The TET family of enzymes, discovered just two years ago, plays a key role in the lineage decisions of stem cells. TET enzymes are important for the proper function of embryonic stem cells and may also be valuable players in the development of induced pluripotent stem cells, therapeutic cells that have been artificially reprogrammed back to their pluripotent state.

Embryonic stem cells are special cells that can generate every cell type in an embryo given the right cellular signals.  This feature is called pluripotency.  New work from the lab of Anjana Rao, a professor at the Program in Cellular and Molecular Medicine at Children's Hospital Boston and the Immune Disease Institute (PCMM/IDI), has found that the TET family of enzymes, new enzymes she discovered just two years ago, play a key role in directing embryonic stem cells as they choose to become bone, blood, organs, skin, or brain during development.

The work, described in the Feb 4 Cell Stem Cell, also connects TET enzymes to pluripotency by showing that TET enzymes alter DNA methylation.  This "epigenetic" modification of DNA is known, among other things, to be associated with pluripotency.  Because of this connection, these findings may lead to improved techniques for generating induced pluripotent stem cells (iPS cells), differentiated cells obtained from adults that have been reprogrammed back to a pluripotent state to be used therapeutically.

The story begins in 2009, when Rao discovered the TET family of enzymes and its ability to modify methylated DNA.  In an adult cell, methylated DNA acts as a strategic cover to prevent the wrong genes from expressing themselves.  For example, genes that are important for the function of neurons in the brain have to be turned off by DNA methylation in a muscle cell.

In mammals, methylation occurs just on cytosine, the C in the familiar list of DNA bases A, G, C, and T.  Methylated cytosine, called 5-methylcytosine (5mC), has been dubbed the "fifth base," a novel nucleic acid base tacked on to the well-known four.  Rao found that TET alters methylation by converting this fifth base into yet another new base, 5-hydroxymethylcytosine (5hmC).

Prior to this finding, researchers had thought that 5hmC was a byproduct of oxidative damage in mammals.  That is, they thought it was abnormal.  However, the discovery of an active enzymatic conversion inside the cell, powered by TET enzymes, suggested that the presence of 5hmC was no accident.  It represented a deliberate DNA modification.

Since embryonic stem cells have more dramatic DNA methylation changes than adult cells, Rao and first author Kian Koh, a research fellow at PCMM/IDI, set out to understand the roles of TET and 5hmC in these cells.

Koh's experiments showed that Tet1 and Tet2 are present in embryonic stem cells and are responsible for the high levels of 5hmC in these cells.  Koh connected the TET enzymes to pluripotency by showing that Tet1 and Tet2 work downstream of a key gene regulation network known to be involved in maintaining pluripotency.  He decreased the levels of Oct4 and Sox2, proteins that drive this network, in cultured embryonic stem cells using RNA-interference and observed a corresponding drop in Tet1 and Tet2 and a rise in Tet3 gene expression.

Further experiments showed that Tet1 influences the lineage decisions of stem cells even after they lose pluripotency and begin to differentiate into muscle, blood and so on.  "In order for an embryo to be normal, all of the cells in the embryo need to make the correct decisions," said Rao.  "We found that Tet1 regulates some of these decisions."

For instance, very early in embryonic development, when the embryo is just a small mass of undifferentiated cells, each cell has to make the decision whether to be part of the placenta or the inner cell mass, which gives rise to the embryo.  In cultures of cells in which Tet1 was knocked down using RNA-interference, Koh found that stem cells were more likely than normal to become trophectoderm cells, cells that form the placenta.

Koh also found that a lack of Tet1 influences lineage decisions further along in embryonic development, based on experiments with teratomas performed in collaboration with the laboratory of George Daley, a professor at Children's Hospital, Boston.  A teratoma is a laboratory-grown tumor that contains a balance of all the cell types in an embryo.  Teratomas formed from cells lacking Tet1, however, contained an overabundance of endoderm cells, which form internal organs, and mesoderm cells, which form blood, bone and cartilage, but a reduction of ectoderm, which form skin and neurons.  "They look very different from wild-type teratomas," said Koh.

"These findings directly link genetic and epigenetic control and suggest an important role for 5hmC in embryonic stem cell maintenance and differentiation," said Jorn Walter, an epigeneticist at Saarland University in Saarbrucken, Germany, in a Cell Stem Cell Preview.  Not only is 5hmC not an accidental DNA modification, this work suggests it may be a key to unlocking one of the most powerful functions an embryonic stem cell can have.

Rao and her team are planning future experiments with knockout mice to address the limitations of using RNA-interference, which does not completely eliminate the proteins it blocks.  Moreover, said Rao, the methods for measuring 5hmC are becoming more powerful and will allow more details about these complex interactions to emerge.

By connecting TET and 5hmC to pluripotency, these findings may influence work in the area of iPS cells, cells that have been artificially reprogrammed from fully differentiated cells back to a pluripotent state.  One avenue of future research in Rao's lab will explore whether TET influences the formation of iPS cells and, in turn, the therapeutic potential of iPS cells.

Koh KP, Yabuuchi A, Rao S, Huang Y, Cunniff K, Nardone J, Laiho A, Tahiliani M, Sommer CA, Mostoslavsky G, Lahesmaa R, Orkin SH, Rodig SJ, Daley GQ, Rao A.  "Tet1 and tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells." Cell Stem Cell. 2011 Feb 4;8(2):200-13.