October 07, 2015
In the old days, before the internet, planes or even mass publishing, it was hard to spark a quick, worldwide movement. You simply couldn’t reach out to likeminded people who lived far away.
Nowadays things are very different. With the advent of social media, it is now trivially easy to spread the word. Using Twitter and Facebook, organizers can easily and effectively organize people who live on the other side of the world.
In terms of transcription activation, our friend Saccharomyces cerevisiae seems to be stuck in the old world. Its transcription factors can only turn up nearby genes. This is different from most other eukaryotic beasts, where activation at a distance is routine.
Except maybe yeast isn’t as backward as we think. It may be that yeast has the potential to activate transcription at a distance, but keeps that potential locked away.
In other words, this ability is there but is just prevented from being used. Yeast is keeping social media out of the hands of its genes.
Getting around social media/internet controls is not easy. Pressure might need to be applied at multiple points before people power is finally released through social media. And even when it does happen, it can sometimes be hard to figure out exactly why certain events tipped the balance.
Turns out that both of these are also true for long range transcription activation in yeast. Mutating a single gene was not enough—it took mutations in multiple genes to see any significant effect.
As you can imagine, it would be very tricky to hit all of the right mutations for a polygenic trait in one fell swoop. This is why Reavey and coworkers started their mutant hunt with a strain that could already weakly activate transcription from a distance, a strain in which the SIN4 gene was deleted. Now they just needed additional mutations to make the effect stronger.
In their screen they used a reporter in which the GAL4 upstream activating sequence (UAS) was placed 799 base pairs upstream of the HIS3 reporter. This reporter gives very low levels of activity in a wild type strain. They included a second reporter, the URA3 gene under control of the same upstream sequences, because Reavey and Winston had discovered in previous work using a single reporter that cis acting mutations and chromosomal rearrangements were a frequent source of false positive results.
The researchers put the reporter strain through multiple rounds of mutation with UV light and selection with increasing levels of 3AT, a competitive inhibitor of His3. After each round of selection, they measured mRNA levels for HIS3 and URA3 and chose strains that not only had higher 3AT resistance but also showed more transcription of the reporter genes. In the end they found three strains that survived in the presence of galactose (to turn on the activator) and 10 mM 3AT.
As expected, each strain had multiple mutations. One strain had acquired mutations in the GRR1 and MOT3 genes. To confirm that these were the most important mutations, Reavey and colleagues engineered a fresh strain with just the original sin4 null mutation and the selected grr1-1 and mot3-1 mutations. The fresh strain completely recapitulated the selected strain, showing that these three mutations could unlock yeast’s potential for long-range transcriptional activation.
It makes sense that a grr1 mutation could affect transcriptional activation. Grr1 is a ubiquitin ligase that destabilizes Med3 (also known as Pdg1), a key component of the Mediator complex involved in transcription activation. The researchers provided evidence that this is how the grr1-1 mutation affects the process, by showing that mutating MED3 mimicked the effects of mutating GRR1.
It’s also not too hard to imagine how a mutation in Mot3, a sequence-specific transcriptional activator, could affect transcriptional activation, presumably by changing the expression of a gene under its control.
The results were not so clear-cut for two other strains that were selected. They arose from the same lineage, and each had acquired the ptr3-1 mutation on top of the original sin4 null mutation. One strain went on to further pick up the mit1-1 mutation, while the other got an msn2-1 mutation.
Again it isn’t too surprising that mutations in genes that encode sequence-specific transcriptional activators like Mit1 and Msn2 arose in these strains. But the selection of the ptr3 mutation in these lineages is something of a mystery.
It is hard to imagine how the usual job of Ptr3 in nutrient sensing and transport would be involved in keeping long range transcription activation down. Perhaps the researchers have uncovered a novel function for this gene.
And re-creating these two strains only partially restored the levels of transcription activation at a distance that were seen in the original strains. A little genetic detective work showed that a big reason for this was that both of the selected strains had acquired an extra copy of the chromosome that had the HIS3 reporter, chromosome III.
Reavey and colleagues deleted each of their identified genes to see which ones caused their effect through a loss of function mutation. Deleting GRR1, PTR3, and MSN2 all had the same effect as the original isolated mutations.
The same was not true for MOT3 and MIT1. Deleting either gene actually weakens long range transcription activation, suggesting that these two had their effect through gain of function mutations.
Finally, the researchers showed that the increase in long-distance transcriptional activation was not simply due to a general increase in transcription activation in the selected strains, by showing that their mutants did not have increased activity of a reporter with the GAL4 UAS placed 280 base pairs upstream of HIS3. In fact, if anything, the strains showed decreased activation with this reporter.
So this experimental strategy allowed Reavey and coworkers to identify some of the key genes involved in keeping transcription activation at a distance under control in yeast. In particular, they found compelling evidence that the Mediator complex is an important player. But there is still plenty of work to do. For example, which of the genes regulated by Mit1, Msn2, and Mot3 are important in long range activation? And what on Earth is Ptr3 doing in all of this?
The success of this approach also confirms that doing repeated rounds of selection in yeast is a viable way to select multiple mutants and study polygenic traits. This strategy may prove a boon for studying the many human diseases that are the result of polygenic traits.
Not only can we use yeast to uncover its activation potential, but we can also now potentially use it to uncover new treatments for human disease. Unleashing another awesome yeast power…
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight