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July 22, 2011

UNC researchers find variants of Cytosine DNA base that are modifed by Tet Proteins

For decades, scientists have known that DNA consists of four basic units -- adenine, guanine, thymine and cytosine. In recent history, scientists have expanded that list from four to six. Now researchers from the UNC School of Medicine have discovered the seventh and eighth bases of DNA.

These last two bases – called 5-formylcytosine and 5 carboxylcytosine – are actually versions of cytosine that have been modified by Tet proteins, molecular entities thought to play a role in DNA demethylation and stem cell reprogramming.

The discovery could advance stem cell research by giving a glimpse into the DNA changes – such as the removal of chemical groups through demethylation – that could reprogram adult cells to make them act like stem cells.

Basic DNA bases
1. adenine
2. guanine
3. thymine
4. cytosine
Variant bases of cytosine
5. 5-methylcytosine (methyl group is tacked onto a cytosine)
6. Tet proteins can convert 5 methylC (the fifth base) to 5 hydroxymethylC (sixth)
7. 5-Methylcytosine to 5-Formylcytosine
8. 5-Methylcytosine to 5-Carboxylcytosine


Science - Tet Proteins Can Convert 5-Methylcytosine to 5-Formylcytosine and 5-Carboxylcytosine


5-methylcytosine (5mC) in DNA plays an important role in gene expression, genomic imprinting, and suppression of transposable elements. 5mC can be converted to 5-hydroxymethylcytosine (5hmC) by the Tet proteins. Here, we show that, in addition to 5hmC, the Tet proteins can generate 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) from 5mC in an enzymatic activity–dependent manner. Furthermore, we reveal the presence of 5fC and 5caC in genomic DNA of mouse ES cells and mouse organs. The genomic content of 5hmC, 5fC, and 5caC can be increased or reduced through overexpression or depletion of Tet proteins. Thus, we identify two previously unknown cytosine derivatives in genomic DNA as the products of Tet proteins. Our study raises the possibility that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation.

Much is known about the “fifth base,” 5-methylcytosine, which arises when a chemical tag or methyl group is tacked onto a cytosine. This methylation is associated with gene silencing, as it causes the DNA’s double helix to fold even tighter upon itself. Last year, Zhang’s group reported that Tet proteins can convert 5 methylC (the fifth base) to 5 hydroxymethylC (the sixth base) in the first of a four step reaction leading back to bare-boned cytosine. But try as they might, the researchers could not continue the reaction on to the seventh and eighth bases, called 5 formylC and 5 carboxyC.

The problem, they eventually found, was not that Tet wasn’t taking that second and third step, it was that their experimental assay wasn’t sensitive enough to detect it. Once they realized the limitations of the assay, they redesigned it and were in fact able to detect the two newest bases of DNA. The researchers then examined embryonic stem cells as well as mouse organs and found that both bases can be detected in genomic DNA.

The finding could have important implications for stem cell research, as it could provide researchers with new tools to erase previous methylation patterns to reprogram adult cells. It could also inform cancer research, as it could give scientists the opportunity to reactivate tumor suppressor genes that had been silenced by DNA methylation.

18 pages of supplemental information

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