New & Noteworthy
July 17, 2017
For most people, a move to Tibet or other high altitude places is a real struggle. They suffer the many nasty symptoms of high altitude sickness while they are there.
Some people though, like natives of Tibet or of the Andes, have adapted to the extreme altitudes through natural selection and do just fine. How they adapted is a typical Darwinian story.
Those who happened to have the right set of DNA did better than those who didn’t and so had more kids. Over time, the beneficial DNA became more common until the population was able to tolerate the low oxygen of the higher altitudes. (In Tibet, they may have acquired this helpful DNA by having kids with our close relatives the Denisovans.)
Imagine instead that the story went a bit differently. In a Lamarckian twist, let’s say that people who live in low oxygen were more likely to gain the traits needed to do well in this environment, strictly as a result of there not being enough oxygen around. In other words, the environment would make it more likely for the beneficial changes to happen.
Turns out this might have been the way the story went if people were more like yeast. And if dealing with low oxygen environments relied on a gene near a place in the DNA where replication forks often stalled. And if that the gene was upregulated in low oxygen.
It is under these conditions that Hull and coworkers found that yeast could preferentially develop the right mutations in an environment-dependent way. Instead of low oxygen though, these authors studied the yeast growing in high levels of copper.
This last point is important because it hints at how yeast can increase the likelihood of beneficial mutations at CUP1 in the presence of copper. The increased transcription of the CUP1 genes increases the likelihood of a change in the copy number of these genes. Those yeast with increased copy number thrive in their new copper-tainted environment.
Now of course not every gene ends up with an increase in beneficial mutations just because it is induced. No, the gene also seems to have to be near where a DNA replication fork is more likely to stall. It is this combination of high rates of transcription and stalled DNA replication that can lead to changes in gene copy number.
The first thing the authors did was to map out where replication forks tend to stall in the yeast genome. These sites are marked by the presence of S139-phosphorylated histone H2A (γH2a).
Using chromatin immunoprecipitation sequencing (ChIP-seq) for γH2a they showed that likely stalling spots were within 1,000 base pairs of around 7% of the genes of Saccharomyces cerevisiae. These genes tend to be expressed at low levels under optimal conditions and higher levels under less ideal growth conditions. One of these genes is CUP1.
It is well known that when yeast are put into a high copper environment, the end result is yeast with more copies of the CUP1 gene. But this could simply represent the few cells that happened to have extra copies that then outgrow their compatriots with fewer copies. Ordinary old Darwinian selection.
What these authors wanted to show is that it is increased transcription that leads to increased copy number and not the selective pressure. They get at this a couple of different ways.
In the first approach, they introduce multiple copies of a Gal-inducible reporter at the CUP1 locus. In this system there is increased transcription in the presence of galactose, but no selection.
They found multiple colonies with changes in the copy number of the reporter gene with galactose and no differences in copy number with glucose. So, transcription matters.
The second way they attacked this problem was by killing off any daughter cells to get rid of the problem of selection. In this strategy copy number mutants do not get a chance to outgrow their lower copy number sisters. Only the original mother cells remain.
Any increase in copy number would not be due to run of the mill Darwinian selection. Instead, they would be due to the presence of the factor in the environment the cells need to adapt to. And this is just what these authors saw happen.
They eliminated daughter cells using a mother-enrichment system in which daughter cells are killed in the presence of beta-estradiol. They treated a population of cells with beta-estradiol and then put half in normal media and half in media with 1 mM copper sulfate.
They found about a 9-fold increase in the number of copy number variants (CNV) in the presence of copper (27% CNV events compared to 3%). They did follow up experiments to show that copper was not acting as a mutagen—the copper had to induce transcription to cause the copy number variation. And judging by the bud scars, it looks like the cells divided more in the absence of copper, meaning this 9-fold increase is an underestimate.
So growing in the presence of copper increased mutations in the CUP1 gene that allowed the yeast to grow better in copper. Calling Doctor Lamarck!
We don’t have time to go into the rest of the experiments they did to flesh out their findings. For example, they show that this copy number variation can still happen even when they use tandem arrays that are much shorter than the usual 13 CUP1 copies. And that deletion of the H3K56 acetyltransferase RTT109 completely eliminates the effect of copper on the expansion of CUP1. And so much more! Anyone interested should definitely read the article.
These findings show us another example of Lamarckian selection. The environment itself causes the adaptations needed to prosper and these adaptations can be passed on. Not quite the ancestors of giraffes passing on their stretched necks to the next generation but still pretty cool.
The awesome power of yeast genetics again shows us something new about how life adapts and evolves. #APOYG
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
July 5, 2017
Some people (like me) have no sense of direction. Send me to the store and who knows where I’ll end up!
Tools like maps, a GPS system, and my iPhone all help to make sure I get to where I need to be. And seat belts, airbags and working brakes keep me safe while I am getting there.
Histones are similar. These proteins, which help to organize and run our DNA, can get lost without a variety of helpers to show them the way. They also need to be kept safe as they travel.
Instead of an iPhone, histones get to where they need to go with the help of histone chaperones like Nap1p and the FACT complex. A new study by Hodges and coworkers in GENETICS helps to figure out which parts of histones interact with the FACT complex and, to a lesser extent, Nap1p.
Turns out that a few residues in an acidic patch on H2A/H2B dimers are critical for interacting with FACT. This makes sense given that previous work had shown that this acidic patch interacts with other proteins (although no one had shown it interacts with the FACT complex or Nap1p). Hodges and coworkers also identified other residues outside of the acidic patch that were important for FACT complex binding.
The first step was to analyze residues in this patch known to be lethal when mutated to alanine—H2A: Y58, E62, D91, and H2B: L109. The authors used co-immunoprecipitation (co-IP) assays against either Nap1p or Spt16p (a subunit of the FACT complex) to identify which, if any of these essential residues, was important for interacting with these histone chaperones.
As expected, wild type Nap1p and Spt16p interacted with both H2A and H2B in their assay. Of all of the essential residues of the acidic patch, only L109 on H2B significantly affected H2B’s ability to interact with the FACT complex. There was about a 4-fold decrease in the amount of Spt16p brought down with H2B: L109A compared to wild-type H2B in these experiments.
The authors decided to broaden their search for residues important for interactions by looking at those in the acidic patch that were not lethal when mutated to alanine. Recent work suggested two other residues, H2A: E57 and H2A: E93, might be important for getting the FACT complex to actively transcribed genes in yeast. Hodges and coworkers were able to confirm the importance of H2A: E57 in their co-IP experiments.
They now expanded to other residues on H2A and H2B that have been shown or hypothesized to be important for binding to the FACT complex—H2A: R78A, and H2B: Y45A, M62E. These authors found that only H2B: M62E significantly impacted binding to the FACT complex (H2B: Y45A had a small effect). They also found that H2A: R78A affected binding to Nap1p, but not the FACT complex.
OK, so there is good co-IP data that H2A: E57, H2B: L109A, and H2B: M62E each affect binding of the FACT complex to the H2A/H2B dimer. These authors also provide good evidence that these mutants affect nucleosome occupancy and have nucleosome-based effects on transcription as well.
They used chromosomal immunoprecipitation linked to qPCR (ChIP-qPCR) against H2A and H2B to show decreased occupancy of H2A and H2B with the H2B: L109A mutant at four different promoters. Occupancy was around 3-4 fold lower than with wild type H2B.
They were also able to show that some of their H2A and H2B mutations mimicked the effects of partial loss of function mutations in the Spt16p part of the FACT complex. For example, just like mutations in SPT16 make a cell more sensitive to hydroxyurea, so too do H2B: Y45A, H2B: M62E, and H2A: E57A. These three mutants also induce cryptic transcription from the FLO8 gene like an Spt16 mutant.
Key residues in the acidic patch of H2A/H2B are critical for making sure histones get to the right place in the genome. Mutating them is similar to me not hearing the direction I need to go from my iPhone. Just like a histone not able to hang onto its chaperone, I will end up at the wrong place and not able to do what I needed to do.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
June 27, 2017
In the Terminator franchise, the U.S. creates an artificial intelligence (AI)-based defense system called Skynet to, among other things, react more quickly to threats than any general or politician could. What starts out as an interesting idea almost dooms mankind to extinction once Skynet becomes conscious and decides to eliminate its greatest threat—humans.
Our friend Saccharomyces cerevisiae has its own version of Skynet for when it is “attacked” by too many salt ions. No, the system isn’t conscious and it does not threaten this yeast’s very existence but like Skynet, it is designed to react more quickly than more conventional systems based on gene regulation. It basically buys yeast enough time to allow the cells to more stably adapt to their new high salt environment.
Within 1 -5 minutes of being plunked down into high salt, yeast activates Hog1p, a key MAP kinase. The activated Hog1p heads into the nucleus and within 30-60 minutes, it tweaks the expression of a bunch of genes so the yeast can now better deal with its new environment.
This is a lot of time to be languishing in high salt. Luckily, yeast’s “whole hog” approach to high salt is not limited to just Hog1p. According to a recent study by Jin and coworkers in the Journal of Cell Biology, there is another, faster reaction to the high salt. And at least in these experiments, it is critical for yeast’s survival when it is assaulted by too much salt.
This rapid response involves a signaling lipid found in the vacuole called phosphatidylinositol 3,5-bisphosphate, or PI3,5P2. The amount of this lipid goes way up within just five minutes of the high salt shock.
PI3,5P2 is synthesized in yeast by a single enzyme, Fab1p. It stands to reason that if PI3,5P2 is critical to yeast survival in high salt, then deleting FAB1 should affect yeast’s ability to deal with all of those extra ions in its environment. This is just what Jin and coworkers found.
They compared the viability of wild type, hog1Δ, and fab1Δ strains under normal conditions and after a four hour exposure to 0.9M NaCl (high salt). Under low salt conditions, the fab1Δ strain was less viable than the other two. Around 30% of the fab1Δ yeast were dead.
At high salt, less than 10% of the wild type yeast and around 30% of the hog1Δ yeast were dead after four hours. This compares to the greater than 80% dead fab1Δ yeast.
The next steps in the study were to identify how the high salt increases the amount PI3,5P2. They reasoned that they needed something fast and that kinases just might fit the bill, so they started looking for strains that dealt poorly with high salt in the “…knockout haploid yeast mutant collection of 103 nonessential protein kinases.” They found a likely candidate in Pho85p and further work showed that its partner cyclin Pho80p was also involved.
Both the pho85Δ and pho80Δ strains had enlarged vacuoles (a common phenotype in yeast that cannot make PI3,5P2). More importantly, both strains could not make PI3,5P2 either under normal or high salt conditions and were also less viable than wild type under high salt conditions.
Additional experiments provided strong evidence that Pho85p phosphorylated Fab1p and that this phosphorylated Fab1p was important for synthesizing PI3,5P2 under high salt conditions. The final experiments confirmed that something similar happens in mammalian cells.
Jin and coworkers showed that the Pho80p-Pho85p equivalent in mammalian cells, CDK5-p35, phosphorylates the Fab1p equivalent, PIKfyve, in vitro. They also showed that CDK5-p35 is important for mouse fibroblasts to make more PI3,5P2 when exposed to high salt.
These studies suggest that yeast and probably mammals have at least two systems for dealing with high salt. The first is a rapid increase in PI3,5P2 that protects the cells from the environmental insult which gives the cells time to set up the second system—a longer term, more stable adaptation.
If only the folks in the Terminator world were as smart as yeast and had made Skynet a transient system set up to protect the U.S. while humans had time to respond in a more stable way. Think how much easier Sarah Connor’s life would have been!
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
June 15, 2017
The SGD website (www.yeastgenome.org) and several of its resources will be unavailable on Thursday, July 6, 2017 from 7:00 am to 5:00 pm PDT (10:00 am to 8:00 pm EDT; 2:00 pm to 11:00 pm UTC) for electrical equipment maintenance.
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