Epigenetic Surgery Can Reactivate Genes without Butchering DNA

If DNA is to go under the knife, the cuts should be made with a scalpel, not a cleaver. For example, DNA bases may need to be relieved of the methyl groups they’ve accumulated during gene-silencing reactions. Excising the entire methylated base and replacing it with a demethylated base risks damaging the DNA. It appears, however, that a relatively delicate procedure may suffice to reactivate DNA that has been silenced via methylation.

At Ludwig-Maximilians-Universität (LMU) in Munich, researchers have uncovered a new pathway that can reactivate genes that have been silenced via methylation. Unlike other known pathways, the newfound pathway does not lead to the generation of potentially deleterious intermediates. The new findings appear in the journal Nature Chemical Biology, in an article entitled “5-Formylcytosine to Cytosine Conversion by C–C Bond Cleavage In Vivo.”

According to the study’s authors, the C–C bond cleavage mechanism could be of medical interest, as it suggests a way to reprogram stem cells in a targeted fashion. Such a method would in turn open up new perspectives in regenerative medicine.

In multicellular organisms, every cell contains the complete complement of genetic information characteristic of the particular species. However, in any given cell, only a subset of this comprehensive gene library is actually expressed—and it this selectivity that gives rise to diverse cell types with specific functions.

At the level of the DNA itself, simple chemical modifications of its subunits can determine which genes are active and which are turned off. But gene regulation must also be flexible, which requires that the activation and inactivation of genes should be reversible. This therefore implies that it must also be possible to remove such DNA modifications.

Methylation of one of the four basic building blocks found in the DNA—the nucleotide base known as cytidine—plays an important role in the regulation of gene activity. The attachment of a methyl group (CH₃) to unmethylated cytidine converts it into 5-methylcytidine, which is known to block gene activity.

“Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA,” the authors of the Nature Chemical Biology article explained. “It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites.”

The article’s authors, led by LMU’s Thomas Carell, Ph.D., decided to find out whether this form of demethylation, which would involve direct C–C bond cleavage reactions at fdC and cadC, actually occurred in vivo.

“Here we report the incorporation of synthetic isotope- and (R)-2′-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells,” reported Prof. Carell and colleagues. “Following the fate of these probe molecules using UHPLC–MS/MS [ultra-high-performance liquid chromatography–tandem mass spectrometry] provided quantitative data about the formed reaction products.”

Essentially, the LMU scientists collected data showing that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. The LMU team concluded that fdC undergoes C–C bond cleavage in stem cells, leading to the direct reinstallation of unmodified dC.

Up to now, it had been assumed that the methylated cytidine must be excised from the DNA and replaced by the unmethylated form of the base. This, however, is a risky process, because it requires cutting one or even both of the DNA strands—and unless promptly repaired, DNA breaks can have grave consequences for the cell.

“We have now shown in mouse embryonic stem cells that there is another mode of demethylation that avoids any break in the continuity of the DNA strand,” Carell stated. In this pathway, the attached methyl group is enzymatically oxidized to give rise to 5-formylcytidine, which Carell’s team first detected in mouse stem cells in 2011. They have now used stable isotopes to label 5-formylcytidine in stem cells and shown that it is rapidly converted unmethylated cytidine.

“This mechanism,” Carrell asserted, “allows cells to regulate gene activity at the DNA level without running the risk that the DNA may be damaged in the process.”