Memory Loop Rationally Designed and Synthesized in Yeast

HMS researchers have synthesized a DNA-based memory loop in yeast cells, work that marks a significant step forward in the emerging field of synthetic biology.

After developing a predictive mathematical model and using it to design and construct genes from bits of DNA, researchers in the lab of Pamela Silver, HMS professor of systems biology, fashioned a genetic circuit in which a stimulus launches a recurring function that continues in the cell after the stimulus ends and in daughter cells after the cell divides.

Molecular memory. Using a precise mathematical model they had developed, HMS systems biologists designed and constructed two synthetic genes (A and B), inserting them into yeast cells. One gene was activated by galactose and produced a transcription factor (TF) that activated the second gene, which then expressed a transcription factor that reactivated itself. This functional loop continued after the galactose was withdrawn and was passed on in multiple generations of daughter cells. (An animation of the memory loop may also be viewed online.)

“Synthetic biology is an incredibly exciting field, with more possibilities than many of us can imagine,” said Silver, senior author on the paper, in the Sept. 15 Genes and Development. “While this proof-of-concept experiment is simply one step forward, we’ve established a foundational technology that just might set the standard of what we should expect in subsequent work.” (For video interviews with Silver, see Understanding Synthetic Biology and Applying Synthetic Biology.)

A team in Silver’s lab led by Caroline Ajo-Franklin, now at Lawrence Berkeley National Laboratory, and postdoctoral fellow David Drubin demonstrated that not only could they construct circuits out of genetic material, but they could also develop mathematical models whose predictive abilities match those of any electrical engineering system.

“That’s the litmus test,” said Drubin, “namely, building a biological device that does precisely what you predicted it would do.”

The components of this memory loop were simple: two genes that coded for transcription factors. The researchers placed the genes into yeast cells and exposed the cells to galactose. The first gene, designed to be switched on by the sugar, expressed a transcription factor that activated the second gene.

It was at this point that the feedback loop began. The second gene also produced a transcription factor, but this protein bound to the gene from which it had originated, reactivating it. So the second gene again created the same transcription factor, which looped back and activated the gene again.
The researchers then eliminated the galactose, causing the first synthetic gene to shut off. Even with this gene gone, the feedback loop continued.

“Essentially what happened is that the cell ‘remembered’ that it had been exposed to galactose and continued to pass this memory on to its descendents,” said Ajo-Franklin.

Most important, the researchers’ mathematical model succeeded in guiding their construction of the DNA-based device. The model was derived from in vivo quantitative descriptions they obtained on the behavior of the individual transcription factors, which could be used in constructing the feedback loop. These precise descriptions, when incorporated into the model for the loop, told them which transcription factors would work and under what conditions.
For synthetic biology, this kind of specificity is crucial. “If we ever want to create biological black boxes, that is, gene-based circuits like this one that you can plug into a cell and have perform a specified task, we need levels of mathematical precision as exact as the kind that goes into creating computer chips,” Silver explained.

The researchers are now working to scale up the memory device into a larger, more complex circuit, one that can, for example, respond to DNA damage in cells.

“One day we’d like to have a comprehensive library of these so-called black boxes,” said Drubin. “In the same way you take a component off the shelf and plug it into a circuit and get a predicted reaction, that’s what we’d one day like to do in cells.”

—David Cameron

Connection Traced Between Caloric Restriction and Longevity

For nearly 70 years scientists have known that caloric restriction prolongs life. In everything from yeast to primates, a significant decrease in calories can extend life span by as much as one third. But definitively fingering the mechanisms that drive this longevity has remained elusive.

Reporting in the Sept. 21 Cell, researchers from HMS, in collaboration with scientists from Cornell Medical School and the National Institutes of Health, have discovered two genes in mammalian cells that apparently act as gatekeepers of cellular longevity. When cells experience certain kinds of stress, such as caloric restriction, these genes rev up a defense, which protects the cells against the deleterious effects of aging like insulin resistance, muscle wasting, and neurodegeneration.

“We’ve reason to believe now that these two genes may be potential drug targets for diseases associated with aging,” said David Sinclair, HMS associate professor of pathology and senior author on the paper.

The genes whose role Sinclair and his colleagues illuminated are members of the sirtuin family of deacetylases, SIRT3 and SIRT4. (Last year, the Sinclair lab showed that another sirtuin gene, SIRT1, has a powerful impact on longevity when stimulated by resveratrol, a compound found in red wine.) The action of the two protein products confirms what scientists have suspected for a long time: mitochondria are vital for sustaining the health and longevity of a cell. Since mitochondria are a cell’s power generators, if their stability starts to wane, energy declines, and the cell’s days are numbered. In this paper, Sinclair and his collaborators discovered that SIRT3 and SIRT4 play a vital role in a network that maintains the vitality of mitochondria and keeps cells healthy.

When cells undergo caloric restriction, signals sent in through the membrane activate the gene NAMPT. As levels of the NAMPT protein ramp up, the small molecule NAD begins to amass in the mitochondria. This buildup increases the activity of mitochondrial enzymes created by SIRT3 and SIRT4. As a result, the mitochondria grow stronger, energy output increases, and the cell’s aging process significantly slows down. (Interestingly, this same process also is activated by exercise.)

“We’re not sure yet what particular mechanism is activated by these increased levels of NAD, and as a result, SIRT3 and SIRT4,” said Sinclair, “but we do see that normal cell suicide programs are noticeably attenuated. This is the first time ever that SIRT3 and SIRT4 have been linked to cell survival.

“Mitochondria are the guardians of cell survival,” adds Sinclair. “If we can keep boosting levels of NAD in the mitochondria, which in turn stimulates buckets more of SIRT3 and SIRT4, then for a period of time the cell really needs nothing else.” Sinclair and his colleagues refer to this observation as the mitochondrial oasis.

In recent years, scientists have become increasingly aware of the importance of mitochondrial function in treating disorders associated with aging such as cancer, diabetes, and neurodegenerative diseases. SIRT3 and SIRT4 may now potentially be drug targets in new therapeutic approaches. “Theoretically, we can envision a small molecule that can increase levels of NAD, or SIRT3 and SIRT4 directly, in the mitochondria,” said Sinclair. “Such a molecule could be used for many age-related diseases.” (For a video interview with Sinclair, see The Biology of Aging .)

—David Cameron

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