New research provides insight into how an enzyme that helps regulate aging and other metabolic processes accesses our genetic material to modulate gene expression in the cell. A team led by Penn State researchers has produced images of a sirtuin enzyme bound to a nucleosome – a tight complex of DNA and proteins called histones – showing how the enzyme navigates the nucleosome complex to access both to DNA and histone proteins and clarify how it works. in humans and other animals.

An article describing the results appears on April 14 in the journal Scientists progress.

Sirtuins are a type of enzyme found in organisms ranging from bacteria to humans that play important roles in aging, detecting DNA damage, and suppressing tumors in various cancers. Due to these varied roles, pharmaceutical companies are exploring their potential for biomedical applications. Many efforts have focused on the ability of certain sirtuins to decrease gene expression by suppressing a chemical indicator of histone proteins.

“In our cells, DNA isn’t naked like we see in textbooks; it’s wrapped around proteins called histones in a large complex called the nucleosome,” said Song Tan, Verne M. Willaman Professor of molecular biology at Penn State and author. paper. “This packaging may also contribute signals to turn genes on or off: adding an ‘acetyl’ chemical flag to the histone packaging material turns a gene on, while removing the acetyl flag turns off the gene. Sirtuins can reduce gene activity by suppressing the acetyl flag of histones packaged in nucleosomes. Understanding how sirtuins interact with the nucleosome to suppress this flag could inform future drug discovery efforts.

Previous studies have focused on how sirtuins interact with short, isolated histone segments, in part because these histone “tail” peptides are much easier to work with in the lab. According to Tan, the nucleosome is a hundred times larger than the typical histone peptides used in these studies and is therefore much more complicated to work with.

“We visualized a sirtuin enzyme called SIRT6 on its physiologically relevant substrate – the entire nucleosome,” said Jean-Paul Armache, assistant professor of biochemistry and molecular biology at Penn State and author of the paper. “And we found that SIRT6 interacts with multiple parts of the nucleosome, not just the histone where the acetyl flag needs to be changed.”

Using a powerful type of imaging called cryo-electron microscopy with instruments from the Penn State Cryo-Electron Microscopy Facility, the National Cancer Institute, and the Pacific Northwest Cryo-EM Center, the researchers identified how SIRT6 positions itself on the nucleosome in order to remove an acetyl group from the K9 position on the histone called H3. Follow-up biochemical experiments – in collaboration with the laboratory of Craig Peterson at the University of Massachusetts Chan Medical School – confirmed their results.

The researchers found that SIRT6 binds to the nucleosome using a type of connection called an arginine anchor. This type of binding, described by Tan’s lab in 2014, is used by a variety of proteins that target a particularly acidic area on the surface of the nucleosome. In this case, a structural feature of SIRT6 called an extended loop nestles into a divot in the acid patch, much like a pipe sitting in a ditch.

“The arginine anchor is a common paradigm for the number of chromatin proteins that interact with the nucleosome,” Tan said. “When we mutated the arginine anchor SIRT6, the activity at position K9 was severely affected, supporting a critical role for the arginine anchor of SIRT6. Surprisingly, this mutation also impacted the enzyme activity of SIRT6 at a different position, K56, located much further away.”

Instead of SIRT6 binding to the nucleosome in two different ways to access the two different histone positions, it is possible that SIRT6 binds to access K9 in a way that could also provide access to K56.

“SIRT6 binds to a partially unenveloped nucleosome, with DNA displaced from the end of the nucleosome,” Armache said. “This exposes the K56 position, and it’s possible that SIRT6 can essentially bend to reach this position. We would like to validate this hypothesis in the future. We also hope to explore how SIRT6 works alongside other enzymes and better understand its role. in the DNA damage response.”