New & Noteworthy

How Histones Use FACT(s) to Find Their Way

July 5, 2017


Looking at map

The map is like FACT helping histones get to the right place. from publicdomainpictures.net.

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.

shepherding

Histones need to be shepherded to the genome by histone chaperones. by Mclaire MClaire, Wikimedia Commons

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

Yeast’s Skynet Against Salt

June 27, 2017


Skynet

Like Skynet, PI3,5P2 signaling is a rapid defense response. from Wikipedia.

 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.

Sarah Connor

One way to give Sarah Connor an easier life might have been to make Skynet’s control of defense transient. from cdn.movieweb.com.

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

Network Maintenance at SGD on July 6, 2017

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.

During this brief maintenance period, the main SGD website (www.yeastgenome.org) will be unavailable for use. Other resources affected by the maintenance are listed as follows:

Unavailable Available
Downloads page Genome Browser
SPELL YeastMine
Textpresso YeastPathways
SGD Wiki YeastGFP

We will make every effort to minimize any downtime associated with this maintenance. We apologize for any inconvenience this may cause, and thank you for your patience and understanding.

Making the Best of a Sticky Situation

June 5, 2017


Lemonade stand

Like turning lemons into lemonade, Hope and coworkers turned gummy yeast into a useful strain. from Pixabay.

 Back in 1915, writer Elbert Hubbard coined the phrase, “When life gives you lemons make lemonade.” (His actual quote was “He picked up the lemons that Fate had sent him and started a lemonade-stand.”)

The idea of course is to take something bad and make it into something good. Like, if your research gives you terribly weak glue, invent Post-It notes.

Or as Hope and coworkers show in a new study in GENETICS, when your yeast experiment gets gummed up because the yeast evolves a sticky trait, do additional experiments to learn about the evolution of that complex trait. And “invent” a way to make the yeast less likely to flocculate, or stick together.

This new “invention” will be very useful for anyone trying to evolve yeast to create new products or to study how evolution works. It also showed that for this trait, under these conditions with this strain, there was one major way to get to flocculence—up-regulating the FLO1 gene. This meant they could greatly reduce the risk of this trait popping up by simply deleting FLO1.

Studying evolution in yeast often involves using a chemostat, an automated way to keep the yeast growing through repeated dilutions with fresh media. Scientists use this method to study how yeast evolves under varying conditions over hundreds of generations.

An unfortunate side effect of this method is that it also tends to select for yeast that stick together. These yeast are diluted away less, often meaning they become more common over the generations.

In this study, Hope and coworkers ran 96 chemostats under three different conditions for 300 generations and found that in 34.7% of the cultures, the yeast ended up aggregated. This was even though they used a strain of S288c in which the FLO8 gene was mutated. This strain flocculates less often than wild type!

These authors picked the 23 most aggregated cultures to study in more detail. They found that 2 out of 23 strains aggregated because of a mother/daughter separation defect. The rest were more run-of-the-mill flocculent strains. 

They next used whole genome sequencing to try to identify which genes when mutated caused flocculence in the strains. They saw no FLO8 revertants.

The two strains with a mother/daughter separation defect both had mutations in the ACE2 gene, which encodes an important transcription factor for septation as well as other processes.

Flos

By CBS Television (eBay item photo front photo back) [Public domain], via Wikimedia Commons. Progressive insurance facebook flo advertising failsBy woodleywonderworks, via Flickr
No, these Flos don’t cause flocculence, FLO1 does.

FLO1-related mutations dominated the other 21. They found a couple of different Ty insertions in the promoter region of FLO1 in 12 of the strains and mutations in TUP1 in 5 more. Tup1p is a general repressor known to repress FLO1, so it looks like up-regulating FLO1 leads to flocculent cultures. Other candidate mutations were found in FLO9 and ROX3.

They wanted to try to identify the responsible gene(s) in strains where there was no obvious candidate gene and also to confirm that the genes they identified really caused the trait, so they next did backcrosses between each mutant strain and a wild type strain.

The backcrosses identified two other ways to get flocculence— by mutating either CSE2 or MIT1. The researchers also confirmed that the mutations they found, including these two new ones, were probably the main cause of the flocculence in each individual strain.  This trait co-segregated with the appropriate mutation in a 2:2 pattern for 20/21 strains as is predicted for a single causal mutation.

The results are even more FLO1-heavy than they appear. Further studies showed that ROX3, CSE2, and MIT1 all require a functional FLO1 to see their effects.

So FLO1 appears to be the main route to flocculence in this strain. And the next set of experiments confirmed this.

Hope and coworkers ran 32 wild type and 32 FLO1 knockout strains in separate chemostats for 250 generations and found that 8 wild-type strains flocculated while only 1 FLO1 knockout strain did. Knocking out FLO1 seems to make for a more well-behaved yeast (at least in terms of evolving a flocculent trait in a chemostat).

And the strain can probably be improved upon even more. For example, researchers may want to limit Ty mobility as this was a major way that FLO1 was up-regulated, increase the copy number of key repressors or link those repressors to essential genes. Another possibility is to also mutate FLO9 as its up-regulation was the cause of that one aggregated strain in the FLO1 knockout experiment.

Researchers no longer need to “settle” (subtle, huh?) for big parts of their experiments being hampered by gummy yeast. Hope and coworkers have created a strain that is less likely to flocculate by simply knocking out FLO1. Not as ubiquitously useful as a Post-It note but a potential Godsend for scientists using yeast to understand evolution.

by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

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