Epigenetic enzymes thicken the iPS cell reprogramming plot

Manipulating the enzymes that turn genes on and off could help make the process of reprogramming cells into iPS cells a lot more efficient and safer.

There are several ways to reprogram skin cells into induced pluripotent stem (iPS) cells – cells that behave like embryonic stem cells, and which could help better understand the genetic basis of and develop new treatments for different diseases.

The major methods scientists use now include using viruses to deliver reprogramming genes or using RNAs to produce the necessary proteins without the genes. Different methods have different advantages and disadvantages, and some are more efficient than others.

What’s common across all of the methods is that they rely on four proteins to turn back the cellular clock – c-Myc, Klf4, Oct4, and Sox2. Less understood is whether enzymes that modify chromatin (the DNA-plus-protein package that constitutes our genome) play any role in the reprogramming process. These enzymes manage and control the cell’s epigenetic code – the layer of control that helps cells fine-tune gene expression by adding and removing small chemical tags to genes and proteins.

“During iPS reprogramming, a cell’s epigenetic code gets completely rewritten,” says George Q. Daley, director of the Stem Cell Transplantation Program at Children’s Hospital Boston. “But how the cell’s epigenetic enzymes influence the reprogramming process has been a mystery.”

As reported in Nature earlier this month, a team in the Daley lab led by postdoctoral fellow Tamer Onder recently tried to solve this mystery. Starting with skin cells, they depleted, one by one, 22 enzymes that control whether methyl groups (or “marks”) are attached to DNA or chromatin proteins, a well-known epigenetic mechanism that provides an exquisite layer of control over gene activity. The team then used the altered cells as raw material for generating iPS cells.

Some enzymes turned out to be required for reprogramming – the team could generate few or no iPS cells from skin cells in which they’d been stifled. Silencing other enzymes, however – in particular one called Dot1l – boosted the rate and efficiency of reprogramming significantly.

Dot1l has received much attention lately. It adds methyl marks to certain genes in a way that keeps the cell from accessing and transcribing them, essentially turning them off. Last summer, researchers in the Dana-Farber/Children’s Hospital Cancer Center announced that Dot1l plays a critical role in set of hard-to-treat leukemias called MLL-rearranged leukemias. At the time, the researchers, led by hematologist Scott Armstrong, found they could knock down MLL-driven cancer cells using a drug that blocked Dot1l.

George Daley thinks that by manipulating epigenetic enzymes he can start to replace viruses (between steps 1 and 2) and other biological tools with chemical tools when producing iPS cells. (Y tambe/Wikimedia Commons)

To further probe Dot1l’s role in reprogramming cells, Daley’s team repeated their experiments by silencing Dot1l alone, blocking it with the same drug Armstrong’s team investigated, or cutting Dot1l out of the cell’s genome altogether. They found that in deactivating Dot1l they activated other genes known to drive the reprogramming process, suggesting that Dot1l normally keeps those genes quiet.

By taking Dot1l out of the picture, the team no longer needed to use Klf4 and c-Myc, two of the canonical reprogramming factors to create iPS cells. They could do so using just Oct4 and Sox2, suggesting that it could be possible to generate iPS cells using fewer manipulations, which could make such cells safer for possible clinical use in the future.

“We think turning off enzymes like Dot1l that promote gene silencing may, in the context of reprogramming, help reactivate genes that initiate and maintain a ‘stem’ like state,” says Daley. “These results could significantly enhance the efficiency with which we and other labs can develop new iPS lines, especially disease-specific lines, and also represent a step towards safer chemical, rather than biological, programming.”