The search is on to figure out a role for the recently discovered 5-hydroxymethylcytosine (5hmC) base. While it’s still up in the air if 5hmC will play a main character or just be cast as an extra , this new study in Nature ties the mysterious modification to a demethylation pathway, regulated by Tet proteins, that helps keep embryonic stem cells (ESCs) in the stem zone.
A previous study (Tahiliani, et.al. Science, 2009) showed that Tet1 could convert 5-methylcytosine (5mC) to 5hmC in mammalian DNA, but the tools available at the time limited them to in vitro analysis using thin layer chromatography and mass spec. With access to a new 5hmC antibody from Active Motif, researchers from the University of North Carolina were able to confirm the results of the Tahiliani paper, and then go one step further.
UNC scientist Shinsuke Ito and his colleagues first verified that both Tet1 and Tet2 reduced 5mC levels in vitro, and showed that this activity was conserved in humans and mice. The team went on to show by immunostaining that 5hmC levels were increased as 5mC decreased after expression of Tet proteins in vivo.
With Tet’s enzymatic features confirmed in vivo, Dr. Ito’s team explored possible functions for this mechanism. Tet expression profiles in mouse tissues led them to look closely at ESCs, where they found the following:
- shRNA knockdown of Tet1 and 2 caused morphological changes and lower alkaline phosphatase activity.
- Tet1 knockdown also led to slower ESC growth rate through a self-renewal defect and reduced Nanog expression.
- ChIP-qPCR experiments showed that Tet1 directly targets the Nanog promoter.
- MeDIP analysis showed that Tet1 knockdown increased Nanog methylation.
- Tet1 regulates Nanog by keeping it’s promoter hypomethylated.
Ito put these findings together with work from other recent papers to create new model where Tet proteins maintain ESC self-renewal through hypomethylation of the Nanog promoter and 5hmC acts as a stop-over on the demethyaltion highway, waiting for some unknown enzyme to complete its conversion from 5mC back to cytosine.
Uncover more details about the Tet / 5hmC connection at Nature, July 2010.