Mapping out DNA’s extra bases

5-methylcytosine (L) and 5-hydroxymethylcytosine (R): the two DNA bases you didn't learn about in high school biology. (Image: Wikimedia Commons)

Adenine and thymine, cytosine and guanine. We all learned the names of the four DNA bases in high school biology class. But, just like the list of planets, the list of bases may not be set in stone.

Over the years, epigenetics researchers have identified two alternate forms of cytosine whose biology differs enough from that of their parent base that may count as fifth and sixth DNA bases. These additional cytosines, called 5-methylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc), each have a group of atoms called a methyl group added onto their central ring, a feature normal cytosine lacks.

Apart from making biology textbook editors very unhappy, these two bases may play unique roles in biology. Adding methyl groups (or methylation “marks”) to cytosines and other components of the genome is a well-known epigenetic mechanism that gives the cell exquisite control of gene activity, which in turn greatly influences how the cell will behave. For instance, patterns of methylation marks on the genes of embryonic stem (ES) cells are linked the cells’ ability to develop into more mature cells. And the genomes of cancer cells often have methylation marks in the wrong places.

To help clarify the specific roles of these two new bases, which could help shed light on the nature of what they do, a team of researchers at Children’s Hospital Boston has developed a pair of methods for mapping the locations of two methylated cytosines within a cell’s genome with almost GPS-like accuracy. The methods – published in Nature by Anjana Rao of the Immune Disease Institute and the Program for Cellular and Molecular Medicine at Children’s (and now also at the La Jolla Institute of Immunology and Allergy in California); graduate student Will Pastor and postdoctoral fellows Utz Pape and Nancy Huang in Rao’s lab; and Suneet Agarwal of the Children’s Division of Hematology and Oncology – use markedly different techniques to achieve the same goal: pinpointing where in the genome 5hmc can be found.

“This is one of several recent papers that try to answer the question, ‘Where is 5hmc?’” says Agarwal. “Ours takes advantage of the chemical properties of this unique mark, coupled with the latest in genome-wide sequencing technologies.”

The new work adds to the growing body of knowledge coming out of the Rao lab on the two new cytosines and genetic regulation. Two years ago, Rao’s team found that a family of enzymes called TET turns 5mc into 5hmc. Since then, her lab has also shown that the TET enzymes help control the fate of embryonic stem cells – that is, whether they turn into muscle, bone, blood, etc. – and that TET mutations in leukemia cells impair the enzymes’ catalytic capabilities.

It also raises more questions about 5mc and 5hmc than it answers. “When found at a gene’s transcription start site, 5mc is associated with low expression of that gene. It isn’t completely clear whether that is causal, but it seems like methylation definitely has a role in silencing genes,” explains Pastor about the mapping project. “So there was a thought that 5hmc would likely have a role in activating genes.

“But what we found doesn’t really fit with that idea,” Pastor continues, “or with the idea that 5hmc is a quick intermediate in genes that are being activated. It’s clearly found reproducibly at certain points in the ES cell genome, and it tends to be found more at genes that are silent or at genes that are being negatively regulated by one of TET enzymes. The fact that you find it at transcription start sites does imply that it has a role in transcription, but it may not have been the role that we initially suspected.”

Agarwal wonders whether 5hmc might help get the genes in ES cells ready to respond to signals that determine their developmental fate. “It’s possible that these marks could help to poise genes in the ES cell to respond quickly to particular signals by turning specific genes on or off,” notes Agarwal. “So that if an ES cell receives signals that encourage it to be a lung cell or a cardiomyocyte, it’s ready to go.”

So from new DNA bases to the TET enzymes to the biology of stem cells and cancer – how do the threads tie together? The picture isn’t yet clear. “The bottom line is that the TET enzymes have a clearly defined enzymatic function,” Rao concludes, “but what exactly they do from a biological point of view is still a little obscure. The data suggest that the role of 5hmc is complex.”

Which should give the textbook editors plenty of time.