A Sirt-ain role for cellular differentiation
The epigenome is a collection of physical “amendments” to DNA—things like proteins around which the double helix is wrapped like thread on a spool and chemical tags on the DNA of specific genes that can make them hard to access. This collection of epigenetic factors works together to help give each cell in the body its specific identity by regulating which genes are expressed—it’s a big reason why skin cells don’t get confused with blood cells and why bone cells are full of calcium instead of fat. The epigenome guides differentiation, the process by which embryonic stem cells (ESCs) go from being pluripotent—having the ability to turn into almost any cell type in the body—to taking on one specific identity. But in order for differentiation to happen, the products of a handful of pluripotency genes, which work to maintain the pre-differentiated state of a cell, must be overcome.
In a paper released earlier this week in the journal Nature Cell Biology, a team of researchers from the Broad, in collaboration with Massachusetts General Hospital, identified Sirt6 (an enzyme important for regulating chromatin, the three dimensional sum of DNA and proteins) as a regulator of pluripotency. They found that Sirt6 is essential to a nuanced mechanism for determining cell fate.
An earlier paper from other researchers had identified an epigenetic mark (a chemical tag to histones, those "spool proteins" mentioned earlier) called H3K56ac as important for influencing the ultimate fate of an ESC. “We had been working on Sirt6 and knew it was involved in creating that particular mark,” said Raul Mostoslavsky, an associate member at Broad and lead author on the paper. So he and his team, including post doc Jean-Pierre Etchegaray, began digging in to find out whether Sirt6 was important for differentiation.
First, they knocked out the gene that codes for Sirt6. Doing so resulted in cells that “had serious trouble when we pushed them to differentiate,” said Mostoslavsky. It turns out Sirt6 is a repressor—it blocks the expression of two pluripotency genes known as Oct4 and Sox2, and without it differentiation can’t happen normally. Etchegaray and Mostoslavsky worked with Alon Goren of the Broad Technology Labs (BTL) to look at all of the relevant epigenetic factors at play across the genome—in regions where Sirt6 was and was not present.
“We found an increase in levels of 5hmC—a specific mark on the DNA itself—which is put there by an enzyme called Tet,” said Etchegaray. Before their work, 5hmC was thought to be an intermediate form of another epigenetic mark, but the team showed that in fact it is a specific epigenetic mark important for guiding gene expression and is thus a fully-loaded mark in its own right. This alone is an important contribution of the paper, as the field is still debating whether 5hmC indeed functions as a new mark, rather than an intermediate product, said Mostoslavsky.
In the end, the team revealed the critical pieces of the mechanism through which Sirt6 helps determine stem cell fate: When the chromatin structural protein known as histone H3 is marked with an acetyl group (a combination of hydrogen, carbon, and oxygen atoms), Oct4 and Sox2 are readily expressed and free to do their work of ensuring pluripotency. But when Sirt6 is around, it removes that mark and the genes are shut down. This leads to the regulated marking of genes with 5hmC, which is required for proper cellular development, giving Sirt6 a second, indirect form of control over cell fate decisions. Without Sirt6, 5hmC can’t be removed and cells don’t develop normally.
“I’ve loved working with these guys,” said Goren, who created a systematic and reproducible process for large-scale, genome-wide epigenetic mapping that will be relevant beyond this work, and is now available to the community via the BTL. “They’ve been digging deep into each and every component of the mechanism to yield a deep understanding of the cellular biology.”
The work could also have implications for more efficiently developing induced pluripotent stem cells (iPSCs), which are increasingly being used in the laboratory in place of ESCs for testing drugs and determining disease mechanisms.
Other Broad researchers involved in the work include: Adrianne Gladden.
Paper cited: Etchegaray, J. P., et al. The histone deacetylase SIRT6 controls embryonic stem cell fate via TET-mediated production of 5-hydroxymethylcytosine, Nature Cell Biology (2015). Doi: 10.1038/ncb3147