kriaucionis and Heintz believe it may have 
a positive role in regulating gene expression as well. The reason that this 
nucleotide had not been seen before, the researchers say, is because of the 
methodologies used in most epigenetic experiments. Typically, scientists use a 
procedure called bisulfite sequencing to identify the sites of DNA methylation. 
But this test cannot distinguish between 5-hydroxymethylcytosine and 
5-methylcytosine, a shortcoming that has kept the newly discovered nucleotide 
hidden for years, the researchers say. Its discovery may force investigators to 
revisit earlier work. The Human Epigenome Project, for example, is in the 
process of mapping all of the sites of methylation using bisulfite sequencing. 
"If it turns out in the future 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," says Kriaucionis.

Providing further evidence for their case that 5-hydroxymethylcytosine is a 
serious epigenetic player, a second paper to be published in Science by an 
independent group at Harvard reveals the discovery of genes that produce enzymes 
that specifically convert 5-methylcytosine into 5-hydroxymethylcytosine. These 
enzymes may work in a way analogous to DNA methyltransferase, suggesting a 
dynamic system for regulating gene expression through 5-hydroxymethylcytosine. 
Kriaucionis and Heintz did not know of the other group's work, led by Anjana 
Rao, until earlier this month. "You look at our result, and the beautiful 
studies of the enzymology by Dr. Rao's group, and realize that you are at the 
tip of an iceberg of interesting biology and experimentation," says Heintz, a 
neuroscientist whose research has not focused on epigenetics in the past. "This 
finding of an enzyme that can convert 5-methylcytosine to 
5-hydroxymethylcytosine establishes this new epigenetic mark as a central player 
in the field."

Kriaucionis is now mapping the sites where 5-hydroxymethylcytosine is present in 
the genome, and the researchers plan to genetically modify mice to under- or 
overexpress the newfound nucleotide in specific cell types in order to study its 
effects. "This is a major discovery in the field, and it is certain to be tied 
to neural function in a way that we can decipher," Heintz says.

-- 
Brian Atkins
Singularity Institute for Artificial Intelligence
http://www.singinst.org/