New & Noteworthy

Role of Npl3p in regulating senescence rates

May 06, 2022

Telomeres, regions of repetitive DNA at the terminal ends of linear chromosomes, function as protective caps essential to maintain chromosomal structural integrity. Telomeres shorten with every cell cycle and eventually activate replicative senescence (a checkpoint-mediated cell cycle arrest) once they reach a critical length. Regulation of telomere length is essential as an uncontrolled shortening of telomeres can cause organismal aging, and the inability to trigger senescence can result in tumor development. In budding yeast, regulatory factors such as TERRA (telomere repeat containing RNA), a RNAPII-transcribed long non-coding RNA at all telomeres, and R-loops promote Homology-Directed Repair (HDR) at critically short telomeres. Whereas at long telomeres, RNase H2 and Rat1p are recruited and function to degrade TERRA and R loops during S phase.

An interesting new study by Perez-Martinez L et al. in EMBO identifies a telomere-binding protein, Npl3p, to stabilize R-loops at critically short telomeres to prevent premature senescence. The authors propose that at short telomeres, TERRA recruits Npl3p, and the bound protein stabilizes R-loops by limiting access to degrading enzymes like Rat1p and RNAse H2. As a result, the stabilized R-loops promote HDR, and telomeres are elongated to prevent early senescence. Conversely, the absence of Npl3p causes R-loop instability and a defective HDR mechanism, unable to constrain the senescence rates.

The authors additionally show that like TERRA and R-loops, Npl3p levels are regulated in a cell-cycle-dependent manner, with increased accumulation during the early S phase followed by a decline in the late S phase. The study also points to the fact that several proteins or complexes, including Npl3p and Tlc1p, accumulate strongly at short telomeres and in the presence of RNAse A and RNAse H, this interaction is lost.

The study highlights Npl3p as an essential factor in studying the diseases associated with dysregulation of telomere length and senescence rates.

Categories: Research Spotlight

Tags: Homology-Directed Repair, Saccharomyces cerevisiae, senescence, telomere, telomere length

The Sounds of Silencing

June 17, 2015

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For centuries, we thought of the universe as an empty, eerily silent place. Turns out we were dead on when it came to the emptiness, not so much when it came to the silence.

Once we invented devices that could detect electromagnetic radiation—starting with the Tesla coil receiver in the 1890s—we began to realize what a noisy place the universe really is. And now with modern radio telescopes becoming more and more sensitive, we know there is a cacophony of signals out there (although the Search for Extraterrestrial Intelligence has yet to find any non-random patterns).

The ends of chromosomes, telomeres, have also long thought to be largely silent in terms of gene expression. But a new paper in GENETICS by Ellahi and colleagues challenges that idea. 

Much like surveying the universe with a high-powered radio telescope, the researchers used modern techniques to make a comprehensive survey of the telomeric landscape–and saw that the genes were not so silent. Their work revealed that there’s a lot more gene expression going on at telomeres than we thought before.

It also gave us some fascinating insights into the role of the Sir proteins, founding members of the conserved sirtuin family that is implicated in aging and cancer.

Telomeres are special structures that “cap” the ends of linear chromosomes to protect the genes near the ends from being lost during DNA replication, something like aglets, those plastic tips that keep the ends of your shoelaces from fraying. They have characteristic DNA sequence elements that we don’t have space to describe here (but you can find a short summary in SGD).

Classical genetics experiments in Drosophila fruit flies showed that telomeres had a silencing effect on the genes near them, and early work in yeast seemed to confirm this. Reporter genes became transcriptionally silenced when they were placed near artificial constructs that mimicked telomere sequences.

This early work was solid, but had a few limitations.  The artificial telomere constructs were, well, artificial; some of the reporter genes encoded enzymes that had an effect on overall cellular metabolism, such as Ura3; and the studies tended to look at just one or a few telomeres.

To get the whole story, Ellahi and colleagues decided to look very carefully at the telomeric universe of S. cerevisiae. First, they used ChIP-seq to look at the physical locations of three proteins, Sir2, Sir3, and Sir4, on chromosomes near the telomeres.

These proteins, first characterized and named Silent Information Regulators for their role in silencing yeast’s mating type cassettes, had been seen to also mediate telomeric silencing. Scientists had hypothesized that they might be present at telomeres in a gradient, strongly repressing genes close to the chromosomal ends and petering out with increasing distance from the telomere. 

Ellahi and coworkers re-analyzed recent ChIP-seq data from their group to find where the Sir proteins were binding within the first and last 20 kb regions of every chromosome. These 20 kb regions included the telomere and the so-called subtelomeric region where genes are thought to be silenced. They found all three Sir proteins at all 32 natural telomeres.

However, the Sir proteins were not uniformly distributed across the telomeres, but rather occupied distinct positions. Typically, all three were in the same position, as would be expected since they form a complex. And they were definitely not in a gradient along the telomere.

Next the researchers asked whether gene expression was truly silenced in that subtelomeric region. They used mRNA-seq to measure gene expression from the ends of chromosomes in wild type or sir2, sir3, or sir4 null mutants.

They found that contrary to expectations, there is actually a lot of transcription going on near telomeres, even in the closest 5 kb region. The levels are lower than in other parts of the genome, but that can be partly explained by the fact that open reading frames are less dense in these regions. And only 6% of genes are silenced in a Sir-dependent manner.

The sensitivity of mRNA-seq allowed Ellahi and colleagues to uncover new patterns of gene expression in this work. They were able to detect very low-level transcription from some of the telomeric repetitive elements. Also, because the SIR genes are involved in mating type regulation, the mRNA-seq data from the sir mutants revealed a whole new set of genes that are differentially expressed in different cell types (haploids of mating types a and α, or a/α diploids).

The researchers point out that their work raises the question of why the cell would use the Sir proteins to repress transcription of a few subtelomeric genes. Wouldn’t it be more straightforward if these genes just had weaker promoters to keep their expression low?

They hypothesize that Sir repression could actually be part of a stress response mechanism, allowing a few important genes to be turned on strongly when needed. This idea could have intriguing implications for the role of Sir family proteins in aging and cancer in larger organisms. 

So, neither the universe nor the ends of our chromosomes are as silent as we thought. But unlike the disappointed SETI researchers, biologists studying everything from yeast to humans can now build on this large quantity of meaningful data from S. cerevisiae telomeres. 

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, silencing, telomere

Telomerator is the Chromosomal Terminator

December 10, 2014

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A simple tool for adding telomeres to linear DNA:

First there was the Terminator. Now, in a new study published in PNAS by Mitchell and Boeke, we have the telomerator.

Instead of being a homicidal robot from the future bent on killing Sarah Connor, the telomerator is a tool that lets scientists easily turn circular DNA into stable chromosomes in the yeast Saccharomyces cerevisiae. While less splashy, this bit of synthetic biology is definitely cool in its own way (and much less dangerous!).

The system that Mitchell and Boeke created is very clever. They first inserted the intron from the ACT1 gene into the middle of the URA3 gene. The URA3 gene was still functional, as ura3 mutants could use it to grow on medium lacking uracil.  

They next inserted a sequence into the middle of the intron that consisted of an 18 base pair I-SceI cleavage site flanked on each side by around 40 base pairs of yeast telomere repeats (called Telomere Seed Sequences or TeSSs). This construct still allowed ura3 mutants to grow in the absence of added uracil.

The final step was to introduce the homing endonuclease I-SceI to the cell so that it cut the circular DNA precisely between the two TeSSs. The idea is that when you add the homing endonuclease, the newly linearized piece of DNA ends up with the telomere seeds on each end. Telomerase adds more repeats to the seeds until the DNA has proper telomeres. Voilà, a chromosome is born.

The URA3 gene part of the plasmid is important for selecting cells with the linearized DNA. Basically a circularized DNA will grow on medium lacking uracil but fail to grow on medium with 5-FOA, while the linearized DNA will do the opposite. In other words, the process of linearization should destroy the URA3 gene. And that’s just what they found.

Previous work had shown that to be stable in yeast, a chromosome needs to be at least 90 kilobases (kb) or so long. This is why they tested their new telomerator in synIXR, a synthetic yeast chromosome that is about 100 kb in length. This chromosome has 52 genes from the right arm of chromosome 9, two genes from the left arm, around 10 kb of nonessential BAC DNA, the native centromere CEN9, and a LEU2 marker. 

Mitchell and Boeke inserted the telomerator sequence into two different locations in the BAC part of the circularized synIXR and found that adding I-SceI appeared to linearize the DNA. In both cases they found that around 100 out of 200 cells were resistant to 5-FOA and unable to grow in the absence of uracil but could still grow in the absence of leucine. This is just what we would predict if we cut the DNA in the middle of the URA3 gene and created a stable piece of linear DNA.

They next wanted to use this tool to study the effects of telomeric DNA on nearby genes. We would predict that because of telomeric silencing, genes near a telomere will be downregulated. Any genes that affect growth when turned down should quickly become evident.

To accomplish this they inserted the telomerator three base pairs downstream of each of the 54 genes on synIXR, generating 54 new plasmids. After activating the telomerator by expressing the I-SceI nuclease, they used pulsed field gel electrophoresis to confirm that 51 of the 54 synthetic chromosomes had indeed been linearized.

As expected, they found that putting a telomere near a gene sometimes has profound effects. For example, when they linearized DNA where the telomerator was 3’ of either YIR014W, MRS1, or YIR020C-B, they got no growth. They also found many more effects on the growth rate at both 30° C and 37° C at many different, “telomerized” genes. The implication is that when these genes are near telomeric DNA, they no longer function at a high enough level for the yeast to grow well or in some cases to even survive.

To confirm that the effects they saw were due to telomeric silencing, Mitchell and Boeke tested each linearized DNA in a sir2 mutant, a key player in this form of silencing. Mutating sir2 reversed the effects of placing a telomere near the gene, further supporting the idea that the newly created chromosome ends are like normal telomeres because they undergo the same Sir2-mediated silencing.

Finally, the researchers tested the stability of the newly created chromosomes by selecting for Ura⁺ revertants from six individual cultures with different linearized molecules. They failed to select any revertants in which the DNA had recircularized, showing that the linear chromosomes are stable.

So in contrast to the Terminator, who sliced and diced his victims randomly, the telomerator will allow synthetic biologists to create linear chromosomes with precisely positioned telomeres. This study proved the concept, and this tool will be incredibly useful in the future, both in yeast and potentially in other eukaryotes. Both the Terminator and the telomerator can say, “I’ll be back”!

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

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, synthetic biology, telomere

Not too long, not too short

October 02, 2014

Have you ever noticed that the length of your hair is just right for a small window of time? Too short, and you feel a little exposed – too long, and it gets in your face.

Like your hair, the telomeres at the ends of your chromosomes have a length that is just right for them (and you). Telomeres are non-coding sequences of DNA added to the ends of chromosomes that protect the important DNA there from being lost. Telomeres that are too long may contribute to cancer; while telomeres that are too short are associated with aging.

It makes sense, then, that telomere length should be carefully regulated (just as, in a perfect world, you might want to keep your hair the perfect length all the time). The enzyme that adds the non-coding DNA sequences to the end of chromosomes, telomerase, is composed of highly conserved subunits. In yeast, this enzyme is a quaternary complex composed of the regulatory subunits Est1 and Est3, the catalytic subunit Est2, and the RNA template component, TLC1.

Previously, it was thought that telomerase was primed for action whenever it was needed in the cell. But this does not seem to be the case.

In a recent publication in Genes & Development, Tucey et al. showed that, in addition to the active quaternary telomerase complex, two subcomplexes – a preassembly complex and a disassembled complex – were also present in the cell. Each of these subcomplexes lacked one subunit compared with the active telomerase, and the missing subunit was different for each subcomplex.

So, how did the authors discover these subcomplexes? Using a special immunoprecipitation protocol, one of the first things the researchers noticed was that Est1, Est2, and the TLC1 RNA were associated with each other throughout G1 and S phase, but that Est3 was missing from the complex during G1 phase and present only at very low levels throughout S phase. They called this Est1-TLC1-Est2 complex the “preassembly complex.”

In fact, Est3 was only appreciably associated with the preassembly complex during G2/M and late in the cell cycle. And, even then, only 25% of the preassembly complex was associated with Est3 to form the active quaternary complex. So the presence of the active holoenzyme was regulated with respect to the cell cycle.

Since Est3 was not always bound to the preassembly complex, the authors set out to determine what was required for Est3 binding. They found that neither Est1 nor Est2 could bind Est3 if it was unable to bind TLC1 RNA. Thus, the RNA component of the presassembly complex was necessary for its two other subunits to form an active telomerase by binding to Est3.

In addition, they determined that both Est1 and Est2 contained binding surfaces for Est3, and that both of these surfaces were required for the full association of Est3 with the preassembly complex. So Est3 interacts directly with each of the other protein subunits to form the active telomerase.

There was still one more question that intrigued Tucey and coworkers – Est3 appeared to exist in excess compared with the other subunits, so why did it have such limited association with the rest of the telomerase subunits? This suggested some sort of inhibitory regulation of Est3.

Indeed, the authors uncovered an Est3 mutant, Est3-S113Y, which appeared to have lost the ability to be inhibited. This mutant exhibited elongated telomeres and increased association with telomerase, and was associated with the preassembly complex in G1 phase rather than being restricted to late in the cell cycle. This mutation lies directly adjacent to Est3’s binding domain, leading the authors to conclude that Est3 is regulated by a “toggle switch” that specifies whether or not it can bind to the preassembly complex.

As mentioned previously, the authors also saw evidence of a second subcomplex during their studies. When they drilled down on the components, they identified a “disassembly complex” that lacks only Est2, in contrast to the preassembly complex that lacks Est3. They determined that this subcomplex is inactive and requires the prior formation of the quaternary complex since its formation requires Est2 binding to TLC1, just as is observed for the preassembly complex.

Given the cell’s desire to have telomeres that aren’t too long and aren’t too short, it makes sense that the enzyme that lengthens them is regulated. There is a toggle switch that signals formation of the active complex and, once formed, the active complex is transient, dissociating its catalytic subunit to become inactive. This regulation ensures that telomerase is active only late in the cell cycle. Just as you might work to keep your hair just the right length, the cell regulates telomerase to keep the lengths of telomeres from getting out of control.

by Selina Dwight, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: aging, protein complex, telomerase, telomere

Affecting the Shelf Life of Chromosomes

December 19, 2013

Just like the chicken or milk you buy at a store, chromosomes have a shelf life too.  Of course, chromosomes don’t spoil because of growing bacteria.  Instead, they go bad because they lose a little of the telomeres at their ends each time they are copied.  Once these telomeres get too short, the chromosome stops working and the cell dies.

Turns out food and chromosomes have another thing in common—the rates of spoilage of both can be affected by their environment.   For example, we all know that chicken will last longer if you store it in a refrigerator and that it will go bad sooner if you leave it out on the counter on a hot day.  In a new study out in PLoS Genetics, Romano and coworkers show a variety of ways that the loss of telomeres can be slowed down or sped up in the yeast S. cerevisiae.  And importantly, they also show that some forms of environmental stress have no effect.

The authors looked at the effect of thirteen different environments on telomere length over 100-400 generations.  They found that caffeine, high temperature and low levels of hydroxyurea lead to shortened telomeres, while alcohol and acetic acid lead to longer telomeres.  It seems that for a long life, yeast should lay off the espresso and and try to avoid fevers, while enjoying those martinis and sauerbraten.

Romano and coworkers also found a number of conditions that had no effect on telomere length, with the most significant being oxidative stress.  In contrast, previous studies in humans had suggested that the oxidative stress associated with emotional stress contributed to increased telomere loss; given these results, this may need to be looked at again.  In any event, yeast can deal with the stresses of modern life with little or no impact on their telomere length.

The authors next set out to identify the genes that are impacted by these stressors.  They focused on four different conditions—two that led to decreased telomere length, high temperature and caffeine, one that led to longer telomeres, ethanol, and one that had no effect, hydrogen peroxide.  As a first step they identified key genes by comparing genome-wide transcript levels under each condition.  They then went on to look at the effect of each stressor on strains deleted for each of the genes they identified.

Not surprisingly, the most important genes were those involved with the enzyme telomerase.  This enzyme is responsible for adding to the telomeres at the ends of chromosomes.  Without something like this, eukaryotes, with their linear chromosomes, would have disappeared long ago.

A key gene they identified was RIF1, encoding a negative regulator of telomerase.  Deleting this gene led to decreased effects of ethanol and caffeine, suggesting that this gene is key to each stressor’s effects.  The same was not true of high temperature—the strain deleted for RIF1 responded normally to high temperature.  So high temperature works through a different mechanism.

Digging deeper into this pathway, Romano and coworkers found that Rap1p was the central player in ethanol’s ability to lengthen telomeres.  This makes sense, as the ability of Rif1p to negatively regulate telomerase depends upon its interaction with Rap1p. 

The increase in telomere length by ethanol was not just dependent on genes associated with telomerase either.  The authors identified a number of other genes involved, including DOA4, SNF7, and DID4. 

Caffeine, like ethanol, affected telomere length through Rif1p-Rap1p but with an opposite effect.  As caffeine is known to be an inhibitor of phosphatydylinositol-3 kinase related kinases, the authors looked at whether known kinases in the telomerase pathway were involved in caffeine-dependent telomere shortening.  They found that when they deleted both TEL1 and MEC1, caffeine no longer affected telomere length. 

The authors were not so lucky in their attempts to tease out the mechanism of the ability of high temperature to shorten telomeres.  They were not able to identify any single deletions that eliminated this effect of high temperature.

Whatever the mechanisms, the results presented in this study are important for a couple of different reasons.  First off, they obviously teach us more about how telomere length is maintained.  But this is more than a dry, academic finding.

Given that many of the 400 or so genes involved in maintaining telomere length are evolutionarily conserved, these results may also translate to humans too.  This matters because telomere length is involved in a number of diseases and aging.

Studies like this may help us identify novel genes to target in diseases like cancer.  And they may help us better understand how lifestyle choices can affect your telomeres and so your health.  So if you have a cup of coffee, be sure to spike it with alcohol! 

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

Categories: Research Spotlight

Tags: environmental stress, Saccharomyces cerevisiae, telomere

Alternative Ways to Increase a Cell’s Shelf Life

June 05, 2013

Like milk or eggs, most cells with linear chromosomes have a shelf life. Each time these cells divide, they lose a little off the end of their chromosomes. Eventually, too much is lost and the cells crap out. Or, to use a more scientific term, they become senescent.

But this is not the fate of every cell. Some cells, like those that go on to become sperm or eggs, use a reverse transcriptase called telomerase to extend their telomeres as part of their normal life cycle. And they aren’t the only ones. Around 85% of cancers hijack the telomerase and use it for their own nefarious ends.

The other 15% of cancers use a variety of different mechanisms to keep their telomeres from getting too short (Cesare and Reddel, 2010). All these different ways are lumped together in a single category called alternative lengthening of telomeres or ALT. The telomeres are lengthened in these cells by recombination with other telomeres, either those on other chromosomes or those that exist as shed, extrachromasomal bits. 

While telomere extension may keep cells alive, it can sometimes be a double-edged sword. A double stranded DNA break is usually recognized as DNA damage. However, if the break happens near a telomere seed (a sequence that looks like a telomere), then the DNA damage response can be suppressed and the end can be extended into a new telomere, in a process called chromosome healing. But now the cell could be in trouble, with new, partial chromosomes being created and getting pulled this way and that.

In a new study out in GENETICS, Lai and Heierhorst decided to investigate whether chromosome healing happens in yeast cells that have stayed alive because of ALT.  What they found was that chromosome healing at telomere seeds was suppressed in these post-senescence survivors.

They created these ALT dependent, post-senescence survivors from an est2 mutant strain that lacked the catalytic subunit of telomerase.  Without telomerase, the only way for these cells to survive is by using ALT. 

In the first experiment, they looked at whether the post-senescence survivors could create a new telomere by chromosome healing.  The authors used a galactose inducible HO endonuclease to create a double stranded break near an 81 base pair sequence known to be a telomere seed sequence in wild type. 

Broken DNA usually signals cells to pause the cell cycle until the damage is repaired. This is known as the DNA damage checkpoint. During chromosome healing in wild type, this checkpoint is suppressed so the chromosome break isn’t recognized as DNA damage.

In the post-senescence survivors, even after 21 hours there was no evidence of a telomere forming.  They didn’t suppress the DNA damage checkpoint either.

Lai and Heierhorst determined that these ALT-dependent cells could still repair a different break that was not near a telomere seed sequence. They just couldn’t repair the break at the telomere seed. And this wasn’t because the DNA damage checkpoint was active. When they prevented the checkpoint by using a rad53 mutant, the telomere still wasn’t repaired.

Instead, the post-senescence survivors eventually repaired the break by some other mechanism, generating lots of differing products in the process. When they repaired breaks at sites that were not telomere seeds, they were able to use homologous recombination. But homologous recombination was suppressed at the telomere seed site.

Since ALT is used in cancer cells, and happens most often in some of the least-curable types of cancer, whatever we can learn about the process in yeast is valuable. It may give us clues on how to change the expiration date of those cancer cells to “ASAP”.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, DNA damage checkpoint, Saccharomyces cerevisiae, telomere

When Heat is Not the Burning Issue

May 04, 2012

When is a temperature sensitive mutation not a temperature sensitive mutation? When the process being studied is affected by temperature too.

Mutations conferring temperature sensitivity – that is, a phenotype that only appears at higher- or lower-than-normal growth temperature due to the loss or alteration of function of a gene product at that temperature – have for decades been an invaluable tool in dissecting many biological processes in yeast and other model organisms. Now there is reason to question whether some temperature sensitive phenotypes might actually be the result of a more complex interaction between multiple genes.

This synthetic genetic interaction is easy to mistake for the effect of a single mutation. A scientist starts out with a mutation that weakens a gene in a certain process. Unfortunately for the scientist, the process being studied is itself weakened by higher temperatures. The effect of the higher temperature combines with the effect of the mutation to shut down the process. The mutation looks temperature sensitive even when it isn’t.

And this does not appear to be merely a theoretical concern. As Paschini and coworkers show in a new study out in GENETICS, something similar may have happened with key mutations used to study telomere function in yeast.

These researchers looked at several mutations but we’ll focus on the work they did with cdc13-1. A key experiment that had been previously done with this mutation dealt with the effects of the loss of cdc13 function in the absence of RAD9.

Basically, researchers had found that prolonged incubation of a cdc13-1/Δrad9 strain at 36° severely compromised viability. These results were used to infer CDC13 function based on its loss at 36°. However, Paschini and coworkers provide compelling data that cdc13-1 behaves equally poorly at 23° and 36°.

First off, they showed that prolonged incubation of wild type yeast at the temperatures used in these studies (36°) resulted in shorter telomeres. They found very little effect on telomere length at 32°.

Next they showed that biochemically, cdc13-1 didn’t behave like a temperature sensitive mutation. Strains with cdc13-1 produced around 4-fold less protein at both 23° and 36° and the protein that was made bound telomeres equally well at both temperatures.

They argue from these two pieces of data that the loss of viability comes from a combination of the compromised cdc13-1 mutation and the effects of higher temperature on telomere function. Something is going on in the rad9 experiment but it is not due to an increased loss of CDC13 function at higher temperatures. There is some other factor involved that is being inhibited.

Of course it could be that cdc13-1 still confers temperature sensitivity, but that they didn’t have the right biochemical assay to see it. To address this issue, they generated five new mutations in cdc13 that behaved more like traditional temperature sensitive mutations.

They focused on one, cdc13-S611L, that was compromised for protein production at temperatures of 32° and above. They then repeated the rad9 double mutant experiment at 32° and 36°. They found that viability was compromised only at 36° even though Cdc13p was equally absent at both 32° and 36°. These results suggest that the loss of viability at 36° is not only the result of cdc13-1.

If this and other results hold up, scientists will need to rethink what previous experiments meant and they may need to modify their models. This should also get other researchers thinking about their temperature sensitive mutations.  It is important to confirm biochemically that a mutation indeed makes a specific gene product temperature sensitive. Because sometimes even if it quacks, it isn’t a duck…

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

Categories: Research Spotlight

Tags: cdc13, synthetic lethal, telomere, temperature sensitive, yeast

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