New & Noteworthy

SGD Summer 2014 Newsletter

July 28, 2014

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This Summer 2014 newsletter is also available on the community wiki. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.

Look for SGD at the Yeast Genetics Meeting in Seattle!

July 23, 2014

SGD staff will be attending the GSA Yeast Genetics Meeting in Seattle, July 29 – August 2, 2014 en force! We will be hosting a Workshop, Posters, and an Exhibit Table. The Workshop, “Computational Tools at SGD,” is on Thursday, July 31 at 1:30 PM in Kane Hall, Room 220. We will be discussing our powerful search tool, YeastMine, what’s new in the realm of Strains and Sequences, and new displays in SGD. Bring your questions and comments – we love feedback!

Follow @yeastgenome and #YEAST14 on Twitter for the latest research being presented at YGM.

Find these SGD staff members, as well as those presenting posters, at the Workshop and the Exhibit table:


Maria Costanzo
Workshop Speaker
Rob Nash
Rob Nash
Workshop Speaker
Ben Hitz
Ben Hitz

Workshop: “Computational Tools at SGD”

Thursday, July 31, 1:30 – 3:00 PM
Kane Hall, Room 220
Featured topics: YeastMine (our powerful search tool), Sequences and Strains update, New data displays at SGD

Posters

In addition to the Workshop, SGD curators will present 4 posters – please stop by and chat with us.

Poster
Date & Time
Poster Title
Presenter
318C Friday, August 1
7:30 – 8:30 PM
HUB Grand Ballroom
Defining the transcriptome of Saccharomyces cerevisiae Edith Wong
Edith Wong
387C Friday, August 1
8:30 – 9:30 PM
HUB Grand Ballroom
Yeast – it simply has a lot to say about human disease Selina Dwight
Selina Dwight
411C Friday, August 1
8:30 – 9:30 PM
HUB Grand Ballroom
Transcriptional regulation and protein complexes in budding yeast Stacia Engel
Stacia Engel
459C Friday, August 1
8:30 – 9:30 PM
HUB Grand Ballroom
Staying current and modern: Overhauling an actively-used model organism database website Kelley Paskov
Kelley Paskov

Exhibit Table

SGD will also have an exhibit table at the YGM. Come by to take a spin on our site, receive a prize for taking a survey, learn about various features of the database, and provide us with feedback as to what we can do to improve SGD. Look for us wearing our SuperBud fleece jackets, and feel free to flag any of us down!

Esa1p, the Balancing Artist

July 15, 2014

In the art of rock balancing, the artist positions large rocks with exquisite precision. If he or she succeeds, the rocks counterbalance each other and stay in seemingly impossible positions to make a surprising and beautiful sculpture. But a little uneven pressure is enough to make the whole thing collapse.

Esa1p keeps the acetylation state of the cell as precisely balanced as these rocks. Image from Wikimedia Commons

It turns out that the cellular acetylation state is just as precisely balanced. In a new GENETICS paper, Torres-Machorro and Pillus identify Esa1p, an acetyltransferase, as the balancing artist in Saccharomyces cerevisiae cells.

Acetylation is an important type of protein modification. Histones, the proteins that interact with DNA to provide structure to chromosomes, are acetylated by histone acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs). Some HATs and HDACs also act on non-histone proteins.

The acetylation state in a cell is a dynamic process.  All those HATs are adding acetyl groups at the same time that HDACs are removing them.  The final level of acetylation depends on the activities of each of these classes of proteins.

Acetylation of histones has been associated with increases in gene expression and deacetylation with decreases.  So to keep gene expression levels in balance, it is very important to keep acetylation balanced as well.  Throwing acetylation patterns just a bit out of whack can have profound consequences on global gene expression that can ultimately lead to cell death. 

The authors focused on one particular HAT, Esa1p, that acetylates histones H4 and H2A and also has non-histone targets. They were intrigued by the fact that yeast cells cannot survive without Esa1p, since no other HAT or HDAC subunit is essential in yeast.

An obvious explanation for lethality is that losing this protein leads to too low a level of acetylation.  They reasoned that if they also knocked out an HDAC, then the overall acetylation levels might increase and so rescue the esa1 null mutant.  And they were right.

Using a plasmid-shuffling method, they created various double mutant strains of esa1 and HDAC genes, and found that a strain that was mutant in esa1 and also in either the SDS3 or DEP1 genes was viable. SDS3 and DEP1 both encode subunits of the Rpd3L HDAC complex.

Torres-Machorro and Pillus next characterized the esa1 sds3 double mutant further.  They found that although the sds3 mutation suppressed the inviability of the esa1 mutant, it did not suppress other phenotypes such as sensitivity to high temperature and DNA damaging agents.

The authors found that the sds3 mutation subtly increased histone H4 acetylation, which was low in the absence of Esa1p.  However, acetylation levels of a different histone, H3, remained high even in the absence of Esa1p. This suggested that the fundamental problem in the esa1 null mutant was an imbalance in the global state of histone acetylation.

To test this hypothesis, the researchers used a variety of different genetic methods to tweak the balance of cellular acetylation in the esa1 sds3 mutant. They created mutations in histones H3 and H4 that made it seem as if acetylation was low or high, and they also mutated other genes for HDAC subunits. It is as if they were passers-by who decided to poke at a balanced rock sculpture to see what it took to bring the whole thing down.

Although the details are too numerous to report here, the results showed that by using these genetic methods to tweak the overall acetylation state of the cell, the fitness of the esa1 sds3 strain could be improved: phenotypes such as slow growth, sensitivity to high temperature or DNA damaging agents, or cell cycle defects were suppressed to some extent by the various manipulations.  This lends support to the hypothesis that Esa1p is the master balancer of acetylation levels in the cell and that this is its essential function.

This balancing act may happen in human cells too. Esa1p has a human ortholog, TIP60, that has been implicated in cancer and other diseases. Like Esa1p, TIP60 is essential and is involved in the DNA damage response.

So yeast teaches us that the acetylation of proteins is balanced on a knife’s edge.  Even the slightest changes can lead to a collapse in global gene regulation, which can have catastrophic effects like cancer. All that we learn about Esa1p, the acetylation balancing artist, may have much broader implications for human health.

Adding Introns to Synthetic Biology’s Toolbox

July 3, 2014

As any good handyman knows, the more tools you have in your tool chest, the better the chance that you can find what you need to solve a problem.  The same goes for synthetic biologists.  The more parts they can mix and match, the more likely they are to engineer the exact level of gene expression they need.

Synthetic biologists have added introns to their tool chest. Image from Wikimedia Commons

In the last few years synthetic biologists have amassed a wide variety of transcription and translation elements that can be combined in different ways to exquisitely tune the level of expression of their gene of interest.  And now, in a new study out in PLOS Genetics, Yofe and coworkers have added introns to the list of parts available for our favorite yeast Saccharomyces cerevisiae.

Yeast isn’t loaded with introns, but it does have a reasonable number that can be co-opted for synthetic biology.  The authors inserted 240 of these introns individually into the same position near the 5’ end of the yellow fluorescent protein (YFP) gene and monitored the level of fluorescence of each individual strain over a 24 hour period.  They chose the 5’ end of the gene because yeast has a bias for introns being located there.

The authors found that these reporters spanned a 100-fold range of gene expression, that every intron caused a decrease in the level of gene expression, and that even though many of these introns respond to environmental stimuli in their natural context, their effect on gene expression here was immune to the environmental changes the authors tested.  Taken together, these results suggest that introns could be used in yeast systems for dampening over-exuberant gene expression in ways that are independent of growth conditions.  If all of this holds up, introns will prove to be very useful tools indeed.

Yofe and coworkers next wanted to use this library to figure out some of the rules for why some introns cause lowered activity compared to others.  The simplest possibility, that longer introns cause a larger decrease in gene expression, turned out not to be true.  There was no correlation between the size of the intron and its effect on the level of fluorescence. 

Next they scanned the sequences of their constructs to look for elements that might increase or decrease splicing efficiency.  These splicing regulatory elements (SREs) are better understood in larger eukaryotes, but there is evidence that they are important in yeast as well.  The authors identified a number of intron splicing enhancers (ISEs) and intron splicing silencers (ISSs) that were highly enriched near the splice sites. 

To confirm that these sequences did in fact affect splicing efficiency (and hence gene expression), they showed that mutating the enhancer motif TTTATGCT to the silencer motif TTTGTGTA in two reporters resulted in a 22% and a 13% decrease in gene expression.  This proof of principle experiment suggests that future synthetic biologists may be able to further tweak the expression of their genes by manipulating these SREs.

In a final set of experiments the authors used the library to identify rules that can be used to predict how inserting various introns into different positions will affect a gene’s activity.  They found that the most important features were the presence of SREs and the RNA structures at the intron-exon junction.  Synthetic biologists should be able to use these rules to intelligently design their reporter systems.

These experiments are the first step towards adding introns to the ever growing set of tools available to synthetic biologists for modulating gene expression.  We are getting closer to figuring out how genes are controlled and being able to use that knowledge to our advantage.  Or to put it another way, we have taken another baby step towards being able to control a gene as well as a yeast cell does.

Next Page »