New & Noteworthy
Make Some More Room for Lamarck
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.
Lamarck’s theories are only mostly dead which means they are slightly alive. from the Brick In the Sky blog (https://thebrickinthesky.wordpress.com/).
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.
One way that yeast deal with toxic levels of copper is to turn up the CUP1 gene. Or more precisely, turn up their tandem arrays of multiple copies of CUP1.
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.
Lamarck was only mostly wrong when it comes to evolution. Occasionally beasts can pass on traits they acquired over their lifetime. From Wikimedia Commons.
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
Categories: Research Spotlight
Tags: ChIP-seq, copper, copy number variants, CUP1-1, CUP1-2, Darwin, histone, Lamarck, replication fork, RTT109
How Histones Use FACT(s) to Find Their Way
July 05, 2017
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.
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
Categories: Research Spotlight