New & Noteworthy
June 5, 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”.
May 1, 2013
When a cell goes cancerous, its chromosomes get seriously messed up. Pieces get deleted, duplicated, mixed and matched. One of the worst things that can happen, in terms of a cell keeping its chromosomes together, is that a chromosome ends up with two centromeres.
A centromere is the part of a chromosome that gets attached to the spindle so it can be moved to the right place during cell division. When there are two centromeres, both get attached and something has to give if the chromosome is pulled in two different directions. Often this means that the DNA of the chromosome breaks between the two centromeres.
This isn’t as simple as the rope breaking during a tug-of-war, though. A chromosome can withstand around 480 piconewtons of force before breaking, but the force exerted by the spindle that breaks the chromosome between the centromeres is just one piconewton or less. Clearly something else is going on to create those breaks!
In a new study out in GENETICS, Song and coworkers looked more closely at what happens when a dicentric chromosome breaks. They used a diploid strain of S. cerevisiae to show that where the DNA breaks is not random. In their experiments, the break tended to happen within 10 kilobases (kb) of the “foreign” centromere.
They used a previously described system where a conditional centromere was placed 50 kb from the normal centromere on chromosome III. This conditional centromere is only turned on in the absence of galactose. They then mated this strain to an unrelated one, resulting in a diploid with a high degree of heterozygosity. In other words, the chromosomes from each strain were different at lots of different places.
Song and coworkers streaked diploids from isolated colonies to a plate lacking galactose and then investigated how the yeast managed to resolve its double centromere issue. Two key ways that the yeast could eliminate the additional centromere involve crossing over between sister chromatids or break-induced repair. They focused on these as it is relatively easy to identify the DNA breakpoint. Because the two chromosomes in each pair are so different, they just needed to look for a loss of heterozygosity. In other words, where did the chromosomes become the same?
When they looked through 27 colonies, they found that the breaks weren’t randomly spread between the centromeres. Surprisingly, about half of them happened very near the conditional centromere. To make sure that there wasn’t something special about these sequences, they looked at two different strains with the conditional centromere located in different places on chromosome V instead of III. They obtained similar results.
Since the force exerted by the spindle isn’t enough to break the chromosome, there must be enzymes involved in creating the DNA breaks. But why do they prefer the region near the conditional centromere? One possibility is that the DNA there is stretched and is more open to enzymes. As the chromosome is being pulled apart, an enzyme gets into this region and manages to cut the DNA.
Although we don’t have time to go into it here, the paper also has a lot to say about the variety of ways that a diploid cell resolves its extra centromere in a way that allows it to survive. And that will inform the study of chromosome dynamics in all kinds of cells.
March 21, 2013
At first our favorite small eukaryote, S. cerevisiae, might not seem like a great model for cancer studies. After all, budding yeast can’t tell us anything about some of the pathways that go wrong in cancer, like growth factor signaling. And it clearly can’t help explain what happens in specific tissues of the human body. But in other ways, it actually turns out to be a great model.
For example, all the details of cell cycle control were originally worked out in yeast. And now a whole new batch of genes has been found that influence a phenomenon, chromosome instability (CIN), that is important in both yeast and cancer cells.
As the name implies, chromosomes are unstable in cells suffering from CIN. Big chunks of DNA are lost, or break off and fuse to different chromosomes, turning the genome into an aneuploid mess. And this mess has consequences.
CIN can cause new mutations or make old ones have a stronger effect. Eventually these mutations can affect genes that are important for keeping a cell’s growth in line. Once these are compromised, a tumor cell is born.
Since CIN is pretty common in yeast, we might be able to better understand it in cancer cells by studying it in yeast. The Hieter lab at the University of British Columbia has come up with a powerful screen to get yeast to confess why it CINs.
A previous study from the group set the stage by finding a large group of mutants that have CIN phenotypes, implying that those genes are involved in keeping chromosome structure stable. In a new paper in G3: Genes, Genomes, Genetics, van Pel et al. uncovered the network of interactions among the genes in this set, using synthetic genetic array (SGA) technology. And they confirmed that the human homologs of some of these genes interact in the same way as in yeast, making them potential targets for cancer therapies.
The idea behind SGA studies is that if two proteins are involved in the same process, then a strain carrying mutations in both of their genes will be much worse off than a strain carrying either single mutation. In the worst case, the double mutant will be dead. This is known as a synthetic lethal interaction.
Yeast is a great model for doing these sorts of studies on a very large scale. We can construct networks showing how lots of different genes interact, and most importantly, find the genes that are central to many interactions. These “hubs” are likely to be the key players in those processes.
The researchers looked specifically for interactions between genes that are involved with CIN in yeast and are also similar to human cancer-related genes. They came up with various interaction hubs that will be interesting research subjects for a long time to come. In this study, they focused on one of these: genes involved with the DNA replication fork.
One of these in particular, CTF4, is a hub for both physical and genetic interactions. Unfortunately, Ctf4p doesn’t look like a good target for chemotherapy. It’s thought to act as a scaffold, and lacks any known activity that could potentially be inhibited by a drug. However, the interaction network around CTF4 that van Pel et al. uncovered suggests another way to target this hub. If a gene that interacts with CTF4 itself has a synthetic lethal interaction with another gene, and we could re-create the synthetic lethal phenotype in a cancer cell, we might be able to knock out the whole process. And that is just what they found in human cells.
First the authors identified a couple of human genes that were predicted from the yeast screen to be close to human CTF4 in the interaction network and to have a synthetic lethal interaction with each other. They then lowered the expression of one using small interfering RNA (siRNA), and reduced the activity of the other with a known inhibitor. Neither treatment alone had much effect, but combining them significantly reduced cell viability.
Since cancer cells frequently carry mutations in CIN genes, it should be possible to create a synthetic lethal interaction, guided by the yeast interaction network, where one partner is mutated in cancer cells (equivalent to using siRNA in this study) and the other partner is inhibited with a drug. Since it relies on a cancer-specific mutation, this approach has the potential to selectively target cancer cells while not disturbing normal cells, the ultimate goal for chemotherapy.
March 5, 2013
Cancer often gets going with chromosome instability. Basically a cell gets a mutation that causes its chromosomes to mutate at a higher rate. Now it and any cells that come from it build mutations faster and faster until they hit on the right combination to make the cell cancerous. An accelerating avalanche of mutations has led to cancer.
There are plenty of obvious candidates for the genes that start these avalanches: genes like those involved in segregating chromosomes and repairing DNA, for example. But there are undoubtedly sleeper genes that no one has really thought of. In a new study out in GENETICS, Minaker and coworkers have used the yeast S. cerevisiae to identify three of these genes — GPN1 (previously named NPA3), GPN2, and GPN3.
A mutation in any one of these genes leads to chromosomal problems. For example, mutations in GPN1 and GPN2 cause defects in sister chromatid cohesion and mutations in GPN3 confer a visible chromosome transmission defect. All of the mutants also show increased sensitivity to hydroxyurea and ultraviolet light, two potent mutagens. And if two of the genes are mutated at once, these defects become more severe. Clearly, mutating GPN1, GPN2, and/or GPN3 leads to an increased risk for even more mutations!
What makes this surprising is what these genes actually do in a cell. They are responsible for getting RNA polymerase II (RNAPII) and RNA polymerase III (RNAPIII) into the nucleus and assembled properly. This was known before for GPN1, but here the authors show that in gpn2 and gpn3 mutants, RNAPII and RNAPIII subunits also fail to get into the nucleus. Genetic and physical interactions between all three GPN proteins suggest that they work together in overlapping ways to get enough RNAPII and RNAPIII chugging away in the nucleus.
So it looks like having too little RNAPII and RNAPIII in the nucleus causes chromosome instability. This is consistent with previous work that shows that mutations in many of the RNAPII subunits have similar effects. Still, these genes would not be the first ones most scientists would look at when trying to find causes of chromosomal instability. Score another point for unbiased screens in yeast leading to a better understanding of human disease.
June 1, 2012
One reason cancer is so tricky to treat has to do with its adaptability. It can quickly try out new genetic combinations until it hits upon one that can survive whatever treatment a doctor is currently throwing at it. The result is return of the cancer after remission.
One way cancer is able to change its genetics so rapidly has to do with chromosome instability. The number of chromosomes in a cancer cell is much less stable than in a normal cell. This allows the cancer cell to constantly explore a wide range of chromosomal combinations.
It is still an open question how this dynamic instability happens. The gene-centric theory suggests that mutations in key genes are the main driving force. The chromosome-centric model says that having the wrong number of chromosomes is the critical component.
Distinguishing between these two models using cancer cells has proven difficult because these cells always have mutated genes. There is simply no way to look at just chromosome numbers in this system. This is where yeast can help.
In a recent paper published in PLoS Genetics, Zhu and coworkers used yeast to explore whether altered chromosome number was sufficient to explain chromosome instability. They found that chromosome numbers alone can explain some but not all of chromosomal instability.
The authors created various chromosomal combinations in yeast by sporulating isogenic triploid yeast cells. These cells had different numbers of genetically identical chromosomes. They then explored the stability of each chromosome number combination using both FACS and qPCR.
What they found was that chromosome number certainly impacted chromosomal stability. Chromosome number became less and less stable as the chromosome number veered further and further from the haploid state. Of course, once the cells became diploid, stability returned.
The authors explain this with the idea that there is only so much cellular machinery to move chromosomes to the proper place during mitosis. As more and more chromosomes are added to the cell, the machinery becomes increasingly taxed, resulting in more and more errors.
But once the diploid state is reached, all the genes are present to make twice as much mitotic machinery. Now stable chromosome segregation can happen.
This was the broad pattern Zhu and coworkers observed but it certainly wasn’t the whole story. The authors found islands of stability in the chromosomal chaos.
For example, very often when there were equal numbers of chromosome VII (ChrVII) and chromosome X (ChrX), the chromosome number was more stable than predicted. They explored this further and found evidence that suggested that at least part of this was due to the MAD1 gene on ChrVII and the MAD2 gene on ChrX.
Stable chromosome numbers required that these genes be present in a 1:1 ratio. Once the ratio strayed from one, chromosomal instability increased. But these genes don’t explain everything. There were unstable combinations where the MAD1/MAD2 ratio was correct. As might be expected, there are other gene combinations that can lead to instability as well.
So incorrect chromosome number alone can explain the chromosomal instability seen in cancer cells. But genes clearly play a role too, as evidenced by the islands of stability and the MAD1 gene and MAD2 genes. As usual, reality is probably a combination of the two models.
So it looks like chromosome number does play an important role in chromosomal instability. Too many chromosomes may overtax the mitotic machinery so that chromosomes end up mis-segregated.